US20160374949A9 - Peptide/particle delivery systems - Google Patents

Peptide/particle delivery systems Download PDF

Info

Publication number
US20160374949A9
US20160374949A9 US13/272,042 US201113272042A US2016374949A9 US 20160374949 A9 US20160374949 A9 US 20160374949A9 US 201113272042 A US201113272042 A US 201113272042A US 2016374949 A9 US2016374949 A9 US 2016374949A9
Authority
US
United States
Prior art keywords
seq
human
group
peptide
microparticle
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
US13/272,042
Other versions
US9717694B2 (en
US20120114759A1 (en
Inventor
Jordan Jamieson Green
Aleksander S. Popel
Joel Chaim Sunshine
Ron B. Shmueli
Stephany Yi Tzeng
Kristen Lynn Kozielski
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Johns Hopkins University
Original Assignee
Johns Hopkins University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from PCT/US2010/035127 external-priority patent/WO2010132879A2/en
Application filed by Johns Hopkins University filed Critical Johns Hopkins University
Priority to US13/272,042 priority Critical patent/US9717694B2/en
Assigned to THE JOHNS HOPKINS UNIVERSITY reassignment THE JOHNS HOPKINS UNIVERSITY ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: GREEN, JORDAN JAMIESON, SHMUELI, RON B., SUNSHINE, JOEL CHAIM, TZENG, STEPHANY YI, ZOZIELSKI, KRISTEN LYNN, POPEL, ALEKSANDER S.
Publication of US20120114759A1 publication Critical patent/US20120114759A1/en
Priority to US14/438,353 priority patent/US20190209690A9/en
Publication of US20160374949A9 publication Critical patent/US20160374949A9/en
Priority to US15/645,337 priority patent/US10786463B2/en
Publication of US9717694B2 publication Critical patent/US9717694B2/en
Application granted granted Critical
Assigned to NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT reassignment NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT CONFIRMATORY LICENSE (SEE DOCUMENT FOR DETAILS). Assignors: JOHNS HOPKINS UNIVERSITY
Priority to US17/149,583 priority patent/US20210220287A1/en
Active legal-status Critical Current
Adjusted expiration legal-status Critical

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5021—Organic macromolecular compounds
    • A61K9/5026—Organic macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T428/00—Stock material or miscellaneous articles
    • Y10T428/29—Coated or structually defined flake, particle, cell, strand, strand portion, rod, filament, macroscopic fiber or mass thereof
    • Y10T428/2982—Particulate matter [e.g., sphere, flake, etc.]

Definitions

  • Biomaterials have the potential to significantly impact medicine as delivery systems for imaging agents, biosensors, drugs, and genes.
  • the presently disclosed subject matter provides polymeric nanoparticles, microparticles, and gels for delivering cargo, e.g., a therapeutic agent, such as a peptide, to a target, e.g., a cell, and their use for treating multiple diseases, including angiogenesis-dependent diseases, such as age-related macular degeneration and cancer.
  • cargo e.g., a therapeutic agent, such as a peptide
  • target e.g., a cell
  • angiogenesis-dependent diseases such as age-related macular degeneration and cancer.
  • the presently disclosed subject matter provides a bioreducible, hydrolytically degradable polymer of formula (Ia):
  • n is an integer from 1 to 10,000;
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 are each independently selected from the group consisting of hydrogen, branched and unbranched alkyl, branched and unbranched alkenyl, branched and unbranched alkynyl, aryl, halogen, hydroxyl, alkoxy, carbamoyl, carboxyl ester, carbonyldioxyl, amide, thiohydroxyl, alkylthioether, amino, alkylamino, dialkylamino, trialkylamino, cyano, ureido, a substituted alkanoyl group, cyclic, cyclic aromatic, heterocyclic, and aromatic heterocyclic groups, each of which may be substituted with at least one substituent selected from the group consisting of branched or unbranched alkyl, branched and unbranched alkenyl, branched and unbranched alkynyl, amino,
  • R 1 can be present or absent and when present the compound of formula (I) further comprises a counter ion selected from the group consisting of chloride, fluoride, bromide, iodide, sulfate, nitrate, fumarate, acetate, carbonate, stearate, laurate, and oleate; and
  • At least one R comprises a backbone of a diacrylate having the following structure:
  • X 1 and X 2 are each independently substituted or unsubstituted C 2 -C 20 alkylene, and wherein each X 1 and X 2 can be the same or different.
  • the presently disclosed subject matter provides a nanoparticle, microparticle, or gel comprising a compound of formula (I):
  • n is an integer from 1 to 10,000;
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 are each independently selected from the group consisting of hydrogen, branched and unbranched alkyl, branched and unbranched alkenyl, branched and unbranched alkynyl, aryl, halogen, hydroxyl, alkoxy, carbamoyl, carboxyl ester, carbonyldioxyl, amide, thiohydroxyl, alkylthioether, amino, alkylamino, dialkylamino, trialkylamino, cyano, ureido, a substituted alkanoyl group, cyclic, cyclic aromatic, heterocyclic, and aromatic heterocyclic groups, each of which may be substituted with at least one substituent selected from the group consisting of branched or unbranched alkyl, branched and unbranched alkenyl, branched and unbranched alkynyl, amino,
  • R 1 can be present or absent and when present the compound of formula (I) further comprises a counter ion selected from the group consisting of chloride, fluoride, bromide, iodide, sulfate, nitrate, fumarate, acetate, carbonate, stearate, laurate, and oleate; and
  • R, R′, and R′′ comprise a reducible or degradable linkage, and wherein each R, R′, or R′′ can independently be the same or different;
  • the compound of formula (I) must also comprise one or more of the following characteristics:
  • each R group is different
  • each R′′ group is different
  • each R′′ group is not the same as any of R′, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 ;
  • the R′′ groups degrade through a different mechanism than the ester-containing R groups, wherein the degradation of the R′′ group is selected from the group consisting of a bioreducible mechanism or an enzymatically degradable mechanism; and/or
  • the compound of formula (I) comprises a substructure of a larger cross-linked polymer, wherein the larger cross-linked polymer comprises different properties from compound of formula (I);
  • an anti-angiogenic peptide selected from the group consisting of an anti-angiogenic peptide, an anti-lymphangiogenic peptide, an anti-tumorigenic peptide, and an anti-permeability peptide.
  • the presently disclosed subject matter provides a multilayer particle comprising a core and one or more layers, wherein the core comprises a material selected from the group consisting of a compound of formula (I), a gold nanoparticle, an inorganic nanoparticle, an organic polymer, and the one or more layers comprise a material selected from the group consisting of a compound of formula (I), an organic polymer, one or more peptides, and one or more additional biological agents.
  • the presently disclosed subject matter provides a microparticle comprising a compound of formula (I), poly(lactide-co-glycolide) (PLGA), or combinations thereof.
  • the presently disclosed subject matter provides a method for stabilizing a suspension of nanoparticles and/or microparticles of formula (I), the method comprising: (a) providing a suspension of nanoparticles and/or microparticles of formula (I); (b) admixing a lyroprotectant with the suspension; (c) freezing the suspension for a period of time; and (d) lyophilizing the suspension for a period of time.
  • the presently disclosed subject matter provides a pellet or scaffold comprising one or more lyophilized particle, wherein the one or more lyophilized particle comprises a compound of formula (I).
  • the presently disclosed subject matter provides a method of treating a disease or condition, the method comprising administering to a subject in need of treatment thereof a therapeutically effective amount of a nanoparticle, microparticle, gel, or multilayer particle comprising a compound of formula (I), wherein the nanoparticle, microparticle, gel, or multilayer particle further comprises a therapeutic agent specific for the disease or condition to be treated.
  • the disease or condition comprises an angiogenesis-dependent disease or condition, including, but not limited to, cancer and age-related macular degeneration.
  • the disease or condition is a non-angiogenic disease or condition.
  • the therapeutic agent encapsulated with the presently disclosed particles can be selected from the group consisting of gene, DNA, RNA, siRNA, miRNA, is RNA, agRNA, smRNA, a nucleic acid, a peptide, a protein, a chemotherapeutic agent, a hydrophobic drug, a small molecule drug, and combinations thereof.
  • FIG. 1 is an illustration of the presently disclosed multilayer particles
  • FIG. 2 is a scheme for producing hydrogels comprising the presently disclosed materials.
  • FIG. 3 shows a scheme for producing stable nanoparticle suspensions
  • FIGS. 4A-4D show representative polymer structures tuned to peptide cargos
  • FIGS. 5A and 5B show representative formation and sizing of polymer/peptide nanoparticles (by nanoparticle tracking analysis on a Nanosight LM10);
  • FIG. 6 shows DEAH peptide release by 336 nanoparticles at 4° C. (above) and 37° C. (below);
  • FIG. 7 shows HUVEC viability/proliferation assays with polymer/SP6001/DEAH peptide
  • FIG. 8 shows HUVEC migration assays with 336 polymer/DEAH peptide
  • FIG. 9 shows in vivo 336 polymer nanoparticle/SP6001 DEAH peptide
  • FIG. 10 shows (top) Particle size and (bottom) cell viability effects of various polymer/SP2012 nanoparticles as compared to peptide only of non-cytotoxic polymers
  • FIG. 11 shows polymer/peptide formulations for alternative peptides
  • FIG. 12 shows data for FITC-tagged bovine serum albumin (BSA) mixed with a macromer solution containing 10% (w/v) PEGDA (Mn ⁇ 270 Da) with various amounts of B4S4, dissolved in a 1:1 (v/v) mixture of DMSO and PBS;
  • BSA bovine serum albumin
  • FIG. 13 shows an SEM of increasing B4S4 from top [0.2% w/w] to bottom [5% w/w]);
  • FIG. 14 shows the size distribution of appropriately freeze-dried particles (bottom left, right-most histogram) remains the same as freshly-prepared particles (bottom left, left-most histogram). Freeze-dried particles also remain more stable in serum-containing medium than freshly-prepared particles (upper left). Using DNA-loaded nanoparticles, transfection efficiency is comparable between fresh particles and particles lyophilized with sucrose (right) even after 3 months of storage;
  • FIG. 15 is Left: brightfield+GFP+DsRed, showing presence of cells (green) being transfected with DsRed (red) on a bone scaffold (brightfield). Right: GFP and DsRed shown only;
  • FIG. 17 demonstrates the incorporation of DNA-loaded nanoparticles into natural and synthetic scaffolds, disks, microparticles, and hydrogels;
  • FIG. 18 demonstrates transfection of GFP + glioblastoma cells with scrambled (control) siRNA (top panels) or siRNA against GFP (bottom);
  • FIGS. 19A-19C show activity of R6-series polymers at delivering siRNA to knockdown GFP signal in GB cells; % Knockdown of GFP expression in GFP+ glioblastoma cells transfected with siRNA against GFP, normalized to cells transfected with scrambled siRNA, using various BR6 polymers as a transfection agent: (A) transfection with acrylate-terminated BR6 polymers with either S3, S4 or S5 as the side chain; (B) transfection with E10 end-capped versions of the polymers in Figure A; and (C) GFP fluorescence images of cells transfected with BR6-S4-Ac complexed scrambled RNA (top) vs. siRNA against GFP (bottom);
  • FIG. 20 shows gel retardation assay of siRNA with BR6-S5-E10 at varying ratios of polymer to RNA.
  • the polymer effectively retards siRNA (top), but in the presence of 5 mM glutathione siRNA is released immediately (bottom).
  • FIG. 21 shows that an E10-endcapped polymer (top) retards siRNA efficiently, but upon addition of 5 mM glutathione, siRNA is immediately released (bottom). Numbers refer to the w/w ratio of polymer-to-siRNA in all cases;
  • FIG. 22 shows that the same base polymer as shown in FIG. 25 with a different endcap (E7, 1-(3-aminopropyl)-4-methylpiperazine) also retards siRNA (top) but is not affected by application of glutathione (bottom);
  • E7 1-(3-aminopropyl)-4-methylpiperazine
  • FIG. 23 provides gel permeation chromatography data of BR6 polymerized with S4 at a BR6:S4 ratio of 1.2:1 at 90° C. for 24 hours, before and after end-capping with E7;
  • FIG. 24 shows that knockdown efficiency also is affected by molecular weight of the polymer.
  • 1.2:1, 1.1:1, and 1.05:1 refer to the ratio of reactants in the base polymer step growth reaction;
  • FIG. 25 demonstrates combined DNA (RFP) and siRNA delivery (against GFP) in GB;
  • FIG. 26 shows that siRNA knockdown is affected by the endcap (E), base polymer (increasing hydrophobicity from L to R within each E), and molecular weight (increasing L to R within each base polymer);
  • FIG. 27 shows 4410, 200 w/w (blue line on above graph), 8 days after transfection: Left: hMSCs treated with scrambled control; Right: hMSCs treated with siRNA;
  • FIG. 28 demonstrates that in variable molecular weight embodiments, polymer molecular weight is between 4.00-10.00 kDa for siRNA delivery;
  • FIG. 29 demonstrates the use of the presently disclosed materials for DNA delivery
  • FIG. 30 shows GB Transfection
  • FIG. 31 shows 551 GB cells cultured as neurospheres (undifferentiated).
  • FIG. 32 demonstrates that, for a DNA delivery application, in some embodiments, polymer molecular weight is between 3.00-10.0 kDa;
  • FIG. 33 provides representative characteristics exhibited by the presently disclosed biodegradable polymers
  • FIG. 34 demonstrates the delivery of DNA to GB bulk tumor cells for representative biomaterials
  • FIG. 35 demonstrates the transfection of genes to BCSC for representative presently disclosed biomaterials
  • FIG. 36 demonstrates the delivery of DNA to fetal (healthy) cells
  • FIG. 37 demonstrates the delivery of DNA to BCSCs
  • FIGS. 38 and 39 demonstrate the delivery of apoptosis-inducing genes in BCSCs
  • FIG. 40 shows that particles lyophilized with sucrose and used immediately are as effective in transfection as freshly prepared particles
  • FIGS. 41 and 42 demonstrate the use of the presently disclosed materials and methods for long-term gene delivery
  • FIG. 43 demonstrates siRNA delivery to GB cells
  • FIGS. 44 and 45 provide a comparison of siRNA vs. DNA delivery in GB cells
  • FIG. 46 depicts a strategy of combining nanoparticles within microparticles to extend release further.
  • PLGA or blends of PLGA with the presently disclosed polymers are used to form microparticles by single or double emulsion;
  • FIG. 47 shows DEAH-FITC release from microparticles comprising a presently disclosed polymer and a peptide
  • FIG. 48 shows slow extended release from microparticles containing nanoparticles that contain peptides
  • FIG. 49 shows in vivo effects of microparticle formulations in both the CNV and rho/VEGF model over time.
  • compositions of matter, methods of formulation, and methods of treatment utilizing drug delivery systems comprising one or more degradable polymers and one or more biological agents.
  • the polymers described in these systems must be biodegradable. Mechanisms for this degradability include, but are not limited to, hydrolytic degradation, enzymatic degradation, and disulfide reduction.
  • the biological agents described in these systems include, but are not limited to, therapeutic or diagnostic agents, such as small molecules, peptides, proteins, DNA, siRNA, miRNA, is RNA, contrast agents, and other agents one skilled in the field would wish to encapsulate.
  • therapeutic or diagnostic agents such as small molecules, peptides, proteins, DNA, siRNA, miRNA, is RNA, contrast agents, and other agents one skilled in the field would wish to encapsulate.
  • biological therapeutic agents that are sensitive to degradation and sized approximately 10,000-25,000 Da, including siRNA and peptides, are suitable for use with the presently disclosed materials.
  • Peptide drugs in polymeric delivery systems are useful for various therapeutic and diagnostic applications. Some embodiments of the presently disclosed subject matter are useful for treating angiogenesis-dependent diseases including, but not limited to, age-related macular degeneration (AMD) and cancer.
  • AMD age-related macular degeneration
  • One particular embodiment of the presently disclosed subject matter includes specific peptide sequences, as well as methods of formulating, stabilizing, and administering these peptides as single agents or as combinations of peptides via polymeric nanoparticle-based, microparticle-based, gel-based, or conjugate-based delivery systems.
  • the presently disclosed nanoparticles, microparticles, and gels can be used to deliver cargo, for example a therapeutic agent, such as a peptide or protein, to a target, for example, a cell.
  • a therapeutic agent such as a peptide or protein
  • the cargo delivered by the presently disclosed nanoparticles, microparticles, and gels can act, in some embodiments, as a therapeutic agent or a biosensor agent.
  • Combinations of polymeric materials and cargo, for example a single peptide or combination of peptides can be formulated by the presently disclosed methods, which allows for the control, or tuning, of the time scale for delivery.
  • the presently disclosed polymeric materials can be used to form self-assembled electrostatic complexes, micelles, polymersomes, emulsion-based particles, and other particle formulations known to one of ordinary skill in the art. Nanoparticles formed from the presently disclosed polymeric materials can be formulated into larger microparticles to further extend duration and timing of release. Lyophilized formulations that can maintain longer shelf life and stability also are described.
  • the presently disclosed particles can be administered as a powder, cream, ointment, implant, or other reservoir device.
  • the presently disclosed nanoparticles, microparticles, and gels can be used to treat many diseases and conditions including, but not limited to, all types of cancers, ophthalmic diseases, cardiovascular diseases, and the like.
  • the disease or condition treated by the presently disclosed nanoparticles, microparticles, and gels include breast cancer and age-related macular degeneration.
  • the presently disclosed materials offer several advantages for use in delivering cargo, e.g., a therapeutic agent, such as a peptide or siRNA, to a target, e.g., a cell. Such advantages include a slower degradation in the extracellular environment and a quicker degradation in the intracellular environment. Further, the method of synthesis allows for diversity of monomer starting materials and corresponding facile permutations of polymer structure.
  • the presently disclosed materials can be used to form self-assembled nanoparticles, blended microparticles, gels, and bioconjugates.
  • the presently disclosed polymers also have the following advantages compared to other drug delivery polymers known in the art: a higher polymerization than with disulfide acrylamides, which is important for various applications because it can be used to tune both binding/encapsulation and release; two time scales for degradation (hydrolytic degradation in water and disulfide reduction due to glutathione inside the cell), which facilitates drug release and reduces potential cytotoxicity; tunable structural diversity, with hydrophobic, hydrophilic, and charged moieties to aid in encapsulation of a target biological agent; and, usefulness for drug delivery, including high siRNA delivery, even without end-modification of the polymer.
  • polyesters have been shown previously to form nanoparticles in the presence of biological agents, such as nucleic acids, and facilitate their entry into a cell. In such materials, release of the nucleic acid is modulated by hydrolytic degradation of the polyester polymer.
  • the addition of a bioreducible disulfide moiety into the backbone of these polymers can specifically target release to the reducing intracellular environment.
  • a library of bioreducible polyesters can be synthesized by oxidizing and acrylating various mercapto-alcohols (representative diacrylates formed from the presently disclosed synthetic process are shown in Scheme 1 below), then reacting with amine side chains.
  • amine-containing molecules can be reacted to terminal groups of the polymer.
  • this amine-containing molecule also contains poly(ethylene glycol) (PEG) or a targeting ligand.
  • PEG poly(ethylene glycol)
  • the disulfide acrylates are not reacted with amines, but are instead polymerized through other mechanisms including, but not limited to, free radical polymerization to form network polymers and gels.
  • oligomers are first formed and then the oligomers are polymerized to form block co-polymers or gels.
  • n is an integer from 1 to 10,000;
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 are each independently selected from the group consisting of hydrogen, branched and unbranched alkyl, branched and unbranched alkenyl, branched and unbranched alkynyl, aryl, halogen, hydroxyl, alkoxy, carbamoyl, carboxyl ester, carbonyldioxyl, amide, thiohydroxyl, alkylthioether, amino, alkylamino, dialkylamino, trialkylamino, cyano, ureido, a substituted alkanoyl group, cyclic, cyclic aromatic, heterocyclic, and aromatic heterocyclic groups, each of which may be substituted with at least one substituent selected from the group consisting of branched or unbranched alkyl, branched and unbranched alkenyl, branched and unbranched alkynyl, amino,
  • R 1 can be present or absent and when present the compound of formula (I) further comprises a counter ion selected from the group consisting of chloride, fluoride, bromide, iodide, sulfate, nitrate, fumarate, acetate, carbonate, stearate, laurate, and oleate; and
  • At least one R comprises a backbone of a diacrylate having the following structure:
  • X 1 and X 2 are each independently substituted or unsubstituted C 2 -C 20 alkylene, and wherein each X 1 and X 2 can be the same or different.
  • the bioreducible, hydrolytically degradable polymer of claim 1 wherein at least one R comprises a backbone of a diacrylate selected from the group consisting of:
  • R, R′, and R′′ groups are defined immediately herein below as for compounds disclosed in International PCT Patent Application Publication No. WO/2010/132879 for “Multicomponent Degradable Cationic Polymers,” to Green et al., which is incorporated herein by reference in its entirety.
  • Multicomponent degradable cationic polymers suitable for the delivery of peptides to a target are disclosed in International PCT Patent Application Publication No. WO/2010/132879 for “Multicomponent Degradable Cationic Polymers,” to Green et al., which is incorporated herein by reference in its entirety.
  • Such polymers in addition to the presently disclosed polymers can be used to deliver cargo, e.g., a therapeutic agent, to a target, e.g., a cell.
  • the presently disclosed subject matter generally provides multicomponent degradable cationic polymers.
  • the presently disclosed polymers have the property of biphasic degradation. Modifications to the polymer structure can result in a change in the release of therapeutic agents, which can occur over multiple time scales.
  • the presently disclosed polymers include a minority structure, e.g., an endcapping group, which differs from the majority structure comprising most of the polymer backbone.
  • the bioreducible oligomers form block copolymers with hydrolytically degradable oligomers.
  • the end group/minority structure comprises an amino acid or chain of amino acids, while the backbone degrades hydrolytically and/or is bioreducible.
  • small changes in the monomer ratio used during polymerization in combination with modifications to the chemical structure of the end-capping groups used post-polymerization, can affect the efficacy of delivery of a therapeutic agent to a target.
  • changes in the chemical structure of the polymer either in the backbone of the polymer or end-capping groups, or both, can change the efficacy of target delivery to a cell.
  • small changes to the molecular weight of the polymer or changes to the endcapping groups of the polymer, while leaving the main chain, i.e., backbone, of the polymer the same can enhance or decrease the overall delivery of the target to a cell.
  • R groups that comprise the backbone or main chain of the polymer can be selected to degrade via different biodegradation mechanisms within the same polymer molecule. Such mechanisms include, but are not limited to, hydrolytic, bioreducible, enzymatic, and/or other modes of degradation.
  • compositions can be prepared according to Scheme 2:
  • At least one of the following groups R, R′, and R′′ contain reducible linkages and, for many of the presently disclosed materials, additional modes of degradation also are present. More generally, R′ can be any group that facilitates solubility in water and/or hydrogen bonding, for example, OH, NH, and SH. Representative degradable linkages include, but are not limited to:
  • end group structures i.e., R′′ groups in Scheme 2, for the presently disclosed cationic polymers are distinct and separate from the backbone structures (R) structures, the side chain structures (R′), and end group structures of the intermediate precursor molecule for a given polymeric material.
  • the presently disclosed subject matter includes a nanoparticle, microparticle, or gel comprising a compound of formula (I):
  • n is an integer from 1 to 10,000;
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 are each independently selected from the group consisting of hydrogen, branched and unbranched alkyl, branched and unbranched alkenyl, branched and unbranched alkynyl, aryl, halogen, hydroxyl, alkoxy, carbamoyl, carboxyl ester, carbonyldioxyl, amide, thiohydroxyl, alkylthioether, amino, alkylamino, dialkylamino, trialkylamino, cyano, ureido, a substituted alkanoyl group, cyclic, cyclic aromatic, heterocyclic, and aromatic heterocyclic groups, each of which may be substituted with at least one substituent selected from the group consisting of branched or unbranched alkyl, branched and unbranched alkenyl, branched and unbranched alkynyl, amino,
  • R 1 can be present or absent and when present the compound of formula (I) further comprises a counter ion selected from the group consisting of chloride, fluoride, bromide, iodide, sulfate, nitrate, fumarate, acetate, carbonate, stearate, laurate, and oleate; and
  • R, R′, and R′′ comprise a reducible or degradable linkage, and wherein each R, R′, or R′′ can independently be the same or different;
  • the compound of formula (I) must also comprise one or more of the following characteristics:
  • each R group is different
  • each R′′ group is different
  • each R′′ group is not the same as any of R′, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 9 , and R 9 ;
  • the R′′ groups degrade through a different mechanism than the ester-containing R groups, wherein the degradation of the R′′ group is selected from the group consisting of a bioreducible mechanism or an enzymatically degradable mechanism; and/or
  • the compound of formula (I) comprises a substructure of a larger cross-linked polymer, wherein the larger cross-linked polymer comprises different properties from compound of formula (I);
  • an anti-angiogenic peptide selected from the group consisting of an anti-angiogenic peptide, an anti-lymphangiogenic peptide, an anti-tumorigenic peptide, and an anti-permeability peptide.
  • n is an integer from 1 to 1,000; in some embodiments, n is an integer from 1 to 100; in some embodiments, n is an integer from 1 to 30; in some embodiments, n is an integer from 5 to 20; in some embodiments, n is an integer from 10 to 15; and in some embodiments, n is an integer from 1 to 10.
  • the reducible or degradable linkage comprising R, R′, and R′′ is selected from the group consisting of an ester, a disulfide, an amide, an anhydride or a linkage susceptible to enzymatic degradation, subject to the proviso provided hereinabove.
  • R comprises a backbone of a diacrylate selected from the group consisting of:
  • R′ comprises a side chain derived from compound selected from the group consisting of:
  • R′′ comprises an end group derived from a compound selected from the group consisting of
  • the compound of formula (I) is subject to the further proviso that if at least one R group comprises an ester linkage, then the R′′ groups impart one or more of the following characteristics to the compound of formula (I): independent control of cell-specific uptake and/or intracellular delivery of a particle; independent control of endosomal buffering and endosomal escape; independent control of DNA release; triggered release of an active agent; modification of a particle surface charge; increased diffusion through a cytoplasm of a cell; increased active transport through a cytoplasm of a cell; increased nuclear import within a cell; increased transcription of an associated DNA within a cell; increased translation of an associated DNA within a cell; increased persistence of an associated therapeutic agent within a cell, wherein the therapeutic agent is selected from the group consisting of DNA, RNA, a peptide or a protein.
  • any poly(beta-amino ester) specifically disclosed or claimed in U.S. Pat. No. 6,998,115; U.S. Pat. No. 7,427,394; U.S. patent application publication no. US2005/0265961; and U.S. patent publication no. US2010/0036084, each of which is incorporated herein by reference in its entirety, is explicitly excluded from the presently disclosed compounds of formula (I).
  • the reducible or degradable linkage comprising R, R′, and R′′ is selected from the group consisting of an ester, a disulfide, an amide, an anhydride or a linkage susceptible to enzymatic degradation, subject to the above-mentioned provisos.
  • n is an integer from 1 to 1,000; in other embodiments, n is an integer from 1 to 100; in other embodiments, n is an integer from 1 to 30; in other embodiments, n is an integer from 5 to 20; in other embodiments, n is an integer from 10 to 15; and in other embodiments, n is an integer from 1 to 10.
  • R′′ can be an oligomer as described herein, e.g., one fully synthesized primary amine-terminated oligomer, and can be used as a reagent during the second reaction step of Scheme 2. This process can be repeated iteratively to synthesize increasingly complex molecules.
  • R′′ can comprise a larger biomolecule including, but not limited to, poly(ethyleneglycol) (PEG), a targeting ligand, including, but not limited to, a sugar, a small molecule, an antibody, an antibody fragment, a peptide sequence, or other targeting moiety known to one skilled in the art; a labeling molecule including, but not limited to, a small molecule, a quantum dot, a nanoparticle, a fluorescent molecule, a luminescent molecule, a contrast agent, and the like; and a branched or unbranched, substituted or unsubstituted alkyl chain.
  • PEG poly(ethyleneglycol)
  • a targeting ligand including, but not limited to, a sugar, a small molecule, an antibody, an antibody fragment, a peptide sequence, or other targeting moiety known to one skilled in the art
  • a labeling molecule including, but not limited to, a small molecule, a quantum dot, a nanoparticle, a
  • the branched or unbranched, substituted or unsubstituted alkyl chain is about 2 to about 5 carbons long; in some embodiments, the alkyl chain is about 6 to about 8 carbons long; in some embodiments, the alkyl chain is about 9 to about 12 carbons long; in some embodiments, the alkyl chain is about 13 to about 18 carbons long; in some embodiments, the alkyl chain is about 19 to about 30 carbons long; in some embodiments, the alkyl chain is greater than about 30 carbons long.
  • both R′′ groups i.e., the end groups of the polymer, comprise alkyl chains.
  • only one R′′ group comprises an alkyl chain.
  • at least one alkyl chain is terminated with an amino (NH 2 ) group.
  • the at least one alkyl chain is terminated with a hydroxyl (OH) group.
  • the PEG has a molecular weight of about 5 kDa or less; in some embodiments, the PEG has a molecular weight of about 5 kDa to about 10 kDa; in some embodiments, the PEG has a molecular weight of about 10 kDa to about 20 kDa; in some embodiments, the PEG has a molecular weight of about 20 kDa to about 30 kDa; in some embodiments, the PEG is greater than 30 kDa.
  • both R′′ groups comprise PEG. In other embodiments, only one R′′ group comprises PEG.
  • one R′′ group is PEG and the other R′′ group is a targeting ligand and/or labeling molecule as defined herein above.
  • one R′′ group is an alkyl chain and the other R′′ group is a targeting ligand and/or labeling molecule.
  • Representative monomers used to synthesize the presently disclosed cationic polymers include, but are not limited to, those provided immediately herein below.
  • the presently disclosed subject matter is not limited to the representative monomers disclosed herein, but also includes other structures that one skilled in the art could use to create similar biphasic degrading cationic polymers.
  • a particular biodegradable polymer can be tuned through varying the constituent monomers used to form the backbone (designated as “B” groups), side-chains (designated as “S” groups), and end-groups (designated as “E” groups) of the polymer.
  • the presently disclosed cationic polymers comprise a polyalcohol structure, i.e., the side chain represented by R′ in Scheme 2 comprises an alcohol.
  • end group structures (R′′) and the backbone structures (R) are defined as above and the side chain must contain at least one hydroxyl (OH) group.
  • the presently disclosed cationic polymer comprises a specific poly(ester amine) structure with secondary non-hydrolytic modes of degradation.
  • the cationic polymer comprises a polyester that degrades through ester linkages (hydrolytic degradation) that is further modified to comprise bioreducible groups as end (R′′) groups.
  • bioreducible end groups in such embodiments include, but are not limited to:
  • the presently disclosed cationic polymer comprises a specific poly(ester amine alcohol) structure with secondary non-hydrolytic modes of degradation.
  • the cationic polymer comprises a specific structure where a polyester that degrades through ester linkages (hydrolytic degradation) is modified to contain bioreducible groups as end groups.
  • the presently disclosed cationic polymer comprises a specific poly(amido amine) structure having disulfide linking groups in the polymer backbone and an independent, non-reducible amine contacting group at the terminal ends of the polymer.
  • R 1 and R 2 are alkyl chains.
  • the alkyl chain is 1-2 carbons long; in some embodiments, the alkyl chain is 3-5 carbons long; in some embodiments, the alkyl chain is 6-8 carbons long; in some embodiments, the alkyl chain is 9-12 carbons long; in some embodiments, the alkyl chain is 13-18 carbons long; in some embodiments, the alkyl chain is 19-30 carbons long; and in some embodiments, the alkyl chain is greater than 30 carbons long
  • Suitable non-reducible amino R′′ groups for such embodiments include, but are not limited to:
  • the presently disclosed cationic polymers comprise a specific poly(amido amine alcohol) structure having disulfide linking groups in the polymer backbone and an independent non-reducible amine contacting group at the terminal ends of the polymer.
  • the presently disclosed cationic polymer comprises a copolymer of representative oligomers as described hereinabove.
  • Such embodiments include, but are not limited to, a poly(amido amine) structure having disulfides in the polymer backbone and an independently degradable (non-reducible) group at least one end of the polymer.
  • Such embodiments also include using a cross-linker to add bioreducible linkages to hydrolytically degradable materials and also provide for higher molecular weight materials.
  • a representative example of this embodiment, along with suitable monomers is as follows:
  • the presently disclosed polymer is selected from the group consisting of:
  • R substituent groups that make up the presently disclosed polymers degrade via different biodegradation mechanisms within the same polymer. These biodegradation mechanisms can include hydrolytic, bioreducible, enzymatic, and/or other modes of degradation; (b) the ends of the polymer include a minority structure that differs from the majority structure that comprises most of the polymer backbone; (c) in several embodiments, the side chain molecules contain hydroxyl (OH)/alcohol groups.
  • the backbone is bioreducible and the end groups of the polymer degrade hydrolytically; (b) the backbone degrades hydrolytically and the end groups are bioreducible; and (c) hydrolytically degradable oligomers are cross-linked with a bioreducible cross-linker; (d) bioreducible oligomers form block copolymers with hydrolytically degradable oligomers; and (e) the end group/minority structure comprises an amino acid or chain of amino acids, whereas the backbone degrades hydrolytically and/or is bioreducible.
  • One way to synthesize the presently disclosed materials is by the conjugate addition of amine-containing molecules to acrylates or acrylamides. This reaction can be done neat or in a solvent, such as DMSO or THF. Reactions can take place at a temperature ranging from about room temperature up to about 90° C. and can have a duration from about a few hours to about a few weeks.
  • the presently disclosed methods can be used to create linear or branched polymers.
  • the molecular weight (MW) has a range from about 1 kDa to about 5 kDa, in other embodiments, the MW has a range from about 5 kDa to about 10 kDa, in other embodiments the MW has a range from about 10 kDa to about 15 kDa, in other embodiments, the MW has a range from about 15 kDa to about 25 kDa, in other embodiments, the MW has a range from about 25 kDa to about 50 kDa, and in other embodiments, the MW has a range from about 50 kDa to about 100 kDa.
  • the polymer forms a network, gel, and/or scaffold of apparent molecular weight greater than 100 kDa.
  • the presently disclosed subject matter provides hydrolytic and bioreducible polymeric particle formulations for the delivery of one or more peptides to a target.
  • the particles are nanoparticles and, in other embodiments, they are microparticles.
  • the presently disclosed approach includes degradable nanoparticles, microparticles, and gels that release a peptide, which is capable of therapeutic activity through multiple modes of action.
  • the presently disclosed peptides can simultaneously inhibit: (1) endothelial cell proliferation; (2) endothelial cell adhesion, (3) endothelial cell migration, (4) tumor cell proliferation, (5) tumor cell adhesion, and (6) tumor cell migration.
  • the presently disclosed nanoparticles, microparticles, and gels When combined with such peptides, the presently disclosed nanoparticles, microparticles, and gels: (1) protect and increase the persistence of the peptides that would otherwise be rapidly cleared in vivo; (2) allow passive targeting of tumor vasculature via nanoparticle biophysical properties to enable enhanced efficacy at the target site of action; (3) enable extended peptide release and minimized dosing schedules for affected patients; and (4) facilitate a continuous peptide concentration rather than a pulsatile profile that would be caused by bolus injections and fast clearance.
  • microparticles have similar benefits to the nanoparticles except that they also persist longer and have an easier route for clinical administration.
  • another advantage of the presently disclosed nanoparticles is that they are better able to passively target the peptides to tumor vasculature than are the microparticles. Representative embodiments of the presently disclosed microparticles are provided in Example 10, herein below.
  • one or more peptides which can be the same or different, can be combined, e.g., encapsulated, directly or individually into different nanoparticles that then can be combined into the same microparticles.
  • Selected polymers are able to encapsulate selected peptides possessing varied chemical properties. Changes to polymer structure, including small changes to the ends of the polymer only, can vary biophysical properties of these particles. These properties can be important to tune for effective in vivo peptide delivery.
  • a small subset of the potential polymer library was screened to measure the effect of encapsulating the antiangiogenic peptides chemokinostatin-1 and pentastatin-1 within polymeric particles compared to unencapsulated, free peptides. Polymeric encapsulation of peptides enhanced the ability of the peptides to inhibit the proliferation of endothelial cells.
  • An example of representative polymers encapsulating peptides is provided in Scheme 5.
  • particles synthesized and composed as described above are then used as a “core” inner particle for future coatings to create multi-component (also referred to herein as multi-layer) particles.
  • cores such as an inorganic nanoparticles (like gold) or soft polymeric nanoparticles, for example, as disclosed in International PCT Patent Application Publication No. WO/2010/132879 for “Multicomponent Degradable Cationic Polymers,” to Green et al., which is incorporated herein by reference in its entirety.
  • the core particle is then coated with charged polymers as described above, peptides as described above, and other biological agents. Exemplary embodiments of multilayer particles are illustrated in FIG. 1 .
  • Multilayering can be mediated by electrostatic forces and alternate cationic and anionic layers can be used to incorporate additional peptides and biological agents.
  • Polyelectrolytes including degradable polymers and peptides, also are used to provide structure to the multilayers.
  • Multilayers can release drugs, peptides, and biological agents from the particle due to hydrolytic degradation, enzyme activity, disulfide reduction, and/or diffusion.
  • the presently disclosed subject matter provides photocrosslinked gels for controlled release of cargo, including, but not limited to peptides and proteins. Such gels can be tuned for release of other drugs.
  • a solution of acrylate-terminated polymers is made using either acrylate-terminated polymers, such as poly( ⁇ -amino esters) (PBAEs), poly(ethylene glycol)diacrylate (PEGDA), small crosslinkers including, but not limited to, 1,4-butanediol diacrylate, or a mixture of the above.
  • PBAEs poly( ⁇ -amino esters)
  • PEGDA poly(ethylene glycol)diacrylate
  • small crosslinkers including, but not limited to, 1,4-butanediol diacrylate, or a mixture of the above.
  • a variety of solvents can be used, including water, PBS, and DMSO, to encapsulate drugs within them. Addition of a small amount (0.05% w/v) of photoinitiator and exposure to long-wave UV light for a period of time, e.g., 5-15 min at 1-3 mW, causes formation of a drug-loaded gel.
  • the gel swelling properties can vary with pH by taking advantage of the PBAE portions, which can be reversibly protonated. Changing ratios of PBAE to PEGDA and the addition of crosslinkers changes swelling properties by changing pore size or overall hydrophobicity. For example, doping in increasing amounts of a more hydrophobic PBAE (B4S4) into a network of hydrophilic PEGDA causes the release kinetics to slow when measuring protein release.
  • B4S4 more hydrophobic PBAE
  • the presently disclosed subject matter provides a method of keeping DNA or other cargo stable and functional after storage. For example, freeze-drying often causes denaturation of biological molecules or irreversible aggregation and inactivation of nanoparticles.
  • sucrose as a lyoprotectant at a final concentration of, for example, 7.5-45 mg/mL
  • the presently disclosed subject matter demonstrates that particles can be freeze dried and stored, for example, at 4° C. or ⁇ 20° C. for extended periods, e.g., months, without significant change in physicochemical or biological properties.
  • formulations when stored dry, also might be stable at ambient temperatures up to 40° C.
  • the presently disclosed process allows particles to be prepared in advance and used much more easily in a clinical setting.
  • the presently disclosed subject matter also demonstrates that particles can be concentrated in this way much more highly than would be possible with free polymer, which may be advantageous for dose adjustment in clinical or pre-clinical models.
  • the presently disclosed nanoparticles can be stored in a dry form and can be used in gene delivery via three-dimensional (3D) constructs. While DNA is used as a cargo in this example, other cargos of interest to one skilled in the art including, but not limited to, siRNA, peptides, protein, imaging agents, and the like, can be used, as well. In other embodiments, DNA-loaded nanoparticles were incorporated into natural and synthetic scaffolds, disks, microparticles, and hydrogels for various potential applications.
  • angiogenesis which facilitates the supply of the growing tumor with oxygen and nutrients
  • lymphangiogenesis which facilitates the spreading of cancer cells through the lymphatics
  • cancer cell proliferation a critical role in the growth of tumors and antiangiogenic therapies have the potential to treat cancer, either alone or in combination with conventional chemotherapies, by starving tumors of oxygen and nutrients.
  • antiangiogenic therapies have the potential to treat cancer, either alone or in combination with conventional chemotherapies, by starving tumors of oxygen and nutrients.
  • the presently disclose'd subject matter provides peptides derived from several classes of proteins that are effective at preventing angiogenesis.
  • the presently disclosed subject matter provides other peptides that are able to inhibit cancer through additional mechanisms including, but not limited to, antilymphangiogenesis and apoptosis.
  • the presently disclosed biomaterials facilitate delivery of combinations of these peptides in an engineered fashion to synergistically kill cancer or treat other diseases, in particular, other angiogenesis-dependent diseases. More particularly, the presently disclosed subject matter provides an effective array of safe, biodegradable polymers for use in forming peptide-containing nanoparticles, microparticles, gels, and conjugates.
  • the presently disclosed biomaterials can be used to construct particles, gels, and conjugates that vary in their biophysical properties and in biological properties, such as tumor accumulation and peptide release.
  • the presently disclosed formulations work through one or more of the following mechanisms: antiangiogenesis; inhibition of human endothelial cell proliferation and migration; inhibition of lymphatic endothelial cell proliferation and migration; and promotion of cancer apoptosis, as well as other mechanisms.
  • the presently disclosed materials and methods can safely, effectively, and relatively inexpensively treat age-related macular degeneration (AMD), cancer, and other diseases.
  • AMD age-related macular degeneration
  • siRNA is a promising technology to silence the activity of many biological targets in many diseases including cancer, cardiovascular diseases, infectious diseases, neurological diseases, ophthalmic diseases, and others. In some cases, siRNA can be used to reach previously undruggable targets.
  • the method of delivery and examples described herein for siRNA delivery apply equally to other similar RNA molecules including, but not limited to is RNA, agRNA, saRNA, and miRNA.
  • the presently disclosed strategy combines more effective and multimodal therapeutic agents with nanomedicine to provide a delivery system to enhance their therapeutic effect. More particularly, the presently disclosed subject matter provides a single system that incorporates multimodal therapeutic activity, including, but not limited to, antiangiogenic activity, antilymphangiogenic activity, and apoptotic activity, and can be effective in limiting both tumor growth and metastasis.
  • small peptides possess many advantageous characteristics as therapeutic agents, including high specificity and low toxicity. Reichert J. Development trends for peptide therapeutics. Tufts Center for the Study of Drug Development 2008 [cited 2010; Available from: http://www.peptidetherapeutics.org/PTF_Summary_2008.pdf].
  • the main disadvantage of small peptides as therapeutic agents is their short half-life.
  • the presently disclosed subject matter capitalizes on the advantages of peptide agents by developing novel antiangiogenic, antilymphangiogenic, and apoptotic peptides targeting multiple pathways, and overcoming the disadvantages by designing a multi-agent nanocarrier system.
  • peptides are much easier to produce and are more scalable and less immunogenic than full-length proteins, they are eliminated from the body more quickly.
  • the presently disclosed subject matter can increase and sustain residence time, increase accumulation in tumor vasculature, and maximize the therapeutic effects of such peptides.
  • the presently disclosed subject matter combines biomaterial synthesis, sustained drug delivery, and anti-cancer peptide creation to provide nanoparticle-, microparticle-, and gel-based systems for sustained peptide delivery.
  • the presently disclosed biodegradable biomaterials can be tuned for the encapsulation, protection, and sustained release of each type of peptide.
  • the use of the presently disclosed nanoparticles, microparticles, and gels limits toxicity because they can extravasate from the leaky neovasculature of the tumors and be trapped in the interstitium of the tumor once the anti-angiogenic compounds kill or normalize the vasculature.
  • the presently disclosed subject matter demonstrates that effective biomaterials for anti-cancer peptide nanoparticles, microparticles, and gels can be fabricated. Multiple anti-cancer peptides and other peptides can be combined within the same particle for multimodal peptide delivery, as well as multimodal therapy with other active agents including, but not limited to, other peptides, nucleic acids, proteins, small molecules, and the like.
  • the presently disclosed subject matter provides peptides that work through multiple biological mechanisms in combination with the presently disclosed biomaterials, including multilayer and multi-peptide nanoparticle formulations.
  • An array of biodegradable polymers can be used to encapsulate peptides to create nanoparticles having varied biophysical properties and release kinetics.
  • Each peptide can have a specialized subset of materials employed for its encapsulation.
  • differing chemical structures can be synthesized by the conjugate addition of amines to acrylates or acrylamides of differing structure.
  • the polymer structure can be tuned through variation to the backbone, side chain, end-group, hydrophobicity, and degradability.
  • hydrophobic core particles first are constructed by self-assembly, for example between the somatotropin-derived peptide, the collagen IV-derived peptide, and a hydrophobic polymer. These nanoparticles are then coated by charged biodegradable polymers and peptides following a particle coating and layer-by-layer technique that modifies techniques previously described. Green J J, Chiu E, Leshchiner E S, Shi J, Langer R, Anderson D G. Electrostatic ligand coatings of nanoparticles enable ligand-specific gene delivery to human primary cells. Nano Lett 2007; 7(4):874-9; Shmueli R B, Anderson D G, Green J J.
  • peptides can self-assemble with the presently disclosed polymers in an aqueous buffer due to physical, hydrophobic, and electrostatic forces.
  • Zhang S Uludag H. Nanoparticulate systems for growth factor delivery. Pharm Res 2009; 26(7):1561-80.
  • peptide-containing micelles can be formed by synthetic polymer-mPEG (e.g., E15 from FIG. 4 ) block copolymers.
  • polymer/peptide particle sizes can be tuned from approximately 50 nm to approximately 500 nm.
  • peptides can be encapsulated by a double emulsion procedure.
  • droplets of aqueous buffer containing peptide are dispersed in the hydrophobic polymer phase and then the polymer phase is itself dispersed in another aqueous phase to form the polymeric particles.
  • Jain R A The manufacturing techniques of various drug loaded biodegradable poly(lactide-co-glycolide) (PLGA) devices. Biomaterials 2000; 21(23):2475-90.
  • blends of novel hydrophobic polymers and poly(lactic-co-glycolic acid) also can be made to form particles with unique degradation properties.
  • peptides have been developed that show either anti-proliferative or anti-migratory activity or both on endothelial cells. These peptides appear to function through distinct mechanisms of action and have been tested both in vitro and in vivo in tumor xenografts and in ocular mouse models.
  • peptides include a 24-mer peptide NGRKACLNPASPIVKKIIEKMLNS derived from the CXC chemokine protein GRO- ⁇ /CXCL1 and a collagen IV derived and modified 20-mer peptide LRRFSTMPFMF-Abu-NINNV-Abu-NF as a highly potent anti-proliferative and anti-migratory peptide targeting ⁇ v ⁇ 1 integrins on both endothelial and tumor cells; here Abu is the 2-Aminobutyric acid introduced in the sequence to facilitate translation to human.
  • EIELVEEEPPF 11-mer anti-angiogenic peptide EIELVEEEPPF derived from the serpin domain of DEAH box polypeptide also has been identified that shows significant inhibition of MDA-MB-231 tumor xenograft growth.
  • Representative peptides suitable for encapsulation with the presently disclosed biomaterials include those disclosed in International PCT Patent Application Publication Number WO2007/033215 A2 for “Compositions Having Antiangiogenic Activity and Uses Thereof,” to Popel et al., published Mar. 22, 2007; International PCT Patent Application Publication Number WO2008/085828 A2 for “Peptide Modulators of Angiogenesis and Use Thereof,” to Popel, published Jul. 17, 2008; U.S. Provisional Patent Application No. 61/421,706, filed Dec. 12, 2010, which is commonly owned; and U.S. Provisional Patent Application No. 61/489,500, filed May 24, 2011, which also is commonly owned, each of which is incorporated herein by reference in its entirety.
  • peptide suitable for use in the presently disclosed subject matter are disclosed in Tables 1-10 of International PCT Patent Application Publication Number WO2008/085828 A2 for “Peptide Modulators of Angiogenesis and Use Thereof,” to Popel, published Jul. 17, 2008, which is incorporated herein by reference in its entirety.
  • the presently disclosed subject matter provides a nanoparticle, microparticle, or gel comprising one or more peptides, wherein the one or more peptide is selected from the group consisting of an isolated peptide or analog thereof comprising one of the following amino acid sequences:
  • TSP Motif W-X(2)-C-X(3)-C-X(2)-G, CXC Motif: G-X(3)-C-L Collagen Motif: C-N-X(3)-V-C Collagen Motif: P-F-X(2)-C Somatotropin Motif: L-X(3)-L-L-X(3)-S-X-L Serpin Motif: L-X(2)-E-E-X-P
  • X denotes a variable amino acid and the number in parentheses denotes the number of variable amino acids
  • W denotes tryptophan
  • C denotes cysteine
  • G denotes glycine
  • V denotes valine
  • L denotes leucine
  • P is proline
  • the peptide reduces blood vessel formation in a cell, tissue or organ.
  • the one or more peptide comprises an amino acid sequence shown in Table 1-6, 8 and 9.
  • the one or more peptide comprises an isolated peptide or analog thereof having at least 85% identity to an amino acid sequence shown in Table 1-10.
  • the one or more peptide comprises an amino acid sequence shown in Table 1-10. In yet other embodiments, the one or more peptide consists essentially of an amino acid sequence shown in Table 1-10.
  • the one or more peptide comprises an isolated peptide or analog thereof comprising or consisting essentially of a sequence having at least 85% amino acid sequence identity to an amino acid sequence selected from the group consisting of:
  • the one or more peptide comprises an isolated peptide or analog thereof comprising or consisting essentially of a sequence having at least 85% amino acid sequence identity to an amino acid sequence selected from the group consisting of:
  • the one or more peptide comprises an isolated peptide or analog thereof comprising or consisting essentially of a sequence having at least 85% amino acid sequence identity to an amino acid sequence selected from the group consisting of:
  • Collagen type IV alpha6 LPRFSTMPFIYCNINEVCHY fibril wherein the peptide reduces blood vessel formation in a cell, tissue or organ.
  • AAH48330.1 188-268 EAACVWCNGEEYRGAVDRTESGRECQRWDLQHPHQHPFEPGK FLDQGLDDNYCRNPDGSERPWCYTTDPQIEREFCDLPRC 2426 Macrophage stim.
  • the presently disclosed subject matter provides a nanoparticle, microparticle, or gel comprising a compound of Formula (I), wherein the one or more peptide is selected from the group consisting of an isolated peptide or analog thereof comprising the amino acid sequence W-X 2 -C-X 3 -C-X 2 -G, wherein X denotes a variable amino acid; W is tryptophan; C is cysteine, G is glycine; and wherein the peptide reduces blood vessel formation in a cell, tissue or organ.
  • the one or more peptide is selected from the group consisting of an isolated peptide or analog thereof comprising the amino acid sequence W-X 2 -C-X 3 -C-X 2 -G, wherein X denotes a variable amino acid; W is tryptophan; C is cysteine, G is glycine; and wherein the peptide reduces blood vessel formation in a cell, tissue or organ.
  • the one or more peptide is selected from the group consisting of an isolated peptide or analog thereof comprising or consisting essentially of a sequence having at least 85% amino acid sequence identity to an amino acid sequence selected from the group consisting of:
  • THSD-1 QPWSQCSATCGDGVRERRR; THSD-3: SPWSPCSGNCSTGKQQRTR; THSD-6: WTRCSSSCGRGVSVRSR; CILP: SPWSKCSAACGQTGVQTRTR; WISP-1: SPWSPCSTSCGLGVSTRI; WISP-2: TAWGPCSTTCGLGMATRV; WISP-3: TKWTPCSRTCGMGISNRV; F-spondin: SEWSDCSVTCGKGMRTRQR; F-spondin: WDECSATCGMGMKKRHR; CTGF: TEWSACSKTCGMGISTRV; fibulin-6: ASWSACSVSCGGGARQRTR; fibulin-6: QPWGTCSESCGKGTQTRAR; fibulin-6: SAWRACSVTCGKGIQKRSR; CYR61: TSWSQCSKTCGTGISTRV; NOVH: TEWTACSKSCGMGFSTRV; UNC5-C: TEWSVCNSRCGRG
  • the one or more peptide is selected from the group consisting of an isolated peptide or analog thereof having at least 85% identity to an amino acid sequence selected from the group consisting of:
  • ENA-78 NGKEICLDPEAPFLKKVIQKILD; CXCL6: NGKQVCLDPEAPFLKKVIQKILDS; CXCL1: NGRKACLNPASPIVKKIIEKMLNS; Gro- ⁇ : NGKKACLNPASPMVQKIIEKIL; Beta thromboglobulin/CXCL7: DGRKICLDPDAPRIKKIVQK KL, Interleukin 8 (IL-8)/CXCL8: DGRELCLDPKENWVQRVVEKF LK, GCP-2: NGKQVCLDPEAPFLKKVIQKILDS,
  • the one or more peptide is selected from the group consisting of an isolated peptide or analog thereof having at least 85% amino acid sequence identity to an amino acid sequence selected from the group consisting of
  • Alpha 6 fibril of type 4 collagen YCNINEVCHYARRND KSYWL; Alpha 5 fibril of type 4 collagen: LRRFSTMPFMFCNIN NVCNF; Alpha 4 fibril of type 4 collagen: AAPFLECQGRQGTCH FFAN; Alpha 4 fibril of type 4 collagen: LPVFSTLPFAYCNIH QVCHY; Alpha 4 fibril of type 4 collagen: YCNIHQVCHYAQRND RSYWL, and Collagen type IV, alpha6 fibril LPRFSTMPFIYCNINE VCHY;
  • peptides suitable for use in the presently disclosed subject matter are disclosed in U.S. Provisional Patent Application No. 61/421,706, filed Dec. 12, 2010, which is commonly owned, and is incorporated herein by reference in its entirety.
  • peptides suitable for use in the presently disclosed subject matter are disclosed in U.S. Provisional Patent Application No. 61/489,500, filed Way 24, 2011, which also is commonly owned, and is incorporated herein by reference in its entirety.
  • substituent refers to the ability, as appreciated by one skilled in this art, to change one functional group for another functional group provided that the valency of all atoms is maintained.
  • substituents may be either the same or different at every position.
  • the substituents also may be further substituted (e.g., an aryl group substituent may have another substituent off it, such as another aryl group, which is further substituted, for example, with fluorine at one or more positions).
  • R groups such as groups R 1 , R 2 , and the like, or variables, such as “m” and “n”
  • R 1 and R 2 can be substituted alkyls, or R 1 can be hydrogen and R 2 can be a substituted alkyl, and the like.
  • R or group will generally have the structure that is recognized in the art as corresponding to a group having that name, unless specified otherwise herein.
  • certain representative “R” groups as set forth above are defined below.
  • hydrocarbon refers to any chemical group comprising hydrogen and carbon.
  • the hydrocarbon may be substituted or unsubstituted. As would be known to one skilled in this art, all valencies must be satisfied in making any substitutions.
  • the hydrocarbon may be unsaturated, saturated, branched, unbranched, cyclic, polycyclic, or heterocyclic.
  • Illustrative hydrocarbons are further defined herein below and include, for example, methyl, ethyl, n-propyl, iso-propyl, cyclopropyl, allyl, vinyl, n-butyl, tert-butyl, ethynyl, cyclohexyl, methoxy, diethylamino, and the like.
  • alkyl refers to C 1-20 inclusive, linear (i.e., “straight-chain”), branched, or cyclic, saturated or at least partially and in some cases fully unsaturated (i.e., alkenyl and alkynyl)hydrocarbon radicals derived from a hydrocarbon moiety containing between one and twenty carbon atoms by removal of a single hydrogen atom.
  • alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, sec-pentyl, iso-pentyl, neopentyl, n-hexyl, sec-hexyl, n-heptyl, n-octyl, n-decyl, n-undecyl, dodecyl, and the like, ethenyl, propenyl, butenyl, pentenyl, hexenyl, octenyl, butadienyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, and allenyl groups.
  • Branched refers to an alkyl group in which a lower alkyl group, such as methyl, ethyl or propyl, is attached to a linear alkyl chain.
  • Lower alkyl refers to an alkyl group having 1 to about 8 carbon atoms (i.e., a C 1-8 alkyl), e.g., 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms.
  • Higher alkyl refers to an alkyl group having about 10 to about 20 carbon atoms, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms.
  • alkyl refers, in particular, to C 1-8 straight-chain alkyls. In other embodiments, “alkyl” refers, in particular, to C 1-8 branched-chain alkyls.
  • Alkyl groups can optionally be substituted (a “substituted alkyl”) with one or more alkyl group substituents, which can be the same or different.
  • alkyl group substituent includes but is not limited to alkyl, substituted alkyl, halo, arylamino, acyl, hydroxyl, aryloxyl, alkoxyl, alkylthio, arylthio, aralkyloxyl, aralkylthio, carboxyl, alkoxycarbonyl, oxo, and cycloalkyl.
  • alkyl chain There can be optionally inserted along the alkyl chain one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms, wherein the nitrogen substituent is hydrogen, lower alkyl (also referred to herein as “alkylaminoalkyl”), or aryl.
  • substituted alkyl includes alkyl groups, as defined herein, in which one or more atoms or functional groups of the alkyl group are replaced with another atom or functional group, including for example, alkyl, substituted alkyl, halogen, aryl, substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino, dialkylamino, sulfate, and mercapto.
  • Cyclic and “cycloalkyl” refer to a non-aromatic mono- or multicyclic ring system of about 3 to about 10 carbon atoms, e.g., 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms.
  • the cycloalkyl group can be optionally partially unsaturated.
  • the cycloalkyl group also can be optionally substituted with an alkyl group substituent as defined herein, oxo, and/or alkylene. There can be optionally inserted along the cyclic alkyl chain one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms,
  • nitrogen substituent is hydrogen, alkyl, substituted alkyl, aryl, or substituted aryl, thus providing a heterocyclic group.
  • Representative monocyclic cycloalkyl rings include cyclopentyl, cyclohexyl, and cycloheptyl.
  • Multicyclic cycloalkyl rings include adamantyl, octahydronaphthyl, decalin, camphor, camphane, and noradamantyl.
  • cycloalkylalkyl refers to a cycloalkyl group as defined hereinabove, which is attached to the parent molecular moiety through an alkyl group, also as defined above.
  • alkyl group also as defined above.
  • examples of cycloalkylalkyl groups include cyclopropylmethyl and cyclopentylethyl.
  • cycloheteroalkyl or “heterocycloalkyl” refer to a non-aromatic ring system, unsaturated or partially unsaturated ring system, such as a 3- to 10-member substituted or unsubstituted cycloalkyl ring system, including one or more heteroatoms, which can be the same or different, and are selected from the group consisting of N, O, and S, and optionally can include one or more double bonds.
  • the cycloheteroalkyl ring can be optionally fused to or otherwise attached to other cycloheteroalkyl rings and/or non-aromatic hydrocarbon rings.
  • Heterocyclic rings include those having from one to three heteroatoms independently selected from oxygen, sulfur, and nitrogen, in which the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized.
  • the term heterocylic refers to a non-aromatic 5-, 6-, or 7-membered ring or a polycyclic group wherein at least one ring atom is a heteroatom selected from O, S, and N (wherein the nitrogen and sulfur heteroatoms may be optionally oxidized), including, but not limited to, a bi- or tri-cyclic group, comprising fused six-membered rings having between one and three heteroatoms independently selected from the oxygen, sulfur, and nitrogen, wherein (i) each 5-membered ring has 0 to 2 double bonds, each 6-membered ring has 0 to 2 double bonds, and each 7-membered ring has 0 to 3 double bonds, (ii) the nitrogen and sulfur heteroatoms may be optionally oxidized, (
  • Representative cycloheteroalkyl ring systems include, but are not limited to pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperidyl, piperazinyl, indolinyl, quinuclidinyl, morpholinyl, thiomorpholinyl, thiadiazinanyl, tetrahydrofuranyl, and the like.
  • alkenyl refers to a monovalent group derived from a C 1-20 inclusive straight or branched hydrocarbon moiety having at least one carbon-carbon double bond by the removal of a single hydrogen atom.
  • Alkenyl groups include, for example, ethenyl (i.e., vinyl), propenyl, butenyl, 1-methyl-2-buten-1-yl, and the like.
  • cycloalkenyl refers to a cyclic hydrocarbon containing at least one carbon-carbon double bond.
  • Examples of cycloalkenyl groups include cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadiene, cyclohexenyl, 1,3-cyclohexadiene, cycloheptenyl, cycloheptatrienyl, and cyclooctenyl.
  • alkynyl refers to a monovalent group derived from a straight or branched C 1-20 hydrocarbon of a designed number of carbon atoms containing at least one carbon-carbon triple bond.
  • alkynyl include ethynyl, 2-propynyl(propargyl), 1-propyne, 3-hexyne, and the like.
  • Alkylene refers to a straight or branched bivalent aliphatic hydrocarbon group having from 1 to about 20 carbon atoms, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms.
  • the alkylene group can be straight, branched or cyclic.
  • the alkylene group also can be optionally unsaturated and/or substituted with one or more “alkyl group substituents.” There can be optionally inserted along the alkylene group one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms (also referred to herein as “alkylaminoalkyl”), wherein the nitrogen substituent is alkyl as previously described.
  • alkylene groups include methylene (—CH 2 —); ethylene (—CH 2 —CH 2 -); propylene (—(CH 2 ) 3 —); cyclohexylene (—C 6 H 10 —); —CH ⁇ CH—CH ⁇ CH—; —CH ⁇ CH—CH 2 —; —(CH 2 ) q —N(R)—(CH 2 ) r —, wherein each of q and r is independently an integer from 0 to about 20, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, and R is hydrogen or lower alkyl; methylenedioxyl (—O—CH 2 —O—); and ethylenedioxyl (—O—(CH 2 ) 2 —O—).
  • An alkylene group can have about 2 to about 3 carbon atoms and can further have 6-20 carbons.
  • aryl is used herein to refer to an aromatic substituent that can be a single aromatic ring, or multiple aromatic rings that are fused together, linked covalently, or linked to a common group, such as, but not limited to, a methylene or ethylene moiety.
  • the common linking group also can be a carbonyl, as in benzophenone, or oxygen, as in diphenylether, or nitrogen, as in diphenylamine.
  • aryl specifically encompasses heterocyclic aromatic compounds.
  • the aromatic ring(s) can comprise phenyl, naphthyl, biphenyl, diphenylether, diphenylamine and benzophenone, among others.
  • aryl means a cyclic aromatic comprising about 5 to about 10 carbon atoms, e.g., 5, 6, 7, 8, 9, or 10 carbon atoms, and including 5- and 6-membered hydrocarbon and heterocyclic aromatic rings.
  • the aryl group can be optionally substituted (a “substituted aryl”) with one or more aryl group substituents, which can be the same or different, wherein “aryl group substituent” includes alkyl, substituted alkyl, alkenyl, alkynyl, aryl, substituted aryl, aralkyl, hydroxyl, alkoxyl, aryloxyl, aralkyloxyl, carboxyl, acyl, halo, haloalkyl, nitro, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, acyloxyl, amino, alkylamino, dialkylamino, trialkylamino, acylamino, aroylamino, carbamoyl, cyano, alkylcarbamoyl, dialkylcarbamoyl, carboxyaldehyde, carboxyl, alkoxycarbonyl, carboxamide, aryl
  • substituted aryl includes aryl groups, as defined herein, in which one or more atoms or functional groups of the aryl group are replaced with another atom or functional group, including for example, alkyl, substituted alkyl, halogen, aryl, substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino, dialkylamino, sulfate, and mercapto.
  • aryl groups include, but are not limited to, cyclopentadienyl, phenyl, furan, thiophene, pyrrole, pyran, pyridine, imidazole, benzimidazole, isothiazole, isoxazole, pyrazole, pyrazine, triazine, pyrimidine, quinoline, isoquinoline, indole, carbazole, and the like.
  • heteroaryl and “aromatic heterocycle” and “aromatic heterocyclic” are used interchangeably herein and refer to a cyclic aromatic radical having from five to ten ring atoms of which one ring atom is selected from sulfur, oxygen, and nitrogen; zero, one, or two ring atoms are additional heteroatoms independently selected from sulfur, oxygen, and nitrogen; and the remaining ring atoms are carbon, the radical being joined to the rest of the molecule via any of the ring atoms, such as, for example, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, and the like.
  • Aromatic heterocyclic groups can be unsubstituted or substituted with substituents selected from the group consisting of branched and unbranched alkyl, alkenyl, alkynyl, haloalkyl, alkoxy, thioalkoxy, amino, alkylamino, dialkylamino, trialkylamino, acylamino, cyano, hydroxy, halo, mercapto, nitro, carboxyaldehyde, carboxy, alkoxycarbonyl, and carboxamide.
  • heterocyclic and aromatic heterocyclic groups that may be included in the compounds of the invention include: 3-methyl-4-(3-methylphenyl)piperazine, 3 methylpiperidine, 4-(bis-(4-fluorophenyl)methyl)piperazine, 4-(diphenylmethyl)piperazine, 4(ethoxycarbonyl)piperazine, 4-(ethoxycarbonylmethyl)piperazine, 4-(phenylmethyl)piperazine, 4-(1-phenylethyl)piperazine, 4-(1,1-dimethylethoxycarbonyl)piperazine, 4-(2-(bis-(2-propenyl)amino)ethyl)piperazine, 4-(2-(diethylamino)ethyl)piperazine, 4-(2-chlorophenyl)piperazine, 4(2-cyanophenyl)piperazine, 4-(2-ethoxyphenyl)piperazine, 4-(2-ethylphenyl)piperazine, 4-(2-flu
  • a ring structure for example, but not limited to a 3-carbon, a 4-carbon, a 5-carbon, a 6-carbon, a 7-carbon, and the like, aliphatic and/or aromatic cyclic compound, including a saturated ring structure, a partially saturated ring structure, and an unsaturated ring structure, comprising a substituent R group, wherein the R group can be present or absent, and when present, one or more R groups can each be substituted on one or more available carbon atoms of the ring structure.
  • the presence or absence of the R group and number of R groups is determined by the value of the variable “n,” which is an integer generally having a value ranging from 0 to the number of carbon atoms on the ring available for substitution.
  • n is an integer generally having a value ranging from 0 to the number of carbon atoms on the ring available for substitution.
  • Each R group if more than one, is substituted on an available carbon of the ring structure rather than on another R group.
  • a dashed line representing a bond in a cyclic ring structure indicates that the bond can be either present or absent in the ring. That is, a dashed line representing a bond in a cyclic ring structure indicates that the ring structure is selected from the group consisting of a saturated ring structure, a partially saturated ring structure, and an unsaturated ring structure.
  • acyl refers to an organic acid group wherein the —OH of the carboxyl group has been replaced with another substituent and has the general formula RC( ⁇ O)—, wherein R is an alkyl, alkenyl, alkynyl, aryl, carbocylic, heterocyclic, or aromatic heterocyclic group as defined herein).
  • R is an alkyl, alkenyl, alkynyl, aryl, carbocylic, heterocyclic, or aromatic heterocyclic group as defined herein).
  • acyl specifically includes arylacyl groups, such as an acetylfuran and a phenacyl group. Specific examples of acyl groups include acetyl and benzoyl.
  • alkoxyl or “alkoxy” are used interchangeably herein and refer to a saturated (i.e., alkyl-O—) or unsaturated (i.e., alkenyl-O— and alkynyl-O—) group attached to the parent molecular moiety through an oxygen atom, wherein the terms “alkyl,” “alkenyl,” and “alkynyl” are as previously described and can include C 1 -C 20 inclusive, linear, branched, or cyclic, saturated or unsaturated oxo-hydrocarbon chains, including, for example, methoxyl, ethoxyl, propoxyl, isopropoxyl, n-butoxyl, sec-butoxyl, t-butoxyl, and n-pentoxyl, neopentoxy, n-hexoxy, and the like.
  • alkoxyalkyl refers to an alkyl-O-alkyl ether, for example, a methoxyethyl or an ethoxymethyl group.
  • Aryloxyl refers to an aryl-O— group wherein the aryl group is as previously described, including a substituted aryl.
  • aryloxyl as used herein can refer to phenyloxyl or hexyloxyl, and alkyl, substituted alkyl, halo, or alkoxyl substituted phenyloxyl or hexyloxyl.
  • Alkyl refers to an aryl-alkyl-group wherein aryl and alkyl are as previously described, and included substituted aryl and substituted alkyl.
  • exemplary aralkyl groups include benzyl, phenylethyl, and naphthylmethyl.
  • Alkyloxyl refers to an aralkyl-O— group wherein the aralkyl group is as previously described.
  • An exemplary aralkyloxyl group is benzyloxyl.
  • Alkoxycarbonyl refers to an alkyl-O—CO— group.
  • exemplary alkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl, butyloxycarbonyl, and t-butyloxycarbonyl.
  • Aryloxycarbonyl refers to an aryl-O—CO— group.
  • exemplary aryloxycarbonyl groups include phenoxy- and naphthoxy-carbonyl.
  • Alkoxycarbonyl refers to an aralkyl-O—CO— group.
  • An exemplary aralkoxycarbonyl group is benzyloxycarbonyl.
  • Carbamoyl refers to an amide group of the formula —CONH 2 .
  • Alkylcarbamoyl refers to a R′RN—CO— group wherein one of R and R′ is hydrogen and the other of R and R′ is alkyl and/or substituted alkyl as previously described.
  • Dialkylcarbamoyl refers to a R′RN—CO— group wherein each of R and R′ is independently alkyl and/or substituted alkyl as previously described.
  • carbonyldioxyl refers to a carbonate group of the formula —O—CO—OR.
  • acyloxyl refers to an acyl-O— group wherein acyl is as previously described.
  • amino refers to the —NH 2 group and also refers to a nitrogen containing group as is known in the art derived from ammonia by the replacement of one or more hydrogen radicals by organic radicals.
  • acylamino and alkylamino refer to specific N-substituted organic radicals with acyl and alkyl substituent groups respectively.
  • alkylamino, dialkylamino, and trialkylamino refer to one, two, or three, respectively, alkyl groups, as previously defined, attached to the parent molecular moiety through a nitrogen atom.
  • alkylamino refers to a group having the structure —NHR′ wherein R′ is an alkyl group, as previously defined;
  • dialkylamino refers to a group having the structure —NR′R′′, wherein R′ and R′′ are each independently selected from the group consisting of alkyl groups.
  • trialkylamino refers to a group having the structure —NR′R′′R′′′, wherein R′, R′′, and R′′′ are each independently selected from the group consisting of alkyl groups. Additionally, R′, R′′, and/or R′′′ taken together may optionally be —(CH 2 ) k — where k is an integer from 2 to 6. Examples include, but are not limited to, methylamino, dimethylamino, ethylamino, diethylamino, diethylaminocarbonyl, methylethylamino, iso-propylamino, piperidino, trimethylamino, and propylamino.
  • alkylthioether and thioalkoxyl refer to a saturated (i.e., alkyl-S—) or unsaturated (i.e., alkenyl-S— and alkynyl-S—) group attached to the parent molecular moiety through a sulfur atom.
  • thioalkoxyl moieties include, but are not limited to, methylthio, ethylthio, propylthio, isopropylthio, n-butylthio, and the like.
  • “Acylamino” refers to an acyl-NH— group wherein acyl is as previously described.
  • “Aroylamino” refers to an aroyl-NH— group wherein aroyl is as previously described.
  • carbonyl refers to the —(C ⁇ O)— group.
  • carboxyl refers to the —COOH group. Such groups also are referred to herein as a “carboxylic acid” moiety.
  • halo refers to fluoro, chloro, bromo, and iodo groups.
  • hydroxyl refers to the —OH group.
  • hydroxyalkyl refers to an alkyl group substituted with an —OH group.
  • mercapto refers to the —SH group.
  • oxo refers to a compound described previously herein wherein a carbon atom is replaced by an oxygen atom.
  • nitro refers to the —NO 2 group.
  • thio refers to a compound described previously herein wherein a carbon or oxygen atom is replaced by a sulfur atom.
  • thiohydroxyl or thiol refers to a group of the formula —SH.
  • ureido refers to a urea group of the formula —NH—CO—NH 2 .
  • the term “monomer” refers to a molecule that can undergo polymerization, thereby contributing constitutional units to the essential structure of a macromolecule or polymer.
  • a “polymer” is a molecule of high relative molecule mass, the structure of which essentially comprises the multiple repetition of unit derived from molecules of low relative molecular mass, i.e., a monomer.
  • an “oligomer” includes a few monomer units, for example, in contrast to a polymer that potentially can comprise an unlimited number of monomers. Dimers, trimers, and tetramers are non-limiting examples of oligomers.
  • the term “nanoparticle,” refers to a particle having at least one dimension in the range of about 1 nm to about 1000 nm, including any integer value between 1 nm and 1000 nm (including about 1, 2, 5, 10, 20, 50, 60, 70, 80, 90, 100, 200, 500, and 1000 nm and all integers and fractional integers in between).
  • the nanoparticle has at least one dimension, e.g., a diameter, of about 100 nm.
  • the nanoparticle has a diameter of about 200 nm.
  • the nanoparticle has a diameter of about 500 nm.
  • the nanoparticle has a diameter of about 1000 nm (1 ⁇ m).
  • the particle also can be referred to as a “microparticle.
  • the term “microparticle” includes particles having at least one dimension in the range of about one micrometer ( ⁇ m), i.e., 1 ⁇ 10 ⁇ 6 meters, to about 1000 ⁇ m.
  • the term “particle” as used herein is meant to include nanoparticles and microparticles.
  • nanoparticles suitable for use with the presently disclosed methods can exist in a variety of shapes, including, but not limited to, spheroids, rods, disks, pyramids, cubes, cylinders, nanohelixes, nanosprings, nanorings, rod-shaped nanoparticles, arrow-shaped nanoparticles, teardrop-shaped nanoparticles, tetrapod-shaped nanoparticles, prism-shaped nanoparticles, and a plurality of other geometric and non-geometric shapes.
  • the presently disclosed nanoparticles have a spherical shape.
  • a “subject” can include a human subject for medical purposes, such as for the treatment of an existing condition or disease or the prophylactic treatment for preventing the onset of a condition or disease, or an animal subject for medical, veterinary purposes, or developmental purposes.
  • Suitable animal subjects include mammals including, but not limited to, primates, e.g., humans, monkeys, apes, and the like; bovines, e.g., cattle, oxen, and the like; ovines, e.g., sheep and the like; caprines, e.g., goats and the like; porcines, e.g., pigs, hogs, and the like; equines, e.g., horses, donkeys, zebras, and the like; felines, including wild and domestic cats; canines, including dogs; lagomorphs, including rabbits, hares, and the like; and rodents, including mice, rats, and the like.
  • mammals including, but not limited to, primates, e.g., humans, monkeys, apes, and the like; bovines, e.g., cattle, oxen, and the like; ovines, e.g., sheep and the like; cap
  • an animal may be a transgenic animal.
  • the subject is a human including, but not limited to, fetal, neonatal, infant, juvenile, and adult subjects.
  • a “subject” can include a patient afflicted with or suspected of being afflicted with a condition or disease.
  • the terms “subject” and “patient” are used interchangeably herein.
  • association When two entities are “associated with” one another as described herein, they are linked by a direct or indirect covalent or non-covalent interaction. Preferably, the association is covalent. Desirable non-covalent interactions include hydrogen bonding, van der Waals interactions, hydrophobic interactions, magnetic interactions, electrostatic interactions, etc.
  • Biocompatible The term “biocompatible”, as used herein is intended to describe compounds that are not toxic to cells. Compounds are “biocompatible” if their addition to cells in vitro results in less than or equal to 20% cell death, and their administration in vivo does not induce inflammation or other such adverse effects.
  • Biodegradable As used herein, “biodegradable” compounds are those that, when introduced into cells, are broken down by the cellular machinery or by hydrolysis into components that the cells can either reuse or dispose of without significant toxic effect on the cells (i.e., fewer than about 20% of the cells are killed when the components are added to cells in vitro). The components preferably do not induce inflammation or other adverse effects in vivo. In certain preferred embodiments, the chemical reactions relied upon to break down the biodegradable compounds are uncatalyzed.
  • the “effective amount” of an active agent or drug delivery device refers to the amount necessary to elicit the desired biological response.
  • the effective amount of an agent or device may vary depending on such factors as the desired biological endpoint, the agent to be delivered, the composition of the encapsulating matrix, the target tissue, and the like.
  • Peptide or “protein”: A “peptide” or “protein” comprises a string of at least three amino acids linked together by peptide bonds.
  • the terms “protein” and “peptide” may be used interchangeably.
  • Peptide may refer to an individual peptide or a collection of peptides. Inventive peptides preferably contain only natural amino acids, although non-natural amino acids (i.e., compounds that do not occur in nature but that can be incorporated into a polypeptide chain) and/or amino acid analogs as are known in the art may alternatively be employed.
  • one or more of the amino acids in an inventive peptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc.
  • a chemical entity such as a carbohydrate group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc.
  • the modifications of the peptide lead to a more stable peptide (e.g., greater half-life in vivo). These modifications may include cyclization of the peptide, the incorporation of D-amino acids, etc. None of the modifications should substantially interfere with the desired biological activity of the peptide.
  • Polynucleotide or oligonucleotide Polynucleotide or oligonucleotide refers to a polymer of nucleotides. Typically, a polynucleotide comprises at least three nucleotides.
  • the polymer may include natural nucleosides (i.e., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, C5-propynylcytidine, C5-propynyluridine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, and 2-thiocytidine), chemically modified bases, biologically modified bases
  • Small molecule refers to organic compounds, whether naturally-occurring or artificially created (e.g., via chemical synthesis) that have relatively low molecular weight and that are not proteins, polypeptides, or nucleic acids. Typically, small molecules have a molecular weight of less than about 1500 g/mol. Also, small molecules typically have multiple carbon-carbon bonds.
  • Known naturally-occurring small molecules include, but are not limited to, penicillin, erythromycin, taxol, cyclosporin, and rapamycin.
  • Known synthetic small molecules include, but are not'limited to, ampicillin, methicillin, sulfamethoxazole, and sulfonamides.
  • analog is meant a chemical compounds having a structure that is different from the general structure of a reference agent, but that functions in a manner similar to the reference agent.
  • a peptide analog having a variation in sequence or having a modified amino acid.
  • TSP thrombospondin derived peptide
  • a peptide comprising a TSP motif: W-X(2)-C-X(3)-C-X(2)-G.
  • Exemplary TSP derived peptides are shown in Tables 1 and 2. If desired, the peptide includes at least about 5, 10, 20, 30, 40, 50 or more amino acids that flank the carboxy or amino terminus of the motif in the naturally occurring amino acid sequence of the peptide.
  • TSP1 derived peptides include, for example, those derived from proteins WISP-1
  • CXC derived peptide is meant a peptide comprising a CXC Motif: G-X(3)-C-L.
  • Exemplary CXC derived peptides are shown in Table 3. If desired, the peptide includes at least about 5, 10, 20, 30, 40, 50 or more amino acids that flank the carboxy or amino terminus of the motif in the naturally occurring amino acid sequence.
  • CXC derived peptides include, for example, those derived from proteins GRO- ⁇ /CXCL1 (NGRKACLNPASPIVKKIIEKMLNS), GRO- ⁇ /MIP-21 ⁇ /CXCL3 (NGKKACLNPASPMVQKEEKIL), and ENA-78/CXCL5 (NGKEICLDPEAPFLKKVIQKILD).
  • Collagen IV derived peptide is meant a peptide comprising a C—N—X(3)-V-C or P—F-X(2)-C collagen motif. Exemplary collagen IV derived peptides are shown in Table 5. If desired, the peptide includes at least about 5, 10, 20, 30, 40, 50 or more amino acids that flank the carboxy or amino terminus of the motif in the naturally occurring amino acid sequence. Type IV collagen derived peptides include, for example, LRRFSTMPFMFCNINNVCNF and FCNINNVCNFASRNDYSYWL, and LPRFSTMPFIYCNINEVCHY.
  • Somatotropin derived peptide is meant a peptide comprising a Somatotropin Motif: L-X(3)-L-L-X(3)-S—X-L.
  • Exemplary somatotropin derived peptides are shown in Table 8. If desired, the peptide includes at least about 5, 10, 20, 30, 40, 50 or more amino acids that flank the carboxy or amino terminus of the motif in the naturally occurring amino acid sequence.
  • Serpin derived peptide is meant a peptide comprising a Serpin Motif: L-X(2)-E-E-X—P. Exemplary serpin derived peptides are shown in Table 9. If desired, the peptide includes at least about 5, 10, 20, 30, 40, 50 or more amino acids that flank the carboxy or amino terminus of the motif in the naturally occurring amino acid sequence.
  • Beta 1 integrin is meant a polypeptide that binds a collagen IV derived peptide or that has at least about 85% identity to NP_596867 or a fragment thereof.
  • Beta 3 integrin is meant a polypeptide that binds a collagen IV derived peptide or that has at least about 85% identity to P05106 or a fragment thereof.
  • CD36 is meant a CD36 glycoprotein that binds to a thrombospondin-derived peptide or that has at least about 85% identity to NP_001001548 or a fragment thereof.
  • CD36 is described, for example, by Oquendo et al., “CD36 directly mediates cytoadherence of Plasmodium falciparum parasitized erythrocytes,” Cell 58: 95-101, 1989.
  • CD47 is meant a CD47 glycoprotein that binds to a thrombospondin-derived peptides or that has at least about 85% identity to NP_000315 or a fragment thereof.
  • CD47 is described, for example, by Han et al., “CD47, a ligand for the macrophage fusion receptor, participates in macrophage multinucleation.” J. Biol. Chem. 275: 37984-37992, 2000.
  • CXCR3 is meant a G protein coupled receptor or fragment thereof having at least about 85% identity to NP_001495.
  • CXCR3 is described, for example, by Trentin et al., “The chemokine receptor CXCR3 is expressed on malignant B cells and mediates chemotaxis.” J. Clin. Invest. 104: 115-121, 1999.
  • blood vessel formation is meant the dynamic process that includes one or more steps of blood vessel development and/or maturation, such as angiogenesis, vasculogenesis, formation of an immature blood vessel network, blood vessel remodeling, blood vessel stabilization, blood vessel maturation, blood vessel differentiation, or establishment of a functional blood vessel network.
  • blood vessel development and/or maturation such as angiogenesis, vasculogenesis, formation of an immature blood vessel network, blood vessel remodeling, blood vessel stabilization, blood vessel maturation, blood vessel differentiation, or establishment of a functional blood vessel network.
  • angiogenesis is meant the growth of new blood vessels originating from existing blood vessels. Angiogenesis can be assayed by measuring the total length of blood vessel segments per unit area, the functional vascular density (total length of perfused blood vessel per unit area), or the vessel volume density (total of blood vessel volume per unit volume of tissue).
  • vasculogenesis is meant the development of new blood vessels originating from stem cells, angioblasts, or other precursor cells.
  • blood vessel stability is meant the maintenance of a blood vessel network.
  • alteration is meant a change in the sequence or in a modification (e.g., a post-translational modification) of a gene or polypeptide relative to an endogeneous wild-type reference sequence.
  • ameliorate is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.
  • antibody is meant any immunoglobulin polypeptide, or fragment thereof, having immunogen binding ability.
  • cancer in an animal refers to the presence of cells possessing characteristics typical of cancer-causing cells, for example, uncontrolled proliferation, loss of specialized functions, immortality, significant metastatic potential, significant increase in anti-apoptotic activity, rapid growth and proliferation rate, and certain characteristic morphology and cellular markers.
  • cancer cells will be in the form of a tumor; such cells may exist locally within an animal, or circulate in the blood stream as independent cells, for example, leukemic cells.
  • disease is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ.
  • fragment is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide.
  • a fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.
  • isolated nucleic acid molecule is meant a nucleic acid (e.g., a DNA) that is free of the genes, which, in the naturally occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene.
  • the term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences.
  • the term includes an RNA molecule which is transcribed from a DNA molecule, as well as a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.
  • an “isolated polypeptide” is meant a polypeptide of the invention that has been separated from components that naturally accompany it.
  • the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated.
  • the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the invention.
  • An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.
  • marker any protein or polynucleotide having an alteration in expression level or activity that is associated with a disease or disorder.
  • “By “neoplasia” is meant a disease that is caused by or results in inappropriately high levels of cell division, inappropriately low levels of apoptosis, or both. Solid tumors, hematological disorders, and cancers are examples of neoplasias.
  • operably linked is meant that a first polynucleotide is positioned adjacent to a second polynucleotide that directs transcription of the first polynucleotide when appropriate molecules (e.g., transcriptional activator proteins) are bound to the second polynucleotide.
  • appropriate molecules e.g., transcriptional activator proteins
  • peptide is meant any fragment of a polypeptide. Typically peptide lengths vary between 5 and 1000 amino acids (e.g., 5, 10, 15, 20, 25, 50, 100, 200, 250, 500, 750, and 1000).
  • polypeptide is meant any chain of amino acids, regardless of length or post-translational modification.
  • promoter is meant a polynucleotide sufficient to direct transcription.
  • reduce is meant a decrease in a parameter (e.g., blood vessel formation) as detected by standard art known methods, such as those described herein. As used herein, reduce includes a 10% change, preferably a 25% change, more preferably a 40% change, and even more preferably a 50% or greater change.
  • substantially identical is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein).
  • a reference amino acid sequence for example, any one of the amino acid sequences described herein
  • nucleic acid sequence for example, any one of the nucleic acid sequences described herein.
  • such a sequence is at least 60%, more preferably 80% or 85%, and even more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.
  • Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e ⁇ 3 and e ⁇ 100 indicating a closely related sequence.
  • sequence analysis software for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin
  • Sequence identity in the context of two nucleic acid or polypeptide sequences includes reference to the residues in the two sequences which are the same when aligned for maximum correspondence over a specified comparison window, and can take into consideration additions, deletions and substitutions.
  • percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (for example, charge or hydrophobicity) and therefore do not deleteriously change the functional properties of the molecule.
  • sequences differ in conservative substitutions the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences which differ by such conservative substitutions are said to have sequence similarity.
  • Percentage of sequence identity means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions, substitutions, or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions, substitutions, or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • substantially identical or “homologous” in their various grammatical forms in the context of polynucleotides means that a polynucleotide comprises a sequence that has a desired identity, for example, at least 60% identity, preferably at least 70% sequence identity, more preferably at least 80%, still more preferably at least 90% and even more preferably at least 95%, compared to a reference sequence using one of the alignment programs described using standard parameters.
  • a desired identity for example, at least 60% identity, preferably at least 70% sequence identity, more preferably at least 80%, still more preferably at least 90% and even more preferably at least 95%, compared to a reference sequence using one of the alignment programs described using standard parameters.
  • a desired identity for example, at least 60% identity, preferably at least 70% sequence identity, more preferably at least 80%, still more preferably at least 90% and even more preferably at least 95%.
  • nucleotide sequences are substantially identical if two molecules hybridize to each other under stringent conditions. However, nucleic acids which do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This may occur, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
  • One indication that two nucleic acid sequences are substantially identical is that the polypeptide which the first nucleic acid encodes is immunologically cross reactive with the polypeptide encoded by the second nucleic acid, although such cross-reactivity is not required for two polypeptides to be deemed substantially identical.
  • An “expression vector” is a nucleic acid construct, generated recombinantly or synthetically, bearing a series of specified nucleic acid elements that enable transcription of a particular gene in a host cell. Typically, gene expression is placed under the control of certain regulatory elements, including constitutive or inducible promoters, tissue-preferred regulatory elements, and enhancers.
  • a “recombinant host” may be any prokaryotic or eukaryotic cell that contains either a cloning vector or expression vector. This term also includes those prokaryotic or eukaryotic cells that have been genetically engineered to contain the cloned gene(s) in the chromosome or genome of the host cell.
  • operably linked is used to describe the connection between regulatory elements and a gene or its coding region. That is, gene expression is typically placed under the control of certain regulatory elements, including constitutive or inducible promoters, tissue-specific regulatory elements, and enhancers. Such a gene or coding region is said to be “operably linked to” or “operatively linked to” or “operably associated with” the regulatory elements, meaning that the gene or coding region is controlled or influenced by the regulatory element.
  • a “reference sequence” is a defined sequence used as a basis for sequence comparison.
  • a reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.
  • the length of the reference polypeptide sequence will generally be at least about 5, 10, or 15 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, about 100 amino acids, or about 150 amino acids.
  • the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides about 300 nucleotides or about 450 nucleotides or any integer thereabout or therebetween.
  • Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math., 2: 482, 1981; by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol., 48: 443, 1970; by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci.
  • the BLAST family of programs which can be used for database similarity searches includes: BLASTN for nucleotide query sequences against nucleotide database sequences; BLASTX for nucleotide query sequences against protein database sequences; BLASTP for protein query sequences against protein database sequences; TBLASTN for protein query sequences against nucleotide database sequences; and TBLASTX for nucleotide query sequences against nucleotide database sequences.
  • sequence identity/similarity values refer to the value obtained using the BLAST 2.0 suite of programs, or their successors, using default parameters (Altschul et al., Nucleic Acids Res, 2:3389-3402, 1997). It is to be understood that default settings of these parameters can be readily changed as needed in the future.
  • BLAST searches assume that proteins can be modeled as random sequences. However, many real proteins comprise regions of nonrandom sequences which may be homopolymeric tracts, short-period repeats, or regions enriched in one or more amino acids. Such low-complexity regions may be aligned between unrelated proteins even though other regions of the protein are entirely dissimilar.
  • a number of low-complexity filter programs can be employed to reduce such low-complexity alignments. For example, the SEG (Wooten and Federhen, Comput. Chem., 17:149-163, 1993) and XNU (Clayerie and States, Comput. Chem., 17:191-1, 1993) low-complexity filters can be employed alone or in combination.
  • the terms “treat,” “treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.
  • a “tumor,” as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues.
  • the terms “prevent,” “preventing,” “prevention,” “prophylactic treatment” and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.
  • the term “about,” when referring to a value can be meant to encompass variations of, in some embodiments, ⁇ 100% in some embodiments ⁇ 50%, in some embodiments ⁇ 20%, in some embodiments ⁇ 10%, in some embodiments ⁇ 5%, in some embodiments ⁇ 1%, in some embodiments ⁇ 0.5%, and in some embodiments ⁇ 0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods or employ the disclosed compositions.
  • the reaction was carried out for 24 hrs, then the TEA HCl precipitate was removed by filtration, and the solvent was removed by rotary evaporation.
  • the product was dissolved in 100 mL dichloromethane and washed five times with 200 mL of an aqueous solution of 0.2 M Na 2 CO 3 and three times with distilled water. The solution was dried with NaSO 4 and the solvent was removed by rotary evaporation.
  • Base monomer BR6 was polymerized with side chain monomers S3, S4, and S5 at a base:side chain ratio of 1.2:1 by weight without solvent at 90° C. for 24 hrs while stirring.
  • base polymer was dissolved in anhydrous dimethyl sulfoxide at 100 mg/mL with 0.2 mM end-cap. The reaction was allowed to proceed for 1 hr at room temperature while shaking.
  • FIG. 5 are shown representative formation and sizing of polymer/peptide nanoparticles (by nanoparticle tracking analysis on a Nanosight LM10).
  • Selected peptides and PBAEs were diluted in 25 mM sodium acetate buffer and then together in different weight-to-weight ratios.
  • w/w is unity, 1:1, in other embodiments there is an excess of polymer to peptide.
  • this ratio is 5:1, in other embodiments between 1:1-10:1, in other embodiments it is 10:1 to 20:1.
  • FIG. 6 both a 5:1 and 1:1 ratio is shown.
  • NanoSight nanoparticle tracking software and analysis individual particles were tracked in order to determine the average size distribution of the particles.
  • hydrophilic peptides DEAH Box poly8
  • PBAEs 336
  • there were very few background particles that is very few particles of peptide or PBAE only.
  • a noticeable nanoparticle distribution was observed, with an average size ranging from 100-150 nm.
  • hydrophobic peptides and PBAEs they have the possibility of aggregating with themselves.
  • a shift in the mean can be observed as a way to detect difference in nanoparticle formation.
  • DEAH peptide release by 336 nanoparticles at 4° C. (above) and 37° C. (below).
  • Changing polymer to peptide formulation ratios and concentrations are key to tune release. Slowing the reaction rate of degradation of the liable polymer bonds extends release from nanoparticles. This is shown by change in temperature, but could also be accomplished by increasing hydrophobicity of the polymer, increasing the molecular weight between liable ester groups, or other modifications known by someone in the art; FITC labeled DEAH peptide and 336 polymer were mixed and incubated for up to 10 minutes in sodium acetate buffer.
  • HUVEC viability/proliferation assays with polymer/SP6001/DEAH peptide; the CellTiter 96® AQ ueous One Solution Cell Proliferation assay was used to see the effect of both peptide and polymer on cell proliferation and viability.
  • Polymers at the right concentrations have minimal cytotoxic effect on the cells, such as 336 below 100 uM.
  • the individual peptides and Polymers were diluted in sodium acetate buffer and added to HUVECs in a 96-well plate. After incubating for a few days, the assay substrate was added and then incubated for a few hours at 37° C. Absorbance measurements were performed using a plate reader.
  • HUVEC migration assays with 336 polymer/DEAH peptide. These nanoparticles inhibit endothelial migration in addition to proliferation and viability. Peptide-polymer nanoparticles were made as described previously. Samples were added to HUVEC cells and migration was measured using the ACEA time course cell migration system. Nanoparticle formulations at a total peptide concentration of 20 uM were able to inhibit migration more than any peptide only at 20 ⁇ M.
  • FIG. 9 is shown in vivo 336 polymer nanoparticle/SP6001 DEAH peptide; Peptide-336 polymer nanoparticles were formulated as previously described and intravitreously injected to test in vivo efficacy.
  • ACNV laser mouse model was used on C57 BL/6 female mice. The mice receive laser eye treatments on day zero, followed by the intravitreous injections. Mice are then perfused with fluorescein labeled dextran on day 14 and choroidal flat mounts (bottom) were analyzed via fluorescence microscopy. On day 14, both the peptide only and nanoparticles formulations significantly reduced angiogenesis in the eye (top) and did so to a similar extent. This suggests that all peptide was released from nanoparticles by day 14.
  • FIG. 10 is shown (top) Particle size and (bottom) cell viability effects of various polymer/SP2012 nanoparticles as compared to peptide only of non-cytotoxic polymers; a range of polymer structures were mixed with SP2000 series peptides, in a similar manner as described above. Similar sizing is found with peptides from the same class with similar structural properties. For example, SP2000, SP2012, SP2024, SP2034, and SP2036 can be encapsulated similarly to each other with the same polymers, but different from peptides from other classes such as SP6001. Sizing was performed using the Malzern Zetasizer. Size strongly depends on polymer choice.
  • Pep-pol/pol/SP2012 refers to the change in cell proliferation/viability due to the peptide/polymer nanoparticle formulation divided by any change in cell proliferation/viability from the same dose of polymer by itself and this quantity divided by the change in cell proliferation/viability by delivering the same amount of peptide SP2012 as a bolus);
  • FIG. 11 shows polymer/peptide formulations for alternative peptides.
  • Peptide-polymer formulations made as described previously. Here two different classes of peptides are used. Experiments performed in a 96-well plate, with final results obtained using the same cell viability/proliferation assay as described previously. An increased effect (decreased metabolic activity) is observed for the nanoparticle formulations over the free peptide.
  • FITC-tagged bovine serum albumin (BSA) was mixed with a macromer solution containing 10% (w/v) PEGDA (Mn-270 Da) with various amounts of B4S4, dissolved in a 1:1 (v/v) mixture of DMSO and PBS.
  • Irgacure 2959 was added at 0.05% (w/v), and the solution was briefly vortexed and immediately polymerized to form gels. The gels were incubated at 37° C. in 1 ⁇ PBS with shaking. PBS was removed at each time point to measure fluorescence.
  • the observed slowed release is due to two factors: first, increased overall hydrophobicity can decrease the movement of water in and out of the gel, reducing degradation rate and protein release. Furthermore, this method of mixing relatively hydrophobic diacrylates with hydrophilic diacrylates in a co-solvent (mixture of water and DMSO) that can dissolve both types of polymer causes the spontaneous formation of micro-emulsions within the gel (see SEM in FIG. 13 ; increasing B4S4 from top [0.2% w/w] to bottom [5% w/w]). Similar to traditionally studied controlled-release microparticles, these microparticles within photopolymerized gels could serve as another way to tune the release of an encapsulated peptide, protein, or drug.
  • a co-solvent mixture of water and DMSO
  • nanoparticles were formed by mixing PBAE and DNA in 25 mM sodium acetate buffer (pH 5) at a 30:1 polymer:DNA ratio (w/w). After 10 min of incubation, sucrose solution was added at various concentrations. The particles were mixed, then frozen at ⁇ 80° C. for 1 hr and lyophilized for 48 hr. They then were used for transfection or sizing or were stored at either room temperature, 4° C. or ⁇ 20° C. and tested at various timepoints.
  • the size distribution of appropriately freeze-dried particles remains the same as freshly-prepared particles (bottom left, left-most histogram). Freeze-dried particles also remain more stable in serum-containing medium than freshly-prepared particles (upper left).
  • transfection efficiency is comparable between fresh particles and particles lyophilized with sucrose (right) even after 3 months of storage. Modifying type of sugar and concentration of sugar modulates the stability of the degradable nanoparticles.
  • DNA nanoparticles were prepared by mixing DNA and polymer in a sodium acetate buffer. Sucrose was added for a final concentration of 15 mg/mL, and the solution was used to coat the surface of a trabecular bone construct. This construct was then lyophilized for 2 days before being seeded with primary human cells ( ⁇ 50% GFP + for ease of visualization). Referring now to FIG. 15 , DsRed expression was observed within 24 hr, indicating that the nanoparticles remained functional and able to transfect cells in this new system.
  • Lyophilized nanoparticles also can be mixed with PLGA microparticles to form a larger construct that can be more easily manipulated and also can tune controlled release properties.
  • DsRed DNA-containing nanoparticles were compressed into a pellet with PLGA microparticles. This pellet was then placed within a well containing primary human glioblastoma cells ( ⁇ 20% GFP + for ease of visualization through the opaque pellet).
  • DNA-loaded nanoparticles have been incorporated into natural and synthetic scaffolds, disks, microparticles, and hydrogels.
  • Reducible functional groups mediate successful siRNA-delivery, including transfection.
  • GFP + primary human glioblastoma cells were seeded in 96-well plates at a density of 10 4 cells/well in complete culture medium (DMEM/F-12 with 10% FBS and 1% antibiotic-antimycotic) and allowed to adhere overnight. Just before transfection, the culture medium was changed to serum-free medium. Particles were prepared by diluting polymer and siRNA both in 25 mM sodium acetate buffer (pH 5), then mixing them at a 100:1 polymer:siRNA ratio (w/w).
  • Nanoparticles formed spontaneously after 10 min of incubation and were added to the cells in medium at a 1:5 ratio (v/v) and a final concentration of 60 nM.
  • Each polymer/siRNA treatment group was paired with a control group using a scrambled siRNA sequence (scrRNA).
  • scrRNA scrambled siRNA sequence
  • Cells were incubated with the particles for 4 hr. The medium and particles were then aspirated and replaced with complete medium.
  • GFP expression was measured using a Synergy 2 multiplate fluorescence reader (Biotek). Background fluorescence was measured from GFP cells in medium and was subtracted from all other readings. Knockdown was calculated by normalizing GFP fluorescence (excitation 485 nm, emission 528 nm) from the siRNA-treated cells to the scrRNA-treated cells. Medium was changed every 3 days.
  • the reducible disulfide bond in the endgroup E10 drastically improves siRNA delivery and gene knockdown.
  • FIG. 18 GFP + glioblastoma cells were transfected with scrambled (control) siRNA (top panels) or siRNA against GFP (bottom).
  • E10 cystamine dihydrochloride
  • FIGS. 1A-1C the activity of R6-series polymers at delivering siRNA to knockdown GFP signal is GB cells is further demonstrated.
  • FIG. 20 a gel retardation assay of siRNA with BR6-S5-E10 at varying ratios of polymer to RNA is shown.
  • the polymer effectively retards siRNA (top), but in the presence of 5 mM glutathione siRNA is released immediately (bottom).
  • an E10-endcapped polymer retards siRNA efficiently, but upon addition of 5 mM glutathione, siRNA is immediately released (bottom). Numbers refer to the w/w ratio of polymer-to-siRNA in all cases.
  • knockdown efficiency also is affected by molecular weight of the polymer.
  • 1.2:1, 1.1:1, and 1.05:1 refer to the ratio of reactants in the base polymer step growth reaction, which affects the ultimate molecular weight.
  • Top 4310 formulations were able to achieve greater knockdown over time compared to commercially available reagents like Lipofectamine 2000 (Lipo).
  • siRNA knockdown is affected by the endcap (E), base polymer (increasing hydrophobicity from L to R within each E), and molecular weight (increasing L to R within each base polymer).
  • E endcap
  • base polymer increasing hydrophobicity from L to R within each E
  • molecular weight increasing L to R within each base polymer.
  • One endcap that shows high knockdown even at lower molecular weights is E10, which is strikingly more effective than the other endcaps tested for the same base polymers.
  • Other PBAEs were also highly effective when synthesized at high molecular weight.
  • FIG. 27 is shown 4410, 200 w/w (blue line on above graph), 8 days after transfection: Left: hMSCs treated with scrambled control; Right: hMSCs treated with siRNA.
  • polymer molecular weight is between 4.00-10.00 kDa for siRNA delivery.
  • the presently disclosed biomaterial can be used for other forms of delivery, for example DNA delivery.
  • DNA transfection shows some similar trends compared with siRNA, but with different optimal endcaps. Specific polymer structure is critical to determine which polymers are effective for DNA delivery or siRNA delivery or both. Both DNA and siRNA transfection depend less on MW with high polymer hydrophobicity. High GFP DNA delivery was achieved using PBAEs, with transfection in 10% serum and at 5 ⁇ g DNA/mL. Referring now to FIG. 30 , several formulations with up to 90% transfection and high (>90%) viability are shown.
  • GB transfection is demonstrated. More particularly, 551 GB cells cultured as neurospheres (undifferentiated). They were plated in monolayer on laminin 24 hr before transfection with DsRed DNA using 447 LG (red). 48 hr after transfection, they were stained for nestin (blue). Red and blue overlaid (left) show that transfection occurred in nestin+ cells (nestin only: right).
  • polymer molecular weight is between 3.00-10.0 kDa.
  • the presently disclosed subject matter demonstrates in vivo activity for selected peptides in DIVAA angioreactors and a lung cancer xenograft model, Koskimaki J E, Karagiannis E D, Tang B C, Hammers H, Watkins D N, Pili R, et al. Pentastatin-1, a collagen IV derived 20-mer peptide, suppresses tumor growth in a small cell lung cancer xenograft model. BMC Cancer 2010; 10:29, and in a breast cancer xenograft model using MDA-MB-231 cells.
  • FIG. 33D Representative data showing the activity of free peptide and peptide encapsulated in the presently disclosed polymeric particles are shown in FIG. 33D , which shows the metabolic activity of free peptides and peptides in polymeric particles.
  • Glioblastoma is a grade IV brain cancer as defined by the WHO and is the most common primary CNS tumor in the United States. Current treatment includes surgical resection, radiotherapy, and chemotherapy. The median survival with treatment is approximately 14 months.
  • BCSCs Brain cancer stem cells possess genetic and morphological features similar to neural stem cells. Small numbers of BCSCs can initiate gliomas. BCSCs are refactory to conventional anti-cancer treatments.
  • Gene delivery typically is accomplished by either vaccine-mediated or polymer mediates techniques.
  • Virus-mediated gene delivery is highly efficient, insertional mutagenesis, and toxicity/immunogenicity.
  • Polymer-mediated gene delivery is chemically versatile, potentially safer than vaccine-mediated gene delivery, but typically is less efficient. See Green et al., 2008. Acc. Chem. Res. 41(6):749-59; Putnam 2006. Nat. Mater. 5(6):439-51.
  • Non-viral, e.g., polymer-mediated gene delivery can be accomplished, in some embodiments, by using poly(beta-amino esters) (PBAEs).
  • PBAEs suitable for use in target delivery can be synthesized in a two-step reaction provided herein below in Scheme 6 and can form nanocomplexes with negatively-charged cargo (e.g., DNA, siRNA) via electrostatic interactions as disclosed, for example, in some embodiments described in International PCT Patent Application Publication No. WO/2010/132879 for “Multicomponent Degradable Cationic Polymers,” to Green et al., which is incorporated herein by reference in its entirety.
  • cargo e.g., DNA, siRNA
  • the presently disclosed subject matter demonstrates the delivery of DNA to GB cells, i.e., bulk tumor (non-stem cells; verifies the efficacy of the presently disclosed methods in BCSCs; demonstrates the delivery of apoptosis-inducing genes in BCSCs; provides practical considerations for translation of the presently disclosed methods; and discusses how the presently disclosed methods can be used in conjunction with other methods for treating GB.
  • FIG. 34 the delivery of DNA to GB bulk tumor cells is demonstrated for representative biomaterials.
  • FIG. 35 the transfection of genes to BCSC is demonstrated for representative presently disclosed biomaterials.
  • FIG. 36 demonstrates the delivery of DNA to fetal (healthy) cells.
  • FIG. 37 also demonstrates the delivery of DNA to BCSCs.
  • the delivery of apoptosis-inducing genes in BCSCs is demonstrated in FIGS. 38 to 39 .
  • PBAEs can be used for highly effective DNA delivery to GB cells, including tumor-initiating stem cells; transfection occurs even in 3D neurospheres in suspension; transfection is much less efficient in non-cancer cells (F34 fetal cells) as compared to GB cells; and transfection with secreted TRAIL causes more death in BCSCs with not significant effect on healthy cells.
  • the presently disclosed methods provide an ease of preparation, e.g., only water needs to be added to the lyophilized nanoparticles, long-term storage, large, consistent batches, manipulation for uses in other devices, and stability in suspension. See scheme in FIG. 3 .
  • FIG. 40 particles lyophilized with sucrose and used immediately are as effective in transfection as freshly prepared particles. Further, no loss in efficiency is observed within three months; and approximately 50% efficiency is retained after six months.
  • FIGS. 41 and 42 Other methods for treatment of GB include siRNA delivery to GB cells ( FIG. 43 ).
  • FIGS. 44 and 45 A comparison of siRNA vs. DNA delivery in GB cells is shown in FIGS. 44 and 45 . More particularly, as shown in FIG. 45 , both 4410 and 447 can form complexes with DNA and siRNA; a higher weight ratio of polymer-to-nucleic acid is needed for siRNA than for DNA; E10 polymers release siRNA immediately, but not DNA, upon addition of glutathione (GSH).
  • GSH glutathione
  • PBAE/nucleic acid nanoparticles can be fabricated in a form that remains stable over time and allow flexibility for clinical use; PBAEs can be used for effective DNA or siRNA delivery to GB-derived BCSCs; and efficient release of cargo is necessary for effective nucleic acid delivery, especially with siRNA.
  • FIG. 46 depicts a strategy of combining nanoparticles within microparticles to extend release further.
  • PLGA or blends of PLGA can be combined with the presently disclosed polymers to form microparticles by double emulsion.
  • FIG. 47 shows release of a representative peptide, DEAH-FITC, from a presently disclosed microparticle.
  • FIG. 48 shows slow extended release from microparticles containing nanoparticles that contain peptides; FITC-DEAH peptide was first mixed with the 336 PBAE to allow for self-assembly into nanoparticles and was then mixed with BSA (middle) or not (bottom) to form an aqueous mixture.
  • This mixture was added to a DCM-PLGA phase and sonicated to form a w/o suspension. This suspension was then added to a PVA solution and homogenized to form the final w/o/w suspension. This mixture was finally added to another PVA solution to allow for the DCM to evaporate and harden the formed microparticle.
  • Different release profiles can potentially be obtained as seen above for the different microparticle formulations. In all cases, there is a long-term release of the peptide. Forming nanoparticles that encapsulate the peptide within the microparticles, extends the release compared to encapsulating peptide directly into microparticles (middle figure).
  • the particles can be designed to have different release depending on the local environment (top figure). In some embodiments, release is constant over time and zero-order with respect to time (bottom figure).
  • FIG. 49 in shown the in vivo effects of microparticle formulations in both the CNV and rhoNEGF model over time.
  • DEAH SP6001-336 PBAE nanoparticle formulation made as described previously.
  • Top Intravitreal injections into CNV model mice as described previously show comparable effects after 14 days, even though only small fraction of peptide is released over that time from microparticles.
  • Bottom Intravitreal injections into CNV model mice as described previously show comparable effects after 14 days, even though only small fraction of peptide is released over that time from microparticles.
  • (Middle) and (Bottom) A genetic model of wet form of age-related macular degeneration in mice used to test long-term effect of microparticles. After 1 week (middle) comparable effects seen in reduction of angiogenesis.
  • microparticle After 8 weeks (bottom), however, while peptide only no longer inhibits angiogenesis, the microparticle still does, as it is still releasing peptide over this time.
  • PLGA is used to form the microparticles used above
  • other polymers including the synthetic polyesters and polyamides described above.
  • blends of these polymer are combined with PLGA to form microparticles with differing environmental sensitivity and release properties; (a) the effect of microparticle (SP-6001) in CNV model mouse; (b) the effect of microparticle (SP-6001) in rhoNEGF (V6) mouse, 1 week after injection; and (c) the effect of microparticle (SP-6001) in rhoNEGF (V6) mouse, 8 weeks after injection.

Abstract

Polymeric nanoparticles, microparticles, and gels for delivering cargo, e.g., a therapeutic agent, such as a peptide, to a target, e.g., a cell, and their use for treating diseases, including angiogenesis-dependent diseases, such as age-related macular degeneration and cancer, are disclosed. Methods for formulating, stabilizing, and administering single peptides or combinations of peptides via polymeric particle and gel delivery systems also are disclosed.

Description

    CROSS-REFERENCE TO RELATED APPLICATIONS
  • This application claims the benefit of U.S. Provisional Application Nos. 61/392,224, filed Oct. 12, 2010; 61/542,995, filed Oct. 4, 2011; and 61/543,046, filed Oct. 4, 2011, each which is incorporated herein by reference in its entirety.
    • BACKGROUND
  • Biomaterials have the potential to significantly impact medicine as delivery systems for imaging agents, biosensors, drugs, and genes. Farokhzad O C. Nanotechnology for drug delivery: the perfect partnership. Expert Opin Drug Deliv 2008; 5(9):927-9; Putnam D. Polymers for gene delivery across length scales. Nat Mater 2006; 5(6):439-51; Brigger I, Dubernet C, Couvreur P. Nanoparticles in cancer therapy and diagnosis. Adv Drug Deliv Rev 2002; 54(5):631-51. Challenges exist, however, in creating a delivery vehicle capable of effective, safe, and controlled release of sensitive biomolecules. Although rapid advances have been made for sustained delivery of small molecule drugs using biotechnology, similar advances have not been made for the delivery of peptides, siRNA, or combinations of biological agents.
    • SUMMARY
  • The presently disclosed subject matter provides polymeric nanoparticles, microparticles, and gels for delivering cargo, e.g., a therapeutic agent, such as a peptide, to a target, e.g., a cell, and their use for treating multiple diseases, including angiogenesis-dependent diseases, such as age-related macular degeneration and cancer. Methods for formulating, stabilizing, and administering single peptides or combinations of peptides via polymeric particle and gel delivery systems, for example, using a controlled release strategy, also are disclosed.
  • In some aspects, the presently disclosed subject matter provides a bioreducible, hydrolytically degradable polymer of formula (Ia):
  • Figure US20160374949A9-20161229-C00001
  • wherein:
  • n is an integer from 1 to 10,000;
  • R1, R2, R3, R4, R5, R6, R7, R8, and R9 are each independently selected from the group consisting of hydrogen, branched and unbranched alkyl, branched and unbranched alkenyl, branched and unbranched alkynyl, aryl, halogen, hydroxyl, alkoxy, carbamoyl, carboxyl ester, carbonyldioxyl, amide, thiohydroxyl, alkylthioether, amino, alkylamino, dialkylamino, trialkylamino, cyano, ureido, a substituted alkanoyl group, cyclic, cyclic aromatic, heterocyclic, and aromatic heterocyclic groups, each of which may be substituted with at least one substituent selected from the group consisting of branched or unbranched alkyl, branched and unbranched alkenyl, branched and unbranched alkynyl, amino, alkylamino, dialkylamino, trialkylamino, aryl, ureido, heterocyclic, aromatic heterocyclic, cyclic, aromatic cyclic, halogen, hydroxyl, alkoxy, cyano, amide, carbamoyl, carboxylic acid, ester, carbonyl, carbonyldioxyl, alkylthioether, and thiohydroxyl groups;
  • wherein R1 can be present or absent and when present the compound of formula (I) further comprises a counter ion selected from the group consisting of chloride, fluoride, bromide, iodide, sulfate, nitrate, fumarate, acetate, carbonate, stearate, laurate, and oleate; and
  • wherein at least one R comprises a backbone of a diacrylate having the following structure:
  • Figure US20160374949A9-20161229-C00002
  • wherein X1 and X2 are each independently substituted or unsubstituted C2-C20 alkylene, and wherein each X1 and X2 can be the same or different.
  • In other aspects, the presently disclosed subject matter provides a nanoparticle, microparticle, or gel comprising a compound of formula (I):
  • Figure US20160374949A9-20161229-C00003
  • wherein:
  • n is an integer from 1 to 10,000;
  • R1, R2, R3, R4, R5, R6, R7, R8, and R9 are each independently selected from the group consisting of hydrogen, branched and unbranched alkyl, branched and unbranched alkenyl, branched and unbranched alkynyl, aryl, halogen, hydroxyl, alkoxy, carbamoyl, carboxyl ester, carbonyldioxyl, amide, thiohydroxyl, alkylthioether, amino, alkylamino, dialkylamino, trialkylamino, cyano, ureido, a substituted alkanoyl group, cyclic, cyclic aromatic, heterocyclic, and aromatic heterocyclic groups, each of which may be substituted with at least one substituent selected from the group consisting of branched or unbranched alkyl, branched and unbranched alkenyl, branched and unbranched alkynyl, amino, alkylamino, dialkylamino, trialkylamino, aryl, ureido, heterocyclic, aromatic heterocyclic, cyclic, aromatic cyclic, halogen, hydroxyl, alkoxy, cyano, amide, carbamoyl, carboxylic acid, ester, carbonyl, carbonyldioxyl, alkylthioether, and thiohydroxyl groups;
  • wherein R1 can be present or absent and when present the compound of formula (I) further comprises a counter ion selected from the group consisting of chloride, fluoride, bromide, iodide, sulfate, nitrate, fumarate, acetate, carbonate, stearate, laurate, and oleate; and
  • at least one of R, R′, and R″ comprise a reducible or degradable linkage, and wherein each R, R′, or R″ can independently be the same or different;
  • under the proviso that when at least one R group comprises an ester linkage of the formula —C(═O)—O— and the compound of formula (I) comprises a poly(beta-amino ester), then the compound of formula (I) must also comprise one or more of the following characteristics:
  • (a) each R group is different;
  • (b) each R″ group is different;
  • (c) each R″ group is not the same as any of R′, R1, R2, R3, R4, R5, R6, R7, R8, and R9;
  • (d) the R″ groups degrade through a different mechanism than the ester-containing R groups, wherein the degradation of the R″ group is selected from the group consisting of a bioreducible mechanism or an enzymatically degradable mechanism; and/or
  • (e) the compound of formula (I) comprises a substructure of a larger cross-linked polymer, wherein the larger cross-linked polymer comprises different properties from compound of formula (I);
  • and one or more peptides selected from the group consisting of an anti-angiogenic peptide, an anti-lymphangiogenic peptide, an anti-tumorigenic peptide, and an anti-permeability peptide.
  • In other aspects, the presently disclosed subject matter provides a multilayer particle comprising a core and one or more layers, wherein the core comprises a material selected from the group consisting of a compound of formula (I), a gold nanoparticle, an inorganic nanoparticle, an organic polymer, and the one or more layers comprise a material selected from the group consisting of a compound of formula (I), an organic polymer, one or more peptides, and one or more additional biological agents. In yet other aspects, the presently disclosed subject matter provides a microparticle comprising a compound of formula (I), poly(lactide-co-glycolide) (PLGA), or combinations thereof.
  • In other aspects, the presently disclosed subject matter provides a method for stabilizing a suspension of nanoparticles and/or microparticles of formula (I), the method comprising: (a) providing a suspension of nanoparticles and/or microparticles of formula (I); (b) admixing a lyroprotectant with the suspension; (c) freezing the suspension for a period of time; and (d) lyophilizing the suspension for a period of time.
  • In further aspects, the presently disclosed subject matter provides a pellet or scaffold comprising one or more lyophilized particle, wherein the one or more lyophilized particle comprises a compound of formula (I).
  • In yet further aspects, the presently disclosed subject matter provides a method of treating a disease or condition, the method comprising administering to a subject in need of treatment thereof a therapeutically effective amount of a nanoparticle, microparticle, gel, or multilayer particle comprising a compound of formula (I), wherein the nanoparticle, microparticle, gel, or multilayer particle further comprises a therapeutic agent specific for the disease or condition to be treated. In some aspects, the disease or condition comprises an angiogenesis-dependent disease or condition, including, but not limited to, cancer and age-related macular degeneration. In other aspects, the disease or condition is a non-angiogenic disease or condition. In certain aspects, the therapeutic agent encapsulated with the presently disclosed particles can be selected from the group consisting of gene, DNA, RNA, siRNA, miRNA, is RNA, agRNA, smRNA, a nucleic acid, a peptide, a protein, a chemotherapeutic agent, a hydrophobic drug, a small molecule drug, and combinations thereof.
  • Certain aspects of the presently disclosed subject matter having been stated hereinabove, which are addressed in whole or in part by the presently disclosed subject matter, other aspects will become evident as the description proceeds when taken in connection with the accompanying Examples and Figures as best described herein below.
    • BRIEF DESCRIPTION OF THE FIGURES
  • Having thus described the presently disclosed subject matter in general terms, reference will now be made to the accompanying Figures, which are not necessarily drawn to scale, and wherein:
  • FIG. 1 is an illustration of the presently disclosed multilayer particles;
  • FIG. 2 is a scheme for producing hydrogels comprising the presently disclosed materials.
  • FIG. 3 shows a scheme for producing stable nanoparticle suspensions;
  • FIGS. 4A-4D show representative polymer structures tuned to peptide cargos;
  • FIGS. 5A and 5B show representative formation and sizing of polymer/peptide nanoparticles (by nanoparticle tracking analysis on a Nanosight LM10);
  • FIG. 6 shows DEAH peptide release by 336 nanoparticles at 4° C. (above) and 37° C. (below);
  • FIG. 7 shows HUVEC viability/proliferation assays with polymer/SP6001/DEAH peptide;
  • FIG. 8 shows HUVEC migration assays with 336 polymer/DEAH peptide;
  • FIG. 9 shows in vivo 336 polymer nanoparticle/SP6001 DEAH peptide;
  • FIG. 10 shows (top) Particle size and (bottom) cell viability effects of various polymer/SP2012 nanoparticles as compared to peptide only of non-cytotoxic polymers;
  • FIG. 11 shows polymer/peptide formulations for alternative peptides;
  • FIG. 12 shows data for FITC-tagged bovine serum albumin (BSA) mixed with a macromer solution containing 10% (w/v) PEGDA (Mn˜270 Da) with various amounts of B4S4, dissolved in a 1:1 (v/v) mixture of DMSO and PBS;
  • FIG. 13 shows an SEM of increasing B4S4 from top [0.2% w/w] to bottom [5% w/w]);
  • FIG. 14 shows the size distribution of appropriately freeze-dried particles (bottom left, right-most histogram) remains the same as freshly-prepared particles (bottom left, left-most histogram). Freeze-dried particles also remain more stable in serum-containing medium than freshly-prepared particles (upper left). Using DNA-loaded nanoparticles, transfection efficiency is comparable between fresh particles and particles lyophilized with sucrose (right) even after 3 months of storage;
  • FIG. 15 is Left: brightfield+GFP+DsRed, showing presence of cells (green) being transfected with DsRed (red) on a bone scaffold (brightfield). Right: GFP and DsRed shown only;
  • FIG. 16 demonstrates that DsRed expression was observed within 4 days and remained very robust even after 12 days: top=1 day, middle=4 days, bottom=12 days after transfection;
  • FIG. 17 demonstrates the incorporation of DNA-loaded nanoparticles into natural and synthetic scaffolds, disks, microparticles, and hydrogels;
  • FIG. 18 demonstrates transfection of GFP+ glioblastoma cells with scrambled (control) siRNA (top panels) or siRNA against GFP (bottom);
  • FIGS. 19A-19C show activity of R6-series polymers at delivering siRNA to knockdown GFP signal in GB cells; % Knockdown of GFP expression in GFP+ glioblastoma cells transfected with siRNA against GFP, normalized to cells transfected with scrambled siRNA, using various BR6 polymers as a transfection agent: (A) transfection with acrylate-terminated BR6 polymers with either S3, S4 or S5 as the side chain; (B) transfection with E10 end-capped versions of the polymers in Figure A; and (C) GFP fluorescence images of cells transfected with BR6-S4-Ac complexed scrambled RNA (top) vs. siRNA against GFP (bottom);
  • FIG. 20 shows gel retardation assay of siRNA with BR6-S5-E10 at varying ratios of polymer to RNA. The polymer effectively retards siRNA (top), but in the presence of 5 mM glutathione siRNA is released immediately (bottom). These data demonstrate the hypothesized intracellular release of siRNA and elucidates the mechanism by which nanoparticles formed using BR6 facilitate strong siRNA transfection and GFP knockdown;
  • FIG. 21 shows that an E10-endcapped polymer (top) retards siRNA efficiently, but upon addition of 5 mM glutathione, siRNA is immediately released (bottom). Numbers refer to the w/w ratio of polymer-to-siRNA in all cases;
  • FIG. 22 shows that the same base polymer as shown in FIG. 25 with a different endcap (E7, 1-(3-aminopropyl)-4-methylpiperazine) also retards siRNA (top) but is not affected by application of glutathione (bottom);
  • FIG. 23 provides gel permeation chromatography data of BR6 polymerized with S4 at a BR6:S4 ratio of 1.2:1 at 90° C. for 24 hours, before and after end-capping with E7;
  • FIG. 24 shows that knockdown efficiency also is affected by molecular weight of the polymer. 1.2:1, 1.1:1, and 1.05:1 refer to the ratio of reactants in the base polymer step growth reaction;
  • FIG. 25 demonstrates combined DNA (RFP) and siRNA delivery (against GFP) in GB;
  • FIG. 26 shows that siRNA knockdown is affected by the endcap (E), base polymer (increasing hydrophobicity from L to R within each E), and molecular weight (increasing L to R within each base polymer);
  • FIG. 27 shows 4410, 200 w/w (blue line on above graph), 8 days after transfection: Left: hMSCs treated with scrambled control; Right: hMSCs treated with siRNA;
  • FIG. 28 demonstrates that in variable molecular weight embodiments, polymer molecular weight is between 4.00-10.00 kDa for siRNA delivery;
  • FIG. 29 demonstrates the use of the presently disclosed materials for DNA delivery;
  • FIG. 30 shows GB Transfection;
  • FIG. 31 shows 551 GB cells cultured as neurospheres (undifferentiated);
  • FIG. 32 demonstrates that, for a DNA delivery application, in some embodiments, polymer molecular weight is between 3.00-10.0 kDa;
  • FIG. 33 provides representative characteristics exhibited by the presently disclosed biodegradable polymers;
  • FIG. 34 demonstrates the delivery of DNA to GB bulk tumor cells for representative biomaterials;
  • FIG. 35 demonstrates the transfection of genes to BCSC for representative presently disclosed biomaterials;
  • FIG. 36 demonstrates the delivery of DNA to fetal (healthy) cells;
  • FIG. 37 demonstrates the delivery of DNA to BCSCs;
  • FIGS. 38 and 39 demonstrate the delivery of apoptosis-inducing genes in BCSCs;
  • FIG. 40 shows that particles lyophilized with sucrose and used immediately are as effective in transfection as freshly prepared particles;
  • FIGS. 41 and 42 demonstrate the use of the presently disclosed materials and methods for long-term gene delivery;
  • FIG. 43 demonstrates siRNA delivery to GB cells;
  • FIGS. 44 and 45 provide a comparison of siRNA vs. DNA delivery in GB cells;
  • FIG. 46 depicts a strategy of combining nanoparticles within microparticles to extend release further. PLGA or blends of PLGA with the presently disclosed polymers are used to form microparticles by single or double emulsion;
  • FIG. 47 shows DEAH-FITC release from microparticles comprising a presently disclosed polymer and a peptide;
  • FIG. 48 shows slow extended release from microparticles containing nanoparticles that contain peptides; and
  • FIG. 49 shows in vivo effects of microparticle formulations in both the CNV and rho/VEGF model over time.
    •  DETAILED DESCRIPTION
  • The presently disclosed subject matter now will be described more fully hereinafter with reference to the accompanying Figures, in which some, but not all embodiments of the inventions are shown. Like numbers refer to like elements throughout. The presently disclosed subject matter may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Indeed, many modifications and other embodiments of the presently disclosed subject matter set forth herein will come to mind to one skilled in the art to which the presently disclosed subject matter pertains having the benefit of the teachings presented in the foregoing descriptions and the associated Figures. Therefore, it is to be understood that the presently disclosed subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims.
    • I. Peptide/Particle Delivery Systems
  • The presently disclosed subject matter provides compositions of matter, methods of formulation, and methods of treatment utilizing drug delivery systems comprising one or more degradable polymers and one or more biological agents. The polymers described in these systems must be biodegradable. Mechanisms for this degradability include, but are not limited to, hydrolytic degradation, enzymatic degradation, and disulfide reduction. The biological agents described in these systems include, but are not limited to, therapeutic or diagnostic agents, such as small molecules, peptides, proteins, DNA, siRNA, miRNA, is RNA, contrast agents, and other agents one skilled in the field would wish to encapsulate. In particular embodiments, biological therapeutic agents that are sensitive to degradation and sized approximately 10,000-25,000 Da, including siRNA and peptides, are suitable for use with the presently disclosed materials.
  • Peptide drugs in polymeric delivery systems are useful for various therapeutic and diagnostic applications. Some embodiments of the presently disclosed subject matter are useful for treating angiogenesis-dependent diseases including, but not limited to, age-related macular degeneration (AMD) and cancer. One particular embodiment of the presently disclosed subject matter includes specific peptide sequences, as well as methods of formulating, stabilizing, and administering these peptides as single agents or as combinations of peptides via polymeric nanoparticle-based, microparticle-based, gel-based, or conjugate-based delivery systems.
  • The presently disclosed nanoparticles, microparticles, and gels can be used to deliver cargo, for example a therapeutic agent, such as a peptide or protein, to a target, for example, a cell. The cargo delivered by the presently disclosed nanoparticles, microparticles, and gels can act, in some embodiments, as a therapeutic agent or a biosensor agent. Combinations of polymeric materials and cargo, for example a single peptide or combination of peptides, can be formulated by the presently disclosed methods, which allows for the control, or tuning, of the time scale for delivery.
  • Further, the presently disclosed polymeric materials can be used to form self-assembled electrostatic complexes, micelles, polymersomes, emulsion-based particles, and other particle formulations known to one of ordinary skill in the art. Nanoparticles formed from the presently disclosed polymeric materials can be formulated into larger microparticles to further extend duration and timing of release. Lyophilized formulations that can maintain longer shelf life and stability also are described. The presently disclosed particles can be administered as a powder, cream, ointment, implant, or other reservoir device.
  • The presently disclosed nanoparticles, microparticles, and gels can be used to treat many diseases and conditions including, but not limited to, all types of cancers, ophthalmic diseases, cardiovascular diseases, and the like. In particular embodiments, the disease or condition treated by the presently disclosed nanoparticles, microparticles, and gels include breast cancer and age-related macular degeneration.
    • A. Bioreducible and Hydrolytically Degradable Two-Component Degradable Polymers
  • The presently disclosed materials offer several advantages for use in delivering cargo, e.g., a therapeutic agent, such as a peptide or siRNA, to a target, e.g., a cell. Such advantages include a slower degradation in the extracellular environment and a quicker degradation in the intracellular environment. Further, the method of synthesis allows for diversity of monomer starting materials and corresponding facile permutations of polymer structure. The presently disclosed materials can be used to form self-assembled nanoparticles, blended microparticles, gels, and bioconjugates. The presently disclosed polymers also have the following advantages compared to other drug delivery polymers known in the art: a higher polymerization than with disulfide acrylamides, which is important for various applications because it can be used to tune both binding/encapsulation and release; two time scales for degradation (hydrolytic degradation in water and disulfide reduction due to glutathione inside the cell), which facilitates drug release and reduces potential cytotoxicity; tunable structural diversity, with hydrophobic, hydrophilic, and charged moieties to aid in encapsulation of a target biological agent; and, usefulness for drug delivery, including high siRNA delivery, even without end-modification of the polymer.
  • Certain polyesters have been shown previously to form nanoparticles in the presence of biological agents, such as nucleic acids, and facilitate their entry into a cell. In such materials, release of the nucleic acid is modulated by hydrolytic degradation of the polyester polymer. The addition of a bioreducible disulfide moiety into the backbone of these polymers, however, can specifically target release to the reducing intracellular environment.
  • Accordingly, a library of bioreducible polyesters can be synthesized by oxidizing and acrylating various mercapto-alcohols (representative diacrylates formed from the presently disclosed synthetic process are shown in Scheme 1 below), then reacting with amine side chains. The structure of a representative bioreducible polyester, e.g., 2,2′-disulfanediylbis(ethane-2,1-diyl)diacrylate (BR6) polymerized with S4, also is shown in Scheme 1.
  • Figure US20160374949A9-20161229-C00004
  • In other embodiments, amine-containing molecules can be reacted to terminal groups of the polymer. In particular embodiments, this amine-containing molecule also contains poly(ethylene glycol) (PEG) or a targeting ligand. In other embodiments, the disulfide acrylates are not reacted with amines, but are instead polymerized through other mechanisms including, but not limited to, free radical polymerization to form network polymers and gels. In other embodiments, oligomers are first formed and then the oligomers are polymerized to form block co-polymers or gels.
  • More particularly, the presently disclosed subject matter provides a bioreducible, hydrolytically degradable polymer of formula (Ia):
  • Figure US20160374949A9-20161229-C00005
  • wherein:
  • n is an integer from 1 to 10,000;
  • R1, R2, R3, R4, R5, R6, R7, R8, and R9 are each independently selected from the group consisting of hydrogen, branched and unbranched alkyl, branched and unbranched alkenyl, branched and unbranched alkynyl, aryl, halogen, hydroxyl, alkoxy, carbamoyl, carboxyl ester, carbonyldioxyl, amide, thiohydroxyl, alkylthioether, amino, alkylamino, dialkylamino, trialkylamino, cyano, ureido, a substituted alkanoyl group, cyclic, cyclic aromatic, heterocyclic, and aromatic heterocyclic groups, each of which may be substituted with at least one substituent selected from the group consisting of branched or unbranched alkyl, branched and unbranched alkenyl, branched and unbranched alkynyl, amino, alkylamino, dialkylamino, trialkylamino, aryl, ureido, heterocyclic, aromatic heterocyclic, cyclic, aromatic cyclic, halogen, hydroxyl, alkoxy, cyano, amide, carbamoyl, carboxylic acid, ester, carbonyl, carbonyldioxyl, alkylthioether, and thiohydroxyl groups;
  • wherein R1 can be present or absent and when present the compound of formula (I) further comprises a counter ion selected from the group consisting of chloride, fluoride, bromide, iodide, sulfate, nitrate, fumarate, acetate, carbonate, stearate, laurate, and oleate; and
  • wherein at least one R comprises a backbone of a diacrylate having the following structure:
  • Figure US20160374949A9-20161229-C00006
  • wherein X1 and X2 are each independently substituted or unsubstituted C2-C20 alkylene, and wherein each X1 and X2 can be the same or different.
  • In some embodiments, the bioreducible, hydrolytically degradable polymer of claim 1, wherein at least one R comprises a backbone of a diacrylate selected from the group consisting of:
  • Figure US20160374949A9-20161229-C00007
  • or co-oligomers comprising combinations thereof, wherein the diacrylate can be the same or different.
  • Additional R, R′, and R″ groups are defined immediately herein below as for compounds disclosed in International PCT Patent Application Publication No. WO/2010/132879 for “Multicomponent Degradable Cationic Polymers,” to Green et al., which is incorporated herein by reference in its entirety.
    • B. Hydrolytic and Bioreducible Polymeric Particle Formulations for Delivery of Peptides.
  • Multicomponent degradable cationic polymers suitable for the delivery of peptides to a target are disclosed in International PCT Patent Application Publication No. WO/2010/132879 for “Multicomponent Degradable Cationic Polymers,” to Green et al., which is incorporated herein by reference in its entirety. Such polymers, in addition to the presently disclosed polymers can be used to deliver cargo, e.g., a therapeutic agent, to a target, e.g., a cell.
  • In some embodiments, the presently disclosed subject matter generally provides multicomponent degradable cationic polymers. In some embodiments, the presently disclosed polymers have the property of biphasic degradation. Modifications to the polymer structure can result in a change in the release of therapeutic agents, which can occur over multiple time scales. In some embodiments, the presently disclosed polymers include a minority structure, e.g., an endcapping group, which differs from the majority structure comprising most of the polymer backbone. In other embodiments, the bioreducible oligomers form block copolymers with hydrolytically degradable oligomers. In yet other embodiments, the end group/minority structure comprises an amino acid or chain of amino acids, while the backbone degrades hydrolytically and/or is bioreducible.
  • As described in more detail herein below, small changes in the monomer ratio used during polymerization, in combination with modifications to the chemical structure of the end-capping groups used post-polymerization, can affect the efficacy of delivery of a therapeutic agent to a target. Further, changes in the chemical structure of the polymer, either in the backbone of the polymer or end-capping groups, or both, can change the efficacy of target delivery to a cell. In some embodiments, small changes to the molecular weight of the polymer or changes to the endcapping groups of the polymer, while leaving the main chain, i.e., backbone, of the polymer the same, can enhance or decrease the overall delivery of the target to a cell. Further, the “R” groups that comprise the backbone or main chain of the polymer can be selected to degrade via different biodegradation mechanisms within the same polymer molecule. Such mechanisms include, but are not limited to, hydrolytic, bioreducible, enzymatic, and/or other modes of degradation.
  • In some embodiments, the presently disclosed compositions can be prepared according to Scheme 2:
  • Figure US20160374949A9-20161229-C00008
  • In some embodiments, at least one of the following groups R, R′, and R″ contain reducible linkages and, for many of the presently disclosed materials, additional modes of degradation also are present. More generally, R′ can be any group that facilitates solubility in water and/or hydrogen bonding, for example, OH, NH, and SH. Representative degradable linkages include, but are not limited to:
  • Figure US20160374949A9-20161229-C00009
  • The end group structures, i.e., R″ groups in Scheme 2, for the presently disclosed cationic polymers are distinct and separate from the backbone structures (R) structures, the side chain structures (R′), and end group structures of the intermediate precursor molecule for a given polymeric material.
  • More particularly, in some embodiments, the presently disclosed subject matter includes a nanoparticle, microparticle, or gel comprising a compound of formula (I):
  • Figure US20160374949A9-20161229-C00010
  • wherein:
  • n is an integer from 1 to 10,000;
  • R1, R2, R3, R4, R5, R6, R7, R8, and R9 are each independently selected from the group consisting of hydrogen, branched and unbranched alkyl, branched and unbranched alkenyl, branched and unbranched alkynyl, aryl, halogen, hydroxyl, alkoxy, carbamoyl, carboxyl ester, carbonyldioxyl, amide, thiohydroxyl, alkylthioether, amino, alkylamino, dialkylamino, trialkylamino, cyano, ureido, a substituted alkanoyl group, cyclic, cyclic aromatic, heterocyclic, and aromatic heterocyclic groups, each of which may be substituted with at least one substituent selected from the group consisting of branched or unbranched alkyl, branched and unbranched alkenyl, branched and unbranched alkynyl, amino, alkylamino, dialkylamino, trialkylamino, aryl, ureido, heterocyclic, aromatic heterocyclic, cyclic, aromatic cyclic, halogen, hydroxyl, alkoxy, cyano, amide, carbamoyl, carboxylic acid, ester, carbonyl, carbonyldioxyl, alkylthioether, and thiohydroxyl groups;
  • wherein R1 can be present or absent and when present the compound of formula (I) further comprises a counter ion selected from the group consisting of chloride, fluoride, bromide, iodide, sulfate, nitrate, fumarate, acetate, carbonate, stearate, laurate, and oleate; and
  • at least one of R, R′, and R″ comprise a reducible or degradable linkage, and wherein each R, R′, or R″ can independently be the same or different;
  • under the proviso that when at least one R group comprises an ester linkage of the formula —C(═O)—O— and the compound of formula (I) comprises a poly(beta-amino ester), then the compound of formula (I) must also comprise one or more of the following characteristics:
  • (a) each R group is different;
  • (b) each R″ group is different;
  • (c) each R″ group is not the same as any of R′, R1, R2, R3, R4, R5, R6, R7, R9, and R9;
  • (d) the R″ groups degrade through a different mechanism than the ester-containing R groups, wherein the degradation of the R″ group is selected from the group consisting of a bioreducible mechanism or an enzymatically degradable mechanism; and/or
  • (e) the compound of formula (I) comprises a substructure of a larger cross-linked polymer, wherein the larger cross-linked polymer comprises different properties from compound of formula (I);
  • and one or more peptides selected from the group consisting of an anti-angiogenic peptide, an anti-lymphangiogenic peptide, an anti-tumorigenic peptide, and an anti-permeability peptide.
  • In some embodiments of the nanoparticle, microparticle, or gel n is an integer from 1 to 1,000; in some embodiments, n is an integer from 1 to 100; in some embodiments, n is an integer from 1 to 30; in some embodiments, n is an integer from 5 to 20; in some embodiments, n is an integer from 10 to 15; and in some embodiments, n is an integer from 1 to 10.
  • In particular embodiments, the reducible or degradable linkage comprising R, R′, and R″ is selected from the group consisting of an ester, a disulfide, an amide, an anhydride or a linkage susceptible to enzymatic degradation, subject to the proviso provided hereinabove.
  • In more particular embodiments, R comprises a backbone of a diacrylate selected from the group consisting of:
  • Figure US20160374949A9-20161229-C00011
    Figure US20160374949A9-20161229-C00012
  • In some embodiments, wherein R′ comprises a side chain derived from compound selected from the group consisting of:
  • Figure US20160374949A9-20161229-C00013
  • In some embodiments, R″ comprises an end group derived from a compound selected from the group consisting of
  • Figure US20160374949A9-20161229-C00014
  • In other embodiments, the compound of formula (I) is subject to the further proviso that if at least one R group comprises an ester linkage, then the R″ groups impart one or more of the following characteristics to the compound of formula (I): independent control of cell-specific uptake and/or intracellular delivery of a particle; independent control of endosomal buffering and endosomal escape; independent control of DNA release; triggered release of an active agent; modification of a particle surface charge; increased diffusion through a cytoplasm of a cell; increased active transport through a cytoplasm of a cell; increased nuclear import within a cell; increased transcription of an associated DNA within a cell; increased translation of an associated DNA within a cell; increased persistence of an associated therapeutic agent within a cell, wherein the therapeutic agent is selected from the group consisting of DNA, RNA, a peptide or a protein.
  • More particularly, any poly(beta-amino ester) specifically disclosed or claimed in U.S. Pat. No. 6,998,115; U.S. Pat. No. 7,427,394; U.S. patent application publication no. US2005/0265961; and U.S. patent publication no. US2010/0036084, each of which is incorporated herein by reference in its entirety, is explicitly excluded from the presently disclosed compounds of formula (I). In particular, the poly(beta-amino ester)s disclosed in U.S. Pat. No. 6,998,115; U.S. Pat. No. 7,427,394; U.S. patent application publication no. US2005/0265961; and U.S. patent publication no. US2010/0036084 are symmetrical, i.e., both R groups as defined in formula (I) herein are the same. In certain embodiments of the presently disclosed compounds of formula (I), when at least one R comprises an ester linkage, the two R groups of formula (I) are not the same, i.e., in such embodiments, the compounds of formula (I) are not symmetrical.
  • In particular embodiments, the reducible or degradable linkage comprising R, R′, and R″ is selected from the group consisting of an ester, a disulfide, an amide, an anhydride or a linkage susceptible to enzymatic degradation, subject to the above-mentioned provisos.
  • Further, in some embodiments of the compound of formula (I), n is an integer from 1 to 1,000; in other embodiments, n is an integer from 1 to 100; in other embodiments, n is an integer from 1 to 30; in other embodiments, n is an integer from 5 to 20; in other embodiments, n is an integer from 10 to 15; and in other embodiments, n is an integer from 1 to 10.
  • In some embodiments, R″ can be an oligomer as described herein, e.g., one fully synthesized primary amine-terminated oligomer, and can be used as a reagent during the second reaction step of Scheme 2. This process can be repeated iteratively to synthesize increasingly complex molecules.
  • In other embodiments, R″ can comprise a larger biomolecule including, but not limited to, poly(ethyleneglycol) (PEG), a targeting ligand, including, but not limited to, a sugar, a small molecule, an antibody, an antibody fragment, a peptide sequence, or other targeting moiety known to one skilled in the art; a labeling molecule including, but not limited to, a small molecule, a quantum dot, a nanoparticle, a fluorescent molecule, a luminescent molecule, a contrast agent, and the like; and a branched or unbranched, substituted or unsubstituted alkyl chain.
  • In some embodiments, the branched or unbranched, substituted or unsubstituted alkyl chain is about 2 to about 5 carbons long; in some embodiments, the alkyl chain is about 6 to about 8 carbons long; in some embodiments, the alkyl chain is about 9 to about 12 carbons long; in some embodiments, the alkyl chain is about 13 to about 18 carbons long; in some embodiments, the alkyl chain is about 19 to about 30 carbons long; in some embodiments, the alkyl chain is greater than about 30 carbons long.
  • In certain embodiments, both R″ groups, i.e., the end groups of the polymer, comprise alkyl chains. In other embodiments, only one R″ group comprises an alkyl chain. In some embodiments, at least one alkyl chain is terminated with an amino (NH2) group. In other embodiments, the at least one alkyl chain is terminated with a hydroxyl (OH) group.
  • In some embodiments, the PEG has a molecular weight of about 5 kDa or less; in some embodiments, the PEG has a molecular weight of about 5 kDa to about 10 kDa; in some embodiments, the PEG has a molecular weight of about 10 kDa to about 20 kDa; in some embodiments, the PEG has a molecular weight of about 20 kDa to about 30 kDa; in some embodiments, the PEG is greater than 30 kDa. In certain embodiments, both R″ groups comprise PEG. In other embodiments, only one R″ group comprises PEG.
  • Further, in some embodiments, one R″ group is PEG and the other R″ group is a targeting ligand and/or labeling molecule as defined herein above. In other embodiments, one R″ group is an alkyl chain and the other R″ group is a targeting ligand and/or labeling molecule.
  • Representative monomers used to synthesize the presently disclosed cationic polymers include, but are not limited to, those provided immediately herein below. The presently disclosed subject matter is not limited to the representative monomers disclosed herein, but also includes other structures that one skilled in the art could use to create similar biphasic degrading cationic polymers. For each type of cargo, a particular biodegradable polymer can be tuned through varying the constituent monomers used to form the backbone (designated as “B” groups), side-chains (designated as “S” groups), and end-groups (designated as “E” groups) of the polymer.
  • Figure US20160374949A9-20161229-C00015
    Figure US20160374949A9-20161229-C00016
    Figure US20160374949A9-20161229-C00017
    Figure US20160374949A9-20161229-C00018
  • In particular embodiments, as depicted in Scheme 4, the presently disclosed cationic polymers comprise a polyalcohol structure, i.e., the side chain represented by R′ in Scheme 2 comprises an alcohol.
  • Figure US20160374949A9-20161229-C00019
  • In such embodiments, the end group structures (R″) and the backbone structures (R) are defined as above and the side chain must contain at least one hydroxyl (OH) group.
  • In yet other embodiments, the presently disclosed cationic polymer comprises a specific poly(ester amine) structure with secondary non-hydrolytic modes of degradation. In such embodiments, the cationic polymer comprises a polyester that degrades through ester linkages (hydrolytic degradation) that is further modified to comprise bioreducible groups as end (R″) groups.
  • Figure US20160374949A9-20161229-C00020
  • Representative bioreducible end groups in such embodiments include, but are not limited to:
  • Figure US20160374949A9-20161229-C00021
  • In some embodiments, the presently disclosed cationic polymer comprises a specific poly(ester amine alcohol) structure with secondary non-hydrolytic modes of degradation. In such embodiments, the cationic polymer comprises a specific structure where a polyester that degrades through ester linkages (hydrolytic degradation) is modified to contain bioreducible groups as end groups.
  • Figure US20160374949A9-20161229-C00022
  • In yet other embodiments, the presently disclosed cationic polymer comprises a specific poly(amido amine) structure having disulfide linking groups in the polymer backbone and an independent, non-reducible amine contacting group at the terminal ends of the polymer.
  • Figure US20160374949A9-20161229-C00023
  • In such embodiments, R1 and R2 are alkyl chains. In some embodiments, the alkyl chain is 1-2 carbons long; in some embodiments, the alkyl chain is 3-5 carbons long; in some embodiments, the alkyl chain is 6-8 carbons long; in some embodiments, the alkyl chain is 9-12 carbons long; in some embodiments, the alkyl chain is 13-18 carbons long; in some embodiments, the alkyl chain is 19-30 carbons long; and in some embodiments, the alkyl chain is greater than 30 carbons long
  • Suitable non-reducible amino R″ groups for such embodiments include, but are not limited to:
  • Figure US20160374949A9-20161229-C00024
    Figure US20160374949A9-20161229-C00025
    Figure US20160374949A9-20161229-C00026
    Figure US20160374949A9-20161229-C00027
    Figure US20160374949A9-20161229-C00028
  • In other embodiments, the presently disclosed cationic polymers comprise a specific poly(amido amine alcohol) structure having disulfide linking groups in the polymer backbone and an independent non-reducible amine contacting group at the terminal ends of the polymer.
  • Figure US20160374949A9-20161229-C00029
  • In yet other embodiments, the presently disclosed cationic polymer comprises a copolymer of representative oligomers as described hereinabove. Such embodiments include, but are not limited to, a poly(amido amine) structure having disulfides in the polymer backbone and an independently degradable (non-reducible) group at least one end of the polymer. Such embodiments also include using a cross-linker to add bioreducible linkages to hydrolytically degradable materials and also provide for higher molecular weight materials. A representative example of this embodiment, along with suitable monomers is as follows:
  • Figure US20160374949A9-20161229-C00030
  • In particular embodiments, the presently disclosed polymer is selected from the group consisting of:
  • Figure US20160374949A9-20161229-C00031
    Figure US20160374949A9-20161229-C00032
    Figure US20160374949A9-20161229-C00033
    Figure US20160374949A9-20161229-C00034
  • Further aspects of the presently disclosed subject matter include: (a) the R substituent groups that make up the presently disclosed polymers degrade via different biodegradation mechanisms within the same polymer. These biodegradation mechanisms can include hydrolytic, bioreducible, enzymatic, and/or other modes of degradation; (b) the ends of the polymer include a minority structure that differs from the majority structure that comprises most of the polymer backbone; (c) in several embodiments, the side chain molecules contain hydroxyl (OH)/alcohol groups.
  • In some embodiments: (a) the backbone is bioreducible and the end groups of the polymer degrade hydrolytically; (b) the backbone degrades hydrolytically and the end groups are bioreducible; and (c) hydrolytically degradable oligomers are cross-linked with a bioreducible cross-linker; (d) bioreducible oligomers form block copolymers with hydrolytically degradable oligomers; and (e) the end group/minority structure comprises an amino acid or chain of amino acids, whereas the backbone degrades hydrolytically and/or is bioreducible.
  • One way to synthesize the presently disclosed materials is by the conjugate addition of amine-containing molecules to acrylates or acrylamides. This reaction can be done neat or in a solvent, such as DMSO or THF. Reactions can take place at a temperature ranging from about room temperature up to about 90° C. and can have a duration from about a few hours to about a few weeks. The presently disclosed methods can be used to create linear or branched polymers. In some embodiments, the molecular weight (MW) has a range from about 1 kDa to about 5 kDa, in other embodiments, the MW has a range from about 5 kDa to about 10 kDa, in other embodiments the MW has a range from about 10 kDa to about 15 kDa, in other embodiments, the MW has a range from about 15 kDa to about 25 kDa, in other embodiments, the MW has a range from about 25 kDa to about 50 kDa, and in other embodiments, the MW has a range from about 50 kDa to about 100 kDa. In other embodiments, the polymer forms a network, gel, and/or scaffold of apparent molecular weight greater than 100 kDa.
  • In particular embodiments, the presently disclosed subject matter provides hydrolytic and bioreducible polymeric particle formulations for the delivery of one or more peptides to a target. In some embodiments of the presently disclosed formulations, the particles are nanoparticles and, in other embodiments, they are microparticles. Some applications are to cancer and others are to ophthalmic diseases.
  • Accordingly, in some embodiments, the presently disclosed approach includes degradable nanoparticles, microparticles, and gels that release a peptide, which is capable of therapeutic activity through multiple modes of action. The presently disclosed peptides can simultaneously inhibit: (1) endothelial cell proliferation; (2) endothelial cell adhesion, (3) endothelial cell migration, (4) tumor cell proliferation, (5) tumor cell adhesion, and (6) tumor cell migration.
  • When combined with such peptides, the presently disclosed nanoparticles, microparticles, and gels: (1) protect and increase the persistence of the peptides that would otherwise be rapidly cleared in vivo; (2) allow passive targeting of tumor vasculature via nanoparticle biophysical properties to enable enhanced efficacy at the target site of action; (3) enable extended peptide release and minimized dosing schedules for affected patients; and (4) facilitate a continuous peptide concentration rather than a pulsatile profile that would be caused by bolus injections and fast clearance.
  • The presently disclosed microparticles have similar benefits to the nanoparticles except that they also persist longer and have an easier route for clinical administration. On the other hand, another advantage of the presently disclosed nanoparticles is that they are better able to passively target the peptides to tumor vasculature than are the microparticles. Representative embodiments of the presently disclosed microparticles are provided in Example 10, herein below.
  • Further, in some embodiments, one or more peptides, which can be the same or different, can be combined, e.g., encapsulated, directly or individually into different nanoparticles that then can be combined into the same microparticles.
    • C. Biodegradable Nanoparticles for Sustained Peptide Delivery
  • Selected polymers are able to encapsulate selected peptides possessing varied chemical properties. Changes to polymer structure, including small changes to the ends of the polymer only, can vary biophysical properties of these particles. These properties can be important to tune for effective in vivo peptide delivery. A small subset of the potential polymer library was screened to measure the effect of encapsulating the antiangiogenic peptides chemokinostatin-1 and pentastatin-1 within polymeric particles compared to unencapsulated, free peptides. Polymeric encapsulation of peptides enhanced the ability of the peptides to inhibit the proliferation of endothelial cells. An example of representative polymers encapsulating peptides is provided in Scheme 5.
  • Figure US20160374949A9-20161229-C00035
  • Name Sequence Theor. pI MW
    DEAH box poly8 EIELVEEEPPF 3.51 1330.45
    Wispostatin-1 SPWSPCSTSCGLGVS 7.80 1838.08
    TRI
    Pentastatin LRRFSTMPFMFCNI 9.02 2454.93
    NNVCNF
    Chemokinostatin NGRKACLNPASPIV 10.03 2625.19
    KKIIEKMLNS
  • In other embodiments, particles synthesized and composed as described above are then used as a “core” inner particle for future coatings to create multi-component (also referred to herein as multi-layer) particles. For other embodiments, other nanoparticles are used as cores, such as an inorganic nanoparticles (like gold) or soft polymeric nanoparticles, for example, as disclosed in International PCT Patent Application Publication No. WO/2010/132879 for “Multicomponent Degradable Cationic Polymers,” to Green et al., which is incorporated herein by reference in its entirety. In each embodiment, the core particle is then coated with charged polymers as described above, peptides as described above, and other biological agents. Exemplary embodiments of multilayer particles are illustrated in FIG. 1.
  • Layering can be mediated by electrostatic forces and alternate cationic and anionic layers can be used to incorporate additional peptides and biological agents. Polyelectrolytes, including degradable polymers and peptides, also are used to provide structure to the multilayers. Multilayers can release drugs, peptides, and biological agents from the particle due to hydrolytic degradation, enzyme activity, disulfide reduction, and/or diffusion.
    • D. Polymeric Gels for Controlled Release of Biological Agents.
  • i. Hydrogels (or “Organogels”) for Protein/Peptide Release
  • In some embodiments, the presently disclosed subject matter provides photocrosslinked gels for controlled release of cargo, including, but not limited to peptides and proteins. Such gels can be tuned for release of other drugs. In some embodiments, for example, as illustrated in FIG. 2, a solution of acrylate-terminated polymers is made using either acrylate-terminated polymers, such as poly(β-amino esters) (PBAEs), poly(ethylene glycol)diacrylate (PEGDA), small crosslinkers including, but not limited to, 1,4-butanediol diacrylate, or a mixture of the above. Because many of these materials are amphiphilic, a variety of solvents can be used, including water, PBS, and DMSO, to encapsulate drugs within them. Addition of a small amount (0.05% w/v) of photoinitiator and exposure to long-wave UV light for a period of time, e.g., 5-15 min at 1-3 mW, causes formation of a drug-loaded gel.
  • The gel swelling properties can vary with pH by taking advantage of the PBAE portions, which can be reversibly protonated. Changing ratios of PBAE to PEGDA and the addition of crosslinkers changes swelling properties by changing pore size or overall hydrophobicity. For example, doping in increasing amounts of a more hydrophobic PBAE (B4S4) into a network of hydrophilic PEGDA causes the release kinetics to slow when measuring protein release.
    • E. Stable Formulations
  • To increase stability of nanoparticles in suspension, especially with hydrolytically-degradable polymers, the presently disclosed subject matter provides a method of keeping DNA or other cargo stable and functional after storage. For example, freeze-drying often causes denaturation of biological molecules or irreversible aggregation and inactivation of nanoparticles. Referring now to FIG. 3, by adding sucrose as a lyoprotectant at a final concentration of, for example, 7.5-45 mg/mL, the presently disclosed subject matter demonstrates that particles can be freeze dried and stored, for example, at 4° C. or −20° C. for extended periods, e.g., months, without significant change in physicochemical or biological properties. Certain formulations, when stored dry, also might be stable at ambient temperatures up to 40° C. Furthermore, the presently disclosed process allows particles to be prepared in advance and used much more easily in a clinical setting. The presently disclosed subject matter also demonstrates that particles can be concentrated in this way much more highly than would be possible with free polymer, which may be advantageous for dose adjustment in clinical or pre-clinical models.
  • F. Inclusion of Lyophilized Nanoparticles into Pellets/Scaffolds for Long-Term Delivery
  • The presently disclosed nanoparticles can be stored in a dry form and can be used in gene delivery via three-dimensional (3D) constructs. While DNA is used as a cargo in this example, other cargos of interest to one skilled in the art including, but not limited to, siRNA, peptides, protein, imaging agents, and the like, can be used, as well. In other embodiments, DNA-loaded nanoparticles were incorporated into natural and synthetic scaffolds, disks, microparticles, and hydrogels for various potential applications.
    • G. Methods of Treating Angiogenesis-Dependent Diseases
  • Although significant progress has been made in treating angiogenesis-dependent diseases, such as cancers, major challenges remain in terms of development of drug resistance, metastasis and overall survival rates. Studies designed to decipher the modes of drug resistance have revealed that tumors are very versatile and use multiple pathways to continue to survive and metastasize. See Chiang A C, Massague J. Molecular basis of metastasis. N Engl J Med 2008; 359(26):2814-23; Gupta G P, Massague J. Cancer metastasis: building a framework. Cell 2006; 127(4):679-95. Resistance has been observed for both cytotoxic and antiangiogenic agents. Thus, multimodal therapeutic design emerges as a promising, and perhaps even a mandatory strategy for treatment of cancer. See Sawyers C L. Cancer: mixing cocktails. Nature 2007; 449(7165):993-6; Dorrell M I, Aguilar E, Scheppke L, Barnett F H, Friedlander M. Combination angiostatic therapy completely inhibits ocular and tumor angiogenesis. Proc Natl Acad Sci USA 2007; 104(3):967-72.
  • The key attributes of tumor growth and metastasis are: angiogenesis, which facilitates the supply of the growing tumor with oxygen and nutrients; lymphangiogenesis, which facilitates the spreading of cancer cells through the lymphatics; and cancer cell proliferation. Angiogenesis, in particular, plays a critical role in the growth of tumors and antiangiogenic therapies have the potential to treat cancer, either alone or in combination with conventional chemotherapies, by starving tumors of oxygen and nutrients. There is a need, however, to find more potent anti-cancer therapeutics, including antiangiogenic therapeutics, as well as delivery systems for these therapeutics. The presently disclosed subject matter can address all of these attributes in a combined system.
  • Many forms of cancer, including breast cancer, are dependent on angiogenesis, the growth of blood vessels. There is a great medical need for the development of a safe, effective, and inexpensive means of antiangiogenic therapy. One promising approach is the use of antiangiogenic peptides as the active agents. In some embodiments, the presently disclose'd subject matter provides peptides derived from several classes of proteins that are effective at preventing angiogenesis. In other embodiments, the presently disclosed subject matter provides other peptides that are able to inhibit cancer through additional mechanisms including, but not limited to, antilymphangiogenesis and apoptosis. In their current form, however, all of these peptides have a short in vivo half-life and they are not suitable for systemic administration or for long-term action. Thus, there is a need to package, protect, and deliver these peptides in a more stable, sustained fashion.
  • Accordingly, the presently disclosed biomaterials facilitate delivery of combinations of these peptides in an engineered fashion to synergistically kill cancer or treat other diseases, in particular, other angiogenesis-dependent diseases. More particularly, the presently disclosed subject matter provides an effective array of safe, biodegradable polymers for use in forming peptide-containing nanoparticles, microparticles, gels, and conjugates. The presently disclosed biomaterials can be used to construct particles, gels, and conjugates that vary in their biophysical properties and in biological properties, such as tumor accumulation and peptide release.
  • The presently disclosed formulations work through one or more of the following mechanisms: antiangiogenesis; inhibition of human endothelial cell proliferation and migration; inhibition of lymphatic endothelial cell proliferation and migration; and promotion of cancer apoptosis, as well as other mechanisms. The presently disclosed materials and methods can safely, effectively, and relatively inexpensively treat age-related macular degeneration (AMD), cancer, and other diseases.
  • Further, siRNA is a promising technology to silence the activity of many biological targets in many diseases including cancer, cardiovascular diseases, infectious diseases, neurological diseases, ophthalmic diseases, and others. In some cases, siRNA can be used to reach previously undruggable targets. The method of delivery and examples described herein for siRNA delivery apply equally to other similar RNA molecules including, but not limited to is RNA, agRNA, saRNA, and miRNA.
    • H. Nanoparticle-Mediated Multimodal Peptide Delivery
  • Conventional anti-angiogenesis treatments have proven to be very expensive with limited clinical success, particularly in breast cancer. The presently disclosed strategy combines more effective and multimodal therapeutic agents with nanomedicine to provide a delivery system to enhance their therapeutic effect. More particularly, the presently disclosed subject matter provides a single system that incorporates multimodal therapeutic activity, including, but not limited to, antiangiogenic activity, antilymphangiogenic activity, and apoptotic activity, and can be effective in limiting both tumor growth and metastasis.
  • Generally, small peptides possess many advantageous characteristics as therapeutic agents, including high specificity and low toxicity. Reichert J. Development trends for peptide therapeutics. Tufts Center for the Study of Drug Development 2008 [cited 2010; Available from: http://www.peptidetherapeutics.org/PTF_Summary_2008.pdf]. The main disadvantage of small peptides as therapeutic agents, however, is their short half-life. The presently disclosed subject matter capitalizes on the advantages of peptide agents by developing novel antiangiogenic, antilymphangiogenic, and apoptotic peptides targeting multiple pathways, and overcoming the disadvantages by designing a multi-agent nanocarrier system.
  • Approximately 25 peptides have been approved by the FDA, however, to date none of these approved peptides are antiangiogenic. Rosca E V, Koskimaki J E, Rivera C G, Pandey N B, Tamiz A P, Popel A S. Anti-angiogenic peptides for cancer therapeutics. Curr Pharm Biotechnol, 12(8):1101-1116 (2011). Several endogenous proteins/polypeptides, including angiostatin, endostatin, proteolytic fragments of collagen IV, pigment epithelium-derived factor, and thrombospondin, have antiangiogenic properties and can induce apoptosis in endothelial cells. Lucas R, Holmgren L, Garcia I, Jimenez B, Mandriota S J, Borlat F, et al. Multiple forms of angiostatin induce apoptosis in endothelial cells. Blood 1998; 92(12):4730-41. These proteins/polypeptides are large, however, and are not ideal for use as therapeutic agents. Further, full length human proteins, although theoretically not foreign to an individual's body, induce an immune response in some individuals.
  • More recently, a bioinformatics approach has allowed identification of candidate antiangiogenic regions of several proteins and synthetic peptides corresponding to those short sequences that possess the ability to suppress proliferation and migration of vascular endothelial cells in vitro and angiogenesis in vivo. Delivering such peptides to a cell and prolonging the duration of their activity, however, remains a challenge.
  • Although peptides are much easier to produce and are more scalable and less immunogenic than full-length proteins, they are eliminated from the body more quickly. The presently disclosed subject matter can increase and sustain residence time, increase accumulation in tumor vasculature, and maximize the therapeutic effects of such peptides. The presently disclosed subject matter combines biomaterial synthesis, sustained drug delivery, and anti-cancer peptide creation to provide nanoparticle-, microparticle-, and gel-based systems for sustained peptide delivery. The presently disclosed biodegradable biomaterials can be tuned for the encapsulation, protection, and sustained release of each type of peptide.
  • The use of the presently disclosed nanoparticles, microparticles, and gels limits toxicity because they can extravasate from the leaky neovasculature of the tumors and be trapped in the interstitium of the tumor once the anti-angiogenic compounds kill or normalize the vasculature. Further, the presently disclosed subject matter demonstrates that effective biomaterials for anti-cancer peptide nanoparticles, microparticles, and gels can be fabricated. Multiple anti-cancer peptides and other peptides can be combined within the same particle for multimodal peptide delivery, as well as multimodal therapy with other active agents including, but not limited to, other peptides, nucleic acids, proteins, small molecules, and the like.
  • More particularly, in some embodiments, the presently disclosed subject matter provides peptides that work through multiple biological mechanisms in combination with the presently disclosed biomaterials, including multilayer and multi-peptide nanoparticle formulations. An array of biodegradable polymers can be used to encapsulate peptides to create nanoparticles having varied biophysical properties and release kinetics. Each peptide can have a specialized subset of materials employed for its encapsulation. Referring now to FIG. 4, differing chemical structures can be synthesized by the conjugate addition of amines to acrylates or acrylamides of differing structure. The polymer structure can be tuned through variation to the backbone, side chain, end-group, hydrophobicity, and degradability. Unlike the polymeric materials disclosed in International PCT Patent Application Publication No. WO/2010/132879 for “Multicomponent Degradable Cationic Polymers,” to Green et al., which are cationic, i.e., positively charged, the presently disclosed polymers can be positively charged, whereas others can be negatively charged, others neutral and hydrophobic, and still others amphiphilic. The diversity of the presently disclosed biomaterials comes from the chemical diversity of the R groups (R, R′, R″) in the biomaterial array and from parameter tuning during particle fabrication.
  • For example, to create the presently disclosed multi-peptide particles, hydrophobic core particles first are constructed by self-assembly, for example between the somatotropin-derived peptide, the collagen IV-derived peptide, and a hydrophobic polymer. These nanoparticles are then coated by charged biodegradable polymers and peptides following a particle coating and layer-by-layer technique that modifies techniques previously described. Green J J, Chiu E, Leshchiner E S, Shi J, Langer R, Anderson D G. Electrostatic ligand coatings of nanoparticles enable ligand-specific gene delivery to human primary cells. Nano Lett 2007; 7(4):874-9; Shmueli R B, Anderson D G, Green J J. Electrostatic surface modifications to improve gene delivery. Expert Opin Drug Deliv 7(4):535-50. Through this process, the charged peptides (serpin-derived and chemokine-derived) can be incorporated into these multilayers. Charged biological agents, such as peptides and nucleic acids, can serve as both the therapeutic agent and the support polyelectrolyte in the presently disclosed systems.
  • In one embodiment, peptides can self-assemble with the presently disclosed polymers in an aqueous buffer due to physical, hydrophobic, and electrostatic forces. Zhang S, Uludag H. Nanoparticulate systems for growth factor delivery. Pharm Res 2009; 26(7):1561-80. In other embodiments, peptide-containing micelles can be formed by synthetic polymer-mPEG (e.g., E15 from FIG. 4) block copolymers. Depending on formulation parameters, polymer/peptide particle sizes can be tuned from approximately 50 nm to approximately 500 nm.
  • As an alternative strategy for polymers in the library that are more hydrophobic or have higher glass transition temperatures, peptides can be encapsulated by a double emulsion procedure. In this method, droplets of aqueous buffer containing peptide are dispersed in the hydrophobic polymer phase and then the polymer phase is itself dispersed in another aqueous phase to form the polymeric particles. Jain R A. The manufacturing techniques of various drug loaded biodegradable poly(lactide-co-glycolide) (PLGA) devices. Biomaterials 2000; 21(23):2475-90. As an alternative technique, blends of novel hydrophobic polymers and poly(lactic-co-glycolic acid) also can be made to form particles with unique degradation properties. Little S R, Lynn D M, Ge Q, Anderson D G, Puram S V, Chen J Z, et al. Poly-beta amino ester-containing microparticles enhance the activity of nonviral genetic vaccines. Proc Natl Acad Sci USA 2004; 101(26):9534-9.
    • I. Peptides for Anti-Angiogenesis, Anti-Lymphangiogenesis, Anti-Tumor, and Anti-Permeability Activity.
  • Several classes of peptides have been developed that show either anti-proliferative or anti-migratory activity or both on endothelial cells. These peptides appear to function through distinct mechanisms of action and have been tested both in vitro and in vivo in tumor xenografts and in ocular mouse models. These peptides include a 24-mer peptide NGRKACLNPASPIVKKIIEKMLNS derived from the CXC chemokine protein GRO-α/CXCL1 and a collagen IV derived and modified 20-mer peptide LRRFSTMPFMF-Abu-NINNV-Abu-NF as a highly potent anti-proliferative and anti-migratory peptide targeting αvβ1 integrins on both endothelial and tumor cells; here Abu is the 2-Aminobutyric acid introduced in the sequence to facilitate translation to human.
  • An 11-mer anti-angiogenic peptide EIELVEEEPPF derived from the serpin domain of DEAH box polypeptide also has been identified that shows significant inhibition of MDA-MB-231 tumor xenograft growth. A somatotropin family peptide LLRISLLLIESWLE (SP5031) derived from transmembrane 45 protein that also has been identified and has anti-proliferative and anti-migratory activity on both endothelial cells and lymphatic endothelial cells. It is believed that this peptide is the first antilymphangiogenic peptide agent. Combining these peptides together can result in a peptide-based system that inhibits angiogenesis by several different mechanisms and also inhibits lymphangiogenesis that has been shown to promote tumor metastasis.
  • Representative peptides suitable for encapsulation with the presently disclosed biomaterials include those disclosed in International PCT Patent Application Publication Number WO2007/033215 A2 for “Compositions Having Antiangiogenic Activity and Uses Thereof,” to Popel et al., published Mar. 22, 2007; International PCT Patent Application Publication Number WO2008/085828 A2 for “Peptide Modulators of Angiogenesis and Use Thereof,” to Popel, published Jul. 17, 2008; U.S. Provisional Patent Application No. 61/421,706, filed Dec. 12, 2010, which is commonly owned; and U.S. Provisional Patent Application No. 61/489,500, filed May 24, 2011, which also is commonly owned, each of which is incorporated herein by reference in its entirety.
  • Accordingly, in some embodiments, peptide suitable for use in the presently disclosed subject matter are disclosed in Tables 1-10 of International PCT Patent Application Publication Number WO2008/085828 A2 for “Peptide Modulators of Angiogenesis and Use Thereof,” to Popel, published Jul. 17, 2008, which is incorporated herein by reference in its entirety.
  • Accordingly, in some embodiments, the presently disclosed subject matter provides a nanoparticle, microparticle, or gel comprising one or more peptides, wherein the one or more peptide is selected from the group consisting of an isolated peptide or analog thereof comprising one of the following amino acid sequences:
  • TSP Motif: W-X(2)-C-X(3)-C-X(2)-G,
    CXC Motif: G-X(3)-C-L
    Collagen Motif: C-N-X(3)-V-C
    Collagen Motif: P-F-X(2)-C
    Somatotropin Motif: L-X(3)-L-L-X(3)-S-X-L
    Serpin Motif: L-X(2)-E-E-X-P
  • wherein X denotes a variable amino acid and the number in parentheses denotes the number of variable amino acids; W denotes tryptophan; C denotes cysteine, G denotes glycine, V denotes valine; L denotes leucine, P is proline, and wherein the peptide reduces blood vessel formation in a cell, tissue or organ.
  • In other embodiments, the one or more peptide comprises an amino acid sequence shown in Table 1-6, 8 and 9.
  • In other embodiments, the one or more peptide comprises an isolated peptide or analog thereof having at least 85% identity to an amino acid sequence shown in Table 1-10.
  • In other embodiments, the one or more peptide comprises an amino acid sequence shown in Table 1-10. In yet other embodiments, the one or more peptide consists essentially of an amino acid sequence shown in Table 1-10.
  • In particular embodiments, the one or more peptide comprises an isolated peptide or analog thereof comprising or consisting essentially of a sequence having at least 85% amino acid sequence identity to an amino acid sequence selected from the group consisting of:
  • Placental Lactogen LLRISLLLIESWLE
    hGH-V LLRISLLLTQSWLE
    GH2 LLHISLLLIQSWLE
    Chorionic somatomammotropin LLRLLLLIESWLE
    Chorionic somatomammotropin LLHISLLLIESRLE
    hormone-like 1
    Transmembrane protein 45A LLRSSLILLQGSWF
    IL-17 receptor C RLRLLTLQSWLL
    Neuropeptide FF receptor 2 LLIVALLFILSWL
    Brush border myosin-I LMRKSQILISSWF

    wherein the peptide reduces blood vessel formation in a cell, tissue or organ.
  • In yet more particular embodiments, the one or more peptide comprises an isolated peptide or analog thereof comprising or consisting essentially of a sequence having at least 85% amino acid sequence identity to an amino acid sequence selected from the group consisting of:
  • DEAH box polypeptide 8 EIELVEEEPPF
    Caspase
    10 AEDLLSEEDPF
    CKIP-1 TLDLIQEEDPS

    wherein the peptide reduces blood vessel formation in a cell, tissue or organ.
  • In further embodiments, the one or more peptide comprises an isolated peptide or analog thereof comprising or consisting essentially of a sequence having at least 85% amino acid sequence identity to an amino acid sequence selected from the group consisting of:
  • Collagen type IV, alpha6 LPRFSTMPFIYCNINEVCHY
    fibril

    wherein the peptide reduces blood vessel formation in a cell, tissue or organ.
  • TABLE 1
    The TSP-1 containing 20-mer with all the possible amino acid substitutions
    AA#1 AA#2 AA#3 AA#4 AA#5 AA#6 AA#7 AA#8 AA#9 AA#10
    S (9) P (13) W (29) S (14) P (9) C (29) S (26) V (7) T (15) C (29)
    T (9) E (5) T (5) A (5) N (2) A (6) S (10)
    G (6) S (3) G (5) Q (4) T (1) R (5) R (3)
    Q (2) A (2) E (2) D (3) K (4) N (1)
    A (1) Q (1) D (1) E (3) G (2)
    K (1) R (1) K (1) S (2)
    A (1) R (1) T (2)
    V (1) E (1)
    AA#11 AA#12 AA#13 AA#14 AA#15 AA#16 AA#17 AA#18 AA#19 AA#20
    G (26) G (10) G (29) V (8) Q (11) T (10) R (26) S (5) R (15) R (1)
    S (2) K (4) I (4) S (7) F (4) S (2) T (5) V (1)
    N (1) R (4) M (3) R (6) K (3) Q (1) V (5)
    M (4) T (3) K (2) Q (3) R (3)
    T (2) H (2) Y (2) S (3) H (3)
    L (2) A (1) A (1) L (2) E (2)
    D (1) E (1) E (1) Q (2)
    S (1) F (1) M (1) A (1)
    P (1) K (1) N (1) I (1)
    R (1) V (1)
    S (1)
    Q (1)
    W (1)
    Y (1)
  • TABLE 2
    TSPs
    Motif: W-X(2)-C-X(3)-C-X(2)-G
    Number of Locations: 166
    Number of Different Proteins: 54
    Accession First
    Number|Protein Amino Last Amino
    # Name acid acid Sequence
    1 O00622|CYR61_HUMAN 236 246 WsqCsktCgtG
    2 O14514|BAI1_HUMAN 270 280 WgeCtrdCggG
    3 O14514|BAI1_HUMAN 363 373 WsvCsstCgeG
    4 O14514|BAI1_HUMAN 418 428 WslCsstCgrG
    5 O14514|BAI1_HUMAN 476 486 WsaCsasCsqG
    6 O14514|BAI1_HUMAN 531 541 WgsCsvtCgaG
    7 O15072|ATS3_HUMAN 975 985 WseCsvtCgeG
    8 O60241|BAI2_HUMAN 306 316 WsvCsltCgqG
    9 O60241|BAI2_HUMAN 361 371 WslCsrsCgrG
    10 O60241|BAI2_HUMAN 416 426 WgpCstsCanG
    11 O60241|BAI2_HUMAN 472 482 WslCsktCdtG
    12 O60242|BAI3_HUMAN 300 310 WstCsvtCgqG
    13 O60242|BAI3_HUMAN 354 364 WslCsftCgrG
    14 O60242|BAI3_HUMAN 409 419 WsqCsvtCsnG
    15 O60242|BAI3_HUMAN 464 474 WsgCsksCdgG
    16 O75173|ATS4_HUMAN 529 539 WgdCsrtCggG
    17 O76076|WISP2_HUMAN 201 211 WgpCsttCglG
    18 O95185|UNC5C_HUMAN 269 279 WsvCnsrCgrG
    19 O95388|WISP1_HUMAN 223 233 WspCstsCglG
    20 O95389|WISP3_HUMAN 216 226 WtpCsrtCgmG
    21 O95450|ATS2_HUMAN 863 873 WspCskpCggG
    22 O95450|ATS2_HUMAN 984 994 WsqCsvtCgnG
    23 P07996|TSP1_HUMAN 388 398 WtsCstsCgnG
    24 P07996|TSP1_HUMAN 444 454 WssCsvtCgdG
    25 P07996|TSP1_HUMAN 501 511 WdiCsvtCggG
    26 P13671|CO6_HUMAN 32 42 WtsCsktCnsG
    27 P13671|CO6_HUMAN 75 85 WqrCpinCllG
    28 P14222|PERF_HUMAN 374 384 WrdCsrpCppG
    29 P27918|PROP_HUMAN 86 96 WapCsvtCseG
    30 P27918|PROP_HUMAN 145 155 WepCsvtCskG
    31 P27918|PROP_HUMAN 202 212 WtpCsasChgG
    32 P29279|CTGF_HUMAN 206 216 WsaCsktCgmG
    33 P35442|TSP2_HUMAN 390 400 WtqCsvtCgsG
    34 P35442|TSP2_HUMAN 446 456 WssCsvtCgvG
    35 P35442|TSP2_HUMAN 503 513 WsaCtvtCagG
    36 P48745|NOV_HUMAN 213 223 WtaCsksCgmG
    37 P49327|FAS_HUMAN 627 637 WeeCkqrCppG
    38 P58397|ATS12_HUMAN 551 561 WshCsrtCgaG
    39 P58397|ATS12_HUMAN 832 842 WteCsvtCgtG
    40 P58397|ATS12_HUMAN 952 962 WseCsvsCggG
    41 P58397|ATS12_HUMAN 1321 1331 WseCsttCglG
    42 P58397|ATS12_HUMAN 1372 1382 WskCsrnCsgG
    43 P58397|ATS12_HUMAN 1431 1441 WsqCsrsCggG
    44 P58397|ATS12_HUMAN 1479 1489 WdlCstsCggG
    45 P59510|ATS20_HUMAN 976 986 WsqCsrsCggG
    46 P59510|ATS20_HUMAN 1031 1041 WseClvtCgkG
    47 P59510|ATS20_HUMAN 1086 1096 WgpCtttCghG
    48 P59510|ATS20_HUMAN 1162 1172 WtpCsvsCgrG
    49 P59510|ATS20_HUMAN 1217 1227 WspCsasCghG
    50 P59510|ATS20_HUMAN 1314 1324 WgsCsssCsgG
    51 P59510|ATS20_HUMAN 1368 1378 WgeCsqtCggG
    52 P59510|ATS20_HUMAN 1427 1437 WtsCsasCgkG
    53 P59510|ATS20_HUMAN 1483 1493 WneCsvtCgsG
    54 P59510|ATS20_HUMAN 1664 1674 WskCsvtCgiG
    55 P82987|ATL3_HUMAN 84 94 WsdCsrtCggG
    56 P82987|ATL3_HUMAN 427 437 WtaCsvsCggG
    57 P82987|ATL3_HUMAN 487 497 WsqCtvtCgrG
    58 P82987|ATL3_HUMAN 573 583 WsaCsttCgpG
    59 P82987|ATL3_HUMAN 712 722 WgpCsatCgvG
    60 P82987|ATL3_HUMAN 768 778 WqqCsrtCggG
    61 P82987|ATL3_HUMAN 828 838 WskCsvsCgvG
    62 P82987|ATL3_HUMAN 1492 1502 WsqCsvsCgeG
    63 P82987|ATL3_HUMAN 1606 1616 WkpCtaaCgrG
    64 Q13591|SEM5A_HUMAN 604 614 WspCsttCgiG
    65 Q13591|SEM5A_HUMAN 662 672 WerCtaqCggG
    66 Q13591|SEM5A_HUMAN 793 803 WsqCsrdCsrG
    67 Q13591|SEM5A_HUMAN 850 860 WtkCsatCggG
    68 Q496M8|CI094_HUMAN 259 269 WsaCtrsCggG
    69 Q6S8J7|POTE8_HUMAN 27 37 WccCcfpCcrG
    70 Q6UXZ4|UNC5D_HUMAN 261 271 WsaCnvrCgrG
    71 Q6UY14|ATL4_HUMAN 53 63 WasCsqpCgvG
    72 Q6UY14|ATL4_HUMAN 732 742 WtsCsrsCgpG
    73 Q6UY14|ATL4_HUMAN 792 802 WsqCsvrCgrG
    74 Q6UY14|ATL4_HUMAN 919 929 WgeCsseCgsG
    75 Q6UY14|ATL4_HUMAN 979 989 WspCsrsCqgG
    76 Q6ZMM2|ATL5_HUMAN 44 54 WtrCsssCgrG
    77 Q76LX8|ATS13_HUMAN 1081 1091 WmeCsvsCgdG
    78 Q86TH1|ATL2_HUMAN 56 66 WtaCsrsCggG
    79 Q86TH1|ATL2_HUMAN 631 641 WseCsrtCgeG
    80 Q86TH1|ATL2_HUMAN 746 756 WgpCsgsCgqG
    81 Q86TH1|ATL2_HUMAN 803 813 WerCnttCgrG
    82 Q86TH1|ATL2_HUMAN 862 872 WseCtktCgvG
    83 Q8IUL8|CILP2_HUMAN 155 165 WgpCsgsCgpG
    84 Q8IZJ1|UNC5B_HUMAN 255 265 WspCsnrCgrG
    85 Q8N6G6|ATL1_HUMAN 42 52 WseCsrtCggG
    86 Q8N6G6|ATL1_HUMAN 385 395 WtaCsssCggG
    87 Q8N6G6|ATL1_HUMAN 445 455 WspCtvtCgqG
    88 Q8TE56|ATS17_HUMAN 552 562 WsmCsrtCgtG
    89 Q8TE56|ATS17_HUMAN 809 819 WegCsvqCggG
    90 Q8TE56|ATS17_HUMAN 870 880 WspCsatCekG
    91 Q8TE56|ATS17_HUMAN 930 940 WsqCsasCgkG
    92 Q8TE56|ATS17_HUMAN 981 991 WstCsstCgkG
    93 Q8TE57|ATS16_HUMAN 595 605 WspCsrtCggG
    94 Q8TE57|ATS16_HUMAN 936 946 WsaCsrtCggG
    95 Q8TE57|ATS16_HUMAN 995 1005 WaeCshtCgkG
    96 Q8TE57|ATS16_HUMAN 1060 1070 WsqCsvtCerG
    97 Q8TE57|ATS16_HUMAN 1135 1145 WsqCtasCggG
    98 Q8TE58|ATS15_HUMAN 848 858 WgpCsasCgsG
    99 Q8TE58|ATS15_HUMAN 902 912 WspCsksCgrG
    100 Q8TE59|ATS19_HUMAN 642 652 WspCsrtCsaG
    101 Q8TE59|ATS19_HUMAN 924 934 WedCdatCggG
    102 Q8TE59|ATS19_HUMAN 985 995 WtpCsrtCgkG
    103 Q8TE59|ATS19_HUMAN 1096 1106 WskCsitCgkG
    104 Q8TE60|ATS18_HUMAN 598 608 WseCsrtCggG
    105 Q8TE60|ATS18_HUMAN 940 950 WstCskaCagG
    106 Q8TE60|ATS18_HUMAN 1000 1010 WsqCsktCgrG
    107 Q8TE60|ATS18_HUMAN 1061 1071 WseCsatCglG
    108 Q8TE60|ATS18_HUMAN 1132 1142 WqqCtvtCggG
    109 Q8WXS8|ATS14_HUMAN 856 866 WapCskaCggG
    110 Q8WXS8|ATS14_HUMAN 977 987 WsqCsatCgeG
    111 Q92947|GCDH_HUMAN 225 235 WarCedgCirG
    112 Q96RW7|HMCN1_HUMAN 4538 4548 WraCsvtCgkG
    113 Q96RW7|HMCN1_HUMAN 4595 4605 WeeCtrsCgrG
    114 Q96RW7|HMCN1_HUMAN 4652 4662 WgtCsesCgkG
    115 Q96RW7|HMCN1_HUMAN 4709 4719 WsaCsvsCggG
    116 Q96RW7|HMCN1_HUMAN 4766 4776 WgtCsrtCngG
    117 Q96RW7|HMCN1_HUMAN 4823 4833 WsqCsasCggG
    118 Q99732|LITAF_HUMAN 116 126 WlsCgslCllG
    119 Q9C0I4|THS7B_HUMAN 49 59 WgrCtgdCgpG
    120 Q9C0I4|THS7B_HUMAN 345 355 WspCsktCrsG
    121 Q9C0I4|THS7B_HUMAN 746 756 WtpCprmCqaG
    122 Q9C0I4|THS7B_HUMAN 1009 1019 WgsCsssCgiG
    123 Q9C0I4|THS7B_HUMAN 1258 1268 WteCsqtCghG
    124 Q9C0I4|THS7B_HUMAN 1381 1391 WstCeltCidG
    125 Q9H324|ATS10_HUMAN 530 540 WgdCsrtCggG
    126 Q9H324|ATS10_HUMAN 808 818 WtkCsaqCagG
    127 Q9H324|ATS10_HUMAN 867 877 WslCsrsCdaG
    128 Q9H324|ATS10_HUMAN 927 937 WseCtpsCgpG
    129 Q9H324|ATS10_HUMAN 986 996 WgeCsaqCgvG
    130 Q9HCB6|SPON1_HUMAN 510 520 WspCsisCgmG
    131 Q9HCB6|SPON1_HUMAN 567 577 WdeCsatCgmG
    132 Q9HCB6|SPON1_HUMAN 623 633 WsdCsvtCgkG
    133 Q9HCB6|SPON1_HUMAN 677 687 WseCnksCgkG
    134 Q9HCB6|SPON1_HUMAN 763 773 WseCtklCggG
    135 Q9NS62|THSD1_HUMAN 349 359 WsqCsatCgdG
    136 Q9P283|SEM5B_HUMAN 615 625 WalCstsCgiG
    137 Q9P283|SEM5B_HUMAN 673 683 WskCssnCggG
    138 Q9P283|SEM5B_HUMAN 804 814 WssCsrdCelG
    139 Q9P283|SEM5B_HUMAN 861 871 WspCsasCggG
    140 Q9P2N4|ATS9_HUMAN 1006 1016 WteCsksCdgG
    141 Q9P2N4|ATS9_HUMAN 1061 1071 WseClvtCgkG
    142 Q9P2N4|ATS9_HUMAN 1116 1126 WvqCsvtCgqG
    143 Q9P2N4|ATS9_HUMAN 1191 1201 WtpCsatCgkG
    144 Q9P2N4|ATS9_HUMAN 1247 1257 WssCsvtCgqG
    145 Q9P2N4|ATS9_HUMAN 1337 1347 WgaCsstCagG
    146 Q9P2N4|ATS9_HUMAN 1391 1401 WgeCtklCggG
    147 Q9P2N4|ATS9_HUMAN 1450 1460 WssCsvsCgrG
    148 Q9P2N4|ATS9_HUMAN 1506 1516 WsqCsvsCgrG
    149 Q9P2N4|ATS9_HUMAN 1564 1574 WqeCtktCgeG
    150 Q9P2N4|ATS9_HUMAN 1621 1631 WseCsvtCgkG
    151 Q9P2N4|ATS9_HUMAN 1686 1696 WgsCsvsCgvG
    152 Q9UHI8|ATS1_HUMAN 568 578 WgdCsrtCggG
    153 Q9UHI8|ATS1_HUMAN 863 873 WgeCsksCelG
    154 Q9UHI8|ATS1_HUMAN 917 927 WssCsktCgkG
    155 Q9UKP4|ATS7_HUMAN 547 557 WsiCsrsCgmG
    156 Q9UKP4|ATS7_HUMAN 924 934 WtkCtvtCgrG
    157 Q9UKP5|ATS6_HUMAN 519 529 WgeCsrtCggG
    158 Q9UKP5|ATS6_HUMAN 801 811 WseCsatCagG
    159 Q9UNA0|ATS5_HUMAN 576 586 WgqCsrsCggG
    160 Q9UNA0|ATS5_HUMAN 884 894 WlaCsrtCdtG
    161 Q9UP79|ATS8_HUMAN 536 546 WgeCsrtCggG
    162 Q9UP79|ATS8_HUMAN 842 852 WseCsstCgaG
    163 Q9UPZ6|THS7A_HUMAN 203 213 WseCsktCgsG
    164 Q9UPZ6|THS7A_HUMAN 780 790 WtsCpssCkeG
    165 Q9UPZ6|THS7A_HUMAN 1044 1054 WsrCsksCgsG
    166 Q9UPZ6|THS7A_HUMAN 1423 1433 WslCqltCvnG
  • TABLE 3
    The C-X-C chemokine 22-mer with all the possible amino acid substitutions
    AA#1 AA#2 AA#3 AA#4 AA#5 AA#6 AA#7 AA#8 AA#9 AA#10 AA#11
    N (4) G (6) R (3) K (3) A (2) C (6) L (6) D (4) P (6) A (2) A (3)
    D (2) K (3) E (2) I (2) N (2) E (2) S (2)
    Q (1) L (1) D (1) E (1)
    V (1) K (1)
    AA#12 AA#13 AA#14 AA#15 AA#16 AA#17 AA#18 AA#19 AA#20 AA#21 AA#22
    P (6) F (2) V (3) K (4) K (5) I (3) I (4) E (3) K (6) I (3) L (6)
    I (1) L (2) Q (2) R (1) V (3) V (2) Q (3) F (1)
    M (1) I (1) K (1)
    R (1) M (1)
    W (1)
  • TABLE 4
    CXCs
    Motif: G-X(3)-C-L
    Number of Locations: 1337
    Number of Different Proteins: 1170
    Accession First
    Number|Protein Amino Last Amino
    # Name acid acid Sequence
    167 O00142|KITM_HUMAN 62 67 GkttCL
    168 O00167|EYA2_HUMAN 361 366 GanlCL
    169 O00220|TR10A_HUMAN 332 337 GeaqCL
    170 O00291|HIP1_HUMAN 699 704 GattCL
    171 O00409|FOXN3_HUMAN 465 470 GirsCL
    172 O00444|PLK4_HUMAN 775 780 GhriCL
    173 O00462|MANBA_HUMAN 744 749 GeavCL
    174 O00468|AGRIN_HUMAN 1549 1554 GdhpCL
    175 O00468|AGRIN_HUMAN 2012 2017 GfvgCL
    176 O00476|NPT4_HUMAN 144 149 GcvcCL
    177 O00488|ZN593_HUMAN 41 46 GlhrCL
    178 O00501|CLD5_HUMAN 10 15 GlvlCL
    179 O00624|NPT3_HUMAN 220 225 GcvcCL
    180 O14514|BAI1_HUMAN 243 248 GpenCL
    181 O14522|PTPRT_HUMAN 736 741 GtplCL
    182 O14548|COX7R_HUMAN 97 102 GtiyCL
    183 O14617|AP3D1_HUMAN 1113 1118 GhhvCL
    184 O14628|ZN195_HUMAN 51 56 GlitCL
    185 O14772|FPGT_HUMAN 515 520 GnktCL
    186 O14773|TPP1_HUMAN 2 7 GlqaCL
    187 O14792|OST1_HUMAN 261 266 GrdrCL
    188 O14817|TSN4_HUMAN 68 73 GfvgCL
    189 O14841|OPLA_HUMAN 1240 1245 GdvfCL
    190 O14842|FFAR1_HUMAN 166 171 GspvCL
    191 O14894|T4S5_HUMAN 100 105 GaiyCL
    192 O14981|BTAF1_HUMAN 608 613 GawlCL
    193 O15021|MAST4_HUMAN 1534 1539 GsheCL
    194 O15031|PLXB2_HUMAN 308 313 GaglCL
    195 O15056|SYNJ2_HUMAN 27 32 GrddCL
    196 O15060|ZBT39_HUMAN 272 277 GtnsCL
    197 O15063|K0355_HUMAN 244 249 GcdgCL
    198 O15067|PUR4_HUMAN 914 919 GlvtCL
    199 O15067|PUR4_HUMAN 1040 1045 GpsyCL
    200 O15084|ANR28_HUMAN 449 454 GnleCL
    201 O15084|ANR28_HUMAN 549 554 GhrlCL
    202 O15084|ANR28_HUMAN 661 666 GhseCL
    203 O15105|SMAD7_HUMAN 293 298 GngfCL
    204 O15146|MUSK_HUMAN 648 653 GkpmCL
    205 O15229|KMO_HUMAN 320 325 GfedCL
    206 O15230|LAMA5_HUMAN 1933 1938 GrtqCL
    207 O15296|LX15B_HUMAN 157 162 GwphCL
    208 O15305|PMM2_HUMAN 5 10 GpalCL
    209 O15354|GPR37_HUMAN 448 453 GcyfCL
    210 O15379|HDAC3_HUMAN 214 219 GryyCL
    211 O15397|IPO8_HUMAN 148 153 GsllCL
    212 O15554|KCNN4_HUMAN 263 268 GkivCL
    213 O43156|K0406_HUMAN 642 647 GkdfCL
    214 O43175|SERA_HUMAN 111 116 GmimCL
    215 O43175|SERA_HUMAN 416 421 GfgeCL
    216 O43184|ADA12_HUMAN 407 412 GmgvCL
    217 O43283|M3K13_HUMAN 133 138 GlfgCL
    218 O43396|TXNL1_HUMAN 32 37 GcgpCL
    219 O43396|TXNL1_HUMAN 144 149 GfdnCL
    220 O43405|COCH_HUMAN 10 15 GlgvCL
    221 O43541|SMAD6_HUMAN 363 368 GsgfCL
    222 O43609|SPY1_HUMAN 219 224 GtcmCL
    223 O43638|FREA_HUMAN 315 320 GltpCL
    224 O43747|AP1G1_HUMAN 65 70 GqleCL
    225 O43820|HYAL3_HUMAN 12 17 GvalCL
    226 O43837|IDH3B_HUMAN 181 186 GvieCL
    227 O43889|CREB3_HUMAN 330 335 GntsCL
    228 O60244|CRSP2_HUMAN 447 452 GnseCL
    229 O60266|ADCY3_HUMAN 44 49 GsclCL
    230 O60266|ADCY3_HUMAN 944 949 GgieCL
    231 O60292|SI1L3_HUMAN 658 663 GekvCL
    232 O60423|AT8B3_HUMAN 238 243 GdvvCL
    233 O60504|VINEX_HUMAN 478 483 GehiCL
    234 O60508|PRP17_HUMAN 320 325 GerrCL
    235 O60613|SEP15_HUMAN 4 9 GpsgCL
    236 O60656|UD19_HUMAN 510 515 GyrkCL
    237 O60662|KBTBA_HUMAN 447 452 GmiyCL
    238 O60669|MOT2_HUMAN 93 98 GllcCL
    239 O60673|DPOLZ_HUMAN 47 52 GqktCL
    240 O60704|TPST2_HUMAN 229 234 GkekCL
    241 O60706|ABCC9_HUMAN 1046 1051 GiflCL
    242 O60883|ETBR2_HUMAN 315 320 GcyfCL
    243 O75037|KI21B_HUMAN 1454 1459 GpvmCL
    244 O75037|KI21B_HUMAN 1617 1622 GltpCL
    245 O75052|CAPON_HUMAN 420 425 GrrdCL
    246 O75077|ADA23_HUMAN 487 492 GggaCL
    247 O75078|ADA11_HUMAN 429 434 GggsCL
    248 O75094|SLIT3_HUMAN 1428 1433 GepyCL
    249 O75095|MEGF6_HUMAN 695 700 GaclCL
    250 O75173|ATS4_HUMAN 19 24 GaqpCL
    251 O75173|ATS4_HUMAN 419 424 GyghCL
    252 O75311|GLRA3_HUMAN 387 392 GmgpCL
    253 O75326|SEM7A_HUMAN 499 504 GchgCL
    254 O75342|LX12B_HUMAN 299 304 GegtCL
    255 O75342|LX12B_HUMAN 552 557 GfprCL
    256 O75346|ZN253_HUMAN 131 136 GlnqCL
    257 O75426|FBX24_HUMAN 119 124 GrrrCL
    258 O75436|VP26A_HUMAN 169 174 GiedCL
    259 O75443|TECTA_HUMAN 1687 1692 GdgyCL
    260 O75445|USH2A_HUMAN 1668 1673 GfvgCL
    261 O75445|USH2A_HUMAN 4401 4406 GqglCL
    262 O75446|SAP30_HUMAN 64 69 GqlcCL
    263 O75508|CLD11_HUMAN 164 169 GavlCL
    264 O75569|PRKRA_HUMAN 268 273 GqyqCL
    265 O75592|MYCB2_HUMAN 1087 1092 GfgvCL
    266 O75636|FCN3_HUMAN 16 21 GgpaCL
    267 O75678|RFPL2_HUMAN 117 122 GcavCL
    268 O75679|RFPL3_HUMAN 56 61 GctvCL
    269 O75689|CENA1_HUMAN 37 42 GvfiCL
    270 O75691|UTP20_HUMAN 2132 2137 GalqCL
    271 O75694|NU155_HUMAN 230 235 GkdgCL
    272 O75843|AP1G2_HUMAN 67 72 GqmeCL
    273 O75886|STAM2_HUMAN 42 47 GakdCL
    274 O75911|DHRS3_HUMAN 168 173 GhivCL
    275 O75916|RGS9_HUMAN 642 647 GsgtCL
    276 O75923|DYSF_HUMAN 378 383 GahfCL
    277 O75923|DYSF_HUMAN 1574 1579 GpqeCL
    278 O75925|PIAS1_HUMAN 431 436 GvdgCL
    279 O75954|TSN9_HUMAN 4 9 GclcCL
    280 O75954|TSN9_HUMAN 68 73 GflgCL
    281 O76000|OR2B3_HUMAN 108 113 GateCL
    282 O76013|K1H6_HUMAN 58 63 GlgsCL
    283 O76064|RNF8_HUMAN 15 20 GrswCL
    284 O76075|DFFB_HUMAN 43 48 GsrlCL
    285 O94759|TRPM2_HUMAN 272 277 GnltCL
    286 O94759|TRPM2_HUMAN 713 718 GkttCL
    287 O94761|RECQ4_HUMAN 543 548 GlppCL
    288 O94779|CNTN5_HUMAN 169 174 GhyqCL
    289 O94779|CNTN5_HUMAN 265 270 GsyiCL
    290 O94779|CNTN5_HUMAN 454 459 GmyqCL
    291 O94829|IPO13_HUMAN 159 164 GqgrCL
    292 O94856|NFASC_HUMAN 312 317 GeyfCL
    293 O94887|FARP2_HUMAN 192 197 GqqhCL
    294 O94900|TOX_HUMAN 22 27 GpspCL
    295 O94907|DKK1_HUMAN 107 112 GvqiCL
    296 O94919|ENDD1_HUMAN 371 376 GiesCL
    297 O94933|SLIK3_HUMAN 898 903 GfvdCL
    298 O94955|RHBT3_HUMAN 386 391 GkinCL
    299 O94956|SO2B1_HUMAN 449 454 GmllCL
    300 O95071|EDD1_HUMAN 531 536 GtqvCL
    301 O95153|RIMB1_HUMAN 79 84 GaeaCL
    302 O95153|RIMB1_HUMAN 1485 1490 GlasCL
    303 O95163|IKAP_HUMAN 472 477 GfkvCL
    304 O95202|LETM1_HUMAN 43 48 GlrnCL
    305 O95210|GET1_HUMAN 285 290 GdheCL
    306 O95239|KIF4A_HUMAN 27 32 GcqmCL
    307 O95248|MTMR5_HUMAN 159 164 GlnvCL
    308 O95248|MTMR5_HUMAN 381 386 GyrwCL
    309 O95255|MRP6_HUMAN 845 850 GalvCL
    310 O95255|MRP6_HUMAN 943 948 GtplCL
    311 O95255|MRP6_HUMAN 992 997 GllgCL
    312 O95256|I18RA_HUMAN 447 452 GyslCL
    313 O95279|KCNK5_HUMAN 122 127 GvplCL
    314 O95294|RASL1_HUMAN 130 135 GqgrCL
    315 O95342|ABCBB_HUMAN 327 332 GfvwCL
    316 O95373|IPO7_HUMAN 147 152 GillCL
    317 O95396|MOCS3_HUMAN 250 255 GvlgCL
    318 O95405|ZFYV9_HUMAN 137 142 GnlaCL
    319 O95477|ABCA1_HUMAN 2120 2125 GrfrCL
    320 O95500|CLD14_HUMAN 178 183 GtllCL
    321 O95551|TTRAP_HUMAN 217 222 GnelCL
    322 O95602|RPA1_HUMAN 1556 1561 GitrCL
    323 O95620|DUS4L_HUMAN 125 130 GygaCL
    324 O95633|FSTL3_HUMAN 88 93 GlvhCL
    325 O95671|ASML_HUMAN 588 593 GeyqCL
    326 O95714|HERC2_HUMAN 717 722 GsthCL
    327 O95714|HERC2_HUMAN 3265 3270 GalhCL
    328 O95714|HERC2_HUMAN 4047 4052 GgkhCL
    329 O95715|SCYBE_HUMAN 68 73 GqehCL
    330 O95780|ZN682_HUMAN 132 137 GlnqCL
    331 O95803|NDST3_HUMAN 815 820 GktkCL
    332 O95858|TSN15_HUMAN 285 290 GtgcCL
    333 O95873|CF047_HUMAN 171 176 GpeeCL
    334 O95886|DLGP3_HUMAN 284 289 GgpfCL
    335 O95967|FBLN4_HUMAN 76 81 GgylCL
    336 O95977|EDG6_HUMAN 333 338 GpgdCL
    337 O96006|ZBED1_HUMAN 221 226 GapnCL
    338 O96008|TOM40_HUMAN 72 77 GacgCL
    339 O96009|NAPSA_HUMAN 350 355 GvrlCL
    340 P00505|AATM_HUMAN 268 273 GinvCL
    341 P00750|TPA_HUMAN 515 520 GplvCL
    342 P00751|CFAB_HUMAN 288 293 GakkCL
    343 P01130|LDLR_HUMAN 314 319 GtneCL
    344 P01133|EGF_HUMAN 741 746 GadpCL
    345 P01266|THYG_HUMAN 2020 2025 GevtCL
    346 P01375|TNFA_HUMAN 26 31 GsrrCL
    347 P01730|CD4_HUMAN 366 371 GmwqCL
    348 P01833|PIGR_HUMAN 437 442 GfywCL
    349 P02775|SCYB7_HUMAN 101 106 GrkiCL
    350 P02776|PLF4_HUMAN 37 42 GdlqCL
    351 P02776|PLF4_HUMAN 79 84 GrkiCL
    352 P02778|SCYBA_HUMAN 70 75 GekrCL
    353 P02787|TRFE_HUMAN 209 214 GafkCL
    354 P02787|TRFE_HUMAN 538 543 GafrCL
    355 P02788|TRFL_HUMAN 213 218 GafkCL
    356 P02788|TRFL_HUMAN 549 554 GafrCL
    357 P03986|TCC_HUMAN 28 33 GtylCL
    358 P04350|TBB4_HUMAN 235 240 GvttCL
    359 P04920|B3A2_HUMAN 751 756 GvvfCL
    360 P05108|CP11A_HUMAN 458 463 GvrqCL
    361 P05141|ADT2_HUMAN 155 160 GlgdCL
    362 P05549|AP2A_HUMAN 371 376 GiqsCL
    363 P06401|PRGR_HUMAN 484 489 GasgCL
    364 P06756|ITAV_HUMAN 905 910 GvaqCL
    365 P07202|PERT_HUMAN 819 824 GgfqCL
    366 P07339|CATD_HUMAN 362 367 GktlCL
    367 P07357|CO8A_HUMAN 117 122 GdqdCL
    368 P07437|TBB5_HUMAN 235 240 GvttCL
    369 P07686|HEXB_HUMAN 483 488 GgeaCL
    370 P07814|SYEP_HUMAN 261 266 GhscCL
    371 P07942|LAMB1_HUMAN 1052 1057 GqclCL
    372 P07988|PSPB_HUMAN 244 249 GicqCL
    373 P08151|GLI1_HUMAN 14 19 GepcCL
    374 P08151|GLI1_HUMAN 828 833 GlapCL
    375 P08243|ASNS_HUMAN 8 13 GsddCL
    376 P08319|ADH4_HUMAN 241 246 GatdCL
    377 P08582|TRFM_HUMAN 212 217 GafrCL
    378 P08582|TRFM_HUMAN 558 563 GafrCL
    379 P08686|CP21A_HUMAN 424 429 GarvCL
    380 P08697|A2AP_HUMAN 139 144 GsgpCL
    381 P08709|FA7_HUMAN 14 19 GlqgCL
    382 P08922|ROS_HUMAN 2248 2253 GdviCL
    383 P09001|RM03_HUMAN 291 296 GhknCL
    384 P09326|CD48_HUMAN 5 10 GwdsCL
    385 P09341|GROA_HUMAN 81 86 GrkaCL
    386 P09848|LPH_HUMAN 1846 1851 GphaCL
    387 P10071|GLI3_HUMAN 1359 1364 GpesCL
    388 P10109|ADX_HUMAN 151 156 GcqiCL
    389 P10145|IL8_HUMAN 73 78 GrelCL
    390 P10635|CP2D6_HUMAN 439 444 GrraCL
    391 P10646|TFPI1_HUMAN 213 218 GpswCL
    392 P10720|PF4V_HUMAN 40 45 GdlqCL
    393 P10720|PF4V_HUMAN 82 87 GrkiCL
    394 P10745|IRBP_HUMAN 328 333 GvvhCL
    395 P11047|LAMC1_HUMAN 903 908 GqceCL
    396 P11362|FGFR1_HUMAN 337 342 GeytCL
    397 P11717|MPRI_HUMAN 231 236 GtaaCL
    398 P12236|ADT3_HUMAN 155 160 GlgdCL
    399 P13473|LAMP2_HUMAN 228 233 GndtCL
    400 P13498|CY24A_HUMAN 45 50 GvfvCL
    401 P13569|CFTR_HUMAN 124 129 GiglCL
    402 P13686|PPA5_HUMAN 215 220 GpthCL
    403 P13804|ETFA_HUMAN 49 54 GevsCL
    404 P13807|GYS1_HUMAN 185 190 GvglCL
    405 P13861|KAP2_HUMAN 354 359 GdvkCL
    406 P14222|PERF_HUMAN 530 535 GggtCL
    407 P14543|NID1_HUMAN 24 29 GpvgCL
    408 P14867|GBRA1_HUMAN 6 11 GlsdCL
    409 P15151|PVR_HUMAN 119 124 GnytCL
    410 P15538|C11B1_HUMAN 446 451 GmrqCL
    411 P15692|VEGFA_HUMAN 168 173 GarcCL
    412 P16109|LYAM3_HUMAN 271 276 GnmiCL
    413 P16112|PGCA_HUMAN 2183 2188 GhviCL
    414 P16581|LYAM2_HUMAN 376 381 GymnCL
    415 P17038|ZNF43_HUMAN 127 132 GfnqCL
    416 P17040|ZNF31_HUMAN 184 189 GnsvCL
    417 P17936|IBP3_HUMAN 66 71 GcgcCL
    418 P18510|IL1RA_HUMAN 87 92 GgkmCL
    419 P18564|ITB6_HUMAN 674 679 GeneCL
    420 P18577|RHCE_HUMAN 306 311 GgakCL
    421 P19099|C11B2_HUMAN 446 451 GmrqCL
    422 P19224|UD16_HUMAN 512 517 GyrkCL
    423 P19367|HXK1_HUMAN 713 718 GdngCL
    424 P19835|CEL_HUMAN 96 101 GdedCL
    425 P19875|MIP2A_HUMAN 81 86 GqkaCL
    426 P19876|MIP2B_HUMAN 81 86 GkkaCL
    427 P19883|FST_HUMAN 252 257 GgkkCL
    428 P20062|TCO2_HUMAN 79 84 GyqqCL
    429 P20273|CD22_HUMAN 691 696 GlgsCL
    430 P20648|ATP4A_HUMAN 108 113 GglqCL
    431 P20701|ITAL_HUMAN 76 81 GtghCL
    432 P20701|ITAL_HUMAN 1150 1155 GdpgCL
    433 P20813|CP2B6_HUMAN 432 437 GkriCL
    434 P20916|MAG_HUMAN 301 306 GvyaCL
    435 P20929|NEBU_HUMAN 4517 4522 GvvhCL
    436 P21554|CNR1_HUMAN 427 432 GdsdCL
    437 P21580|TNAP3_HUMAN 99 104 GdgnCL
    438 P21802|FGFR2_HUMAN 5 10 GrfiCL
    439 P21802|FGFR2_HUMAN 338 343 GeytCL
    440 P21817|RYR1_HUMAN 840 845 GpsrCL
    441 P21860|ERBB3_HUMAN 513 518 GpgqCL
    442 P21964|COMT_HUMAN 30 35 GwglCL
    443 P22064|LTB1S_HUMAN 938 943 GsfrCL
    444 P22064|LTB1S_HUMAN 1359 1364 GsykCL
    445 P22105|TENX_HUMAN 565 570 GrgqCL
    446 P22309|UD11_HUMAN 276 281 GginCL
    447 P22309|UD11_HUMAN 513 518 GyrkCL
    448 P22310|UD14_HUMAN 514 519 GyrkCL
    449 P22314|UBE1_HUMAN 230 235 GvvtCL
    450 P22455|FGFR4_HUMAN 97 102 GrylCL
    451 P22455|FGFR4_HUMAN 220 225 GtytCL
    452 P22455|FGFR4_HUMAN 329 334 GeytCL
    453 P22607|FGFR3_HUMAN 335 340 GeytCL
    454 P22680|CP7A1_HUMAN 330 335 GnpiCL
    455 P22732|GTR5_HUMAN 348 353 GfsiCL
    456 P23142|FBLN1_HUMAN 269 274 GihnCL
    457 P23142|FBLN1_HUMAN 547 552 GgfrCL
    458 P23416|GLRA2_HUMAN 376 381 GmghCL
    459 P23759|PAX7_HUMAN 466 471 GqseCL
    460 P24386|RAE1_HUMAN 395 400 GgiyCL
    461 P24557|THAS_HUMAN 475 480 GprsCL
    462 P24592|IBP6_HUMAN 100 105 GrgrCL
    463 P24593|IBP5_HUMAN 96 101 GrgvCL
    464 P24821|TENA_HUMAN 143 148 GagcCL
    465 P24903|CP2F1_HUMAN 432 437 GrrlCL
    466 P25205|MCM3_HUMAN 239 244 GtyrCL
    467 P25874|UCP1_HUMAN 21 26 GiaaCL
    468 P25940|C05A3_HUMAN 1581 1586 GgetCL
    469 P26374|RAE2_HUMAN 397 402 GgiyCL
    470 P26951|IL3RA_HUMAN 363 368 GleeCL
    471 P27487|DPP4_HUMAN 335 340 GrwnCL
    472 P27540|ARNT_HUMAN 332 337 GskfCL
    473 P27987|IP3KB_HUMAN 284 289 GtrsCL
    474 P28332|ADH6_HUMAN 237 242 GateCL
    475 P28340|DPOD1_HUMAN 709 714 GklpCL
    476 P29274|AA2AR_HUMAN 162 167 GqvaCL
    477 P29353|SHC1_HUMAN 570 575 GselCL
    478 P29459|IL12A_HUMAN 33 38 GmfpCL
    479 P30040|ERP29_HUMAN 153 158 GmpgCL
    480 P30530|UFO_HUMAN 106 111 GqyqCL
    481 P30532|ACHA5_HUMAN 279 284 GekiCL
    482 P30566|PUR8_HUMAN 169 174 GkrcCL
    483 P31323|KAP3_HUMAN 368 373 GtvkCL
    484 P32004|L1CAM_HUMAN 308 313 GeyrCL
    485 P32004|L1CAM_HUMAN 493 498 GryfCL
    486 P32314|FOXN2_HUMAN 319 324 GirtCL
    487 P32418|NAC1_HUMAN 414 419 GtyqCL
    488 P32929|CGL_HUMAN 80 85 GakyCL
    489 P32970|TNFL7_HUMAN 29 34 GlviCL
    490 P33402|GCYA2_HUMAN 284 289 GncsCL
    491 P34913|HYES_HUMAN 258 263 GpavCL
    492 P34981|TRFR_HUMAN 94 99 GyvgCL
    493 P34998|CRFR1_HUMAN 83 88 GyreCL
    494 P35227|PCGF2_HUMAN 316 321 GslnCL
    495 P35251|RFC1_HUMAN 402 407 GaenCL
    496 P35270|SPRE_HUMAN 6 11 GravCL
    497 P35367|HRH1_HUMAN 96 101 GrplCL
    498 P35452|HXD12_HUMAN 176 181 GvasCL
    499 P35498|SCN1A_HUMAN 964 969 GqamCL
    500 P35499|SCN4A_HUMAN 774 779 GqamCL
    501 P35503|UD13_HUMAN 514 519 GyrkCL
    502 P35504|UD15_HUMAN 514 519 GyrkCL
    503 P35555|FBN1_HUMAN 1259 1264 GeyrCL
    504 P35555|FBN1_HUMAN 1385 1390 GsyrCL
    505 P35555|FBN1_HUMAN 1416 1421 GngqCL
    506 P35555|FBN1_HUMAN 1870 1875 GsfyCL
    507 P35555|FBN1_HUMAN 2034 2039 GsfkCL
    508 P35556|FBN2_HUMAN 1303 1308 GeyrCL
    509 P35556|FBN2_HUMAN 1952 1957 GsynCL
    510 P35556|FBN2_HUMAN 1994 1999 GsfkCL
    511 P35556|FBN2_HUMAN 2076 2081 GgfqCL
    512 P35590|TIE1_HUMAN 280 285 GltfCL
    513 P35916|VGFR3_HUMAN 4 9 GaalCL
    514 P35968|VGFR2_HUMAN 638 643 GdyvCL
    515 P36509|UD12_HUMAN 510 515 GyrkCL
    516 P36888|FLT3_HUMAN 99 104 GnisCL
    517 P37058|DHB3_HUMAN 13 18 GllvCL
    518 P38398|BRCA1_HUMAN 949 954 GsrfCL
    519 P38571|LICH_HUMAN 7 12 GlvvCL
    520 P38571|LICH_HUMAN 58 63 GyilCL
    521 P38606|VATA1_HUMAN 390 395 GrvkCL
    522 P38607|VATA2_HUMAN 388 393 GrvkCL
    523 P39059|COFA1_HUMAN 8 13 GqcwCL
    524 P40205|NCYM_HUMAN 100 105 GrppCL
    525 P40939|ECHA_HUMAN 709 714 GfppCL
    526 P41217|OX2G_HUMAN 117 122 GcymCL
    527 P42331|RHG25_HUMAN 4 9 GqsaCL
    528 P42345|FRAP_HUMAN 1479 1484 GrmrCL
    529 P42785|PCP_HUMAN 339 344 GqvkCL
    530 P42830|SCYB5_HUMAN 87 92 GkeiCL
    531 P42892|ECE1_HUMAN 79 84 GlvaCL
    532 P43378|PTN9_HUMAN 334 339 GdvpCL
    533 P43403|ZAP70_HUMAN 113 118 GvfdCL
    534 P43403|ZAP70_HUMAN 245 250 GliyCL
    535 P46379|BAT3_HUMAN 872 877 GlfeCL
    536 P46531|NOTC1_HUMAN 1354 1359 GslrCL
    537 P47775|GPR12_HUMAN 166 171 GtsiCL
    538 P47804|RGR_HUMAN 275 280 GiwqCL
    539 P48048|IRK1_HUMAN 204 209 GgklCL
    540 P48052|CBPA2_HUMAN 12 17 GhiyCL
    541 P48059|PINC_HUMAN 176 181 GelyCL
    542 P48067|SC6A9_HUMAN 457 462 GtqfCL
    543 P48230|T4S4_HUMAN 5 10 GcarCL
    544 P48745|NOV_HUMAN 60 65 GcscCL
    545 P49247|RPIA_HUMAN 100 105 GgggCL
    546 P49327|FAS_HUMAN 1455 1460 GlvnCL
    547 P49588|SYAC_HUMAN 897 902 GkitCL
    548 P49640|EVX1_HUMAN 345 350 GpcsCL
    549 P49641|MA2A2_HUMAN 862 867 GwrgCL
    550 P49646|YYY1_HUMAN 393 398 GetpCL
    551 P49753|ACOT2_HUMAN 296 301 GgelCL
    552 P49903|SPS1_HUMAN 323 328 GlliCL
    553 P49910|ZN165_HUMAN 32 37 GqdtCL
    554 P50851|LRBA_HUMAN 2736 2741 GpenCL
    555 P51151|RAB9A_HUMAN 79 84 GsdcCL
    556 P51168|SCNNB_HUMAN 532 537 GsvlCL
    557 P51589|CP2J2_HUMAN 444 449 GkraCL
    558 P51606|RENBP_HUMAN 37 42 GfftCL
    559 P51674|GPM6A_HUMAN 170 175 GanlCL
    560 P51685|CCR8_HUMAN 150 155 GttlCL
    561 P51790|CLCN3_HUMAN 520 525 GaaaCL
    562 P51790|CLCN3_HUMAN 723 728 GlrqCL
    563 P51793|CLCN4_HUMAN 520 525 GaaaCL
    564 P51793|CLCN4_HUMAN 721 726 GlrqCL
    565 P51795|CLCN5_HUMAN 506 511 GaaaCL
    566 P51795|CLCN5_HUMAN 707 712 GlrqCL
    567 P51800|CLCKA_HUMAN 613 618 GhqqCL
    568 P51801|CLCKB_HUMAN 613 618 GhqqCL
    569 P51957|NEK4_HUMAN 322 327 GegkCL
    570 P52306|GDS1_HUMAN 25 30 GcldCL
    571 P52306|GDS1_HUMAN 265 270 GlveCL
    572 P52429|DGKE_HUMAN 411 416 GtkdCL
    573 P52744|ZN138_HUMAN 48 53 GlnqCL
    574 P52789|HXK2_HUMAN 713 718 GdngCL
    575 P52803|EFNA5_HUMAN 147 152 GrrsCL
    576 P52823|STC1_HUMAN 55 60 GafaCL
    577 P52848|NDST1_HUMAN 824 829 GktkCL
    578 P52849|NDST2_HUMAN 302 307 GkrlCL
    579 P52849|NDST2_HUMAN 823 828 GktrCL
    580 P52961|NAR1_HUMAN 220 225 GiwtCL
    581 P53355|DAPK1_HUMAN 1326 1331 GkdwCL
    582 P54132|BLM_HUMAN 891 896 GiiyCL
    583 P54277|PMS1_HUMAN 837 842 GmanCL
    584 P54750|PDE1A_HUMAN 32 37 GilrCL
    585 P54753|EPHB3_HUMAN 297 302 GegpCL
    586 P54826|GAS1_HUMAN 19 24 GawlCL
    587 P55160|NCKPL_HUMAN 938 943 GpieCL
    588 P55268|LAMB2_HUMAN 501 506 GcdrCL
    589 P55268|LAMB2_HUMAN 1063 1068 GqcpCL
    590 P56192|SYMC_HUMAN 8 13 GvpgCL
    591 P56749|CLD12_HUMAN 63 68 GssdCL
    592 P57077|TAK1L_HUMAN 68 73 GflkCL
    593 P57679|EVC_HUMAN 683 688 GssqCL
    594 P58215|LOXL3_HUMAN 13 18 GlllCL
    595 P58397|ATS12_HUMAN 447 452 GwgfCL
    596 P58418|USH3A_HUMAN 69 74 GscgCL
    597 P58512|CU067_HUMAN 166 171 GfpaCL
    598 P59047|NALP5_HUMAN 64 69 GlqwCL
    599 P59510|ATS20_HUMAN 458 463 GygeCL
    600 P60370|KR105_HUMAN 32 37 GtapCL
    601 P60371|KR106_HUMAN 16 21 GsrvCL
    602 P60409|KR107_HUMAN 16 21 GsrvCL
    603 P60413|KR10C_HUMAN 11 16 GsrvCL
    604 P60602|CT052_HUMAN 38 43 GtfsCL
    605 P61011|SRP54_HUMAN 129 134 GwktCL
    606 P61550|ENT1_HUMAN 343 348 GnasCL
    607 P61619|S61A1_HUMAN 143 148 GagiCL
    608 P62072|TIM10_HUMAN 46 51 GesvCL
    609 P62312|LSM6_HUMAN 32 37 GvlaCL
    610 P62714|PP2AB_HUMAN 161 166 GqifCL
    611 P67775|PP2AA_HUMAN 161 166 GqifCL
    612 P68371|TBB2C_HUMAN 235 240 GvttCL
    613 P69849|NOMO3_HUMAN 507 512 GkvsCL
    614 P78310|CXAR_HUMAN 219 224 GsdqCL
    615 P78324|SHPS1_HUMAN 12 17 GpllCL
    616 P78325|ADAM8_HUMAN 101 106 GqdhCL
    617 P78346|RPP30_HUMAN 253 258 GdedCL
    618 P78357|CNTP1_HUMAN 1205 1210 GfsgCL
    619 P78423|X3CL1_HUMAN 350 355 GllfCL
    620 P78504|JAG1_HUMAN 898 903 GprpCL
    621 P78509|RELN_HUMAN 2862 2867 GhgdCL
    622 P78524|ST5_HUMAN 127 132 GvaaCL
    623 P78549|NTHL1_HUMAN 286 291 GqqtCL
    624 P78559|MAP1A_HUMAN 2433 2438 GpqgCX
    625 P80162|SCYB6_HUMAN 87 92 GkqvCL
    626 P82279|CRUM1_HUMAN 1092 1097 GlqgCL
    627 P83105|HTRA4_HUMAN 10 15 GlgrCL
    628 P98088|MUC5A_HUMAN 853 858 GcprCL
    629 P98095|FBLN2_HUMAN 1047 1052 GsfrCL
    630 P98153|IDD_HUMAN 289 294 GddpCL
    631 P98160|PGBM_HUMAN 3181 3186 GtyvCL
    632 P98161|PKD1_HUMAN 649 654 GaniCL
    633 P98164|LRP2_HUMAN 1252 1257 GhpdCL
    634 P98164|LRP2_HUMAN 3819 3824 GsadCL
    635 P98173|FAM3A_HUMAN 83 88 GpkiCL
    636 P98194|AT2C1_HUMAN 158 163 GdtvCL
    637 Q00872|MYPC1_HUMAN 447 452 GkeiCL
    638 Q00973|B4GN1_HUMAN 408 413 GlgnCL
    639 Q01064|PDE1B_HUMAN 243 248 GmvhCL
    640 Q01433|AMPD2_HUMAN 103 108 GpapCL
    641 Q02246|CNTN2_HUMAN 107 112 GvyqCL
    642 Q02246|CNTN2_HUMAN 203 208 GnysCL
    643 Q02318|CP27A_HUMAN 472 477 GvraCL
    644 Q02985|FHR3_HUMAN 188 193 GsitCL
    645 Q03923|ZNF85_HUMAN 133 138 GlnqCL
    646 Q03923|ZNF85_HUMAN 184 189 GmisCL
    647 Q03924|ZN117_HUMAN 103 108 GlnqCL
    648 Q03936|ZNF92_HUMAN 132 137 GlnqCL
    649 Q03938|ZNF90_HUMAN 132 137 GlnqCL
    650 Q04721|NOTC2_HUMAN 476 481 GgftCL
    651 Q05469|LIPS_HUMAN 716 721 GeriCL
    652 Q06730|ZN33A_HUMAN 530 535 GktfCL
    653 Q06732|ZN11B_HUMAN 531 536 GktfCL
    654 Q07325|SCYB9_HUMAN 70 75 GvqtCL
    655 Q07617|SPAG1_HUMAN 133 138 GsnsCL
    656 Q07954|LRP1_HUMAN 875 880 GdndCL
    657 Q07954|LRP1_HUMAN 3001 3006 GsykCL
    658 Q08629|TICN1_HUMAN 178 183 GpcpCL
    659 Q09428|ABCC8_HUMAN 1073 1078 GivlCL
    660 Q10471|GALT2_HUMAN 535 540 GsnlCL
    661 Q12796|PNRC1_HUMAN 63 68 GdgpCL
    662 Q12805|FBLN3_HUMAN 66 71 GgylCL
    663 Q12809|KCNH2_HUMAN 719 724 GfpeCL
    664 Q12841|FSTL1_HUMAN 48 53 GeptCL
    665 Q12852|M3K12_HUMAN 90 95 GlfgCL
    666 Q12860|CNTN1_HUMAN 110 115 GiyyCL
    667 Q12882|DPYD_HUMAN 988 993 GctlCL
    668 Q12933|TRAF2_HUMAN 387 392 GykmCL
    669 Q12986|NFX1_HUMAN 537 542 GdfsCL
    670 Q13077|TRAF1_HUMAN 302 307 GyklCL
    671 Q13129|RLF_HUMAN 48 53 GlrpCL
    672 Q13200|PSMD2_HUMAN 135 140 GereCL
    673 Q13224|NMDE2_HUMAN 584 589 GynrCL
    674 Q13224|NMDE2_HUMAN 1392 1397 GddqCL
    675 Q13255|MGR1_HUMAN 136 141 GinrCL
    676 Q13275|SEM3F_HUMAN 305 310 GghcCL
    677 Q13308|PTK7_HUMAN 429 434 GyldCL
    678 Q13309|SKP2_HUMAN 107 112 GifsCL
    679 Q13322|GRB10_HUMAN 219 224 GlerCL
    680 Q13370|PDE3B_HUMAN 253 258 GgagCL
    681 Q13371|PHLP_HUMAN 200 205 GcmiCL
    682 Q13387|JIP2_HUMAN 594 599 GlfsCL
    683 Q13410|BT1A1_HUMAN 8 13 GlprCL
    684 Q13444|ADA15_HUMAN 405 410 GmgsCL
    685 Q13470|TNK1_HUMAN 105 110 GglkCL
    686 Q13485|SMAD4_HUMAN 359 364 GdrfCL
    687 Q13554|KCC2B_HUMAN 472 477 GpppCL
    688 Q13591|SEM5A_HUMAN 819 824 GgmpCL
    689 Q13591|SEM5A_HUMAN 876 881 GgdiCL
    690 Q13639|5HT4R_HUMAN 89 94 GevfCL
    691 Q13642|FHL1_HUMAN 23 28 GhhcCL
    692 Q13686|ALKB1_HUMAN 300 305 GlphCL
    693 Q13698|CAC1S_HUMAN 1210 1215 GglyCL
    694 Q13751|LAMB3_HUMAN 449 454 GrclCL
    695 Q13772|NCOA4_HUMAN 97 102 GqfnCL
    696 Q13772|NCOA4_HUMAN 364 369 GnlkCL
    697 Q13795|ARFRP_HUMAN 159 164 GrrdCL
    698 Q13822|ENPP2_HUMAN 21 26 GvniCL
    699 Q13885|TBB2A_HUMAN 235 240 GvttCL
    700 Q14008|CKAP5_HUMAN 109 114 GieiCL
    701 Q14008|CKAP5_HUMAN 1237 1242 GvigCL
    702 Q14114|LRP8_HUMAN 175 180 GnrsCL
    703 Q14114|LRP8_HUMAN 336 341 GlneCL
    704 Q14159|K0146_HUMAN 513 518 GtraCL
    705 Q14264|ENR1_HUMAN 358 363 GeltCL
    706 Q14315|FLNC_HUMAN 1649 1654 GlgaCL
    707 Q14344|GNA13_HUMAN 314 319 GdphCL
    708 Q14392|LRC32_HUMAN 360 365 GslpCL
    709 Q14393|GAS6_HUMAN 138 143 GnffCL
    710 Q14393|GAS6_HUMAN 217 222 GsysCL
    711 Q14435|GALT3_HUMAN 93 98 GerpCL
    712 Q14435|GALT3_HUMAN 513 518 GqplCL
    713 Q14451|GRB7_HUMAN 517 522 GilpCL
    714 Q14520|HABP2_HUMAN 121 126 GrgqCL
    715 Q14524|SCN5A_HUMAN 911 916 GqslCL
    716 Q14566|MCM6_HUMAN 154 159 GtflCL
    717 Q14593|ZN273_HUMAN 100 105 GlnqCL
    718 Q14656|ITBA1_HUMAN 197 202 GvlsCL
    719 Q14669|TRIPC_HUMAN 562 567 GladCL
    720 Q14669|TRIPC_HUMAN 1136 1141 GgaeCL
    721 Q14703|MBTP1_HUMAN 845 850 GdsnCL
    722 Q14714|SSPN_HUMAN 91 96 GiivCL
    723 Q14766|LTB1L_HUMAN 1139 1144 GsfrCL
    724 Q14766|LTB1L_HUMAN 1560 1565 GsykCL
    725 Q14767|LTBP2_HUMAN 990 995 GsytCL
    726 Q14767|LTBP2_HUMAN 1156 1161 GsyqCL
    727 Q14767|LTBP2_HUMAN 1197 1202 GsffCL
    728 Q14767|LTBP2_HUMAN 1238 1243 GsfnCL
    729 Q14767|LTBP2_HUMAN 1324 1329 GsfrCL
    730 Q14767|LTBP2_HUMAN 1366 1371 GsflCL
    731 Q14774|HLX1_HUMAN 483 488 GalgCL
    732 Q14916|NPT1_HUMAN 110 115 GfalCL
    733 Q14916|NPT1_HUMAN 207 212 GcavCL
    734 Q14940|SL9A5_HUMAN 576 581 GsgaCL
    735 Q14957|NMDE3_HUMAN 941 946 GpspCL
    736 Q15021|CND1_HUMAN 730 735 GtiqCL
    737 Q15034|HERC3_HUMAN 145 150 GnwhCL
    738 Q15048|LRC14_HUMAN 281 286 GrftCL
    739 Q15058|KIF14_HUMAN 438 443 GfntCL
    740 Q15061|WDR43_HUMAN 103 108 GtctCL
    741 Q15147|PLCB4_HUMAN 987 992 GgsnCL
    742 Q15155|NOMO1_HUMAN 507 512 GkvsCL
    743 Q15274|NADC_HUMAN 92 97 GpahCL
    744 Q15303|ERBB4_HUMAN 516 521 GpdqCL
    745 Q15334|L2GL1_HUMAN 722 727 GvvrCL
    746 Q15399|TLR1_HUMAN 663 668 GmqiCL
    747 Q15413|RYR3_HUMAN 229 234 GhdeCL
    748 Q15413|RYR3_HUMAN 1656 1661 GlrtCL
    749 Q15418|KS6A1_HUMAN 548 553 GnpeCL
    750 Q15546|PAQRB_HUMAN 185 190 GliyCL
    751 Q15633|TRBP2_HUMAN 321 326 GlcqCL
    752 Q15650|TRIP4_HUMAN 196 201 GsgpCL
    753 Q15652|JHD2C_HUMAN 1864 1869 GfvvCL
    754 Q15735|PI5PA_HUMAN 379 384 GpgrCL
    755 Q15746|MYLK_HUMAN 229 234 GvytCL
    756 Q15746|MYLK_HUMAN 579 584 GtytCL
    757 Q15858|SCN9A_HUMAN 940 945 GqamCL
    758 Q15911|ATBF1_HUMAN 3527 3532 GsyhCL
    759 Q16342|PDCD2_HUMAN 121 126 GesvCL
    760 Q16363|LAMA4_HUMAN 1001 1006 GfvgCL
    761 Q16549|PCSK7_HUMAN 16 21 GlptCL
    762 Q16617|NKG7_HUMAN 15 20 GlmfCL
    763 Q16647|PTGIS_HUMAN 437 442 GhnhCL
    764 Q16787|LAMA3_HUMAN 1526 1531 GvssCL
    765 Q30KQ9|DB111_HUMAN 60 65 GthcCL
    766 Q32MQ0|ZN750_HUMAN 121 126 GthrCL
    767 Q3KNT7|NSN5B_HUMAN 134 139 GaehCL
    768 Q3LI83|KR241_HUMAN 153 158 GqlnCL
    769 Q3SYG4|PTHB1_HUMAN 822 827 GgrlCL
    770 Q3T8J9|GON4L_HUMAN 1740 1745 GcadCL
    771 Q495M9|USH1G_HUMAN 76 81 GhlhCL
    772 Q496M8|CI094_HUMAN 170 175 GefsCL
    773 Q499Z4|ZN672_HUMAN 40 45 GrfrCL
    774 Q4G0F5|VP26B_HUMAN 167 172 GiedCL
    775 Q4KMG0|CDON_HUMAN 93 98 GyyqCL
    776 Q53G59|KLH12_HUMAN 426 431 GviyCL
    777 Q53H47|SETMR_HUMAN 72 77 GtcsCL
    778 Q53R12|T4S20_HUMAN 213 218 GflgCL
    779 Q58EX2|SDK2_HUMAN 469 474 GtytCL
    780 Q5HYK3|COQ5_HUMAN 240 245 GrflCL
    781 Q5IJ48|CRUM2_HUMAN 243 248 GsfrCL
    782 Q5JPE7|NOMO2_HUMAN 507 512 GkvsCL
    783 Q5JQC9|AKAP4_HUMAN 242 247 GkskCL
    784 Q5JVG8|ZN506_HUMAN 132 137 GlkqCL
    785 Q5JWF2|GNAS1_HUMAN 2 7 GvrnCL
    786 Q5JWF2|GNAS1_HUMAN 584 589 GtsgCL
    787 Q5JWF8|CT134_HUMAN 111 116 GccvCL
    788 Q5MJ68|SPDYC_HUMAN 138 143 GkdwCL
    789 Q5NUL3|GP120_HUMAN 72 77 GataCL
    790 Q5SRN2|CF010_HUMAN 117 122 GsikCL
    791 Q5T2D3|OTUD3_HUMAN 72 77 GdgnCL
    792 Q5T5C0|STXB5_HUMAN 322 327 GrrpCL
    793 Q5T751|LCE1C_HUMAN 72 77 GggcCL
    794 Q5T752|LCE1D_HUMAN 68 73 GggcCL
    795 Q5T753|LCE1E_HUMAN 72 77 GggcCL
    796 Q5T754|LCE1F_HUMAN 72 77 GggcCL
    797 Q5T7P2|LCE1A_HUMAN 64 69 GggcCL
    798 Q5T7P3|LCE1B_HUMAN 72 77 GggcCL
    799 Q5TA78|LCE4A_HUMAN 55 60 GggcCL
    800 Q5TA79|LCE2A_HUMAN 64 69 GggcCL
    801 Q5TA82|LCE2D_HUMAN 68 73 GggcCL
    802 Q5TCM9|LCE5A_HUMAN 64 69 GggcCL
    803 Q5TEA3|CT194_HUMAN 465 470 GgngCL
    804 Q5TEJ8|ICB1_HUMAN 39 44 GnecCL
    805 Q5THJ4|VP13D_HUMAN 1215 1220 GslgCL
    806 Q5VST9|OBSCN_HUMAN 3315 3320 GdryCL
    807 Q5VST9|OBSCN_HUMAN 4189 4194 GvqwCL
    808 Q5VST9|OBSCN_HUMAN 5195 5200 GvyrCL
    809 Q5VST9|OBSCN_HUMAN 6425 6430 GvytCL
    810 Q5VT25|MRCKA_HUMAN 1325 1330 GaltCL
    811 Q5VUA4|ZN318_HUMAN 1984 1989 GpspCL
    812 Q5VZ18|SHE_HUMAN 8 13 GasaCL
    813 Q5VZM2|RRAGB_HUMAN 366 371 GpkqCL
    814 Q5W111|CLLD6_HUMAN 50 55 GtggCL
    815 Q5XUX1|FBXW9_HUMAN 184 189 GgslCL
    816 Q5ZPR3|CD276_HUMAN 216 221 GtysCL
    817 Q5ZPR3|CD276_HUMAN 434 439 GtysCL
    818 Q5ZPR3|CD276_HUMAN 472 477 GlsvCL
    819 Q63ZY6|NSN5C_HUMAN 216 221 GaehCL
    820 Q63ZY6|NSN5C_HUMAN 293 298 GkgrCL
    821 Q68CP9|ARID2_HUMAN 566 571 GfykCL
    822 Q6BDS2|URFB1_HUMAN 549 554 GnlfCL
    823 Q6GQQ9|OTU7B_HUMAN 190 195 GdgnCL
    824 Q6GTX8|LAIR1_HUMAN 10 15 GlvlCL
    825 Q6IS24|GLTL3_HUMAN 564 569 GtgrCL
    826 Q6ISS4|LAIR2_HUMAN 10 15 GlvlCL
    827 Q6ISS4|LAIR2_HUMAN 97 102 GlyrCL
    828 Q6N022|TEN4_HUMAN 139 144 GrssCL
    829 Q6NUM9|RETST_HUMAN 366 371 GnarCL
    830 Q6P1M0|S27A4_HUMAN 297 302 GigqCL
    831 Q6P1R4|DUS1L_HUMAN 209 214 GniqCL
    832 Q6P587|FAHD1_HUMAN 96 101 GyalCL
    833 Q6P656|CO026_HUMAN 144 149 GqdfCL
    834 Q6PCB7|S27A1_HUMAN 300 305 GvgqCL
    835 Q6PCT2|FXL19_HUMAN 222 227 GgdaCL
    836 Q6Q0C0|TRAF7_HUMAN 397 402 GpvwCL
    837 Q6Q4G3|LAEVR_HUMAN 794 799 GledCL
    838 Q6TGC4|PADI6_HUMAN 22 27 GteiCL
    839 Q6UB99|ANR11_HUMAN 498 503 GssgCL
    840 Q6UWJ8|C16L2_HUMAN 15 20 GgccCL
    841 Q6UWN5|LYPD5_HUMAN 15 20 GaalCL
    842 Q6UX01|LMBRL_HUMAN 394 399 GncvCL
    843 Q6UX53|MET7B_HUMAN 199 204 GdgcCL
    844 Q6UX65|TMM77_HUMAN 99 104 GilsCL
    845 Q6UXV0|GFRAL_HUMAN 127 132 GmwsCL
    846 Q6UY09|CEA20_HUMAN 226 231 GlyrCL
    847 Q6V0L0|CP26C_HUMAN 455 460 GarsCL
    848 Q6V0L0|CP26C_HUMAN 517 522 GnglCL
    849 Q6VVB1|NHLC1_HUMAN 47 52 GhvvCL
    850 Q6VVX0|CP2R1_HUMAN 444 449 GrrhCL
    851 Q6W4X9|MUC6_HUMAN 1095 1100 GdceCL
    852 Q6WN34|CRDL2_HUMAN 54 59 GlmyCL
    853 Q6ZN16|M3K15_HUMAN 82 87 GarqCL
    854 Q6ZN17|LN28B_HUMAN 103 108 GgspCL
    855 Q6ZRI6|CO039_HUMAN 141 146 GlstCL
    856 Q6ZRQ5|CF167_HUMAN 1116 1121 GilkCL
    857 Q6ZSY5|PPR3F_HUMAN 647 652 GaevCL
    858 Q6ZV89|SH2D5_HUMAN 195 200 GghsCL
    859 Q6ZVD8|PHLPL_HUMAN 5 10 GsrnCL
    860 Q6ZW76|ANKS3_HUMAN 632 637 GqalCL
    861 Q75N90|FBN3_HUMAN 551 556 GsfsCL
    862 Q75N90|FBN3_HUMAN 1217 1222 GghrCL
    863 Q75N90|FBN3_HUMAN 1826 1831 GsymCL
    864 Q75N90|FBN3_HUMAN 1866 1871 GsynCL
    865 Q75N90|FBN3_HUMAN 1908 1913 GsfhCL
    866 Q75N90|FBN3_HUMAN 1990 1995 GsfqCL
    867 Q7L099|RUFY3_HUMAN 37 42 GewlCL
    868 Q7L0J3|SV2A_HUMAN 230 235 GrrqCL
    869 Q7L3T8|SYPM_HUMAN 149 154 GkeyCL
    870 Q7L622|K1333_HUMAN 310 315 GitdCL
    871 Q7LBC6|JHD2B_HUMAN 1049 1054 GfgvCL
    872 Q7LBC6|JHD2B_HUMAN 1388 1393 GrllCL
    873 Q7RTN6|STRAD_HUMAN 294 299 GtvpCL
    874 Q7RTP0|NIPA1_HUMAN 122 127 GklgCL
    875 Q7RTU9|STRC_HUMAN 1077 1082 GacsCL
    876 Q7RTX0|TS1R3_HUMAN 20 25 GaplCL
    877 Q7Z2W7|TRPM8_HUMAN 652 657 GgsnCL
    878 Q7Z333|SETX_HUMAN 1106 1111 GekkCL
    879 Q7Z3K3|POGZ_HUMAN 749 754 GrqtCL
    880 Q7Z3T1|OR2W3_HUMAN 108 113 GgveCL
    881 Q7Z401|MYCPP_HUMAN 948 953 GsadCL
    882 Q7Z460|CLAP1_HUMAN 146 151 GiclCL
    883 Q7Z4S6|KI21A_HUMAN 1493 1498 GpvmCL
    884 Q7Z5G4|GOGA7_HUMAN 68 73 GclaCL
    885 Q7Z5K2|WAPL_HUMAN 850 855 GaerCL
    886 Q7Z713|ANR37_HUMAN 75 80 GsleCL
    887 Q7Z7E8|UB2Q1_HUMAN 36 41 GpgpCL
    888 Q7Z7M0|MEGF8_HUMAN 403 408 GcgwCL
    889 Q7Z7M1|GP144_HUMAN 343 348 GselCL
    890 Q86SG6|NEK8_HUMAN 418 423 GsngCL
    891 Q86SQ6|GP123_HUMAN 1058 1063 GraaCL
    892 Q86SQ6|GP123_HUMAN 1091 1096 GhasCL
    893 Q86T20|CF001_HUMAN 75 80 GvldCL
    894 Q86T65|DAAM2_HUMAN 570 575 GappCL
    895 Q86TX2|ACOT1_HUMAN 234 239 GgelCL
    896 Q86U44|MTA70_HUMAN 479 484 GkehCL
    897 Q86UE6|LRTM1_HUMAN 19 24 GvvlCL
    898 Q86UK0|ABCAC_HUMAN 1251 1256 GwlcCL
    899 Q86UK5|LBN_HUMAN 26 31 GgrgCL
    900 Q86UQ4|ABCAD_HUMAN 4056 4061 GppfCL
    901 Q86UQ4|ABCAD_HUMAN 4932 4937 GsfkCL
    902 Q86UU1|PHLB1_HUMAN 119 124 GcmlCL
    903 Q86UU1|PHLB1_HUMAN 1245 1250 GvdtCL
    904 Q86UV5|UBP48_HUMAN 50 55 GnpnCL
    905 Q86UW9|DTX2_HUMAN 347 352 GlpvCL
    906 Q86V24|ADR2_HUMAN 190 195 GailCL
    907 Q86V71|ZN429_HUMAN 132 137 GlnqCL
    908 Q86VH4|LRTM4_HUMAN 271 276 GtfkCL
    909 Q86WB7|UN93A_HUMAN 178 183 GasdCL
    910 Q86WG5|MTMRD_HUMAN 369 374 GyrsCL
    911 Q86WK7|AMGO3_HUMAN 348 353 GlfvCL
    912 Q86WR7|CJ047_HUMAN 84 89 GgvcCL
    913 Q86X76|NIT1_HUMAN 288 293 GpglCL
    914 Q86XN8|RKHD1_HUMAN 192 197 GtdvCL
    915 Q86Y01|DTX1_HUMAN 345 350 GlpvCL
    916 Q86Y56|HEAT2_HUMAN 271 276 GwllCL
    917 Q86YC3|LRC33_HUMAN 396 401 GlasCL
    918 Q8IU80|TMPS6_HUMAN 503 508 GqpdCL
    919 Q8IUK8|CBLN2_HUMAN 27 32 GcgsCL
    920 Q8IUL8|CILP2_HUMAN 464 469 GcqkCL
    921 Q8IVF6|ANR18_HUMAN 706 711 GykkCL
    922 Q8IVH4|MMAA_HUMAN 96 101 GqraCL
    923 Q8IWB7|WDFY1_HUMAN 200 205 GsvaCL
    924 Q8IWN6|CX052_HUMAN 89 94 GskrCL
    925 Q8IWV2|CNTN4_HUMAN 380 385 GmyqCL
    926 Q8IWY4|SCUB1_HUMAN 342 347 GsfqCL
    927 Q8IX30|SCUB3_HUMAN 337 342 GsfqCL
    928 Q8IXI1|MIRO2_HUMAN 515 520 GqtpCL
    929 Q8IXW0|CK035_HUMAN 268 273 GslpCL
    930 Q8IY26|PPAC2_HUMAN 149 154 GtlyCL
    931 Q8IY49|PAQRA_HUMAN 216 221 GvfyCL
    932 Q8IYB9|ZN595_HUMAN 132 137 GvyqCL
    933 Q8IYG6|LRC56_HUMAN 194 199 GnlvCL
    934 Q8IZ96|CKLF1_HUMAN 112 117 GgslCL
    935 Q8IZD0|SAM14_HUMAN 95 100 GgsfCL
    936 Q8IZE3|PACE1_HUMAN 322 327 GetpCL
    937 Q8IZF4|GP114_HUMAN 521 526 GkllCL
    938 Q8IZJ1|UNC5B_HUMAN 547 552 GtfgCL
    939 Q8IZL8|PELP1_HUMAN 317 322 GlarCL
    940 Q8IZY2|ABCA7_HUMAN 2001 2006 GrfrCL
    941 Q8N122|RPTOR_HUMAN 549 554 GqeaCL
    942 Q8N122|RPTOR_HUMAN 1302 1307 GaisCL
    943 Q8N1F7|NUP93_HUMAN 518 523 GdppCL
    944 Q8N1G0|ZN687_HUMAN 1133 1138 GaqqCL
    945 Q8N283|ANR35_HUMAN 65 70 GlteCL
    946 Q8N283|ANR35_HUMAN 703 708 GlwdCL
    947 Q8N357|CB018_HUMAN 57 62 GefsCL
    948 Q8N3C7|RSNL2_HUMAN 201 206 GavkCL
    949 Q8N3V7|SYNPO_HUMAN 28 33 GsyrCL
    950 Q8N441|FGRL1_HUMAN 334 339 GmyiCL
    951 Q8N442|GUF1_HUMAN 334 339 GdtlCL
    952 Q8N4B4|FBX39_HUMAN 114 119 GllsCL
    953 Q8N5D0|WDTC1_HUMAN 48 53 GcvnCL
    954 Q8N5D6|GBGT1_HUMAN 9 14 GlgfCL
    955 Q8N655|CJ012_HUMAN 468 473 GdvkCL
    956 Q8N6F8|WBS27_HUMAN 160 165 GglvCL
    957 Q8N6T3|ARFG1_HUMAN 38 43 GiwiCL
    958 Q8N6V9|TEX9_HUMAN 3 8 GrslCL
    959 Q8N6Y1|PCD20_HUMAN 27 32 GpfsCL
    960 Q8N6Y1|PCD20_HUMAN 881 886 GiyiCL
    961 Q8N726|CD2A2_HUMAN 160 165 GrarCL
    962 Q8N813|CC056_HUMAN 42 47 GsctCL
    963 Q8N895|ZN366_HUMAN 695 700 GrdeCL
    964 Q8N8A2|ANR44_HUMAN 543 548 GhrqCL
    965 Q8N8A2|ANR44_HUMAN 645 650 GhtlCL
    966 Q8N8Q9|NIPA2_HUMAN 112 117 GkigCL
    967 Q8N8R3|MCATL_HUMAN 133 138 GsldCL
    968 Q8N9B4|ANR42_HUMAN 142 147 GrlgCL
    969 Q8N9B4|ANR42_HUMAN 281 286 GhieCL
    970 Q8N9L9|ACOT4_HUMAN 234 239 GadiCL
    971 Q8NB46|ANR52_HUMAN 434 439 GnveCL
    972 Q8NB46|ANR52_HUMAN 732 737 GcedCL
    973 Q8NB46|ANR52_HUMAN 802 807 GhedCL
    974 Q8NB49|AT11C_HUMAN 110 115 GyedCL
    975 Q8NBJ9|SIDT2_HUMAN 296 301 GmlfCL
    976 Q8NBV4|PPAC3_HUMAN 128 133 GtilCL
    977 Q8NCL4|GALT6_HUMAN 505 510 GtnqCL
    978 Q8NCL4|GALT6_HUMAN 593 598 GsgtCL
    979 Q8NCN4|RN169_HUMAN 67 72 GcagCL
    980 Q8NDX1|PSD4_HUMAN 183 188 GlkcCL
    981 Q8NDX1|PSD4_HUMAN 821 826 GedhCL
    982 Q8NEN9|PDZD8_HUMAN 724 729 GgliCL
    983 Q8NFP4|MDGA1_HUMAN 622 627 GsaaCL
    984 Q8NFP9|NBEA_HUMAN 2819 2824 GpenCL
    985 Q8NFU7|CXXC6_HUMAN 1660 1665 GvtaCL
    986 Q8NG94|O11H1_HUMAN 112 117 GtseCL
    987 Q8NG99|OR7G2_HUMAN 109 114 GlenCL
    988 Q8NGC9|O11H4_HUMAN 118 123 GtteCL
    989 Q8NGH6|O52L2_HUMAN 96 101 GytvCL
    990 Q8NGH7|O52L1_HUMAN 96 101 GyivCL
    991 Q8NGI2|O52N4_HUMAN 95 100 GfdeCL
    992 Q8NGJ0|OR5A1_HUMAN 111 116 GlseCL
    993 Q8NGK5|O52M1_HUMAN 95 100 GldaCL
    994 Q8NGR9|OR1N2_HUMAN 112 117 GldnCL
    995 Q8NGS6|O13C3_HUMAN 108 113 GsteCL
    996 Q8NGT2|O13J1_HUMAN 108 113 GsteCL
    997 Q8NGT5|OR9A2_HUMAN 247 252 GygsCL
    998 Q8NGT9|O2A42_HUMAN 107 112 GhseCL
    999 Q8NGU2|OR9A4_HUMAN 251 256 GygsCL
    1000 Q8NGZ9|O2T10_HUMAN 109 114 GaecCL
    1001 Q8NH09|OR8S1_HUMAN 109 114 GteaCL
    1002 Q8NH19|O10AG_HUMAN 99 104 GgteCL
    1003 Q8NH40|OR6S1_HUMAN 66 71 GnlsCL
    1004 Q8NHA8|OR1FC_HUMAN 50 55 GsdhCL
    1005 Q8NHU2|CT026_HUMAN 158 163 GnipCL
    1006 Q8NHU2|CT026_HUMAN 582 587 GfksCL
    1007 Q8NHW6|OTOSP_HUMAN 8 13 GlalCL
    1008 Q8NHX4|SPTA3_HUMAN 175 180 GsrsCL
    1009 Q8NHY2|RFWD2_HUMAN 628 633 GkpyCL
    1010 Q8NHY3|GA2L2_HUMAN 463 468 GpaeCL
    1011 Q8TB24|RIN3_HUMAN 31 36 GmrlCL
    1012 Q8TB24|RIN3_HUMAN 971 976 GsppCL
    1013 Q8TCB7|METL6_HUMAN 89 94 GvgnCL
    1014 Q8TCN5|ZN507_HUMAN 142 147 GmyrCL
    1015 Q8TCT7|PSL1_HUMAN 262 267 GlysCL
    1016 Q8TCT7|PSL1_HUMAN 329 334 GiafCL
    1017 Q8TCT8|PSL2_HUMAN 321 326 GiafCL
    1018 Q8TD26|CHD6_HUMAN 1627 1632 GnlcCL
    1019 Q8TD43|TRPM4_HUMAN 238 243 GthgCL
    1020 Q8TD43|TRPM4_HUMAN 306 311 GaadCL
    1021 Q8TD43|TRPM4_HUMAN 650 655 GdatCL
    1022 Q8TD43|TRPM4_HUMAN 764 769 GgrrCL
    1023 Q8TDJ6|DMXL2_HUMAN 188 193 GkddCL
    1024 Q8TDM6|DLG5_HUMAN 1672 1677 GvkdCL
    1025 Q8TDN4|CABL1_HUMAN 135 140 GsgpCL
    1026 Q8TDU6|GPBAR_HUMAN 81 86 GywsCL
    1027 Q8TDU9|RL3R2_HUMAN 187 192 GvrlCL
    1028 Q8TDV0|GP151_HUMAN 183 188 GvemCL
    1029 Q8TDX9|PK1L1_HUMAN 317 322 GealCL
    1030 Q8TDY2|RBCC1_HUMAN 897 902 GelvCL
    1031 Q8TDZ2|MICA1_HUMAN 743 748 GhfyCL
    1032 Q8TE49|OTU7A_HUMAN 206 211 GdgnCL
    1033 Q8TE58|ATS15_HUMAN 418 423 GhgdCL
    1034 Q8TE85|GRHL3_HUMAN 429 434 GvkgCL
    1035 Q8TEM1|PO210_HUMAN 1489 1494 GdvlCL
    1036 Q8TF62|AT8B4_HUMAN 282 287 GfliCL
    1037 Q8TF76|HASP_HUMAN 190 195 GtsaCL
    1038 Q8WTV0|SCRB1_HUMAN 319 324 GfcpCL
    1039 Q8WUB8|PHF10_HUMAN 320 325 GhpsCL
    1040 Q8WUM0|NU133_HUMAN 112 117 GgwaCL
    1041 Q8WWQ8|STAB2_HUMAN 1358 1363 GngiCL
    1042 Q8WWQ8|STAB2_HUMAN 2026 2031 GsgqCL
    1043 Q8WWX0|ASB5_HUMAN 179 184 GhheCL
    1044 Q8WWZ1|IL1FA_HUMAN 63 68 GgsrCL
    1045 Q8WXI2|CNKR2_HUMAN 22 27 GlddCL
    1046 Q8WXI7|MUC16_HUMAN 22110 22115 GlitCL
    1047 Q8WXK4|ASB12_HUMAN 75 80 GhlsCL
    1048 Q8WXS8|ATS14_HUMAN 489 494 GyqtCL
    1049 Q8WXS8|ATS14_HUMAN 587 592 GgrpCL
    1050 Q8WYB5|MYST4_HUMAN 244 249 GhpsCL
    1051 Q8WYP5|AHTF1_HUMAN 112 117 GsvlCL
    1052 Q8WYP5|AHTF1_HUMAN 318 323 GnrkCL
    1053 Q8WYP5|AHTF1_HUMAN 526 531 GynrCL
    1054 Q8WZ42|TITIN_HUMAN 4919 4924 GkytCL
    1055 Q8WZ42|TITIN_HUMAN 5147 5152 GsavCL
    1056 Q8WZ42|TITIN_HUMAN 7829 7834 GdysCL
    1057 Q8WZ42|TITIN_HUMAN 16742 16747 GaqdCL
    1058 Q8WZ42|TITIN_HUMAN 20237 20242 GtnvCL
    1059 Q8WZ73|RFFL_HUMAN 81 86 GprlCL
    1060 Q8WZ74|CTTB2_HUMAN 924 929 GfknCL
    1061 Q92481|AP2B_HUMAN 379 384 GiqsCL
    1062 Q92496|FHR4_HUMAN 130 135 GsitCL
    1063 Q92520|FAM3C_HUMAN 82 87 GpkiCL
    1064 Q92527|ANKR7_HUMAN 148 153 GeppCL
    1065 Q92529|SHC3_HUMAN 581 586 GselCL
    1066 Q92546|K0258_HUMAN 248 253 GtvaCL
    1067 Q92583|CCL17_HUMAN 30 35 GrecCL
    1068 Q92621|NU205_HUMAN 950 955 GfveCL
    1069 Q92636|FAN_HUMAN 824 829 GtdgCL
    1070 Q92673|SORL_HUMAN 1415 1420 GpstCL
    1071 Q92750|TAF4B_HUMAN 410 415 GaaiCL
    1072 Q92752|TENR_HUMAN 293 298 GqrqCL
    1073 Q92782|DPF1_HUMAN 256 261 GhpsCL
    1074 Q92783|STAM1_HUMAN 41 46 GpkdCL
    1075 Q92785|REQU_HUMAN 302 307 GhpsCL
    1076 Q92794|MYST3_HUMAN 237 242 GhpsCL
    1077 Q92832|NELL1_HUMAN 618 623 GgfdCL
    1078 Q92854|SEM4D_HUMAN 620 625 GvyqCL
    1079 Q92900|RENT1_HUMAN 370 375 GdeiCL
    1080 Q92932|PTPR2_HUMAN 35 40 GrlgCL
    1081 Q92932|PTPR2_HUMAN 634 639 GliyCL
    1082 Q92947|GCDH_HUMAN 285 290 GpfgCL
    1083 Q92947|GCDH_HUMAN 346 351 GlhaCL
    1084 Q92952|KCNN1_HUMAN 361 366 GkgvCL
    1085 Q92956|TNR14_HUMAN 89 94 GlskCL
    1086 Q92968|PEX13_HUMAN 216 221 GtvaCL
    1087 Q93038|TNR25_HUMAN 66 71 GnstCL
    1088 Q969L2|MAL2_HUMAN 37 42 GafvCL
    1089 Q969P0|IGSF8_HUMAN 402 407 GtyrCL
    1090 Q96A54|ADR1_HUMAN 179 184 GavlCL
    1091 Q96AP0|ACD_HUMAN 269 274 GalvCL
    1092 Q96AQ2|TM125_HUMAN 71 76 GtvlCL
    1093 Q96B26|EXOS8_HUMAN 230 235 GklcCL
    1094 Q96B86|RGMA_HUMAN 311 316 GlylCL
    1095 Q96BD0|SO4A1_HUMAN 698 703 GletCL
    1096 Q96CE8|T4S18_HUMAN 8 13 GclsCL
    1097 Q96CW5|GCP3_HUMAN 190 195 GvgdCL
    1098 Q96D59|RN183_HUMAN 95 100 GhqlCL
    1099 Q96DN5|WDR67_HUMAN 52 57 GtgdCL
    1100 Q96DZ5|CLR59_HUMAN 212 217 GaakCL
    1101 Q96EP1|CHFR_HUMAN 528 533 GcygCL
    1102 Q96EY5|F125A_HUMAN 51 56 GyflCL
    1103 Q96EZ4|MYEOV_HUMAN 232 237 GrraCL
    1104 Q96F46|I17RA_HUMAN 628 633 GsqaCL
    1105 Q96GC6|ZN274_HUMAN 256 261 GttcCL
    1106 Q96H40|ZN486_HUMAN 132 137 GlnqCL
    1107 Q96H96|COQ2_HUMAN 172 177 GvllCL
    1108 Q96I82|KAZD1_HUMAN 249 254 GtyrCL
    1109 Q96IV0|NGLY1_HUMAN 70 75 GaveCL
    1110 Q96IW7|SC22A_HUMAN 234 239 GtaaCL
    1111 Q96J02|ITCH_HUMAN 160 165 GvslCL
    1112 Q96J94|PIWL1_HUMAN 674 679 GlkvCL
    1113 Q96JH7|VCIP1_HUMAN 215 220 GdghCL
    1114 Q96JK2|WDR22_HUMAN 178 183 GepfCL
    1115 Q96JT2|S45A3_HUMAN 27 32 GlevCL
    1116 Q96JT2|S45A3_HUMAN 485 490 GrgiCL
    1117 Q96K31|CH076_HUMAN 98 103 GqarCL
    1118 Q96KC8|DNJC1_HUMAN 228 233 GiwfCL
    1119 Q96KM6|K1196_HUMAN 782 787 GkyrCL
    1120 Q96LC7|SIG10_HUMAN 373 378 GqslCL
    1121 Q96LD4|TRI47_HUMAN 25 30 GhnfCL
    1122 Q96LQ0|CN050_HUMAN 366 371 GeprCL
    1123 Q96ME1|FXL18_HUMAN 352 357 GcvhCL
    1124 Q96ME7|ZN512_HUMAN 320 325 GqpeCL
    1125 Q96ME7|ZN512_HUMAN 438 443 GkykCL
    1126 Q96MU7|YTDC1_HUMAN 485 490 GtqlCL
    1127 Q96MU8|KREM1_HUMAN 53 58 GgkpCL
    1128 Q96NL3|ZN599_HUMAN 373 378 GktfCL
    1129 Q96NX9|DACH2_HUMAN 585 590 GnyyCL
    1130 Q96P11|NSUN5_HUMAN 400 405 GaehCL
    1131 Q96PH1|NOX5_HUMAN 272 277 GcgqCL
    1132 Q96PL5|ERMAP_HUMAN 122 127 GsyrCL
    1133 Q96PP9|GBP4_HUMAN 321 326 GavpCL
    1134 Q96Q04|LMTK3_HUMAN 676 681 GacsCL
    1135 Q96Q15|SMG1_HUMAN 2809 2814 GnvtCL
    1136 Q96Q27|ASB2_HUMAN 101 106 GqvgCL
    1137 Q96Q27|ASB2_HUMAN 135 140 GhldCL
    1138 Q96Q91|B3A4_HUMAN 455 460 GaafCL
    1139 Q96QG7|MTMR9_HUMAN 85 90 GmeeCL
    1140 Q96QS1|TSN32_HUMAN 258 263 GpthCL
    1141 Q96QU8|XPO6_HUMAN 413 418 GyfsCL
    1142 Q96R30|OR2V2_HUMAN 103 108 GlfvCL
    1143 Q96RV3|PCX1_HUMAN 696 701 GtvaCL
    1144 Q96RW7|HMCN1_HUMAN 677 682 GiygCL
    1145 Q96RW7|HMCN1_HUMAN 2546 2551 GrytCL
    1146 Q96RW7|HMCN1_HUMAN 3595 3600 GrytCL
    1147 Q96SM3|CPXM1_HUMAN 262 267 GgapCL
    1148 Q96SQ9|CP2S1_HUMAN 436 441 GkrvCL
    1149 Q96SU4|OSBL9_HUMAN 542 547 GcvsCL
    1150 Q99250|SCN2A_HUMAN 955 960 GqtmCL
    1151 Q99466|NOTC4_HUMAN 216 221 GsfqCL
    1152 Q99466|NOTC4_HUMAN 375 380 GsfsCL
    1153 Q99466|NOTC4_HUMAN 414 419 GstlCL
    1154 Q99466|NOTC4_HUMAN 457 462 GsfnCL
    1155 Q99466|NOTC4_HUMAN 609 614 GaffCL
    1156 Q99466|NOTC4_HUMAN 787 792 GtfsCL
    1157 Q99466|NOTC4_HUMAN 1121 1126 GgpdCL
    1158 Q99466|NOTC4_HUMAN 1872 1877 GggaCL
    1159 Q99558|M3K14_HUMAN 536 541 GhavCL
    1160 Q99611|SPS2_HUMAN 373 378 GlliCL
    1161 Q99678|GPR20_HUMAN 115 120 GargCL
    1162 Q99741|CDC6_HUMAN 207 212 GktaCL
    1163 Q99758|ABCA3_HUMAN 1590 1595 GqfkCL
    1164 Q99797|PMIP_HUMAN 277 282 GqlkCL
    1165 Q99848|EBP2_HUMAN 52 57 GlkqCL
    1166 Q99867|TBB4Q_HUMAN 235 240 GvttCL
    1167 Q99884|SC6A7_HUMAN 543 548 GllsCL
    1168 Q99973|TEP1_HUMAN 1464 1469 GpfaCL
    1169 Q99973|TEP1_HUMAN 1486 1491 GarlCL
    1170 Q99973|TEP1_HUMAN 1720 1725 GisaCL
    1171 Q99973|TEP1_HUMAN 2595 2600 GsusCL
    1172 Q99996|AKAP9_HUMAN 3063 3068 GllnCL
    1173 Q9BQ08|RSNB_HUMAN 2 7 GpssCL
    1174 Q9BQG2|NUD12_HUMAN 348 353 GmftCL
    1175 Q9BQR3|PRS27_HUMAN 231 236 GplvCL
    1176 Q9BQS2|SYT15_HUMAN 23 28 GascCL
    1177 Q9BRB3|PIGQ_HUMAN 373 378 GlsaCL
    1178 Q9BRP4|WDR71_HUMAN 206 211 GrsaCL
    1179 Q9BRZ2|TRI56_HUMAN 343 348 GpapCL
    1180 Q9BS86|ZPBP1_HUMAN 346 351 GaktCL
    1181 Q9BT40|SKIP_HUMAN 131 136 GvniCL
    1182 Q9BT51|CU122_HUMAN 6 11 GfshCL
    1183 Q9BTF0|THUM2_HUMAN 407 412 GikkCL
    1184 Q9BTX1|NDC1_HUMAN 310 315 GsdeCL
    1185 Q9BUY5|ZN426_HUMAN 14 19 GdpvCL
    1186 Q9BUY5|ZN426_HUMAN 430 435 GypsCL
    1187 Q9BV38|WDR18_HUMAN 81 86 GpvtCL
    1188 Q9BV38|WDR18_HUMAN 139 144 GgkdCL
    1189 Q9BV73|CP250_HUMAN 806 811 GevrCL
    1190 Q9BV99|LRC61_HUMAN 113 118 GqlqCL
    1191 Q9BVA1|TBB2B_HUMAN 235 240 GvttCL
    1192 Q9BVH7|SIA7E_HUMAN 8 13 GlavCL
    1193 Q9BVK2|ALG8_HUMAN 361 366 GflrCL
    1194 Q9BWT7|CAR10_HUMAN 916 921 GkkhCL
    1195 Q9BWU0|NADAP_HUMAN 185 190 GtsyCL
    1196 Q9BWU0|NADAP_HUMAN 196 201 GcdvCL
    1197 Q9BWV1|BOC_HUMAN 1053 1058 GppcCL
    1198 Q9BXC9|BBS2_HUMAN 26 31 GthpCL
    1199 Q9BXL6|CAR14_HUMAN 850 855 GfkkCL
    1200 Q9BXM7|PINK1_HUMAN 408 413 GgngCL
    1201 Q9BXR0|TGT_HUMAN 50 55 GcriCL
    1202 Q9BXS4|TMM59_HUMAN 229 234 GflrCL
    1203 Q9BXT5|TEX15_HUMAN 1099 1104 GekkCL
    1204 Q9BXU8|FHL17_HUMAN 78 83 GghiCL
    1205 Q9BY15|EMR3_HUMAN 562 567 GctwCL
    1206 Q9BY41|HDAC8_HUMAN 283 288 GigkCL
    1207 Q9BYB4|GNB1L_HUMAN 163 168 GmpmCL
    1208 Q9BYE0|HES7_HUMAN 95 100 GfreCL
    1209 Q9BYJ1|LOXE3_HUMAN 309 314 GqdtCL
    1210 Q9BYK8|PR285_HUMAN 1908 1913 GfslCL
    1211 Q9BYT1|CT059_HUMAN 398 403 GswtCL
    1212 Q9BYX4|IFIH1_HUMAN 265 270 GsusCL
    1213 Q9BZ11|ADA33_HUMAN 400 405 GggaCL
    1214 Q9BZ76|CNTP3_HUMAN 509 514 GfqgCL
    1215 Q9BZ76|CNTP3_HUMAN 1163 1168 GftgCL
    1216 Q9BZC7|ABCA2_HUMAN 2262 2267 GrlrCL
    1217 Q9BZF3|OSBL6_HUMAN 554 559 GrraCL
    1218 Q9BZF9|UACA_HUMAN 79 84 GnleCL
    1219 Q9BZF9|UACA_HUMAN 112 117 GhalCL
    1220 Q9BZH6|BRWD2_HUMAN 79 84 GspyCL
    1221 Q9BZS1|FOXP3_HUMAN 228 233 GraqCL
    1222 Q9BZY9|TRI31_HUMAN 32 37 GhnfCL
    1223 Q9BZZ2|SN_HUMAN 1507 1512 GmyhCL
    1224 Q9C004|SPY4_HUMAN 197 202 GtcmCL
    1225 Q9C0A0|CNTP4_HUMAN 1163 1168 GftgCL
    1226 Q9C0C6|K1737_HUMAN 47 52 GsseCL
    1227 Q9GZK3|OR2B2_HUMAN 108 113 GsteCL
    1228 Q9GZR3|CFC1_HUMAN 144 149 GalhCL
    1229 Q9GZY1|PBOV1_HUMAN 118 123 GlecCL
    1230 Q9H013|ADA19_HUMAN 400 405 GggmCL
    1231 Q9H093|NUAK2_HUMAN 587 592 GpgsCL
    1232 Q9H0A0|NAT10_HUMAN 654 659 GrfpCL
    1233 Q9H0B3|K1683_HUMAN 578 583 GkirCL
    1234 Q9H0J9|PAR12_HUMAN 272 277 GdqiCL
    1235 Q9H0M4|ZCPW1_HUMAN 249 254 GfgqCL
    1236 Q9H172|ABCG4_HUMAN 588 593 GdltCL
    1237 Q9H195|MUC3B_HUMAN 545 550 GqcaCL
    1238 Q9H1B7|CN004_HUMAN 294 299 GgpaCL
    1239 Q9H1D0|TRPV6_HUMAN 10 15 GlilCL
    1240 Q9H1K4|GHC2_HUMAN 47 52 GmidCL
    1241 Q9H1M3|DB129_HUMAN 23 28 GlrrCL
    1242 Q9H1M4|DB127_HUMAN 50 55 GrycCL
    1243 Q9H1P6|CT085_HUMAN 107 112 GlnkCL
    1244 Q9H1R3|MYLK2_HUMAN 240 245 GqalCL
    1245 Q9H1V8|S6A17_HUMAN 421 426 GldpCL
    1246 Q9H221|ABCG8_HUMAN 421 426 GaeaCL
    1247 Q9H228|EDG8_HUMAN 347 352 GlrrCL
    1248 Q9H252|KCNH6_HUMAN 571 576 GfpeCL
    1249 Q9H2D1|MFTC_HUMAN 64 69 GilhCL
    1250 Q9H2G2|SLK_HUMAN 1208 1213 GeseCL
    1251 Q9H2M9|RBGPR_HUMAN 387 392 GesiCL
    1252 Q9H2S1|KCNN2_HUMAN 371 376 GkgvCL
    1253 Q9H2X9|S12A5_HUMAN 602 607 GmslCL
    1254 Q9H2Y7|ZF106_HUMAN 975 980 GegnCL
    1255 Q9H324|ATS10_HUMAN 422 427 GlglCL
    1256 Q9H324|ATS10_HUMAN 556 561 GgkyCL
    1257 Q9H3D4|P73L_HUMAN 557 562 GcssCL
    1258 Q9H3R1|NDST4_HUMAN 814 819 GktkCL
    1259 Q9H4F1|SIA7D_HUMAN 29 34 GlplCL
    1260 Q9H5U8|CX045_HUMAN 403 408 GfdsCL
    1261 Q9H5V8|CDCP1_HUMAN 373 378 GcfvCL
    1262 Q9H6E5|TUT1_HUMAN 15 20 GfrcCL
    1263 Q9H6R4|NOL6_HUMAN 391 396 GislCL
    1264 Q9H792|SG269_HUMAN 1661 1666 GilqCL
    1265 Q9H7F0|AT133_HUMAN 109 114 GhavCL
    1266 Q9H7M9|GI24_HUMAN 142 147 GlycCL
    1267 Q9H808|TLE6_HUMAN 315 320 GpdaCL
    1268 Q9H8X2|IPPK_HUMAN 110 115 GyamCL
    1269 Q9H9S3|S61A2_HUMAN 143 148 GagiCL
    1270 Q9HAF5|CO028_HUMAN 120 125 GvrmCL
    1271 Q9HAS0|NJMU_HUMAN 123 128 GcyyCL
    1272 Q9HAT1|LMA1L_HUMAN 8 13 GplfCL
    1273 Q9HAV4|XPO5_HUMAN 266 271 GaaeCL
    1274 Q9HAW7|UD17_HUMAN 510 515 GyrkCL
    1275 Q9HAW8|UD110_HUMAN 510 515 GyrkCL
    1276 Q9HAW9|UD18_HUMAN 510 515 GyrkCL
    1277 Q9HBX8|LGR6_HUMAN 550 555 GvlgCL
    1278 Q9HBZ2|ARNT2_HUMAN 295 300 GskyCL
    1279 Q9HC07|TM165_HUMAN 138 143 GlmtCL
    1280 Q9HC84|MUC5B_HUMAN 780 785 GklsCL
    1281 Q9HC84|MUC5B_HUMAN 1281 1286 GlgaCL
    1282 Q9HCC6|HES4_HUMAN 113 118 GfheCL
    1283 Q9HCC9|ZFY28_HUMAN 555 560 GatnCL
    1284 Q9HCE9|TM16H_HUMAN 541 546 GgrrCL
    1285 Q9HCM2|PLXA4_HUMAN 990 995 GkqpCL
    1286 Q9HCM4|E41L5_HUMAN 111 116 GspyCL
    1287 Q9HCU4|CELR2_HUMAN 1308 1313 GgytCL
    1288 Q9HCU4|CELR2_HUMAN 1757 1762 GfrgCL
    1289 Q9HCU4|CELR2_HUMAN 1917 1922 GsptCL
    1290 Q9NNW5|WDR6_HUMAN 460 465 GvvaCL
    1291 Q9NP73|GT281_HUMAN 82 87 GagsCL
    1292 Q9NP90|RAB9B_HUMAN 79 84 GadcCL
    1293 Q9NPA1|KCMB3_HUMAN 121 126 GkypCL
    1294 Q9NPA3|M1IP1_HUMAN 58 63 GsggCL
    1295 Q9NPD7|NRN1_HUMAN 37 42 GfsdCL
    1296 Q9NPF8|CENA2_HUMAN 41 46 GifiCL
    1297 Q9NPG4|PCD12_HUMAN 807 812 GwdpCL
    1298 Q9NPH5|NOX4_HUMAN 51 56 GlglCL
    1299 Q9NQ25|SLAF7_HUMAN 3 8 GsptCL
    1300 Q9NQ30|ESM1_HUMAN 125 130 GtgkCL
    1301 Q9NQ75|CT032_HUMAN 50 55 GwwkCL
    1302 Q9NQB0|TF7L2_HUMAN 492 497 GegsCL
    1303 Q9NQQ7|S35C2_HUMAN 302 307 GfalCL
    1304 Q9NQS5|GPR84_HUMAN 195 200 GifyCL
    1305 Q9NQU5|PAK6_HUMAN 662 667 GlpeCL
    1306 Q9NR09|BIRC6_HUMAN 511 516 GanpCL
    1307 Q9NR61|DLL4_HUMAN 204 209 GnlsCL
    1308 Q9NR63|CP26B_HUMAN 437 442 GvrtCL
    1309 Q9NR81|ARHG3_HUMAN 203 208 GwlpCL
    1310 Q9NR99|MXRA5_HUMAN 2414 2419 GnytCL
    1311 Q9NRI5|DISC1_HUMAN 23 28 GsrdCL
    1312 Q9NRX5|SERC1_HUMAN 19 24 GsapCL
    1313 Q9NS15|LTBP3_HUMAN 846 851 GsyrCL
    1314 Q9NS40|KCNH7_HUMAN 722 727 GfpeCL
    1315 Q9NS62|THSD1_HUMAN 419 424 GislCL
    1316 Q9NSD7|RL3R1_HUMAN 243 248 GeelCL
    1317 Q9NSI6|BRWD1_HUMAN 204 209 GsddCL
    1318 Q9NSN8|SNTG1_HUMAN 242 247 GiiqCL
    1319 Q9NST1|ADPN_HUMAN 24 29 GatrCL
    1320 Q9NST1|ADPN_HUMAN 97 102 GlckCL
    1321 Q9NT68|TEN2_HUMAN 858 863 GlvdCL
    1322 Q9NU22|MDN1_HUMAN 427 432 GrgdCL
    1323 Q9NUB4|CT141_HUMAN 156 161 GlafCL
    1324 Q9NUP1|CNO_HUMAN 67 72 GyaaCL
    1325 Q9NVE7|PANK4_HUMAN 304 309 GqlaCL
    1326 Q9NVG8|TBC13_HUMAN 38 43 GglrCL
    1327 Q9NVX2|NLE1_HUMAN 474 479 GkdkCL
    1328 Q9NW08|RPC2_HUMAN 765 770 GfgrCL
    1329 Q9NWT1|PK1IP_HUMAN 83 88 GtitCL
    1330 Q9NWU5|RM22_HUMAN 142 147 GrgqCL
    1331 Q9NWZ3|IRAK4_HUMAN 255 260 GddlCL
    1332 Q9NX02|NALP2_HUMAN 139 144 GnviCL
    1333 Q9NXJ0|M4A12_HUMAN 106 111 GivlCL
    1334 Q9NXR5|ANR10_HUMAN 69 74 GkleCL
    1335 Q9NXR5|ANR10_HUMAN 103 108 GhpqCL
    1336 Q9NXS3|BTBD5_HUMAN 293 298 GlfaCL
    1337 Q9NXW9|ALKB4_HUMAN 19 24 GirtCL
    1338 Q9NY15|STAB1_HUMAN 122 127 GhgtCL
    1339 Q9NY15|STAB1_HUMAN 177 182 GdgsCL
    1340 Q9NY15|STAB1_HUMAN 752 757 GngaCL
    1341 Q9NY15|STAB1_HUMAN 1256 1261 GssrCL
    1342 Q9NY15|STAB1_HUMAN 1991 1996 GsgqCL
    1343 Q9NY15|STAB1_HUMAN 2250 2255 GfhlCL
    1344 Q9NY33|DPP3_HUMAN 515 520 GlylCL
    1345 Q9NY35|CLDND_HUMAN 213 218 GwsfCL
    1346 Q9NY46|SCN3A_HUMAN 956 961 GqtmCL
    1347 Q9NY91|SC5A4_HUMAN 507 512 GtgsCL
    1348 Q9NY99|SNTG2_HUMAN 14 19 GrqgCL
    1349 Q9NYJ7|DLL3_HUMAN 235 240 GecrCL
    1350 Q9NYQ6|CELR1_HUMAN 168 173 GrpiCL
    1351 Q9NYQ7|CELR3_HUMAN 2070 2075 GsdsCL
    1352 Q9NYQ8|FAT2_HUMAN 3908 3913 GfegCL
    1353 Q9NYQ8|FAT2_HUMAN 4285 4290 GggpCL
    1354 Q9NYW6|TA2R3_HUMAN 104 109 GvlyCL
    1355 Q9NZ56|FMN2_HUMAN 1694 1699 GkeqCL
    1356 Q9NZ71|RTEL1_HUMAN 47 52 GktlCL
    1357 Q9NZ94|NLGN3_HUMAN 19 24 GrslCL
    1358 Q9NZH0|GPC5B_HUMAN 164 169 GlalCL
    1359 Q9NZH7|IL1F8_HUMAN 68 73 GkdlCL
    1360 Q9NZL3|ZN224_HUMAN 550 555 GwasCL
    1361 Q9NZR2|LRP1B_HUMAN 866 871 GdddCL
    1362 Q9NZR2|LRP1B_HUMAN 2987 2992 GtykCL
    1363 Q9NZV5|SEPN1_HUMAN 273 278 GavaCL
    1364 Q9P0K1|ADA22_HUMAN 429 434 GggaCL
    1365 Q9P0K7|RAI14_HUMAN 64 69 GhveCL
    1366 Q9P0L1|ZN167_HUMAN 617 622 GlskCL
    1367 Q9P0M9|RM27_HUMAN 84 89 GknkCL
    1368 Q9P0U3|SENP1_HUMAN 531 536 GvhwCL
    1369 Q9P0X4|CAC1I_HUMAN 290 295 GrecCL
    1370 Q9P203|BTBD7_HUMAN 265 270 GnqnCL
    1371 Q9P255|ZN492_HUMAN 143 148 GlnqCL
    1372 Q9P273|TEN3_HUMAN 142 147 GrssCL
    1373 Q9P273|TEN3_HUMAN 1590 1595 GtngCL
    1374 Q9P275|UBP36_HUMAN 824 829 GsetCL
    1375 Q9P283|SEM5B_HUMAN 589 594 GgldCL
    1376 Q9P283|SEM5B_HUMAN 887 892 GediCL
    1377 Q9P298|HIG1B_HUMAN 34 39 GlggCL
    1378 Q9P2B2|FPRP_HUMAN 844 849 GllsCL
    1379 Q9P2C4|TM181_HUMAN 406 411 GerkCL
    1380 Q9P2E3|ZNFX1_HUMAN 1162 1167 GqlfCL
    1381 Q9P2I0|CPSF2_HUMAN 759 764 GlegCL
    1382 Q9P2J9|PDP2_HUMAN 125 130 GvasCL
    1383 Q9P2J9|PDP2_HUMAN 298 303 GmwsCL
    1384 Q9P2N4|ATS9_HUMAN 490 495 GygeCL
    1385 Q9P2P6|STAR9_HUMAN 715 720 GeadCL
    1386 Q9P2R3|ANFY1_HUMAN 720 725 GpggCL
    1387 Q9P2R7|SUCB1_HUMAN 316 321 GnigCL
    1388 Q9P2S2|NRX2A_HUMAN 1061 1066 GfqgCL
    1389 Q9UBD9|CLCF1_HUMAN 10 15 GmlaCL
    1390 Q9UBE0|ULE1A_HUMAN 338 343 GiveCL
    1391 Q9UBG0|MRC2_HUMAN 50 55 GlqgCL
    1392 Q9UBG0|MRC2_HUMAN 89 94 GtmqCL
    1393 Q9UBG0|MRC2_HUMAN 938 943 GdqrCL
    1394 Q9UBG7|RBPSL_HUMAN 56 61 GvrrCL
    1395 Q9UBG7|RBPSL_HUMAN 326 331 GtylCL
    1396 Q9UBH0|IL1F5_HUMAN 63 68 GgsqCL
    1397 Q9UBM4|OPT_HUMAN 124 129 GlptCL
    1398 Q9UBP5|HEY2_HUMAN 125 130 GfreCL
    1399 Q9UBS8|RNF14_HUMAN 258 263 GqvqCL
    1400 Q9UBY5|EDG7_HUMAN 37 42 GtffCL
    1401 Q9UBY8|CLN8_HUMAN 145 150 GflgCL
    1402 Q9UDX3|S14L4_HUMAN 250 255 GnpkCL
    1403 Q9UDX3|S14L4_HUMAN 351 356 GsltCL
    1404 Q9UDX4|S14L3_HUMAN 250 255 GnpkCL
    1405 Q9UGF7|O12D3_HUMAN 62 67 GnlsCL
    1406 Q9UGI6|KCNN3_HUMAN 525 530 GkgvCL
    1407 Q9UGU5|HM2L1_HUMAN 567 572 GplaCL
    1408 Q9UHA7|IL1F6_HUMAN 69 74 GlnlCL
    1409 Q9UHC6|CNTP2_HUMAN 1174 1179 GftgCL
    1410 Q9UHD0|IL19_HUMAN 24 29 GlrrCL
    1411 Q9UHI8|ATS1_HUMAN 458 463 GhgeCL
    1412 Q9UHW9|S12A6_HUMAN 687 692 GmsiCL
    1413 Q9UHX3|EMR2_HUMAN 742 747 GctwCL
    1414 Q9UIA9|XPO7_HUMAN 933 938 GccsCL
    1415 Q9UIE0|N230_HUMAN 286 291 GksfCL
    1416 Q9UIF8|BAZ2B_HUMAN 627 632 GmqwCL
    1417 Q9UIF9|BAZ2A_HUMAN 1006 1011 GpeeCL
    1418 Q9UIH9|KLF15_HUMAN 117 122 GehfCL
    1419 Q9UIR0|BTNL2_HUMAN 337 342 GqyrCL
    1420 Q9UK10|ZN225_HUMAN 466 471 GwasCL
    1421 Q9UK11|ZN223_HUMAN 294 299 GksfCL
    1422 Q9UK12|ZN222_HUMAN 263 268 GksfCL
    1423 Q9UK13|ZN221_HUMAN 488 493 GwasCL
    1424 Q9UK13|ZN221_HUMAN 572 577 GwasCL
    1425 Q9UK99|FBX3_HUMAN 189 194 GlkyCL
    1426 Q9UKB1|FBW1B_HUMAN 281 286 GsvlCL
    1427 Q9UKP4|ATS7_HUMAN 443 448 GwglCL
    1428 Q9UKP5|ATS6_HUMAN 545 550 GgkyCL
    1429 Q9UKQ2|ADA28_HUMAN 500 505 GkghCL
    1430 Q9UKU0|ACSL6_HUMAN 104 109 GngpCL
    1431 Q9UL25|RAB21_HUMAN 121 126 GneiCL
    1432 Q9ULB1|NRX1A_HUMAN 1048 1053 GfqgCL
    1433 Q9ULL4|PLXB3_HUMAN 1191 1196 GrgeCL
    1434 Q9ULV0|MYO5B_HUMAN 1496 1501 GtvpCL
    1435 Q9UM47|NOTC3_HUMAN 1228 1233 GgfrCL
    1436 Q9UM82|SPAT2_HUMAN 37 42 GsdeCL
    1437 Q9UMF0|ICAM5_HUMAN 879 884 GeavCL
    1438 Q9UMW8|UBP18_HUMAN 61 66 GqtcCL
    1439 Q9UNA0|ATS5_HUMAN 467 472 GhgnCL
    1440 Q9UNA0|ATS5_HUMAN 525 530 GqmvCL
    1441 Q9UNI1|ELA1_HUMAN 208 213 GplhCL
    1442 Q9UP79|ATS8_HUMAN 421 426 GhgdCL
    1443 Q9UP79|ATS8_HUMAN 562 567 GgryCL
    1444 Q9UP95|S12A4_HUMAN 622 627 GmslCL
    1445 Q9UPA5|BSN_HUMAN 1765 1770 GspvCL
    1446 Q9UPZ6|THS7A_HUMAN 881 886 GiheCL
    1447 Q9UQ05|KCNH4_HUMAN 213 218 GgsrCL
    1448 Q9UQ49|NEUR3_HUMAN 380 385 GlfgCL
    1449 Q9UQ52|CNTN6_HUMAN 96 101 GmyqCL
    1450 Q9UQD0|SCN8A_HUMAN 949 954 GqamCL
    1451 Q9Y219|JAG2_HUMAN 907 912 GwkpCL
    1452 Q9Y236|OSGI2_HUMAN 480 485 GvtrCL
    1453 Q9Y263|PLAP_HUMAN 721 726 GkaqCL
    1454 Q9Y278|OST2_HUMAN 51 56 GaprCL
    1455 Q9Y297|FBW1A_HUMAN 344 349 GsvlCL
    1456 Q9Y2H6|FNDC3_HUMAN 790 795 GivtCL
    1457 Q9Y2L6|FRM4B_HUMAN 871 876 GsqrCL
    1458 Q9Y2P5|S27A5_HUMAN 345 350 GilgCL
    1459 Q9Y2P5|S27A5_HUMAN 452 457 GkmsCL
    1460 Q9Y2Q1|ZN257_HUMAN 132 137 GlnqCL
    1461 Q9Y2T5|GPR52_HUMAN 205 210 GfivCL
    1462 Q9Y385|UB2J1_HUMAN 87 92 GkkiCL
    1463 Q9Y3B6|CN122_HUMAN 38 43 GeclCL
    1464 Q9Y3C8|UFC1_HUMAN 112 117 GgkiCL
    1465 Q9Y3I1|FBX7_HUMAN 71 76 GdliCL
    1466 Q9Y3N9|OR2W1_HUMAN 108 113 GsveCL
    1467 Q9Y3R4|NEUR2_HUMAN 160 165 GpghCL
    1468 Q9Y3S2|ZN330_HUMAN 182 187 GqhsCL
    1469 Q9Y485|DMXL1_HUMAN 187 192 GkddCL
    1470 Q9Y485|DMXL1_HUMAN 2862 2867 XrnvCL
    1471 Q9Y493|ZAN_HUMAN 1152 1157 GtatCL
    1472 Q9Y4C0|NRX3A_HUMAN 1014 1019 GfqgCL
    1473 Q9Y4F1|FARP1_HUMAN 820 825 GvphCL
    1474 Q9Y4K1|AIM1_HUMAN 1473 1478 GhypCL
    1475 Q9Y4W6|AFG32_HUMAN 31 36 GeqpCL
    1476 Q9Y535|RPC8_HUMAN 43 48 GlciCL
    1477 Q9Y561|LRP12_HUMAN 241 246 GnidCL
    1478 Q9Y574|ASB4_HUMAN 86 91 GhveCL
    1479 Q9Y575|ASB3_HUMAN 291 296 GhedCL
    1480 Q9Y5F7|PCDGL_HUMAN 729 734 GtcaCL
    1481 Q9Y5J3|HEY1_HUMAN 126 131 GfreCL
    1482 Q9Y5N5|HEMK2_HUMAN 45 50 GveiCL
    1483 Q9Y5Q5|CORIN_HUMAN 424 429 GdqrCL
    1484 Q9Y5R5|DMRT2_HUMAN 130 135 GvvsCL
    1485 Q9Y5R6|DMRT1_HUMAN 153 158 GsnpCL
    1486 Q9Y5S2|MRCKB_HUMAN 1374 1379 GsvqCL
    1487 Q9Y5W8|SNX13_HUMAN 73 78 GvpkCL
    1488 Q9Y616|IRAK3_HUMAN 395 400 GldsCL
    1489 Q9Y644|RFNG_HUMAN 203 208 GagfCL
    1490 Q9Y662|OST3B_HUMAN 7 12 GgrsCL
    1491 Q9Y666|S12A7_HUMAN 622 627 GmslCL
    1492 Q9Y6H5|SNCAP_HUMAN 361 366 GhaeCL
    1493 Q9Y6I4|UBP3_HUMAN 449 454 GpesCL
    1494 Q9Y6N6|LAMC3_HUMAN 885 890 GqcsCL
    1495 Q9Y6R1|S4A4_HUMAN 512 517 GaifCL
    1496 Q9Y6R7|FCGBP_HUMAN 1661 1666 GqgvCL
    1497 Q9Y6R7|FCGBP_HUMAN 2388 2393 GqcgCL
    1498 Q9Y6R7|FCGBP_HUMAN 2862 2867 GqgvCL
    1499 Q9Y6R7|FCGBP_HUMAN 3589 3594 GqcgCL
    1500 Q9Y6R7|FCGBP_HUMAN 4063 4068 GqgvCL
    1501 Q9Y6R7|FCGBP_HUMAN 4790 4795 GqcgCL
    1502 Q9Y6R7|FCGBP_HUMAN 4852 4857 GcgrCL
    1503 Q9Y6R7|FCGBP_HUMAN 5032 5037 GcpvCL
  • TABLE 5
    Collagens
    Motif: C-N-X(3)-V-C
    Number of Locations: 24
    Number of Different Proteins: 24
    Accession First
    Number|Protein Amino Last Amino
    # Name acid acid Sequence
    1504 O14514|BAI1_HUMAN 400 406 CNnsaVC
    1505 O75093|SLIT1_HUMAN 507 513 CNsdvVC
    1506 O75534|CSDE1_HUMAN 733 739 CNvwrVC
    1507 P02462|CO4A1_HUMAN 1505 1511 CNinnVC
    1508 P08572|CO4A2_HUMAN 1549 1555 CNpgdVC
    1509 P09758|TACD2_HUMAN 119 125 CNqtsVC
    1510 P25391|LAMA1_HUMAN 751 757 CNvhgVC
    1511 P29400|CO4A5_HUMAN 1521 1527 CNinnVC
    1512 P53420|CO4A4_HUMAN 1525 1531 CNihqVC
    1513 P83110|HTRA3_HUMAN 48 54 CNcclVC
    1514 Q01955|CO4A3_HUMAN 1505 1511 CNvndVC
    1515 Q13625|ASPP2_HUMAN 1002 1008 CNnvqVC
    1516 Q13751|LAMB3_HUMAN 572 578 CNrypVC
    1517 Q14031|CO4A6_HUMAN 1527 1533 CNineVC
    1518 Q8WWQ8|STAB2_HUMAN 1970 1976 CNnrgVC
    1519 Q96GX1|TECT2_HUMAN 642 648 CNrneVC
    1520 Q99965|ADAM2_HUMAN 621 627 CNdrgVC
    1521 Q9BX93|PG12B_HUMAN 112 118 CNqldVC
    1522 Q9BYD5|CNFN_HUMAN 32 38 CNdmpVC
    1523 Q9H013|ADA19_HUMAN 659 665 CNghgVC
    1524 Q9HBG6|IF122_HUMAN 436 442 CNllvVC
    1525 Q9P2R7|SUCB1_HUMAN 152 158 CNqvlVC
    1526 Q9UBX1|CATF_HUMAN 89 95 CNdpmVC
    1527 Q9UKF2|ADA30_HUMAN 638 644 CNtrgVC
  • TABLE 6
    Collagens
    Motif: P-F-X2-C
    Number of Locations: 306
    Number of Different Proteins: 288
    Accession
    Number|Protein First Last
    # Name Aminoacid Aminoacid Sequence
    1528 O00116|ADAS_HUMAN 561 565 PFstC
    1529 O00182|LEG9_HUMAN 98 102 PFdlC
    1530 O00206|TLR4_HUMAN 702 706 PFqlC
    1531 O00270|GPR31_HUMAN 2 6 PFpnC
    1532 O00398|P2Y10_HUMAN 288 292 PFclC
    1533 O00507|USP9Y_HUMAN 259 263 PFgqC
    1534 O14646|CHD1_HUMAN 450 454 PFkdC
    1535 O14843|FFAR3_HUMAN 84 88 PFilC
    1536 O14978|ZN263_HUMAN 547 551 PFseC
    1537 O15015|ZN646_HUMAN 880 884 PFlcC
    1538 O15031|PLXB2_HUMAN 611 615 PFydC
    1539 O15037|K0323_HUMAN 423 427 PFtlC
    1540 O15453|NBR2_HUMAN 9 13 PFlpC
    1541 O15529|GPR42_HUMAN 84 88 PFilC
    1542 O43556|SGCE_HUMAN 207 211 PFssC
    1543 O60299|K0552_HUMAN 308 312 PFaaC
    1544 O60343|TBCD4_HUMAN 89 93 PFlrC
    1545 O60431|OR1I1_HUMAN 93 97 PFvgC
    1546 O60449|LY75_HUMAN 1250 1254 PFqnC
    1547 O60481|ZIC3_HUMAN 331 335 PFpgC
    1548 O60486|PLXC1_HUMAN 618 622 PFtaC
    1549 O60494|CUBN_HUMAN 3302 3306 PFsiC
    1550 O60603|TLR2_HUMAN 669 673 PFklC
    1551 O60656|UD19_HUMAN 149 153 PFdnC
    1552 O60706|ABCC9_HUMAN 627 631 PFesC
    1553 O75152|ZC11A_HUMAN 23 27 PFrhC
    1554 O75197|LRP5_HUMAN 317 321 PFytC
    1555 O75419|CC45L_HUMAN 444 448 PFlyC
    1556 O75473|LGR5_HUMAN 547 551 PFkpC
    1557 O75478|TAD2L_HUMAN 38 42 PFflC
    1558 O75581|LRP6_HUMAN 304 308 PFyqC
    1559 O75794|CD123_HUMAN 147 151 PFihC
    1560 O75882|ATRN_HUMAN 969 973 PFgqC
    1561 O76031|CLPX_HUMAN 313 317 PFaiC
    1562 O95006|OR2F2_HUMAN 93 97 PFqsC
    1563 O95007|OR6B1_HUMAN 285 289 PFiyC
    1564 O95149|SPN1_HUMAN 195 199 PFydC
    1565 O95202|LETM1_HUMAN 51 55 PFgcC
    1566 O95409|ZIC2_HUMAN 336 340 PFpgC
    1567 O95450|ATS2_HUMAN 569 573 PFgsC
    1568 O95759|TBCD8_HUMAN 67 71 PFsrC
    1569 O95841|ANGL1_HUMAN 276 280 PFkdC
    1570 O95886|DLGP3_HUMAN 98 102 PFdtC
    1571 P02461|CO3A1_HUMAN 80 84 PFgeC
    1572 P02462|CO4A1_HUMAN 1501 1505 PFlfC
    1573 P02462|CO4A1_HUMAN 1612 1616 PFieC
    1574 P08151|GLI1_HUMAN 173 177 PFptC
    1575 P08572|CO4A2_HUMAN 1545 1549 PFlyC
    1576 P08572|CO4A2_HUMAN 1654 1658 PFieC
    1577 P08581|MET_HUMAN 534 538 PFvqC
    1578 P09172|DOPO_HUMAN 136 140 PFgtC
    1579 P0C0L4|CO4A_HUMAN 731 735 PFlsC
    1580 P0C0L5|CO4B_HUMAN 731 735 PFlsC
    1581 P15309|PPAP_HUMAN 157 161 PFrnC
    1582 P17021|ZNF17_HUMAN 350 354 PFycC
    1583 P18084|ITB5_HUMAN 546 550 PFceC
    1584 P20645|MPRD_HUMAN 3 7 PFysC
    1585 P20851|C4BB_HUMAN 130 134 PFpiC
    1586 P20933|ASPG_HUMAN 13 17 PFllC
    1587 P21673|SAT1_HUMAN 50 54 PFyhC
    1588 P21854|CD72_HUMAN 222 226 PFftC
    1589 P22309|UD11_HUMAN 152 156 PFlpC
    1590 P22362|CCL1_HUMAN 29 33 PFsrC
    1591 P22681|CBL_HUMAN 417 421 PFcrC
    1592 P23942|RDS_HUMAN 210 214 PFscC
    1593 P24043|LAMA2_HUMAN 2679 2683 PFegC
    1594 P24043|LAMA2_HUMAN 3083 3087 PFrgC
    1595 P24903|CP2F1_HUMAN 483 487 PFqlC
    1596 P25098|ARBK1_HUMAN 252 256 PFivC
    1597 P25490|TYY1_HUMAN 386 390 PFdgC
    1598 P25929|NPY1R_HUMAN 117 121 PFvqC
    1599 P26718|NKG2D_HUMAN 52 56 PFffC
    1600 P26927|HGFL_HUMAN 439 443 PFdyC
    1601 P27987|IP3KB_HUMAN 869 873 PFfkC
    1602 P29400|CO4A5_HUMAN 1517 1521 PFmfC
    1603 P29400|CO4A5_HUMAN 1628 1632 PFieC
    1604 P34896|GLYC_HUMAN 244 248 PFehC
    1605 P35504|UD15_HUMAN 153 157 PFhlC
    1606 P35523|CLCN1_HUMAN 26 30 PFehC
    1607 P35626|ARBK2_HUMAN 252 256 PFivC
    1608 P36383|CXA7_HUMAN 205 209 PFyvC
    1609 P36508|ZNF76_HUMAN 258 262 PFegC
    1610 P36509|UD12_HUMAN 149 153 PFdnC
    1611 P36894|BMR1A_HUMAN 57 61 PFlkC
    1612 P41180|CASR_HUMAN 538 542 PFsnC
    1613 P42338|PK3CB_HUMAN 650 654 PFldC
    1614 P42575|CASP2_HUMAN 141 145 PFpvC
    1615 P45974|UBP5_HUMAN 528 532 PFssC
    1616 P46531|NOTC1_HUMAN 1411 1415 PFyrC
    1617 P48637|GSHB_HUMAN 405 409 PFenC
    1618 P49257|LMAN1_HUMAN 471 475 PFpsC
    1619 P49888|ST1E1_HUMAN 79 83 PFleC
    1620 P50052|AGTR2_HUMAN 315 319 PFlyC
    1621 P50876|UB7I4_HUMAN 273 277 PFvlC
    1622 P51606|RENBP_HUMAN 376 380 PFkgC
    1623 P51617|IRAK1_HUMAN 195 199 PFpfC
    1624 P51689|ARSD_HUMAN 581 585 PFcsC
    1625 P51690|ARSE_HUMAN 576 580 PFplC
    1626 P52740|ZN132_HUMAN 369 373 PFecC
    1627 P52747|ZN143_HUMAN 318 322 PFegC
    1628 P53420|CO4A4_HUMAN 1521 1525 PFayC
    1629 P53420|CO4A4_HUMAN 1630 1634 PFleC
    1630 P53621|COPA|HUMAN 1165 1169 PFdiC
    1631 P54198|HIRA_HUMAN 215 219 PFdeC
    1632 P54793|ARSF_HUMAN 570 574 PFclC
    1633 P54802|ANAG_HUMAN 401 405 PFiwC
    1634 P55157|MTP_HUMAN 823 827 PFlvC
    1635 P62079|TSN5_HUMAN 183 187 PFscC
    1636 P78357|CNTP1_HUMAN 926 930 PFvgC
    1637 P78527|PRKDC_HUMAN 2853 2857 PFvsC
    1638 P81133|SIM1_HUMAN 200 204 PFdgC
    1639 P98088|MUC5A_HUMAN 290 294 PFkmC
    1640 Q01955|CO4A3_HUMAN 1501 1505 PFlfC
    1641 Q01955|CO4A3_HUMAN 1612 1616 PFleC
    1642 Q02817|MUC2_HUMAN 597 601 PFgrC
    1643 Q02817|MUC2_HUMAN 1375 1379 PFglC
    1644 Q02817|MUC2_HUMAN 4916 4920 PFywC
    1645 Q03395|ROM1_HUMAN 213 217 PFscC
    1646 Q07912|ACK1_HUMAN 293 297 PFawC
    1647 Q12830|BPTF_HUMAN 2873 2877 PFyqC
    1648 Q12836|ZP4_HUMAN 238 242 PFtsC
    1649 Q12866|MERTK_HUMAN 313 317 PFrnC
    1650 Q12950|FOXD4_HUMAN 291 295 PFpcC
    1651 Q12968|NFAC3_HUMAN 327 331 PFqyC
    1652 Q13191|CBLB_HUMAN 409 413 PFcrC
    1653 Q13258|PD2R_HUMAN 4 8 PFyrC
    1654 Q13356|PPIL2_HUMAN 38 42 PFdhC
    1655 Q13607|OR2F1_HUMAN 93 97 PFqsC
    1656 Q13753|LAMC2_HUMAN 409 413 PFgtC
    1657 Q13936|CAC1C_HUMAN 2179 2183 PFvnC
    1658 Q14031|CO4A6_HUMAN 1523 1527 PFiyC
    1659 Q14031|CO4A6_HUMAN 1632 1636 PFieC
    1660 Q14137|BOP1_HUMAN 400 404 PFptC
    1661 Q14330|GPR18_HUMAN 247 251 PFhiC
    1662 Q14643|ITPR1_HUMAN 526 530 PFtdC
    1663 Q15042|RB3GP_HUMAN 267 271 PFgaC
    1664 Q15389|ANGP1_HUMAN 282 286 PFrdC
    1665 Q15583|TGIF_HUMAN 269 273 PFhsC
    1666 Q15583|TGIF_HUMAN 314 318 PFslC
    1667 Q15761|NPY5R_HUMAN 128 132 PFlqC
    1668 Q15915|ZIC1_HUMAN 305 309 PFpgC
    1669 Q16363|LAMA4_HUMAN 1788 1792 PFtgC
    1670 Q16572|VACHT_HUMAN 517 521 PFdeC
    1671 Q16586|SGCA_HUMAN 205 209 PFstC
    1672 Q16773|KAT1_HUMAN 123 127 PFfdC
    1673 Q16878|CDO1_HUMAN 160 164 PFdtC
    1674 Q2TBC4|CF049_HUMAN 298 302 PFstC
    1675 Q49AM1|MTER3_HUMAN 28 32 PFlaC
    1676 Q53FE4|CD017_HUMAN 77 81 PFanC
    1677 Q53G59|KLH12_HUMAN 240 244 PFirC
    1678 Q53T03|RBP22_HUMAN 517 521 PFpvC
    1679 Q5IJ48|CRUM2_HUMAN 762 766 PFrgC
    1680 Q5T442|CXA12_HUMAN 241 245 PFfpC
    1681 Q5VYX0|RENAL_HUMAN 310 314 PFlaC
    1682 Q5W0N0|CI057_HUMAN 89 93 PFhgC
    1683 Q6NSW7|NANP8_HUMAN 239 243 PFynC
    1684 Q6P2Q9|PRP8_HUMAN 1892 1896 PFqaC
    1685 Q6PRD1|GP179_HUMAN 232 236 PFleC
    1686 Q6TCH4|PAQR6_HUMAN 95 99 PFasC
    1687 Q6UB98|ANR12_HUMAN 1949 1953 PFsaC
    1688 Q6UB99|ANR11_HUMAN 2552 2556 PFsaC
    1689 Q6UXZ4|UNC5D_HUMAN 766 770 PFtaC
    1690 Q7Z434|MAVS_HUMAN 431 435 PFsgC
    1691 Q7Z6J6|FRMD5_HUMAN 87 91 PFtmC
    1692 Q7Z7G8|VP13B_HUMAN 441 445 PFfdC
    1693 Q7Z7G8|VP13B_HUMAN 1423 1427 PFrnC
    1694 Q7Z7M1|GP144_HUMAN 352 356 PFlcC
    1695 Q86SJ6|DSG4_HUMAN 523 527 PFtfC
    1696 Q86SQ6|GP123_HUMAN 863 867 PFiiC
    1697 Q86T65|DAAM2_HUMAN 548 552 PFacC
    1698 Q86V97|KBTB6_HUMAN 355 359 PFlcC
    1699 Q86XI2|CNDG2_HUMAN 1043 1047 PFsrC
    1700 Q86YT6|MIB1_HUMAN 909 913 PFimC
    1701 Q8IUH2|CREG2_HUMAN 152 156 PFgnC
    1702 Q8IWU5|SULF2_HUMAN 745 749 PFcaC
    1703 Q8IWV8|UBR2_HUMAN 1514 1518 PFlkC
    1704 Q8IWX5|SGPP2_HUMAN 257 261 PFflC
    1705 Q8IX07|FOG1_HUMAN 293 297 PFpqC
    1706 Q8IX29|FBX16_HUMAN 287 291 PFplC
    1707 Q8IXT2|DMRTD_HUMAN 224 228 PFttC
    1708 Q8IZF5|GP113_HUMAN 62 66 PFpaC
    1709 Q8IZQ8|MYCD_HUMAN 403 407 PFqdC
    1710 Q8IZW8|TENS4_HUMAN 423 427 PFttC
    1711 Q8N0W3|FUK_HUMAN 100 104 PFddC
    1712 Q8N122|RPTOR_HUMAN 1033 1037 PFtpC
    1713 Q8N1G1|REXO1_HUMAN 278 282 PFgsC
    1714 Q8N1G2|K0082_HUMAN 790 794 PFhiC
    1715 Q8N201|INT1_HUMAN 1573 1577 PFpaC
    1716 Q8N475|FSTL5_HUMAN 61 65 PFgsC
    1717 Q8N567|ZCHC9_HUMAN 182 186 PFakC
    1718 Q8N7R0|NANG2_HUMAN 166 170 PFynC
    1719 Q8N8U9|BMPER_HUMAN 234 238 PFgsC
    1720 Q8N9L1|ZIC4_HUMAN 207 211 PFpgC
    1721 Q8NB16|MLKL_HUMAN 411 415 PFqgC
    1722 Q8NG11|TSN14_HUMAN 183 187 PFscC
    1723 Q8NGC3|O10G2_HUMAN 98 102 PFggC
    1724 Q8NGC4|O10G3_HUMAN 94 98 PFggC
    1725 Q8NGJ1|OR4D6_HUMAN 165 169 PFpfC
    1726 Q8NH69|OR5W2_HUMAN 93 97 PFygC
    1727 Q8NH85|OR5R1_HUMAN 93 97 PFhaC
    1728 Q8NHU2|CT026_HUMAN 442 446 PFntC
    1729 Q8NHY3|GA2L2_HUMAN 359 363 PFlrC
    1730 Q8N151|BORIS_HUMAN 369 373 PFqcC
    1731 Q8TCB0|IFI44_HUMAN 246 250 PFilC
    1732 Q8TCE9|PPL13_HUMAN 88 92 PFelC
    1733 Q8TCT7|PSL1_HUMAN 275 279 PFgkC
    1734 Q8TD94|KLF14_HUMAN 198 202 PFpgC
    1735 Q8TF76|HASP_HUMAN 474 478 PFshC
    1736 Q8WW14|CJ082_HUMAN 22 26 PFlsC
    1737 Q8WW38|FOG2_HUMAN 299 303 PFpqC
    1738 Q8WWG1|NRG4_HUMAN 32 36 PFcrC
    1739 Q8WWZ7|ABCA5_HUMAN 361 365 PFchC
    1740 Q8WXT5|FX4L4_HUMAN 295 299 PFpcC
    1741 Q8WYR1|PI3R5_HUMAN 814 818 PFavC
    1742 Q8WZ42|TITIN_HUMAN 31091 31095 PFpiC
    1743 Q8WZ60|KLHL6_HUMAN 432 436 PFhnC
    1744 Q92485|ASM3B_HUMAN 41 45 PFqvC
    1745 Q92793|CBP_HUMAN 1279 1283 PFvdC
    1746 Q92838|EDA_HUMAN 328 332 PFlqC
    1747 Q92995|UBP13_HUMAN 540 544 PFsaC
    1748 Q93008|USP9X_HUMAN 251 255 PFgqC
    1749 Q96F10|SAT2_HUMAN 50 54 PFyhC
    1750 Q96FV3|TSN17_HUMAN 185 189 PFscC
    1751 Q96IK0|TM101_HUMAN 27 31 PFwgC
    1752 Q96L50|LLR1_HUMAN 344 348 PFhlC
    1753 Q96L73|NSD1_HUMAN 456 460 PFedC
    1754 Q96P88|GNRR2_HUMAN 184 188 PFtqC
    1755 Q96PZ7|CSMD1_HUMAN 2139 2143 PFprC
    1756 Q96R06|SPAG5_HUMAN 378 382 PFstC
    1757 Q96RG2|PASK_HUMAN 542 546 PFasC
    1758 Q96RJ0|TAAR1_HUMAN 266 270 PFfiC
    1759 Q96RQ9|OXLA_HUMAN 32 36 PFekC
    1760 Q96SE7|ZN347_HUMAN 798 802 PFsiC
    1761 Q96T25|ZIC5_HUMAN 470 474 PFpgC
    1762 Q99666|RGPD8_HUMAN 517 521 PFpvC
    1763 Q99698|LYST_HUMAN 254 258 PFdlC
    1764 Q99726|ZNT3_HUMAN 51 55 PFhhC
    1765 Q9BSE5|SPEB_HUMAN 204 208 PFrrC
    1766 Q9BWQ6|YIPF2_HUMAN 124 128 PFwiC
    1767 Q9BXC9|BBS2_HUMAN 530 534 PFqvC
    1768 Q9BXJ4|C1QT3_HUMAN 18 22 PFclC
    1769 Q9BXK1|KLF16_HUMAN 130 134 PFpdC
    1770 Q9BZE2|PUS3_HUMAN 261 265 PFqlC
    1771 Q9C0C4|SEM4C_HUMAN 719 723 PFrpC
    1772 Q9C0E2|XPO4_HUMAN 50 54 PFavC
    1773 Q9C0I4|THS7B_HUMAN 1482 1486 PFsyC
    1774 Q9GZN6|S6A16_HUMAN 271 275 PFflC
    1775 Q9GZU2|PEG3_HUMAN 1330 1334 PFyeC
    1776 Q9GZZ0|HXD1_HUMAN 162 166 PFpaC
    1777 Q9H0A6|RNF32_HUMAN 344 348 PFhaC
    1778 Q9H0B3|K1683_HUMAN 326 330 PFqiC
    1779 Q9H267|VP33B_HUMAN 189 193 PFpnC
    1780 Q9H2J1|CI037_HUMAN 102 106 PFekC
    1781 Q9H3H5|GPT_HUMAN 77 81 PFlnC
    1782 Q9H8V3|ECT2_HUMAN 239 243 PFqdC
    1783 Q9H9S0|NANOG_HUMAN 239 243 PFynC
    1784 Q9H9V4|RN122_HUMAN 3 7 PFqwC
    1785 Q9HAQ2|KIF9_HUMAN 291 295 PFrqC
    1786 Q9HAW7|UD17_HUMAN 149 153 PFdaC
    1787 Q9HAW8|UD110_HUMAN 149 153 PFdtC
    1788 Q9HAW9|UD18_HUMAN 149 153 PFdaC
    1789 Q9HBX8|LGR6_HUMAN 412 416 PFkpC
    1790 Q9NQW8|CNGB3_HUMAN 309 313 PFdiC
    1791 Q9NRZ9|HELLS_HUMAN 273 277 PFlvC
    1792 Q9NTG7|SIRT3_HUMAN 30 34 PFqaC
    1793 Q9NWZ5|UCKL1_HUMAN 370 374 PFqdC
    1794 Q9NY30|BTG4_HUMAN 98 102 PFevC
    1795 Q9NYM4|GPR83_HUMAN 342 346 PFiyC
    1796 Q9NYV6|RRN3_HUMAN 561 565 PFdpC
    1797 Q9NYW1|TA2R9_HUMAN 190 194 PFilC
    1798 Q9NYW3|TA2R7_HUMAN 193 197 PFcvC
    1799 Q9NZ56|FMN2_HUMAN 716 720 PFsdC
    1800 Q9NZ71|RTEL1_HUMAN 495 499 PFpvC
    1801 Q9NZD2|GLTP_HUMAN 31 35 PFfdC
    1802 Q9P2N4|ATS9_HUMAN 596 600 PFgtC
    1803 Q9UBR1|BUP1_HUMAN 124 128 PFafC
    1804 Q9UBS0|KS6B2_HUMAN 344 348 PFrpC
    1805 Q9UET6|RRMJ1_HUMAN 234 238 PFvtC
    1806 Q9UHD4|CIDEB_HUMAN 37 41 PFrvC
    1807 Q9UKA4|AKA11_HUMAN 917 921 PFshC
    1808 Q9ULC3|RAB23_HUMAN 230 234 PFssC
    1809 Q9ULJ3|ZN295_HUMAN 125 129 PFptC
    1810 Q9ULK4|CRSP3_HUMAN 1086 1090 PFpnC
    1811 Q9ULL4|PLXB3_HUMAN 24 28 PFglC
    1812 Q9ULV8|CBLC_HUMAN 387 391 PFcrC
    1813 Q9UM47|NOTC3_HUMAN 1357 1361 PFfrC
    1814 Q9UNQ2|DIMT1_HUMAN 146 150 PFfrC
    1815 Q9Y3D5|RT18C_HUMAN 86 90 PFtgC
    1816 Q9Y3F1|TA6P_HUMAN 25 29 PFpsC
    1817 Q9Y3R5|CU005_HUMAN 255 259 PFytC
    1818 Q9Y450|HBS1L_HUMAN 487 491 PFrlC
    1819 Q9Y493|ZAN_HUMAN 1364 1368 PFetC
    1820 Q9Y493|ZAN_HUMAN 1751 1755 PFsqC
    1821 Q9Y493|ZAN_HUMAN 2556 2560 PFaaC
    1822 Q9Y548|YIPF1_HUMAN 123 127 PFwiC
    1823 Q9Y5L3|ENP2_HUMAN 324 328 PFsrC
    1824 Q9Y5P8|2ACC_HUMAN 272 276 PFqdC
    1825 Q9Y664|KPTN_HUMAN 143 147 PFqlC
    1826 Q9Y678|COPG_HUMAN 226 230 PFayC
    1827 Q9Y6E0|STK24_HUMAN 371 375 PFsqC
    1828 Q9Y6R7|FCGBP_HUMAN 683 687 PFavC
    1829 Q9Y6R7|FCGBP_HUMAN 1074 1078 PFreC
    1830 Q9Y6R7|FCGBP_HUMAN 1888 1892 PFttC
    1831 Q9Y6R7|FCGBP_HUMAN 3089 3093 PFttC
    1832 Q9Y6R7|FCGBP_HUMAN 4290 4294 PFttC
    1833 Q9Y6R7|FCGBP_HUMAN 5059 5063 PFatC
  • TABLE 7A
    Table of the amino acid sequences of the peptides
    predicted similar to Growth Hormone
    Peptide
    Protein Name Peptide Location sequence
    Placental Lactogen AAA98621(101-114) LLRISLLLIESWLE
    hGH-V AAB59548(101-114) LLRISLLLTQSWLE
    GH2 CAG46722(101-114) LLHISLLLIQSWLE
    Chorionic AAA52116(101-113) LLRLLLLIESWLE
    somatomammotropin
    Chorionic AAI19748(12-25) LLHISLLLIESRLE
    somatomammotropin
    hormone-like 1
    Transmembrane NP_060474(181-194) LLRSSLILLQGSWF
    protein 45A
    IL-17 receptor C Q8NAC3(376-387) RLRLLTLQSWLL
    Neuropeptide Q9Y5X5(378-390) LLIVALLFILSWL
    FF receptor 2
    Brush border AAC27437(719-731) LMRKSQILISSWF
    myosin-I
  • TABLE 7B
    Table of the amino acid sequences of the peptides
    predicted similar to PEDF.
    Peptide
    Protein Name Peptide Location sequence
    DEAH box polypeptide AAH47327(438-448) EIELVEEEPPF
    8
    Caspase 10 CAD32371(67-77) AEDLLSEEDPF
    CKIP-1 CAI14263(66-76) TLDLIQEEDPS
  • TABLE 8
    Amino acid sequences of peptides that contain the
    somatotropin motif.
    Somatotropins
    Motif: L-X(3)-L-L-X(3)-S-X-L
    Number of Locations: 139
    Number of Different Proteins: 139
    Accession
    # Number|Protein Name First Aminoacid Last Aminoacid Sequence
    1834 O14569|C56D2_HUMAN 164 175 LvgyLLgsaSlL
    1835 O15287|FANCG_HUMAN 416 427 LceeLLsrtSsL
    1836 O15482|TEX28_HUMAN 338 349 LatvLLvfvStL
    1837 O43914|TYOBP_HUMAN 11 22 LllpLLlavSgL
    1838 O60609|GFRA3_HUMAN 15 26 LmllLLlppSpL
    1839 O75844|FACE1_HUMAN 279 290 LfdtLLeeySvL
    1840 O95747|OXSR1_HUMAN 90 101 LvmkLLsggSvL
    1841 P01241|SOMA_HUMAN 102 113 LrisLLliqSwL
    1842 P01242|SOM2_HUMAN 102 113 LrisLLliqSwL
    1843 P01243|CSH_HUMAN 102 113 LrisLLlieSwL
    1844 P02750|A2GL_HUMAN 83 94 LpanLLqgaSkL
    1845 P03891|NU2M_HUMAN 149 160 LnvsLLltlSiL
    1846 P04201|MAS_HUMAN 151 162 LvcaLLwalScL
    1847 P05783|K1C18_HUMAN 338 349 LngiLLhleSeL
    1848 P07359|GP1BA_HUMAN 3 14 LlllLLllpSpL
    1849 P09848|LPH_HUMAN 35 46 LtndLLhnlSgL
    1850 P11168|GTR2_HUMAN 136 147 LvgaLLmgfSkL
    1851 P12034|FGF5_HUMAN 3 14 LsflLLlffShL
    1852 P13489|RINI_HUMAN 247 258 LcpgLLhpsSrL
    1853 P14902|I23O_HUMAN 196 207 LlkaLLeiaScL
    1854 P16278|BGAL_HUMAN 135 146 LpawLLekeSiL
    1855 P19838|NFKB1_HUMAN 558 569 LvrdLLevtSgL
    1856 P22079|PERL_HUMAN 512 523 LvrgLLakkSkL
    1857 P23276|KELL_HUMAN 53 64 LilgLLlcfSvL
    1858 P24394|IL4RA_HUMAN 4 15 LcsgLLfpvScL
    1859 P29320|EPHA3_HUMAN 5 16 LsilLLlscSvL
    1860 P31512|FMO4_HUMAN 524 535 LaslLLickSsL
    1861 P35270|SPRE_HUMAN 26 37 LlasLLspgSvL
    1862 P41250|SYG_HUMAN 20 31 LpprLLarpSlL
    1863 P42575|CASP2_HUMAN 114 125 LedmLLttlSgL
    1864 P46721|SO1A2_HUMAN 396 407 LleyLLyflSfL
    1865 P51665|PSD7_HUMAN 201 212 LnskLLdirSyL
    1866 P59531|T2R12_HUMAN 188 199 LisfLLsliSlL
    1867 P69849|NOMO3_HUMAN 1180 1191 LiplLLqltSrL
    1868 P98161|PKD1_HUMAN 82 93 LdvgLLanlSaL
    1869 P98171|RHG04_HUMAN 153 164 LqdeLLevvSeL
    1870 P98196|AT11A_HUMAN 1077 1088 LaivLLvtiSlL
    1871 Q08431|MFGM_HUMAN 10 21 LcgaLLcapSlL
    1872 Q08AF3|SLFN5_HUMAN 533 544 LvivLLgfkSfL
    1873 Q12952|FOXL1_HUMAN 293 304 LgasLLaasSsL
    1874 Q13275|SEM3F_HUMAN 2 13 LvagLLlwaSlL
    1875 Q13394|MB211_HUMAN 300 311 LngiLLqliScL
    1876 Q13609|DNSL3_HUMAN 8 19 LlllLLsihSaL
    1877 Q13619|CUL4A_HUMAN 213 224 LlrsLLgmlSdL
    1878 Q13620|CUL4B_HUMAN 349 360 LlrsLLsmlSdL
    1879 Q14406|CSHL_HUMAN 84 95 LhisLLlieSrL
    1880 Q14667|K0100_HUMAN 8 19 LlvlLLvalSaL
    1881 Q15155|NOMO1_HUMAN 1180 1191 LiplLLqltSrL
    1882 Q15760|GPR19_HUMAN 279 290 LilnLLfllSwL
    1883 Q53RE8|ANR39_HUMAN 166 177 LacdLLpcnSdL
    1884 Q5FWE3|PRRT3_HUMAN 586 597 LatdLLstwSvL
    1885 Q5GH73|XKR6_HUMAN 630 641 LlyeLLqyeSsL
    1886 Q5GH77|XKR3_HUMAN 194 205 LnraLLmtfSlL
    1887 Q5JPE7|NOMO2_HUMAN 1180 1191 LiplLLqltSrL
    1888 Q5JWR5|DOP1_HUMAN 506 517 LpqlLLrmiSaL
    1889 Q5UIP0|RIF1_HUMAN 2413 2424 LsknLLaqiSaL
    1890 Q5VTE6|ANGE2_HUMAN 175 186 LsqdLLednShL
    1891 Q5VU43|MYOME_HUMAN 1932 1943 LreaLLssrShL
    1892 Q5VYK3|ECM29_HUMAN 1296 1307 LipaLLeslSvL
    1893 Q68D06|SLN13_HUMAN 554 565 LvivLLgfrSlL
    1894 Q6GYQ0|GRIPE_HUMAN 641 652 LwddLLsvlSsL
    1895 Q6NTF9|RHBD2_HUMAN 166 177 LvpwLLlgaSwL
    1896 Q6ZMH5|S39A5_HUMAN 217 228 LavlLLslpSpL
    1897 Q6ZMZ3|SYNE3_HUMAN 532 543 LhnsLLqrkSkL
    1898 Q6ZVD8|PHLPL_HUMAN 313 324 LfpiLLceiStL
    1899 Q6ZVE7|GOT1A_HUMAN 23 34 LfgtLLyfdSvL
    1900 Q70J99|UN13D_HUMAN 927 938 LrveLLsasSlL
    1901 Q7Z3Z4|PIWL4_HUMAN 139 150 LriaLLyshSeL
    1902 Q7Z6Z7|HUWE1_HUMAN 841 852 LqegLLqldSiL
    1903 Q7Z7L1|SLN11_HUMAN 554 565 LvivLLgfrSlL
    1904 Q86SM5|MRGRG_HUMAN 223 234 LlnfLLpvfSpL
    1905 Q86U44|MTA70_HUMAN 78 89 LekkLLhhlSdL
    1906 Q86UQ4|ABCAD_HUMAN 3182 3193 LlnsLLdivSsL
    1907 Q86WI3|NLRC5_HUMAN 1485 1496 LlqsLLlslSeL
    1908 Q86YC3|LRC33_HUMAN 263 274 LffpLLpqySkL
    1909 Q8IYK4|GT252_HUMAN 9 20 LawsLLllsSaL
    1910 Q8IYS0|GRM1C_HUMAN 485 496 LesdLLieeSvL
    1911 Q8IZL8|PELP1_HUMAN 33 44 LrllLLesvSgL
    1912 Q8IZY2|ABCA7_HUMAN 1746 1757 LftlLLqhrSqL
    1913 Q8N0X7|SPG20_HUMAN 322 333 LfedLLrqmSdL
    1914 Q8N6M3|CT142_HUMAN 33 44 LagsLLkelSpL
    1915 Q8N816|TMM99_HUMAN 96 107 LlpcLLgvgSwL
    1916 Q8NBM4|PDHL1_HUMAN 15 26 LsksLLlvpSaL
    1917 Q8NCG7|DGLB_HUMAN 555 566 LtqpLLgeqSlL
    1918 Q8NFR9|I17RE_HUMAN 80 91 LcqhLLsggSgL
    1919 Q8NGE3|O10P1_HUMAN 9 20 LpefLLlgfSdL
    1920 Q8TCV5|WFDC5_HUMAN 8 19 LlgaLLavgSqL
    1921 Q8TDL5|LPLC1_HUMAN 165 176 LriqLLhklSfL
    1922 Q8TE82|S3TC1_HUMAN 1025 1036 LegqLLetiSqL
    1923 Q8TEQ8|PIGO_HUMAN 857 868 LvflLLflqSfL
    1924 Q8TEZ7|MPRB_HUMAN 127 138 LlahLLqskSeL
    1925 Q8WWN8|CEND3_HUMAN 1481 1492 LeeqLLqelSsL
    1926 Q8WZ84|OR8D1_HUMAN 43 54 LgmiLLiavSpL
    1927 Q92535|PIGC_HUMAN 253 264 LfalLLmsiScL
    1928 Q92538|GBF1_HUMAN 1224 1235 LrilLLmkpSvL
    1929 Q92743|HTRA1_HUMAN 262 273 LpvlLLgrsSeL
    1930 Q92935|EXTL1_HUMAN 19 30 LllvLLggfSlL
    1931 Q93074|MED12_HUMAN 401 412 LqtiLLccpSaL
    1932 Q96DN6|MBD6_HUMAN 740 751 LgasLLgdlSsL
    1933 Q96GR4|ZDH12_HUMAN 48 59 LtflLLvlgSlL
    1934 Q96HP8|T176A_HUMAN 29 40 LaklLLtccSaL
    1935 Q96K12|FACR2_HUMAN 380 391 LmnrLLrtvSmL
    1936 Q96KP1|EXOC2_HUMAN 339 350 LldkLLetpStL
    1937 Q96MX0|CKLF3_HUMAN 40 51 LkgrLLlaeSgL
    1938 Q96Q45|AL2S4_HUMAN 387 398 LvvaLLvglSwL
    1939 Q96QZ0|PANX3_HUMAN 136 147 LssdLLfiiSeL
    1940 Q96RQ9|OXLA_HUMAN 269 280 LpraLLsslSgL
    1941 Q9BY08|EBPL_HUMAN 178 189 LipgLLlwqSwL
    1942 Q9BZ97|TTY13_HUMAN 30 41 LclmLLlagScL
    1943 Q9H1Y0|ATG5_HUMAN 85 96 LlfdLLassSaL
    1944 Q9H254|SPTN4_HUMAN 1422 1433 LdkkLLhmeSqL
    1945 Q9H330|CI005_HUMAN 430 441 LgkfLLkvdSkL
    1946 Q9H4I8|SEHL2_HUMAN 175 186 LlqrLLksnShL
    1947 Q9HCN3|TMEM8_HUMAN 200 211 LpqtLLshpSyL
    1948 Q9NQ34|TMM9B_HUMAN 4 15 LwggLLrlgSlL
    1949 Q9NR09|BIRC6_HUMAN 1400 1411 LlkaLLdnmSfL
    1950 Q9NRA0|SPHK2_HUMAN 296 307 LgldLLlncSlL
    1951 Q9NRU3|CNNM1_HUMAN 156 167 LgalLLlalSaL
    1952 Q9NTT1|U2D3L_HUMAN 99 110 LskvLLsicSlL
    1953 Q9NVH2|INT7_HUMAN 623 634 LridLLqafSqL
    1954 Q9NVM9|CL011_HUMAN 350 361 LtnfLLngrSvL
    1955 Q9NZD1|GPC5D_HUMAN 60 71 LptqLLfllSvL
    1956 Q9P2E9|RRBP1_HUMAN 1226 1237 LrqlLLesqSqL
    1957 Q9P2G4|K1383_HUMAN 397 408 LlnaLLvelSlL
    1958 Q9P2V4|LRIT1_HUMAN 541 552 LpltLLvccSaL
    1959 Q9UDY8|MALT1_HUMAN 33 44 LrepLLrrlSeL
    1960 Q9UEW8|STK39_HUMAN 138 149 LvmkLLsggSmL
    1961 Q9UGN4|CM35H_HUMAN 188 199 LlllLLvgaSlL
    1962 Q9UHD4|CIDEB_HUMAN 189 200 LghmLLgisStL
    1963 Q9UIG8|SO3A1_HUMAN 270 281 LcgaLLffsSlL
    1964 Q9UPA5|BSN_HUMAN 353 364 LgasLLtqaStL
    1965 Q9UPX8|SHAN2_HUMAN 609 620 LtgrLLdpsSpL
    1966 Q9Y239|NOD1_HUMAN 318 329 LsgkLLkgaSkL
    1967 Q9Y2I2|NTNG1_HUMAN 526 537 LlttLLgtaSpL
    1968 Q9Y2U2|KCNK7_HUMAN 92 103 LpsaLLfaaSiL
    1969 Q9Y2Y8|PRG3_HUMAN 7 18 LpflLLgtvSaL
    1970 Q9Y586|MB212_HUMAN 300 311 LngiLLqliScL
    1971 Q9Y5X0|SNX10_HUMAN 106 117 LqnaLLlsdSsL
    1972 Q9Y5X5|NPFF2_HUMAN 379 390 LivaLLfilSwL
  • TABLE 9
    Table of the amino acid sequences of the peptides
    identified to contain the serpin motif.
    Serpins
    Motif: L-X(2)-E-E-X-P
    Number of Locations: 314
    Number of Different Proteins: 302
    First
    Accession Amino Last
    # Number|Protein Name acid Amino acid Sequence
    1973 O00160|MYO1F_HUMAN 744 751 LglEErPe
    1974 O00507|USP9Y_HUMAN 2474 2481 LcpEEePd
    1975 O00625|PIR_HUMAN 134 141 LksEEiPk
    1976 O14641|DVL2_HUMAN 20 27 LdeEEtPy
    1977 O14686|MLL2_HUMAN 2819 2826 LgpEErPp
    1978 O14709|ZN197_HUMAN 193 200 LsqEEnPr
    1979 O14795|UN13B_HUMAN 1499 1506 LgnEEgPe
    1980 O15013|ARHGA_HUMAN 199 206 LssEEpPt
    1981 O15055|PER2_HUMAN 994 1001 LqlEEaPe
    1982 O15528|CP27B_HUMAN 297 304 LfrEElPa
    1983 O15534|PER1_HUMAN 987 994 LqlEElPr
    1984 O43390|HNRPR_HUMAN 12 19 LkeEEePm
    1985 O60216|RAD21_HUMAN 504 511 LppEEpPn
    1986 O60237|MYPT2_HUMAN 339 346 LyeEEtPk
    1987 O60346|PHLPP_HUMAN 483 490 LeaEEkPl
    1988 O60779|S19A2_HUMAN 259 266 LnmEEpPv
    1989 O60885|BRD4_HUMAN 913 920 LedEEpPa
    1990 O75128|COBL_HUMAN 1064 1071 LerEEkPs
    1991 O75420|PERQ1_HUMAN 334 341 LeeEEePs
    1992 O75787|RENR_HUMAN 116 123 LfsEEtPv
    1993 O75914|PAK3_HUMAN 5 12 LdnEEkPp
    1994 O94933|SLIK3_HUMAN 227 234 LqlEEnPw
    1995 O94966|UBP19_HUMAN 1251 1258 LeaEEePv
    1996 O94986|CE152_HUMAN 847 854 LknEEvPv
    1997 O94991|SLIK5_HUMAN 230 237 LqlEEnPw
    1998 O95153|RIMB1_HUMAN 915 922 LngEEcPp
    1999 O95279|KCNK5_HUMAN 443 450 LagEEsPq
    2000 O95712|PA24B_HUMAN 772 779 LkiEEpPs
    2001 O95881|TXD12_HUMAN 94 101 LedEEePk
    2002 O96018|APBA3_HUMAN 116 123 LhcEEcPp
    2003 O96024|B3GT4_HUMAN 217 224 LhsEEvPl
    2004 P04275|VWF_HUMAN 1012 1019 LqvEEdPv
    2005 P05160|F13B_HUMAN 18 25 LyaEEkPc
    2006 P06858|LIPL_HUMAN 279 286 LlnEEnPs
    2007 P07237|PDIA1_HUMAN 307 314 LkkEEcPa
    2008 P07949|RET_HUMAN 1033 1040 LseEEtPl
    2009 P08519|APOA_HUMAN 3880 3887 LpsEEaPt
    2010 P09769|FGR_HUMAN 497 504 LdpEErPt
    2011 P10745|IRBP_HUMAN 708 715 LvvEEaPp
    2012 P11532|DMD_HUMAN 2255 2262 LlvEElPl
    2013 P14317|HCLS1_HUMAN 352 359 LqvEEePv
    2014 P16150|LEUK_HUMAN 369 376 LkgEEePl
    2015 P17025|ZN182_HUMAN 79 86 LevEEcPa
    2016 P17600|SYN1_HUMAN 239 246 LgtEEfPl
    2017 P18583|SON_HUMAN 1149 1156 LppEEpPt
    2018 P18583|SON_HUMAN 1160 1167 LppEEpPm
    2019 P18583|SON_HUMAN 1171 1178 LppEEpPe
    2020 P19484|TFEB_HUMAN 350 357 LpsEEgPg
    2021 P21333|FLNA_HUMAN 1034 1041 LprEEgPy
    2022 P21802|FGFR2_HUMAN 33 40 LepEEpPt
    2023 P22001|KCNA3_HUMAN 152 159 LreEErPl
    2024 P31629|ZEP2_HUMAN 772 779 LvsEEsPs
    2025 P34925|RYK_HUMAN 578 585 LdpEErPk
    2026 P36955|PEDF_HUMAN 39 46 LveEEdPf
    2027 P40189|IL6RB_HUMAN 787 794 LdsEErPe
    2028 P42898|MTHR_HUMAN 598 605 LyeEEsPs
    2029 P48729|KC1A_HUMAN 266 273 LrfEEaPd
    2030 P51512|MMP16_HUMAN 165 172 LtfEEvPy
    2031 P52746|ZN142_HUMAN 750 757 LgaEEnPl
    2032 P53370|NUDT6_HUMAN 284 291 LtvEElPa
    2033 P53801|PTTG_HUMAN 167 174 LfkEEnPy
    2034 P53804|TTC3_HUMAN 2001 2008 LltEEsPs
    2035 P55285|CADH6_HUMAN 116 123 LdrEEkPv
    2036 P55289|CAD12_HUMAN 117 124 LdrEEkPf
    2037 P56645|PER3_HUMAN 929 936 LlqEEmPr
    2038 P59797|SELV_HUMAN 163 170 LlpEEdPe
    2039 Q01826|SATB1_HUMAN 409 416 LrkEEdPk
    2040 Q04725|TLE2_HUMAN 200 207 LveEErPs
    2041 Q06330|SUH_HUMAN 7 14 LpaEEpPa
    2042 Q06889|EGR3_HUMAN 24 31 LypEEiPs
    2043 Q07157|ZO1_HUMAN 1155 1162 LrhEEqPa
    2044 Q13072|BAGE1_HUMAN 19 26 LmkEEsPv
    2045 Q13087|PDIA2_HUMAN 497 504 LptEEpPe
    2046 Q13255|GRM1_HUMAN 995 1002 LtaEEtPl
    2047 Q13315|ATM_HUMAN 954 961 LpgEEyPl
    2048 Q13439|GOGA4_HUMAN 2092 2099 LeqEEnPg
    2049 Q13596|SNX1_HUMAN 265 272 LekEElPr
    2050 Q13634|CAD18_HUMAN 446 453 LdrEEtPw
    2051 Q14028|CNGB1_HUMAN 137 144 LmaEEnPp
    2052 Q14126|DSG2_HUMAN 117 124 LdrEEtPf
    2053 Q14204|DYHC_HUMAN 3973 3980 LwsEEtPa
    2054 Q14315|FLNC_HUMAN 1738 1745 LphEEePs
    2055 Q14524|SCN5A_HUMAN 46 53 LpeEEaPr
    2056 Q14554|PDIA5_HUMAN 166 173 LkkEEkPl
    2057 Q14562|DHX8_HUMAN 411 418 LskEEfPd
    2058 Q14562|DHX8_HUMAN 441 448 LveEEpPf
    2059 Q14573|ITPR3_HUMAN 315 322 LaaEEnPs
    2060 Q14674|ESPL1_HUMAN 613 620 LspEEtPa
    2061 Q14676|MDC1_HUMAN 145 152 LtvEEtPr
    2062 Q14684|RRP1B_HUMAN 244 251 LsaEEiPe
    2063 Q15021|CND1_HUMAN 1179 1186 LgvEEePf
    2064 Q15735|PI5PA_HUMAN 189 196 LasEEqPp
    2065 Q15788|NCOA1_HUMAN 982 989 LimEErPn
    2066 Q15878|CAC1E_HUMAN 797 804 LnrEEaPt
    2067 Q2TAL6|VWC2_HUMAN 179 186 LctEEgPl
    2068 Q32MZ4|LRRF1_HUMAN 82 89 LrvEErPe
    2069 Q32P28|P3H1_HUMAN 215 222 LysEEqPq
    2070 Q3KNS1|PTHD3_HUMAN 96 103 LpeEEtPe
    2071 Q3ZCX4|ZN568_HUMAN 100 107 LeqEEePw
    2072 Q495W5|FUT11_HUMAN 144 151 LlhEEsPl
    2073 Q52LD8|RFTN2_HUMAN 123 130 LviEEcPl
    2074 Q53GL0|PKHO1_HUMAN 189 196 LiqEEdPs
    2075 Q53GL0|PKHO1_HUMAN 289 296 LraEEpPt
    2076 Q53GL7|PAR10_HUMAN 693 700 LeaEEpPd
    2077 Q53H47|SETMR_HUMAN 499 506 LdqEEaPk
    2078 Q567U6|CCD93_HUMAN 300 307 LsaEEsPe
    2079 Q580R0|CB027_HUMAN 41 48 LelEEaPe
    2080 Q587I9|SFT2C_HUMAN 136 143 LrcEEaPs
    2081 Q5H9T9|CN155_HUMAN 427 434 LlpEEaPr
    2082 Q5H9T9|CN155_HUMAN 697 704 LpaEEtPi
    2083 Q5H9T9|CN155_HUMAN 736 743 LltEEfPi
    2084 Q5JUK9|GGED1_HUMAN 38 45 LqqEEpPi
    2085 Q5JXB2|UE2NL_HUMAN 58 65 LlaEEyPm
    2086 Q5MCW4|ZN569_HUMAN 60 67 LeqEEePw
    2087 Q5SYB0|FRPD1_HUMAN 553 560 LikEEqPp
    2088 Q5THJ4|VP13D_HUMAN 2943 2950 LtgEEiPf
    2089 Q5VYS4|CM033_HUMAN 293 300 LesEEtPn
    2090 Q5VZP5|DUS27_HUMAN 942 949 LrtEEkPp
    2091 Q5VZY2|PPC1A_HUMAN 247 254 LkkEErPt
    2092 Q63HR2|TENC1_HUMAN 564 571 LddEEqPt
    2093 Q66K74|MAP1S_HUMAN 777 784 LgaEEtPp
    2094 Q68CZ1|FTM_HUMAN 1181 1188 LpaEEtPv
    2095 Q68DD2|PA24F_HUMAN 470 477 LyqEEnPa
    2096 Q6BDS2|URFB1_HUMAN 1304 1311 LedEEiPv
    2097 Q6DCA0|AMERL_HUMAN 183 190 LtrEElPk
    2098 Q6DN90|IQEC1_HUMAN 263 270 LhtEEaPa
    2099 Q6DT37|MRCKG_HUMAN 1264 1271 LvpEElPp
    2100 Q6HA08|ASTL_HUMAN 62 69 LilEEtPe
    2101 Q6IFS5|HSN2_HUMAN 298 305 LnqEElPp
    2102 Q6NUN7|CK063_HUMAN 74 81 LdeEEsPr
    2103 Q6P2Q9|PRP8_HUMAN 1852 1859 LpvEEqPk
    2104 Q6P5W5|S39A4_HUMAN 473 480 LvaEEsPe
    2105 Q6P6B1|CH047_HUMAN 249 256 LgkEEqPq
    2106 Q6PD74|P34_HUMAN 141 148 LspEElPe
    2107 Q6PI48|SYDM_HUMAN 488 495 LpkEEnPr
    2108 Q6PJ61|FBX46_HUMAN 246 253 LrkEErPg
    2109 Q6S8J7|POTE8_HUMAN 307 314 LtsEEePq
    2110 Q6SZW1|SARM1_HUMAN 396 403 LlgEEvPr
    2111 Q6UX39|AMTN_HUMAN 114 121 LssEElPq
    2112 Q6ZMY3|SPOC1_HUMAN 184 191 LskEEpPg
    2113 Q6ZN11|ZN793_HUMAN 60 67 LeqEEaPw
    2114 Q6ZNL6|FGD5_HUMAN 382 389 LraEEnPm
    2115 Q6ZV29|PLPL7_HUMAN 854 861 LhrEEgPa
    2116 Q70CQ4|UBP31_HUMAN 527 534 LpqEEqPl
    2117 Q70SY1|CR3L2_HUMAN 153 160 LekEEpPl
    2118 Q7L8C5|SYT13_HUMAN 229 236 LaeEElPt
    2119 Q7Z3E5|ARMC9_HUMAN 570 577 LnsEElPd
    2120 Q7Z410|TMPS9_HUMAN 691 698 LacEEaPg
    2121 Q86SP6|GP149_HUMAN 217 224 LcsEEpPr
    2122 Q86V87|RAI16_HUMAN 496 503 LdlEEdPy
    2123 Q86VQ0|CF152_HUMAN 428 435 LerEEkPe
    2124 Q86W50|MET10_HUMAN 454 461 LsqEEnPe
    2125 Q86Y13|DZIP3_HUMAN 1192 1199 LlpEEfPg
    2126 Q86Y27|BAGE5_HUMAN 19 26 LmkEEsPv
    2127 Q86Y28|BAGE4_HUMAN 19 26 LmkEEsPv
    2128 Q86Y29|BAGE3_HUMAN 19 26 LmkEEsPv
    2129 Q86Y30|BAGE2_HUMAN 19 26 LmkEEsPv
    2130 Q8IU99|FA26C_HUMAN 315 322 LgqEEpPl
    2131 Q8IUA0|WFDC8_HUMAN 217 224 LqdEEcPl
    2132 Q8IV63|VRK3_HUMAN 438 445 LtyEEkPp
    2133 Q8IWY9|CDAN1_HUMAN 948 955 LlpEEtPa
    2134 Q8IXI1|MIRO2_HUMAN 24 31 LvgEEfPe
    2135 Q8IXI2|MIRO1_HUMAN 24 31 LvsEEfPe
    2136 Q8IYS5|OSCAR_HUMAN 122 129 LvtEElPr
    2137 Q8IZ26|ZNF34_HUMAN 251 258 LhtEEkPy
    2138 Q8IZH2|XRN1_HUMAN 1143 1150 LfdEEfPg
    2139 Q8IZP0|ABI1_HUMAN 7 14 LleEEiPs
    2140 Q8N201|INT1_HUMAN 1587 1594 LlqEEePl
    2141 Q8N309|LRC43_HUMAN 373 380 LlvEEsPe
    2142 Q8N3C0|HELC1_HUMAN 451 458 LsfEEkPv
    2143 Q8N3C0|HELC1_HUMAN 1579 1586 LatEEdPk
    2144 Q8N475|FSTL5_HUMAN 786 793 LkaEEwPw
    2145 Q8N4L2|TM55A_HUMAN 132 139 LisEEqPa
    2146 Q8N752|KC1AL_HUMAN 266 273 LrfEEvPd
    2147 Q8NC74|CT151_HUMAN 178 185 LrgEEkPa
    2148 Q8NE71|ABCF1_HUMAN 701 708 LrmEEtPt
    2149 Q8NEG5|ZSWM2_HUMAN 43 50 LlrEEePe
    2150 Q8NEM7|FA48A_HUMAN 115 122 LdaEElPp
    2151 Q8NEZ4|MLL3_HUMAN 3046 3053 LllEEqPl
    2152 Q8NEZ4|MLL3_HUMAN 4023 4030 LvkEEpPe
    2153 Q8NFM7|I17RD_HUMAN 702 709 LgeEEpPa
    2154 Q8NFP4|MDGA1_HUMAN 489 496 LplEEtPd
    2155 Q8NHJ6|LIRB4_HUMAN 60 67 LdkEEsPa
    2156 Q8NI51|BORIS_HUMAN 120 127 LwlEEgPr
    2157 Q8TBH0|ARRD2_HUMAN 387 394 LysEEdPn
    2158 Q8TDX9|PK1L1_HUMAN 1101 1108 LsaEEsPg
    2159 Q8TE68|ES8L1_HUMAN 408 415 LspEEgPp
    2160 Q8TER0|SNED1_HUMAN 1083 1090 LrgEEhPt
    2161 Q8WU49|CG033_HUMAN 8 15 LslEEcPw
    2162 Q8WUA2|PPIL4_HUMAN 16 23 LytEErPr
    2163 Q8WUI4|HDAC7_HUMAN 943 950 LveEEePm
    2164 Q8WWN8|CEND3_HUMAN 1456 1463 LgqEErPp
    2165 Q8WZ42|TITIN_HUMAN 12132 12139 LvvEElPv
    2166 Q8WZ42|TITIN_HUMAN 13832 13839 LfvEEiPv
    2167 Q92538|GBF1_HUMAN 1062 1069 LqrEEtPs
    2168 Q92738|US6NL_HUMAN 51 58 LheEElPd
    2169 Q92765|SFRP3_HUMAN 134 141 LacEElPv
    2170 Q92851|CASPA_HUMAN 70 77 LlsEEdPf
    2171 Q92888|ARHG1_HUMAN 390 397 LepEEpPg
    2172 Q93008|USP9X_HUMAN 2466 2473 LcpEEePd
    2173 Q969V6|MKL1_HUMAN 497 504 LvkEEgPr
    2174 Q96B01|R51A1_HUMAN 55 62 LrkEEiPv
    2175 Q96D15|RCN3_HUMAN 192 199 LhpEEfPh
    2176 Q96DC7|TMCO6_HUMAN 219 226 LqaEEaPe
    2177 Q96FT7|ACCN4_HUMAN 90 97 LslEEqPl
    2178 Q96G97|BSCL2_HUMAN 326 333 LseEEkPd
    2179 Q96GW7|PGCB_HUMAN 880 887 LhpEEdPe
    2180 Q96H72|S39AD_HUMAN 340 347 LleEEdPw
    2181 Q96H78|S2544_HUMAN 265 272 LmaEEgPw
    2182 Q96J42|TXD15_HUMAN 42 49 LwsEEqPa
    2183 Q96JI7|SPTCS_HUMAN 1940 1947 LleEEaPd
    2184 Q96JL9|ZN333_HUMAN 80 87 LkpEElPs
    2185 Q96JQ0|PCD16_HUMAN 3106 3113 LyrEEgPp
    2186 Q96MZ0|GD1L1_HUMAN 195 202 LdhEEePq
    2187 Q96NZ9|PRAP1_HUMAN 71 78 LttEEkPr
    2188 Q96PQ6|ZN317_HUMAN 109 116 LeqEEePr
    2189 Q96RE7|BTB14_HUMAN 133 140 LhaEEaPs
    2190 Q96RG2|PASK_HUMAN 1196 1203 LvfEEnPf
    2191 Q96RL1|UIMC1_HUMAN 388 395 LllEEePt
    2192 Q96SB3|NEB2_HUMAN 435 442 LseEEdPa
    2193 Q96SJ8|TSN18_HUMAN 167 174 LdsEEvPe
    2194 Q99102|MUC4_HUMAN 1306 1313 LhrEErPn
    2195 Q99543|DNJC2_HUMAN 68 75 LqlEEfPm
    2196 Q9BQS2|SYT15_HUMAN 36 43 LtyEElPg
    2197 Q9BVI0|PHF20_HUMAN 483 490 LepEEsPg
    2198 Q9BY44|EIF2A_HUMAN 461 468 LheEEpPq
    2199 Q9BY78|RNF26_HUMAN 356 363 LneEEpPg
    2200 Q9BYD3|RM04_HUMAN 221 228 LthEEmPq
    2201 Q9BZA7|PC11X_HUMAN 315 322 LdrEEtPn
    2202 Q9BZA8|PC11Y_HUMAN 347 354 LdrEEtPn
    2203 Q9C009|FOXQ1_HUMAN 227 234 LrpEEaPg
    2204 Q9H095|IQCG_HUMAN 122 129 LitEEgPn
    2205 Q9H0D2|ZN541_HUMAN 149 156 LggEEpPg
    2206 Q9H2C0|GAN_HUMAN 36 43 LdgEEiPv
    2207 Q9H2X9|S12A5_HUMAN 681 688 LrlEEgPp
    2208 Q9H334|FOXP1_HUMAN 291 298 LshEEhPh
    2209 Q9H3T3|SEM6B_HUMAN 26 33 LfpEEpPp
    2210 Q9H579|CT132_HUMAN 138 145 LvqEErPh
    2211 Q9H5V8|CDCP1_HUMAN 788 795 LatEEpPp
    2212 Q9H6F5|CCD86_HUMAN 227 234 LnkEElPv
    2213 Q9H6Z4|RANB3_HUMAN 4 11 LanEEkPa
    2214 Q9H7E9|CH033_HUMAN 94 101 LapEEvPl
    2215 Q9H8Y1|CN115_HUMAN 137 144 LcsEEsPe
    2216 Q9H9E1|ANRA2_HUMAN 13 20 LivEEcPs
    2217 Q9H9F9|ARP5_HUMAN 415 422 LfsEEtPg
    2218 Q9HAV4|XPO5_HUMAN 521 528 LnrEEiPv
    2219 Q9HCE7|SMUF1_HUMAN 364 371 LedEElPa
    2220 Q9NPR2|SEM4B_HUMAN 47 54 LgsEErPf
    2221 Q9NR50|EI2BG_HUMAN 333 340 LcpEEpPv
    2222 Q9NRJ7|PCDBG_HUMAN 200 207 LdrEEePq
    2223 Q9NTN9|SEM4G_HUMAN 203 210 LrtEEtPm
    2224 Q9NUR3|CT046_HUMAN 104 111 LhsEEgPa
    2225 Q9NVR7|TBCC1_HUMAN 138 145 LigEEwPs
    2226 Q9NX46|ARHL2_HUMAN 235 242 LgmEErPy
    2227 Q9NYB9|ABI2_HUMAN 7 14 LleEEiPg
    2228 Q9P1Y5|K1543_HUMAN 827 834 LlaEEtPp
    2229 Q9P1Y5|K1543_HUMAN 938 945 LaqEEaPg
    2230 Q9P2E7|PCD10_HUMAN 316 323 LdyEEsPv
    2231 Q9P2K9|PTHD2_HUMAN 673 680 LevEEePv
    2232 Q9UBB4|ATX10_HUMAN 289 296 LasEEpPd
    2233 Q9UBN6|TR10D_HUMAN 78 85 LkeEEcPa
    2234 Q9UBT6|POLK_HUMAN 251 258 LlfEEsPs
    2235 Q9UGF5|OR5U1_HUMAN 303 310 LskEElPq
    2236 Q9UGL1|JAD1B_HUMAN 879 886 LlsEEtPs
    2237 Q9UHW9|S12A6_HUMAN 743 750 LrlEEgPp
    2238 Q9UIF9|BAZ2A_HUMAN 609 616 LsaEEiPs
    2239 Q9UIG0|BAZ1B_HUMAN 75 82 LlkEEfPa
    2240 Q9ULD6|PDZD6_HUMAN 390 397 LpaEEvPl
    2241 Q9ULG1|INOC1_HUMAN 235 242 LssEEsPr
    2242 Q9ULI4|KI26A_HUMAN 1396 1403 LrgEEePr
    2243 Q9ULQ1|TPC1_HUMAN 29 36 LgqEElPs
    2244 Q9UMS0|NFU1_HUMAN 93 100 LvtEEtPs
    2245 Q9UN72|PCDA7_HUMAN 200 207 LdrEEtPe
    2246 Q9UN73|PCDA6_HUMAN 200 207 LdrEEaPa
    2247 Q9UN74|PCDA4_HUMAN 200 207 LdrEEaPe
    2248 Q9UNA0|ATS5_HUMAN 481 488 LgpEElPg
    2249 Q9UP95|S12A4_HUMAN 678 685 LrlEEgPp
    2250 Q9UPQ7|PZRN3_HUMAN 385 392 LlpEEhPs
    2251 Q9UPV0|CE164_HUMAN 488 495 LatEEePp
    2252 Q9UPW6|SATB2_HUMAN 398 405 LrkEEdPr
    2253 Q9UPW8|UN13A_HUMAN 332 339 LeeEElPe
    2254 Q9UPX6|K1024_HUMAN 371 378 LntEEvPd
    2255 Q9UQ05|KCNH4_HUMAN 761 768 LlgEElPp
    2256 Q9UQ26|RIMS2_HUMAN 201 208 LrnEEaPq
    2257 Q9UQ26|RIMS2_HUMAN 1327 1334 LsfEEsPq
    2258 Q9Y250|LZTS1_HUMAN 293 300 LayEErPr
    2259 Q9Y2I6|NLP_HUMAN 759 766 LelEEpPq
    2260 Q9Y2K7|JHD1A_HUMAN 661 668 LlnEElPn
    2261 Q9Y2L6|FRM4B_HUMAN 438 445 LpsEEdPa
    2262 Q9Y2V3|RX_HUMAN 126 133 LseEEqPk
    2263 Q9Y343|SNX24_HUMAN 87 94 LenEElPk
    2264 Q9Y3I0|CV028_HUMAN 466 473 LvmEEaPe
    2265 Q9Y3L3|3BP1_HUMAN 130 137 LseEElPa
    2266 Q9Y3L3|3BP1_HUMAN 494 501 LasEElPs
    2267 Q9Y3R5|DOP2_HUMAN 1084 1091 LseEElPy
    2268 Q9Y426|CU025_HUMAN 98 105 LsfEEdPr
    2269 Q9Y566|SHAN1_HUMAN 1838 1845 LpwEEgPg
    2270 Q9Y572|RIPK3_HUMAN 352 359 LnlEEpPs
    2271 Q9Y5E2|PCDB7_HUMAN 200 207 LdrEEiPe
    2272 Q9Y5E3|PCDB6_HUMAN 199 206 LdrEEqPq
    2273 Q9Y5E4|PCDB5_HUMAN 200 207 LdrEErPe
    2274 Q9Y5E5|PCDB4_HUMAN 199 206 LdrEEqPe
    2275 Q9Y5E6|PCDB3_HUMAN 200 207 LdrEEqPe
    2276 Q9Y5E7|PCDB2_HUMAN 202 209 LdrEEqPe
    2277 Q9Y5F1|PCDBC_HUMAN 200 207 LdyEErPe
    2278 Q9Y5F2|PCDBB_HUMAN 200 207 LdyEElPe
    2279 Q9Y5F3|PCDB1_HUMAN 200 207 LdrEEqPe
    2280 Q9Y5G1|PCDGF_HUMAN 200 207 LdrEEqPh
    2281 Q9Y5G2|PCDGE_HUMAN 410 417 LdrEEiPe
    2282 Q9Y5H5|PCDA9_HUMAN 200 207 LdrEEtPe
    2283 Q9Y5I2|PCDAA_HUMAN 199 206 LdrEEnPq
    2284 Q9Y5I3|PCDA1_HUMAN 200 207 LdrEEtPe
    2285 Q9Y5Q9|TF3C3_HUMAN 42 49 LsaEEnPd
    2286 Q9Y5R2|MMP24_HUMAN 201 208 LtfEEvPy
  • TABLE 10
    Table containing the amino acid sequence of the
    peptide predicted similar to Tumstatin/Tum4
    Protein Name Peptide Location Peptide sequence
    Collagen CAI40758.1: LPRFSTMPFIYCNINEVCHY
    type IV, 1630-1648
    alpha6 fibril
  • In other embodiments, the following peptides suitable for use with the presently disclosed subject matter are disclosed in Table 1 of International PCT Patent Application Publication Number WO2007/033215 A2 for “Compositions Having Antiangiogenic Activity and Uses Thereof,” to Popel et al., published Mar. 22, 2007, which is incorporated herein by reference in its entirety.
  • TABLE 11
    Anti-Angiogenic Peptide sequences
    SEQ ID
    NO.
    Thrombospondin Containing Proteins
    2287 ADAM-9 Q13443: 649-661 KCHGHGVCNSNKN
    2288 ADAM-12 O43184: 662-675 MQCHGRGVCNNRKN
    2289 ADAMTS-1 Q9UHI8: 566-584 GPWGDCSRTCGGGVQYTMR
    2290 ADAMTS-2 CAA05880.1: 982-998 GPWSQCSVTCGNGTQER
    2291 ADAMTS-3 NP_055058.1: 973-989 GPWSECSVTCGEGTEVR
    2292 ADAMTS-4 CAH72146.1: 527-540 GPWGDCSRTCGGGV
    2293 ADAMTS-4 CAH72146.1: 527-545 GPWGDCSRTCGGGVQFSSR
    2294 ADAMTS-5 NP_008969.1: 882-898 GPWLACSRTCDTGWHTR
    2295 ADAMTS-6 NP_922932.2: 847-860 QPWSECSATCAGGV
    2296 ADAMTS-6 NP_922932.2: 847-863 QPWSECSATCAGGVQRQ
    2297 ADAMTS-7 AAH61631.1: 1576-1592 GPWGQCSGPCGGGVQRR
    2298 ADAMTS-7 AAH61631.1: 828-841 GPWTKCTVTCGRGV
    2299 ADAMTS-8 Q9UP79: 534-547 GPWGECSRTCGGGV
    2300 ADAMTS-8 Q9UP79: 534-552 GPWGECSRTCGGGVQFSHR
    2301 ADAMTS-9 Q9P2N4: 1247-1261 WSSCSVTCGQGRATR
    2302 ADAMTS-9 Q9P2N4: 1335-1351 GPWGACSSTCAGGSQRR
    2303 ADAMTS-9 Q9P2N4: 595-613 SPFGTCSRTCGGGIKTAIR
    2304 ADAMTS-10 Q9H324: 528-546 TPWGDCSRTCGGGVSSSSR
    2305 ADAMTS-12 P58397: 1479-1493 WDLCSTSCGGGFQKR
    2306 ADAMTS-12 P58397: 549-562 SPWSHCSRTCGAGV
    2307 ADAMTS-13 AAQ88485.1: 751-765 WMECSVSCGDGIQRR
    2308 ADAMTS-14 CAI13857.1: 980-994 WSQCSATCGEGIQQR
    2309 ADAMTS-15 CAC86014.1: 900-916 SAWSPCSKSCGRGFQRR
    2310 ADAMTS-16 Q8TE57: 1133-1149 SPWSQCTASCGGGVQTR
    2311 ADAMTS-16 Q8TE57: 1133-1150 SPWSQCTASCGGGVQTRS
    2312 ADAMTS-18 Q8TE60: 1131-1146 PWQQCTVTCGGGVQTR
    2313 ADAMTS-18 Q8TE60: 1131-1147 PWQQCTVTCGGGVQTRS
    2314 ADAMTS-18 Q8TE60: 998-1014 GPWSQCSKTCGRGVRKR
    2315 ADAMTS-18 Q8TE60: 596-614 SKWSECSRTCGGGVKFQER
    2316 ADAMTS-19 CAC84565.1: 1096-1111 WSKCSITCGKGMQSRV
    2317 ADAMTS-20 CAD56159.3: 1478-1494 NSWNECSVTCGSGVQQR
    2318 ADAMTS-20 CAD56159.3: 1309-1326 GPWGQCSSSCSGGLQHRA
    2319 ADAMTS-20 CAD56159.3: 1661-1675 WSKCSVTCGIGIMKR
    2320 ADAMTS-20 CAD56160.2: 564-581 PYSSCSRTCGGGIESATR
    2321 BAI-1 O14514: 361-379 SPWSVCSSTCGEGWQTRTR
    2322 BAI-2 O60241: 304-322 SPWSVCSLTCGQGLQVRTR
    2323 BAI-3 CAI21673.1: 352-370 SPWSLCSFTCGRGQRTRTR
    2324 C6 AAB59433.1: 30-48 TQWTSCSKTCNSGTQSRHR
    2325 CILP AAQ89263.1: 156-175 SPWSKCSAACGQTGVQTRTR
    2326 CILP-2 AAN17826.1: 153-171 GPWGPCSGSCGPGRRLRRR
    2327 CTGF CAC44023.1: 204-221 TEWSACSKTCGMGISTRV
    2328 CYR61 AAR05446.1: 234-251 TSWSQCSKTCGTGISTRV
    2329 Fibulin-6 CAC37630.1: 1574-1592 SAWRACSVTCGKGIQKRSR
    2330 Fibulin-6 CAC37630.1: 1688-1706 QPWGTCSESCGKGTQTRAR
    2331 Fibulin-6 CAC37630.1: 1745-1763 ASWSACSVSCGGGARQRTR
    2332 NOVH AAL92490.1: 211-228 TEWTACSKSCGMGFSTRV
    2333 Papilin NP_775733.2: 33-51 SQWSPCSRTCGGGVSFRER
    2334 Papilin NP_775733.2: 342-359 GPWAPCSASCGGGSQSRS
    2335 Properdin AAP43692.1: 143-161 GPWEPCSVTCSKGTRTRRR
    2336 ROR-1 CAH71706.1: 313-391 CYNSTGVDYRGTVSVTKSGRQCQPWNSQYPHTHTFTALRFPEL
    NGGHSYCRNPGNQKEAPWCFTLDENFKSDLCDIPAC
    2337 ROR-1 CAH71706.1: 310-391 NHKCYNSTGVDYRGTVSVTKSGRQCQPWNSQYPHTHTFTALRF
    PELNGGHSYCRNPGNQKEAPWCFTLDENFKSDLCDIPAC
    2338 ROR-1 CAH71706.1: 311-388 HKCYNSTGVDYRGTVSVTKSGRQCQPWNSQYPHTHTFTALRFP
    ELNGGHSYCRNPGNQKEAPWCFTLDENFKSDLCDI
    2339 ROR-1 CAH71706.1: 311-391 HKCYNSTGVDYRGTVSVTKSGRQCQPWNSQYPHTHTFTALRFP
    ELNGGHSYCRNPGNQKEAPWCFTLDENFKSDLCDIPAC
    2340 ROR-1 CAH71706.1: 312-392 KCYNSTGVDYRGTVSVTKSGRQCQPWNSQYPHTHTFTALRFPE
    LNGGHSYCRNPGNQKEAPWCFTLDENFKSDLCDIPACD
    2341 ROR-2 Q01974: 315-395 QCYNGSGMDYRGTASTTKSGHQCQPWALQHPHSHHLSSTDFPE
    LGGGHAYCRNPGGQMEGPWCFTQNKNVRMELCDVPSCS
    2342 ROR-2 Q01974: 314-391 HQCYNGSGMDYRGTASTTKSGHQCQPWALQHPHSHHLSSTDFP
    ELGGGHAYCRNPGGQMEGPWCFTQNKNVRMELCDV
    2343 ROR-2 Q01974: 314-394 HQCYNGSGMDYRGTASTTKSGHQCQPWALQHPHSHHLSSTDFP
    ELGGGHAYCRNPGGQMEGPWCFTQNKNVRMELCDVPSC
    2344 ROR-2 Q01974: 314-395 HQCYNGSGMDYRGTASTTKSGHQCQPWALQHPHSHHLSSTDFP
    ELGGGHAYCRNPGGQMEGPWCFTQNKNVRMELCDVPSCS
    2345 ROR-2 Q01974: 315-394 QCYNGSGMDYRGTASTTKSGHQCQPWALQHPHSHHLSSTDFPE
    LGGGHAYCRNPGGQMEGPWCFTQNKNVRMELCDVPSC
    2346 Semaphorin 5A NP_003957.1: 660-678 GPWERCTAQCGGGIQARRR
    2347 Semaphorin 5A NP_003957.1: 848-866 SPWTKCSATCGGGHYMRTR
    2348 Semaphorin 5B AAQ88491.1: 916-934 TSWSPCSASCGGGHYQRTR
    2349 SCO-spondin XP_379967.2: 3781-3799 GPWEDCSVSCGGGEQLRSR
    2350 THSD1 AAQ88516.1: 347-365 QPWSQCSATCGDGVRERRR
    2351 THSD3 AAH33140.1: 280-298 SPWSPCSGNCSTGKQQRTR
    2352 THSD6 AAH40620.1: 44-60 WTRCSSSCGRGVSVRSR
    2353 TSP-2 CAI23645.1: 444-462 SPWSSCSVTCGVGNITRIR
    2354 TSP-2 CAI23645.1: 501-519 SPWSACTVTCAGGIRERTR
    2355 TSRC1 AAH27478.1: 140-159 SPWSQCSVRCGRGQRSRQVR
    2356 UNC5C AAH41156.1: 267-285 TEWSVCNSRCGRGYQKRTR
    2357 UNC5D AAQ88514.1: 259-277 TEWSACNVRCGRGWQKRSR
    2358 VSGP/F-spondin BAB18461.1: 567-583 WDECSATCGMGMKKRHR
    2359 VSGP/F-spondin BAB18461.1: 621-639 SEWSDCSVTCGKGMRTRQR
    2360 WISP-1 AAH74841.1: 221-238 SPWSPCSTSCGLGVSTRI
    2361 WISP-2 AAQ89274.1: 199-216 TAWGPCSTTCGLGMATRV
    2362 WISP-3 CAB16556.1: 191-208 TKWTPCSRTCGMGISNRV
    Collagens
    2363 α1CIV CAH74130.1: 1479-1556 NERAHGQDLGTAGSCLRKFSTMPFLFCNINNVCNFASRNDYSY
    WLSTPEPMPMSMAPITGENIRPFISRCAVCEAPAM
    2364 α1CIV CAH74130.1: 1494-1513 LRKFSTMPFLFCNINNVCNF
    2365 α1CIV CAH74130.1: 1504-1523 FCNINNVCNFASRNDYSYWL
    2366 α1CIV CAH74130.1: 1610-1628 SAPFIECHGRGTCNYYANA
    2367 α2CIV CAH71366.1: 1517-1593 QEKAHNQDLGLAGSCLARFSTMPFLYCNPGDVCYYASRNDKSY
    WLSTTAPLPMMPVAEDEIKPYISRCSVCEAPAIA
    2368 α2CIV CAH71366.1: 1542-1561 YCNPGDVCYYASRNDKSYWL
    2369 α2CIV CAH71366.1: 1646-1664 ATPFIECNGGRGTCHYYAN
    2370 α4CIV CAA56943.1: 1499-1575 QEKAHNQDLGLAGSCLPVFSTLPFAYCNIHQVCHYAQRNDRSY
    WLASAAPLPMMPLSEEAIRPYVSRCAVCEAPAQA
    2371 α4CIV CAA56943.1: 1514-1533 LPVFSTLPFAYCNIHQVCHY
    2372 α4CIV CAA56943.1: 1524-1543 YCNIHQVCHYAQRNDRSYWL
    2373 α4CIV CAA56943.1: 1628-1646 AAPFLECQGRQGTCHFFAN
    2374 α5CIV AAC27816.1: 1495-1572 NKRAHGQDLGTAGSCLRRFSTMPFMFCNINNVCNFASRNDYSY
    WLSTPEPMPMSMQPLKGQSIQPFISRCAVCEAPAV
    2375 α5CIV AAC27816.1: 1510-1529 LRRFSTMPFMFCNINNVCNF
    2376 α5CIV AAC27816.1: 1520-1539 FCNINNVCNFASRNDYSYWL
    2377 α5CIV AAC27816.1: 1626-1644 SAPFIECHGRGTCNYYANS
    2378 α6CIV CAI40758.1: 1501-1577 QEKAHNQDLGFAGSCLPRFSTMPFIYCNINEVCHYARRNDKSY
    WLSTTAPIPMMPVSQTQIPQYISRCSVCEAPSQA
    2379 α6CIV CAI40758.1: 1526-1545 YCNINEVCHYARRNDKSYWL
    2380 α6CIV CAI40758.1: 1630-1648 ATPFIECSGARGTCHYFAN
    CXC Chemokines
    2381 ENA-78/CXCL5 AAP35453.1: 86-108 NGKEICLDPEAPFLKKVIQKILD
    2382 ENA-78/CXCL5 AAP35453.1: 48-103 RCVCLQTTQGVHPKMISNLQVFAIGPQCSKVEVVASLKNGKEIC
    LDPEAPFLKKVI
    2383 ENA-78/CXCL5 AAP35453.1: 51-107 CLQTTQGVHPKMISNLQVFAIGPQCSKVEVVASLKNGKEICLDP
    EAPFLKKVIQKIL
    2384 GCP-2/CXCL6 AAH13744.1: 86-109 NGKQVCLDPEAPFLKKVIQKILDS
    2385 GCP-2/CXCL6 AAH13744.1: 47-106 LRCTCLRVTLRVNPKTIGKLQVFPAGPQCSKVEVVASLKNGKQV
    CLDPEAPFLKKVIQKI
    2386 GCP-2/CXCL6 AAH13744.1: 48-103 RCTCLRVTLRVNPKTIGKLQVFPAGPQCSKVEVVASLKNGKQV
    CLDPEAPFLKKVI
    2387 GCP-2/CXCL6 AAH13744.1: 51-107 CLRVTLRVNPKTIGKLQVFPAGPQCSKVEVVASLKNGKQVCLDP
    EAPFLKKVIQKIL
    2388 GRO-α/CXCL1 AAP35526.1: 80-103 NGRKACLNPASPIVKKIIEKMLNS
    2389 GRO-α/CXCL1 AAP35526.1: 42-97 RCQCLQTLQGIHPKNIQSVNVKSPGPHCAQTEVIATLKNGRKAC
    LNPASPIVKKII
    2390 GRO-α/CXCL1 AAP35526.1: 44-101 QCLQTLQGIHPKNIQSVNVKSPGPHCAQTEVIATLKNGRKACLN
    PASPIVKKIIEKML
    2391 Gro-β/CXCL2 AAH15753.1: 42-97 RCQCLQTLQGIHLKNIQSVKVKSPGPHCAQTEVIATLKNGQKAC
    LNPASPMVKKII
    2392 GRO-γ/MIP-2β/CXCL3 AAA63184.1: 79-100 NGKKACLNPASPMVQKIIEKIL
    2393 GRO-γ/MIP-2β/CXCL3 AAA63184.1: 43-100 QCLQTLQGIHLKNIQSVNVRSPGPHCAQTEVIATLKNGKKACLN
    PASPMVQKIIEKIL
    2394 GRO-γ/MIP-2β/CXCL3 AAA63184.1: 41-96 RCQCLQTLQGIHLKNIQSVNVRSPGPHCAQTEVIATLKNGKKAC
    LNPASPMVQKII
    2395 IL-8/CXCL8 AAP35730.1: 35-94 QCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRELCLDP
    KENWVQRVVEKFLK
    2396 IL-8/CXCL8 AAP35730.1: 72-94 DGRELCLDPKENWVQRVVEKFLK
    2397 IP-10/CXCL10 AAH10954.1: 29-86 RCTCISISNQPVNPRSLEKLEIIPASQFCPRVEIIATMKKKGEKRCL
    NPESKAIKNLL
    2398 MIG/CXCL9 Q07325: 32-91 SCISTNQGTIHLQSLKDLKQFAPSPSCEKIEIIATLKNGVQTCLNP
    DSADVKELIKKWEK
    2399 PF-4/CXCL4 AAK29643.1: 43-100 CVKTTSQVRPRHITSLEVIKAGPHCPTAQLIATLKNGRKICLDLQ
    APLYKKIIKKLLE
    2400 THBG-β/CXCL7 AAB46877.1: 100-121 DGRKICLDPDAPRIKKIVQKKL
    2401 THBG-β/CXCL7 AAB46877.1: 62-117 RCMCIKTTSGIHPKNIQSLEVIGKGTHCNQVEVIATLKDGRKICL
    DPDAPRIKKIV
    2402 THBG-β/CXCL7 AAB46877.1: 64-121 MCIKTTSGIHPKNIQSLEVIGKGTHCNQVEVIATLKDGRKICLDP
    DAPRIKKIVQKKL
    Kringle Containing Proteins
    2403 AK-38 protein AAK74187.1: 14-93 DCMFGNGKGYRGKKATTVTGTPCQEWAAQEPHRHSTFIPGTNK
    WAGLEKNYCRNPDGDINGPWCYTMNPRKLFDYCDIPLCA
    2404 AK-38 protein AAK74187.1: 12-94 QDCMFGNGKGYRGKKATTVTGTPCQEWAAQEPHRHSTFIPGTN
    KWAGLEKNYCRNPDGDINGPWCYTMNPRKLFDYCDIPLCA
    2405 AK-38 protein AAK74187.1: 13-90 DCMFGNGKGYRGKKATTVTGTPCQEWAAQEPHRHSTFIPGTNK
    WAGLEKNYCRNPDGDINGPWCYTMNPRKLFDYCDI
    2406 AK-38 protein AAK74187.1: 14-93 CMFGNGKGYRGKKATTVTGTPCQEWAAQEPHRHSTFIPGTNK
    WAGLEKNYCRNPDGDINGPWCYTMNPRKLFDYCDIPLC
    2407 Hageman fct/cf XII AAM97932.1: 216-292 SCYDGRGLSYRGLARTTLSGAPCQPWASEATYRNVTAEQARN
    WGLGGHAFCRNPDNDIRPWCFVLNRDRLSWEYCDL
    2408 Hageman fct/cf XII AAM97932.1: 214-295 KASCYDGRGLSYRGLARTTLSGAPCQPWASEATYRNVTAEQAR
    NWGLGGHAFCRNPDNDIRPWCFVLNRDRLSWEYCDLAQC
    2409 Hageman fct/cf XII AAM97932.1: 215-296 ASCYDGRGLSYRGLARTTLSGAPCQPWASEATYRNVTAEQARN
    WGLGGHAFCRNPDNDIRPWCFVLNRDRLSWEYCDLAQCQ
    2410 HGF P14210: 127-206 NCIIGKGRSYKGTVSITKSGIKCQPWSSMIPHEHSFLPSSYRGKDL
    QENYCRNPRGEEGGPWCFTSNPEVRYEVCDIPQC
    2411 HGF P14210: 127-207 NCIIGKGRSYKGTVSITKSGIKCQPWSSMIPHEHSFLPSSYRGKDL
    QENYCRNPRGEEGGPWCFTSNPEVRYEVCDIPQCS
    2412 HGF P14210: 304-377 ECIQGQGEGYRGTVNTIWNGIPCQRWDSQYPHEHDMTPENFKC
    KDLRENYCRNPDGSESPWCFTTDPNIRVGYC
    2413 HGF P14210: 210-289 ECMTCNGESYRGLMDHTESGKICQRWDHQTPHRHKFLPERYPD
    KGFDDNYCRNPDGQPRPWCYTLDPHTRWEYCAIKTCA
    2414 HGF P14210: 304-383 ECIQGQGEGYRGTVNTIWNGIPCQRWDSQYPHEHDMTPENFKC
    KDLRENYCRNPDGSESPWCFTTDPNIRVGYCSQIPNC
    2415 Hyaluronan binding NP_004123.1: 192-277 DDCYVGDGYSYRGKMNRTVNQHACLYWNSHLLLQENYNMFM
    EDAETHGIGEHNFCRNPDADEKPWCFIKVTNDKVKWEYCDVSA
    CS
    2416 Hyaluronan binding NP_004123.1: 192-276 DDCYVGDGYSYRGKMNRTVNQHACLYWNSHLLLQENYNMFM
    EDAETHGIGEHNFCRNPDADEKPWCFIKVTNDKVKWEYCDVSAC
    2417 KREMEN-1 BAB40969.1: 31-114 ECFTANGADYRGTQNWTALQGGKPCLFWNETFQHPYNTLKYP
    NGEGGLGEHNYCRNPDGDVS-
    PWCYVAEHEDGVYWKYCEIPAC
    2418 KREMEN-1 BAB40969.1: 31-115 ECFTANGADYRGTQNWTALQGGKPCLFWNETFQHPYNTLKYP
    NGEGGLGEHNYCRNPDGDVSPWCYVAEHEDGVYWKYCEIPACQ
    2419 KREMEN-2 BAD97142.1: 35-119 ECFQVNGADYRGHQNRTGPRGAGRPCLFWDQTQQHSYSSASDP
    HGRWGLGAHNFCRNPDGDVQ-PWCYVAETEEGIYWRYCDIPSC
    2420 KREMEN-2 BAD97142.1: 34-119 SECFQVNGADYRGHQNRTGPRGAGRPCLFWDQTQQHSYSSASD
    PHGRWGLGAHNFCRNPDGDVQPWCYVAETEEGIYWRYCDIPSC
    2421 Lp(a) NP_005568.1: 1615-1690 TEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHS
    RTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPG
    2422 Lp(a) NP_005568.1: 3560-3639 QDCYYHYGQSYRGTYSTTVTGRTCQAWSSMTPHQHSRTPENYP
    NAGLTRNYCRNPDAEIRPWCYTMDPSVRWEYCNLTQC
    2423 Lp(a) NP_005568.1: 4123-4201 QCYHGNGQSYRGTFSTTVTGRTCQSWSSMTPHRHQRTPENYPN
    DGLTMNYCRNPDADTGPWCFTMDPSIRWEYCNLTRC
    2424 Lp(a) NP_005568.1: 4225-4308 EQDCMFGNGKGYRGKKATTVTGTPCQEWAAQEPHRHSTFIPGT
    NKWAGLEKNYCRNPDGDINGPWCYTMNPRKLFDYCDIPLCA
    2425 Macrophage stim. 1 AAH48330.1: 188-268 EAACVWCNGEEYRGAVDRTESGRECQRWDLQHPHQHPFEPGK
    FLDQGLDDNYCRNPDGSERPWCYTTDPQIEREFCDLPRC
    2426 Macrophage stim. 1 AAH48330.1: 368-448 QDCYHGAGEQYRGTVSKTRKGVQCQRWSAETPHKPQFTFTSEP
    HAQLEENFCRNPDGDSHGPWCYTMDPRTPFDYCALRRC
    2427 Macrophage stim. 1 AAH48330.1: 368-449 QDCYHGAGEQYRGTVSKTRKGVQCQRWSAETPHKPQFTFTSEP
    HAQLEENFCRNPDGDSHGPWCYTMDPRTPFDYCALRRCA
    2428 Macrophage stim. 1 AAH48330.1: 370-448 CYHGAGEQYRGTVSKTRKGVQCQRWSAETPHKPQFTFTSEPHA
    QLEENFCRNPDGDSHGPWCYTMDPRTPFDYCALRRC
    2429 Thrombin/cf II AAL77436.1: 105-186 EGNCAEGLGTNYRGHVNITRSGIECQLWRSRYPHKPEINSTTHP
    GADLQENFCRNPDSSTTGPWCYTTDPTVRRQECSIPVC
    2430 Thrombin/cf II AAL77436.1: 106-186 GNCAEGLGTNYRGHVNITRSGIECQLWRSRYPHKPEINSTTHPG
    ADLQENFCRNPDSSTTGPWCYTTDPTVRRQECSIPVC
    2431 Thrombin/cf II AAL77436.1: 107-183 NCAEGLGTNYRGHVNITRSGIECQLWRSRYPHKPEINSTTHPGA
    DLQENFCRNPDSSTTGPWCYTTDPTVRRQECSI
    2432 Thrombin/cf II AAL77436.1: 107-186 NCAEGLGTNYRGHVNITRSGIECQLWRSRYPHKPEINSTTHPGA
    DLQENFCRNPDSSTTGPWCYTTDPTVRRQECSIPVC
    2433 tPA AAH95403.1: 214-293 DCYFGNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQ
    ALGLGKHNYCRNPDGDAKPWCHVLKSRRLTWEYCDV
    2434 tPA AAH95403.1: 213-296 SDCYFGNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSA
    QALGLGKHNYCRNPDGDAKPWCHVLKSRRLTWEYCDVPSC
    2435 tPA AAH95403.1: 213-297 SDCYFGNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSA
    QALGLGKHNYCRNPDGDAKPWCHVLKSRRLTWEYCDVPSCS
    2436 tPA AAH95403.1: 214-296 DCYFGNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQ
    ALGLGKHNYCRNPDGDAKPWCHVLKSRRLTWEYCDVPSC
    Somatotropins
    2437 GH-1 NP_000506.2: 26-160 AFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSF
    LQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQ
    FLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPR
    2438 GH-2 CAG46700.1: 26-160 AFPTIPLSRLFDNAMLRARRLYQLAYDTYQEFEEAYILKEQKYSF
    LQNPQTSLCFSESIPTPSNRAKTQQKSNLELLRISLLLIQSWLEPV
    QLLRSVFANSLVYGASDSNVYRHLKDLEEGIQTLMWRLEDGSPR
    2439 Placental lactogen AAP35572.1: 26-160 AVQTVPLSRLFDHAMLQAHRAHQLAIDTYQEFEETYIPKDQKYS
    FLHDSQTSFCFSDSIPTPSNMEETQQKSNLELLRISLLLIESWLEPV
    RFLRSMFANNLVYDTSDSDDYHLLKDLEEGIQTLMGRLEDGSRR
    2440 Somatoliberin AAH62475.1: 26-145 AFPTIPLSRLFDNASLRAHRLHQLAFDTYQEFNPQTSLCFSESIPT
    PSMREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGA
    SDSNVYDLLKDLEEGIQTLMGRLEDGSPR
    TIMPs
    2441 TIMP 3 AAA21815.1: 148-171 ECLWTDMLSNFGYPGYQSKHYACI
    2442 TIMP 4 AAV38433.1: 175-198 ECLWTDWLLERKLYGYQAQHYVCM
  • In particular embodiments, the presently disclosed subject matter provides a nanoparticle, microparticle, or gel comprising a compound of Formula (I), wherein the one or more peptide is selected from the group consisting of an isolated peptide or analog thereof comprising the amino acid sequence W-X2-C-X3-C-X2-G, wherein X denotes a variable amino acid; W is tryptophan; C is cysteine, G is glycine; and wherein the peptide reduces blood vessel formation in a cell, tissue or organ.
  • In some embodiments, the one or more peptide is selected from the group consisting of an isolated peptide or analog thereof comprising or consisting essentially of a sequence having at least 85% amino acid sequence identity to an amino acid sequence selected from the group consisting of:
  • THSD-1: QPWSQCSATCGDGVRERRR;
    THSD-3: SPWSPCSGNCSTGKQQRTR;
    THSD-6: WTRCSSSCGRGVSVRSR;
    CILP: SPWSKCSAACGQTGVQTRTR;
    WISP-1: SPWSPCSTSCGLGVSTRI;
    WISP-2: TAWGPCSTTCGLGMATRV;
    WISP-3: TKWTPCSRTCGMGISNRV;
    F-spondin: SEWSDCSVTCGKGMRTRQR;
    F-spondin: WDECSATCGMGMKKRHR;
    CTGF: TEWSACSKTCGMGISTRV;
    fibulin-6: ASWSACSVSCGGGARQRTR;
    fibulin-6: QPWGTCSESCGKGTQTRAR;
    fibulin-6: SAWRACSVTCGKGIQKRSR;
    CYR61: TSWSQCSKTCGTGISTRV;
    NOVH: TEWTACSKSCGMGFSTRV;
    UNC5-C: TEWSVCNSRCGRGYQKRTR;
    UNC5-D: TEWSACNVRCGRGWQKRSR;
    SCO-spondin: GPWEDCSVSCGGGEQLRSR;
    Properdin: GPWEPCSVTCSKGTRTRRR;
    C6: TQWTSCSKTCNSGTQSRHR;
    ADAMTS-like-4: SPWSQCSVRCGRGQRSRQVR;
    ADAMTS-4: GPWGDCSRTCGGGVQFSSR;
    ADAMTS-8: GPWGECSRTCGGGVQFSHR;
    ADAMTS-16: SPWSQCTASCGGGVQTR;
    ADAMTS-18: SKWSECSRTCGGGVKFQER;
    semaphorin 5A: GPWERCTAQCGGGIQARRR;
    semaphorin 5A: SPWTKCSATCGGGHYMRTR;
    semaphoring 5B: TSWSPCSASCGGGHYQRTR;
    papilin: GPWAPCSASCGGGSQSRS;
    papilin: SQWSPCSRTCGGGVSFRER;
    ADAM-9: KCHGHGVCNS;
    and
    ADAM-12: MQCHGRGVCNNRKN,
  • wherein A is alanine; I is isoleucine; M is methionine; H is histidine; Y is tyrosine; K is lysine; W is tryptophan; C is cysteine, T is threonine, S is serine; N is asparagine; G is glycine; R is arginine; V is valine, P is proline, and Q is glutamine wherein the peptide reduces blood vessel formation in a cell, tissue or organ.
  • In other embodiments, the one or more peptide is selected from the group consisting of an isolated peptide or analog thereof having at least 85% identity to an amino acid sequence selected from the group consisting of:
  • ENA-78: NGKEICLDPEAPFLKKVIQKILD;
    CXCL6: NGKQVCLDPEAPFLKKVIQKILDS;
    CXCL1: NGRKACLNPASPIVKKIIEKMLNS;
    Gro-γ: NGKKACLNPASPMVQKIIEKIL;
    Beta thromboglobulin/CXCL7: DGRKICLDPDAPRIKKIVQK
    KL,
    Interleukin 8 (IL-8)/CXCL8: DGRELCLDPKENWVQRVVEKF
    LK,
    GCP-2: NGKQVCLDPEAPFLKKVIQKILDS,
  • wherein A is alanine; I is isoleucine; F is phenylalanine; D is aspartic acid; M is methionine; H is histidine; Y is tyrosine; K is lysine; W is tryptophan; C is cysteine, T is threonine, S is serine; N is asparagine; G is glycine; R is arginine; V is valine, P is proline, and Q is glutamine; and wherein the peptide reduces blood vessel formation in a cell, tissue or organ.
  • In yet other embodiments, the one or more peptide is selected from the group consisting of an isolated peptide or analog thereof having at least 85% amino acid sequence identity to an amino acid sequence selected from the group consisting of
  • Alpha 6 fibril of type 4 collagen: YCNINEVCHYARRND
    KSYWL;
    Alpha 5 fibril of type 4 collagen: LRRFSTMPFMFCNIN
    NVCNF;
    Alpha 4 fibril of type 4 collagen: AAPFLECQGRQGTCH
    FFAN;
    Alpha 4 fibril of type 4 collagen: LPVFSTLPFAYCNIH
    QVCHY;
    Alpha 4 fibril of type 4 collagen: YCNIHQVCHYAQRND
    RSYWL,
    and
    Collagen type IV, alpha6 fibril LPRFSTMPFIYCNINE
    VCHY;
  • wherein A is alanine; I is isoleucine; F is phenylalanine; D is aspartic acid; M is methionine; H is histidine; Y is tyrosine; K is lysine; W is tryptophan; C is cysteine, T is threonine, S is serine; N is asparagine; G is glycine; R is arginine; V is valine, P is proline, and Q is glutamine wherein the peptide reduces blood vessel formation in a cell, tissue or organ.
  • In other embodiments, peptides suitable for use in the presently disclosed subject matter are disclosed in U.S. Provisional Patent Application No. 61/421,706, filed Dec. 12, 2010, which is commonly owned, and is incorporated herein by reference in its entirety.
  • SEQ ID NO. ID Sequence
    2443 SP2000 LRRFSTMPFMFCNINNVCNF
    2444 SP2002 LRRFSTMPFMFGNINNVGNF
    2445 SP2004 LRRFSTMPFMF
    2446 SP2006 LRRFSTMPFMF-Abu-NINV
    2447 SP2007 LRRFSTMPFMF-Abu
    2448 SP2008 LRRFSTMP
    2449 SP2009 NINNV-Abu-NF
    2450 SP2010 FMF-Abu-NINNV-Abu-NF
    2451 SP2011 STMPFMF-Abu-NINNV-Abu-NF
    2452 SP2012 LRRFSTMPFMF-Abu-NINNV-Abu-NF
    2453 SP2013 LNRFSTMPF
    2454 SP2014 LRRFST-Nle-PF-Nle-F
    2455 SP2015 LRRFSTMPAMF-Abu-NINNV-Abu-NF
    2456 SP2016 LRRFSTMPFAF-Abu-NINNV-Abu-NF
    2457 SP2017 LRRFSTMPFMA-Abu-NINNV-Abu-NF
    2458 SP2018 LRRFSTMPF-Nle-F-Abu-NINNV-Abu-NF
    2459 SP2019 LRRFSTMPFM(4-ClPhen)-Abu-CNINNV-
    Abu-NF
    2460 SP2020 F-Abu-NINNV-Abu-N
    2461 SP2021 F-Abu-NIN
    2462 SP2022 LRRFSTMPFMFSNINNVSNF
    2463 SP2023 LRRFSTMPFMFANINNVANF
    2464 SP2024 LRRFSTMPFMFININNVINF
    2465 SP2025 LRRFSTMPFMFTNINNVTNF
    2466 SP2026 LRRFSTMPFMFC(AllyGly)NINNV
    (AllyGly)NF
    2467 SP2027 LRRFSTMPFMFVNINNVVNF
    2468 SP2028 LRRFSTMPFMF-Abu-NINN
    2469 SP2029 LRRFSTMPFMFTNINV
    2470 SP2030 F-Abu-NINV
    2471 SP2031 FTNINNVTN
    2472 SP2032 LRRFSTMPFMFTNINN
    2473 SP2033 LRRFSTMPFMFININN
    2474 SP2034 LRRFSTMPF-Da-FININNVINF
    2475 SP2035 LRRFSTAPFAFININNVINF
    2476 SP2036 LRRFSTMPFAFININNVINF;

    wherein Abu is 2-aminobutyric acid; Nle is Norleucine; and AllyGly is allyglycine.
  • In other embodiments, peptides suitable for use in the presently disclosed subject matter are disclosed in U.S. Provisional Patent Application No. 61/489,500, filed Way 24, 2011, which also is commonly owned, and is incorporated herein by reference in its entirety.
  • SEQ ID NO. ID Sequence
    2477 SP5001 RLRLLTLQSWLL
    2478 SP5028 LMRKSQILISSWF
    2479 SP5029 LLIVALLFILSWL
    2480 SP5030 LLRLLLLIESWLE
    2481 SP5031 LLRSSLILLQGSWF
    2482 SP5032 LLHISLLLIESRLE
    2483 SP5033 LLRISLLLIESWLE
    • III. Definitions
  • Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this presently described subject matter belongs.
  • While the following terms in relation to compounds of Formulae I-X are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter. These definitions are intended to supplement and illustrate, not preclude, the definitions that would be apparent to one of ordinary skill in the art upon review of the present disclosure.
  • The terms substituted, whether preceded by the term “optionally” or not, and substituent, as used herein, refer to the ability, as appreciated by one skilled in this art, to change one functional group for another functional group provided that the valency of all atoms is maintained. When more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. The substituents also may be further substituted (e.g., an aryl group substituent may have another substituent off it, such as another aryl group, which is further substituted, for example, with fluorine at one or more positions).
  • When the term “independently selected” is used, the substituents being referred to (e.g., R groups, such as groups R1, R2, and the like, or variables, such as “m” and “n”), can be identical or different. For example, both R1 and R2 can be substituted alkyls, or R1 can be hydrogen and R2 can be a substituted alkyl, and the like.
  • A named “R” or group will generally have the structure that is recognized in the art as corresponding to a group having that name, unless specified otherwise herein. For the purposes of illustration, certain representative “R” groups as set forth above are defined below.
  • The term hydrocarbon, as used herein, refers to any chemical group comprising hydrogen and carbon. The hydrocarbon may be substituted or unsubstituted. As would be known to one skilled in this art, all valencies must be satisfied in making any substitutions. The hydrocarbon may be unsaturated, saturated, branched, unbranched, cyclic, polycyclic, or heterocyclic. Illustrative hydrocarbons are further defined herein below and include, for example, methyl, ethyl, n-propyl, iso-propyl, cyclopropyl, allyl, vinyl, n-butyl, tert-butyl, ethynyl, cyclohexyl, methoxy, diethylamino, and the like.
  • As used herein the term “alkyl” refers to C1-20 inclusive, linear (i.e., “straight-chain”), branched, or cyclic, saturated or at least partially and in some cases fully unsaturated (i.e., alkenyl and alkynyl)hydrocarbon radicals derived from a hydrocarbon moiety containing between one and twenty carbon atoms by removal of a single hydrogen atom. Representative alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, sec-pentyl, iso-pentyl, neopentyl, n-hexyl, sec-hexyl, n-heptyl, n-octyl, n-decyl, n-undecyl, dodecyl, and the like, ethenyl, propenyl, butenyl, pentenyl, hexenyl, octenyl, butadienyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, and allenyl groups. “Branched” refers to an alkyl group in which a lower alkyl group, such as methyl, ethyl or propyl, is attached to a linear alkyl chain. “Lower alkyl” refers to an alkyl group having 1 to about 8 carbon atoms (i.e., a C1-8 alkyl), e.g., 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms. “Higher alkyl” refers to an alkyl group having about 10 to about 20 carbon atoms, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. In certain embodiments, “alkyl” refers, in particular, to C1-8 straight-chain alkyls. In other embodiments, “alkyl” refers, in particular, to C1-8 branched-chain alkyls.
  • Alkyl groups can optionally be substituted (a “substituted alkyl”) with one or more alkyl group substituents, which can be the same or different. The term “alkyl group substituent” includes but is not limited to alkyl, substituted alkyl, halo, arylamino, acyl, hydroxyl, aryloxyl, alkoxyl, alkylthio, arylthio, aralkyloxyl, aralkylthio, carboxyl, alkoxycarbonyl, oxo, and cycloalkyl. There can be optionally inserted along the alkyl chain one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms, wherein the nitrogen substituent is hydrogen, lower alkyl (also referred to herein as “alkylaminoalkyl”), or aryl.
  • Thus, as used herein, the term “substituted alkyl” includes alkyl groups, as defined herein, in which one or more atoms or functional groups of the alkyl group are replaced with another atom or functional group, including for example, alkyl, substituted alkyl, halogen, aryl, substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino, dialkylamino, sulfate, and mercapto.
  • “Cyclic” and “cycloalkyl” refer to a non-aromatic mono- or multicyclic ring system of about 3 to about 10 carbon atoms, e.g., 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. The cycloalkyl group can be optionally partially unsaturated. The cycloalkyl group also can be optionally substituted with an alkyl group substituent as defined herein, oxo, and/or alkylene. There can be optionally inserted along the cyclic alkyl chain one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms,
  • wherein the nitrogen substituent is hydrogen, alkyl, substituted alkyl, aryl, or substituted aryl, thus providing a heterocyclic group. Representative monocyclic cycloalkyl rings include cyclopentyl, cyclohexyl, and cycloheptyl. Multicyclic cycloalkyl rings include adamantyl, octahydronaphthyl, decalin, camphor, camphane, and noradamantyl.
  • The term “cycloalkylalkyl,” as used herein, refers to a cycloalkyl group as defined hereinabove, which is attached to the parent molecular moiety through an alkyl group, also as defined above. Examples of cycloalkylalkyl groups include cyclopropylmethyl and cyclopentylethyl.
  • The terms “cycloheteroalkyl” or “heterocycloalkyl” refer to a non-aromatic ring system, unsaturated or partially unsaturated ring system, such as a 3- to 10-member substituted or unsubstituted cycloalkyl ring system, including one or more heteroatoms, which can be the same or different, and are selected from the group consisting of N, O, and S, and optionally can include one or more double bonds. The cycloheteroalkyl ring can be optionally fused to or otherwise attached to other cycloheteroalkyl rings and/or non-aromatic hydrocarbon rings. Heterocyclic rings include those having from one to three heteroatoms independently selected from oxygen, sulfur, and nitrogen, in which the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. In certain embodiments, the term heterocylic refers to a non-aromatic 5-, 6-, or 7-membered ring or a polycyclic group wherein at least one ring atom is a heteroatom selected from O, S, and N (wherein the nitrogen and sulfur heteroatoms may be optionally oxidized), including, but not limited to, a bi- or tri-cyclic group, comprising fused six-membered rings having between one and three heteroatoms independently selected from the oxygen, sulfur, and nitrogen, wherein (i) each 5-membered ring has 0 to 2 double bonds, each 6-membered ring has 0 to 2 double bonds, and each 7-membered ring has 0 to 3 double bonds, (ii) the nitrogen and sulfur heteroatoms may be optionally oxidized, (iii) the nitrogen heteroatom may optionally be quaternized, and (iv) any of the above heterocyclic rings may be fused to an aryl or heteroaryl ring. Representative cycloheteroalkyl ring systems include, but are not limited to pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperidyl, piperazinyl, indolinyl, quinuclidinyl, morpholinyl, thiomorpholinyl, thiadiazinanyl, tetrahydrofuranyl, and the like.
  • The term “alkenyl” as used herein refers to a monovalent group derived from a C1-20 inclusive straight or branched hydrocarbon moiety having at least one carbon-carbon double bond by the removal of a single hydrogen atom. Alkenyl groups include, for example, ethenyl (i.e., vinyl), propenyl, butenyl, 1-methyl-2-buten-1-yl, and the like.
  • The term “cycloalkenyl” as used herein refers to a cyclic hydrocarbon containing at least one carbon-carbon double bond. Examples of cycloalkenyl groups include cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadiene, cyclohexenyl, 1,3-cyclohexadiene, cycloheptenyl, cycloheptatrienyl, and cyclooctenyl.
  • The term “alkynyl” as used herein refers to a monovalent group derived from a straight or branched C1-20 hydrocarbon of a designed number of carbon atoms containing at least one carbon-carbon triple bond. Examples of “alkynyl” include ethynyl, 2-propynyl(propargyl), 1-propyne, 3-hexyne, and the like.
  • “Alkylene” refers to a straight or branched bivalent aliphatic hydrocarbon group having from 1 to about 20 carbon atoms, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. The alkylene group can be straight, branched or cyclic. The alkylene group also can be optionally unsaturated and/or substituted with one or more “alkyl group substituents.” There can be optionally inserted along the alkylene group one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms (also referred to herein as “alkylaminoalkyl”), wherein the nitrogen substituent is alkyl as previously described. Exemplary alkylene groups include methylene (—CH2—); ethylene (—CH2—CH2-); propylene (—(CH2)3—); cyclohexylene (—C6H10—); —CH═CH—CH═CH—; —CH═CH—CH2—; —(CH2)q—N(R)—(CH2)r—, wherein each of q and r is independently an integer from 0 to about 20, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, and R is hydrogen or lower alkyl; methylenedioxyl (—O—CH2—O—); and ethylenedioxyl (—O—(CH2)2—O—). An alkylene group can have about 2 to about 3 carbon atoms and can further have 6-20 carbons.
  • The term “aryl” is used herein to refer to an aromatic substituent that can be a single aromatic ring, or multiple aromatic rings that are fused together, linked covalently, or linked to a common group, such as, but not limited to, a methylene or ethylene moiety. The common linking group also can be a carbonyl, as in benzophenone, or oxygen, as in diphenylether, or nitrogen, as in diphenylamine. The term “aryl” specifically encompasses heterocyclic aromatic compounds. The aromatic ring(s) can comprise phenyl, naphthyl, biphenyl, diphenylether, diphenylamine and benzophenone, among others. In particular embodiments, the term “aryl” means a cyclic aromatic comprising about 5 to about 10 carbon atoms, e.g., 5, 6, 7, 8, 9, or 10 carbon atoms, and including 5- and 6-membered hydrocarbon and heterocyclic aromatic rings.
  • The aryl group can be optionally substituted (a “substituted aryl”) with one or more aryl group substituents, which can be the same or different, wherein “aryl group substituent” includes alkyl, substituted alkyl, alkenyl, alkynyl, aryl, substituted aryl, aralkyl, hydroxyl, alkoxyl, aryloxyl, aralkyloxyl, carboxyl, acyl, halo, haloalkyl, nitro, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, acyloxyl, amino, alkylamino, dialkylamino, trialkylamino, acylamino, aroylamino, carbamoyl, cyano, alkylcarbamoyl, dialkylcarbamoyl, carboxyaldehyde, carboxyl, alkoxycarbonyl, carboxamide, arylthio, alkylthio, alkylene, thioalkoxyl, and mercapto.
  • Thus, as used herein, the term “substituted aryl” includes aryl groups, as defined herein, in which one or more atoms or functional groups of the aryl group are replaced with another atom or functional group, including for example, alkyl, substituted alkyl, halogen, aryl, substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino, dialkylamino, sulfate, and mercapto.
  • Specific examples of aryl groups include, but are not limited to, cyclopentadienyl, phenyl, furan, thiophene, pyrrole, pyran, pyridine, imidazole, benzimidazole, isothiazole, isoxazole, pyrazole, pyrazine, triazine, pyrimidine, quinoline, isoquinoline, indole, carbazole, and the like.
  • The terms “heteroaryl” and “aromatic heterocycle” and “aromatic heterocyclic” are used interchangeably herein and refer to a cyclic aromatic radical having from five to ten ring atoms of which one ring atom is selected from sulfur, oxygen, and nitrogen; zero, one, or two ring atoms are additional heteroatoms independently selected from sulfur, oxygen, and nitrogen; and the remaining ring atoms are carbon, the radical being joined to the rest of the molecule via any of the ring atoms, such as, for example, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, and the like. Aromatic heterocyclic groups can be unsubstituted or substituted with substituents selected from the group consisting of branched and unbranched alkyl, alkenyl, alkynyl, haloalkyl, alkoxy, thioalkoxy, amino, alkylamino, dialkylamino, trialkylamino, acylamino, cyano, hydroxy, halo, mercapto, nitro, carboxyaldehyde, carboxy, alkoxycarbonyl, and carboxamide. Specific heterocyclic and aromatic heterocyclic groups that may be included in the compounds of the invention include: 3-methyl-4-(3-methylphenyl)piperazine, 3 methylpiperidine, 4-(bis-(4-fluorophenyl)methyl)piperazine, 4-(diphenylmethyl)piperazine, 4(ethoxycarbonyl)piperazine, 4-(ethoxycarbonylmethyl)piperazine, 4-(phenylmethyl)piperazine, 4-(1-phenylethyl)piperazine, 4-(1,1-dimethylethoxycarbonyl)piperazine, 4-(2-(bis-(2-propenyl)amino)ethyl)piperazine, 4-(2-(diethylamino)ethyl)piperazine, 4-(2-chlorophenyl)piperazine, 4(2-cyanophenyl)piperazine, 4-(2-ethoxyphenyl)piperazine, 4-(2-ethylphenyl)piperazine, 4-(2-fluorophenyl)piperazine, 4-(2-hydroxyethyl)piperazine, 4-(2-methoxyethyl)piperazine, 4-(2-methoxyphenyl)piperazine, 4-(2-methylphenyl)piperazine, 4-(2-methylthiophenyl)piperazine, 4(2-nitrophenyl)piperazine, 4-(2-nitrophenyl)piperazine, 4-(2-phenylethyl)piperazine, 4-(2-pyridyl)piperazine, 4-(2-pyrimidinyl)piperazine, 4-(2,3-dimethylphenyl)piperazine, 4-(2,4-difluorophenyl)piperazine, 4-(2,4-dimethoxyphenyl)piperazine, 4-(2,4-dimethylphenyl)piperazine, 4-(2,5-dimethylphenyl)piperazine, 4-(2,6-dimethylphenyl)piperazine, 4-(3-chlorophenyl)piperazine, 4-(3-methylphenyl)piperazine, 4-(3-trifluoromethylphenyl)piperazine, 4-(3,4-dichlorophenyl)piperazine, 4-(3,4-dimethoxyphenyl)piperazine, 4-(3,4-dimethylphenyl)piperazine, 4-(3,4-methylenedioxyphenyl)piperazine, 4-(3,4,5-trimethoxyphenyl)piperazine, 4-(3,5-dichlorophenyl)piperazine, 4-(3,5-dimethoxyphenyl)piperazine, 4-(4-(phenylmethoxy)phenyl)piperazine, 4-(4-(3,1-dimethylethyl)phenylmethyl)piperazine, 4-(4-chloro-3-trifluoromethylphenyl)piperazine, 4-(4-chlorophenyl)-3-methylpiperazine, 4-(4-chlorophenyl)piperazine, 4-(4-chlorophenyl)piperazine, 4-(4-chlorophenylmethyl)piperazine, 4-(4-fluorophenyl)piperazine, 4-(4-methoxyphenyl)piperazine, 4-(4-methylphenyl)piperazine, 4-(4-nitrophenyl)piperazine, 4-(4-trifluoromethylphenyl)piperazine, 4-cyclohexylpiperazine, 4-ethyl piperazine, 4-hydroxy-4-(4-chlorophenyl)methylpiperidine, 4-hydroxy-4-phenylpiperidine, 4-hydroxypyrrolidine, 4-methylpiperazine, 4-phenylpiperazine, 4-piperidinylpiperazine, 4-(2-furanyl)carbonyl)piperazine, 4-((1,3-dioxolan-5-yl)methyl)piperazine, 6-fluoro-1,2,3,4-tetrahydro-2-methylquinoline, 1,4-diazacylcloheptane, 2,3-dihydroindolyl, 3,3-dimethylpiperidine, 4,4-ethylenedioxypiperidine, 1,2,3,4-tetrahydroisoquinoline, 1,2,3,4-tetrahydroquinoline, azacyclooctane, decahydroquinoline, piperazine, piperidine, pyrrolidine, thiomorpholine, and triazole. The heteroaryl ring can be fused or otherwise attached to one or more heteroaryl rings, aromatic or non-aromatic hydrocarbon rings, or heterocycloalkyl rings. A structure represented generally by the formula:
  • Figure US20160374949A9-20161229-C00036
  • as used herein refers to a ring structure, for example, but not limited to a 3-carbon, a 4-carbon, a 5-carbon, a 6-carbon, a 7-carbon, and the like, aliphatic and/or aromatic cyclic compound, including a saturated ring structure, a partially saturated ring structure, and an unsaturated ring structure, comprising a substituent R group, wherein the R group can be present or absent, and when present, one or more R groups can each be substituted on one or more available carbon atoms of the ring structure. The presence or absence of the R group and number of R groups is determined by the value of the variable “n,” which is an integer generally having a value ranging from 0 to the number of carbon atoms on the ring available for substitution. Each R group, if more than one, is substituted on an available carbon of the ring structure rather than on another R group. For example, the structure above where n is 0 to 2 would comprise compound groups including, but not limited to:
  • Figure US20160374949A9-20161229-C00037
  • and the like.
  • A dashed line representing a bond in a cyclic ring structure indicates that the bond can be either present or absent in the ring. That is, a dashed line representing a bond in a cyclic ring structure indicates that the ring structure is selected from the group consisting of a saturated ring structure, a partially saturated ring structure, and an unsaturated ring structure.
  • When a named atom of an aromatic ring or a heterocyclic aromatic ring is defined as being “absent,” the named atom is replaced by a direct bond.
  • As used herein, the term “acyl” refers to an organic acid group wherein the —OH of the carboxyl group has been replaced with another substituent and has the general formula RC(═O)—, wherein R is an alkyl, alkenyl, alkynyl, aryl, carbocylic, heterocyclic, or aromatic heterocyclic group as defined herein). As such, the term “acyl” specifically includes arylacyl groups, such as an acetylfuran and a phenacyl group. Specific examples of acyl groups include acetyl and benzoyl.
  • The terms “alkoxyl” or “alkoxy” are used interchangeably herein and refer to a saturated (i.e., alkyl-O—) or unsaturated (i.e., alkenyl-O— and alkynyl-O—) group attached to the parent molecular moiety through an oxygen atom, wherein the terms “alkyl,” “alkenyl,” and “alkynyl” are as previously described and can include C1-C20 inclusive, linear, branched, or cyclic, saturated or unsaturated oxo-hydrocarbon chains, including, for example, methoxyl, ethoxyl, propoxyl, isopropoxyl, n-butoxyl, sec-butoxyl, t-butoxyl, and n-pentoxyl, neopentoxy, n-hexoxy, and the like.
  • The term “alkoxyalkyl” as used herein refers to an alkyl-O-alkyl ether, for example, a methoxyethyl or an ethoxymethyl group.
  • “Aryloxyl” refers to an aryl-O— group wherein the aryl group is as previously described, including a substituted aryl. The term “aryloxyl” as used herein can refer to phenyloxyl or hexyloxyl, and alkyl, substituted alkyl, halo, or alkoxyl substituted phenyloxyl or hexyloxyl.
  • “Aralkyl” refers to an aryl-alkyl-group wherein aryl and alkyl are as previously described, and included substituted aryl and substituted alkyl. Exemplary aralkyl groups include benzyl, phenylethyl, and naphthylmethyl.
  • “Aralkyloxyl” refers to an aralkyl-O— group wherein the aralkyl group is as previously described. An exemplary aralkyloxyl group is benzyloxyl.
  • “Alkoxycarbonyl” refers to an alkyl-O—CO— group. Exemplary alkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl, butyloxycarbonyl, and t-butyloxycarbonyl.
  • “Aryloxycarbonyl” refers to an aryl-O—CO— group. Exemplary aryloxycarbonyl groups include phenoxy- and naphthoxy-carbonyl.
  • “Aralkoxycarbonyl” refers to an aralkyl-O—CO— group. An exemplary aralkoxycarbonyl group is benzyloxycarbonyl.
  • “Carbamoyl” refers to an amide group of the formula —CONH2. “Alkylcarbamoyl” refers to a R′RN—CO— group wherein one of R and R′ is hydrogen and the other of R and R′ is alkyl and/or substituted alkyl as previously described. “Dialkylcarbamoyl” refers to a R′RN—CO— group wherein each of R and R′ is independently alkyl and/or substituted alkyl as previously described.
  • The term carbonyldioxyl, as used herein, refers to a carbonate group of the formula —O—CO—OR.
  • “Acyloxyl” refers to an acyl-O— group wherein acyl is as previously described.
  • The term “amino” refers to the —NH2 group and also refers to a nitrogen containing group as is known in the art derived from ammonia by the replacement of one or more hydrogen radicals by organic radicals. For example, the terms “acylamino” and “alkylamino” refer to specific N-substituted organic radicals with acyl and alkyl substituent groups respectively.
  • The terms alkylamino, dialkylamino, and trialkylamino as used herein refer to one, two, or three, respectively, alkyl groups, as previously defined, attached to the parent molecular moiety through a nitrogen atom. The term alkylamino refers to a group having the structure —NHR′ wherein R′ is an alkyl group, as previously defined; whereas the term dialkylamino refers to a group having the structure —NR′R″, wherein R′ and R″ are each independently selected from the group consisting of alkyl groups. The term trialkylamino refers to a group having the structure —NR′R″R′″, wherein R′, R″, and R′″ are each independently selected from the group consisting of alkyl groups. Additionally, R′, R″, and/or R′″ taken together may optionally be —(CH2)k— where k is an integer from 2 to 6. Examples include, but are not limited to, methylamino, dimethylamino, ethylamino, diethylamino, diethylaminocarbonyl, methylethylamino, iso-propylamino, piperidino, trimethylamino, and propylamino.
  • The terms alkylthioether and thioalkoxyl refer to a saturated (i.e., alkyl-S—) or unsaturated (i.e., alkenyl-S— and alkynyl-S—) group attached to the parent molecular moiety through a sulfur atom. Examples of thioalkoxyl moieties include, but are not limited to, methylthio, ethylthio, propylthio, isopropylthio, n-butylthio, and the like.
  • “Acylamino” refers to an acyl-NH— group wherein acyl is as previously described. “Aroylamino” refers to an aroyl-NH— group wherein aroyl is as previously described.
  • The term “carbonyl” refers to the —(C═O)— group. The term “carboxyl” refers to the —COOH group. Such groups also are referred to herein as a “carboxylic acid” moiety.
  • The terms “halo,” “halide,” or “halogen” as used herein refer to fluoro, chloro, bromo, and iodo groups.
  • The term “hydroxyl” refers to the —OH group.
  • The term “hydroxyalkyl” refers to an alkyl group substituted with an —OH group.
  • The term “mercapto” refers to the —SH group.
  • The term “oxo” refers to a compound described previously herein wherein a carbon atom is replaced by an oxygen atom.
  • The term “nitro” refers to the —NO2 group.
  • The term “thio” refers to a compound described previously herein wherein a carbon or oxygen atom is replaced by a sulfur atom.
  • The term “sulfate” refers to the —SO4 group.
  • The term thiohydroxyl or thiol, as used herein, refers to a group of the formula —SH.
  • The term ureido refers to a urea group of the formula —NH—CO—NH2.
  • Throughout the specification and claims, a given chemical formula or name shall encompass all tautomers, congeners, and optical- and stereoisomers, as well as racemic mixtures where such isomers and mixtures exist.
  • As used herein the term “monomer” refers to a molecule that can undergo polymerization, thereby contributing constitutional units to the essential structure of a macromolecule or polymer.
  • A “polymer” is a molecule of high relative molecule mass, the structure of which essentially comprises the multiple repetition of unit derived from molecules of low relative molecular mass, i.e., a monomer.
  • As used herein, an “oligomer” includes a few monomer units, for example, in contrast to a polymer that potentially can comprise an unlimited number of monomers. Dimers, trimers, and tetramers are non-limiting examples of oligomers.
  • Further, as used herein, the term “nanoparticle,” refers to a particle having at least one dimension in the range of about 1 nm to about 1000 nm, including any integer value between 1 nm and 1000 nm (including about 1, 2, 5, 10, 20, 50, 60, 70, 80, 90, 100, 200, 500, and 1000 nm and all integers and fractional integers in between). In some embodiments, the nanoparticle has at least one dimension, e.g., a diameter, of about 100 nm. In some embodiments, the nanoparticle has a diameter of about 200 nm. In other embodiments, the nanoparticle has a diameter of about 500 nm. In yet other embodiments, the nanoparticle has a diameter of about 1000 nm (1 μm). In such embodiments, the particle also can be referred to as a “microparticle. Thus, the term “microparticle” includes particles having at least one dimension in the range of about one micrometer (μm), i.e., 1×10−6 meters, to about 1000 μm. The term “particle” as used herein is meant to include nanoparticles and microparticles.
  • It will be appreciated by one of ordinary skill in the art that nanoparticles suitable for use with the presently disclosed methods can exist in a variety of shapes, including, but not limited to, spheroids, rods, disks, pyramids, cubes, cylinders, nanohelixes, nanosprings, nanorings, rod-shaped nanoparticles, arrow-shaped nanoparticles, teardrop-shaped nanoparticles, tetrapod-shaped nanoparticles, prism-shaped nanoparticles, and a plurality of other geometric and non-geometric shapes. In particular embodiments, the presently disclosed nanoparticles have a spherical shape.
  • The subject treated by the presently disclosed methods in their many embodiments is desirably a human subject, although it is to be understood that the methods described herein are effective with respect to all vertebrate species, which are intended to be included in the term “subject.” Accordingly, a “subject” can include a human subject for medical purposes, such as for the treatment of an existing condition or disease or the prophylactic treatment for preventing the onset of a condition or disease, or an animal subject for medical, veterinary purposes, or developmental purposes. Suitable animal subjects include mammals including, but not limited to, primates, e.g., humans, monkeys, apes, and the like; bovines, e.g., cattle, oxen, and the like; ovines, e.g., sheep and the like; caprines, e.g., goats and the like; porcines, e.g., pigs, hogs, and the like; equines, e.g., horses, donkeys, zebras, and the like; felines, including wild and domestic cats; canines, including dogs; lagomorphs, including rabbits, hares, and the like; and rodents, including mice, rats, and the like. An animal may be a transgenic animal. In some embodiments, the subject is a human including, but not limited to, fetal, neonatal, infant, juvenile, and adult subjects. Further, a “subject” can include a patient afflicted with or suspected of being afflicted with a condition or disease. Thus, the terms “subject” and “patient” are used interchangeably herein.
  • “Associated with”: When two entities are “associated with” one another as described herein, they are linked by a direct or indirect covalent or non-covalent interaction. Preferably, the association is covalent. Desirable non-covalent interactions include hydrogen bonding, van der Waals interactions, hydrophobic interactions, magnetic interactions, electrostatic interactions, etc.
  • “Biocompatible”: The term “biocompatible”, as used herein is intended to describe compounds that are not toxic to cells. Compounds are “biocompatible” if their addition to cells in vitro results in less than or equal to 20% cell death, and their administration in vivo does not induce inflammation or other such adverse effects.
  • “Biodegradable”: As used herein, “biodegradable” compounds are those that, when introduced into cells, are broken down by the cellular machinery or by hydrolysis into components that the cells can either reuse or dispose of without significant toxic effect on the cells (i.e., fewer than about 20% of the cells are killed when the components are added to cells in vitro). The components preferably do not induce inflammation or other adverse effects in vivo. In certain preferred embodiments, the chemical reactions relied upon to break down the biodegradable compounds are uncatalyzed.
  • “Effective amount”: In general, the “effective amount” of an active agent or drug delivery device refers to the amount necessary to elicit the desired biological response. As will be appreciated by those of ordinary skill in this art, the effective amount of an agent or device may vary depending on such factors as the desired biological endpoint, the agent to be delivered, the composition of the encapsulating matrix, the target tissue, and the like.
  • “Peptide” or “protein”: A “peptide” or “protein” comprises a string of at least three amino acids linked together by peptide bonds. The terms “protein” and “peptide” may be used interchangeably. Peptide may refer to an individual peptide or a collection of peptides. Inventive peptides preferably contain only natural amino acids, although non-natural amino acids (i.e., compounds that do not occur in nature but that can be incorporated into a polypeptide chain) and/or amino acid analogs as are known in the art may alternatively be employed. Also, one or more of the amino acids in an inventive peptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc. In a preferred embodiment, the modifications of the peptide lead to a more stable peptide (e.g., greater half-life in vivo). These modifications may include cyclization of the peptide, the incorporation of D-amino acids, etc. None of the modifications should substantially interfere with the desired biological activity of the peptide.
  • “Polynucleotide” or “oligonucleotide”: Polynucleotide or oligonucleotide refers to a polymer of nucleotides. Typically, a polynucleotide comprises at least three nucleotides. The polymer may include natural nucleosides (i.e., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, C5-propynylcytidine, C5-propynyluridine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, and 2-thiocytidine), chemically modified bases, biologically modified bases (e.g., methylated bases), intercalated bases, modified sugars (e.g., 2′-fluororibose, ribose, 2′-deoxyribose, arabinose, and hexose), or modified phosphate groups (e.g., phosphorothioates and 5′-N-phosphoramidite linkages).
  • “Small molecule”: As used herein, the term “small molecule” refers to organic compounds, whether naturally-occurring or artificially created (e.g., via chemical synthesis) that have relatively low molecular weight and that are not proteins, polypeptides, or nucleic acids. Typically, small molecules have a molecular weight of less than about 1500 g/mol. Also, small molecules typically have multiple carbon-carbon bonds. Known naturally-occurring small molecules include, but are not limited to, penicillin, erythromycin, taxol, cyclosporin, and rapamycin. Known synthetic small molecules include, but are not'limited to, ampicillin, methicillin, sulfamethoxazole, and sulfonamides.
  • By “analog” is meant a chemical compounds having a structure that is different from the general structure of a reference agent, but that functions in a manner similar to the reference agent. For example, a peptide analog having a variation in sequence or having a modified amino acid.
  • By “thrombospondin (TSP) derived peptide” is meant a peptide comprising a TSP motif: W-X(2)-C-X(3)-C-X(2)-G. Exemplary TSP derived peptides are shown in Tables 1 and 2. If desired, the peptide includes at least about 5, 10, 20, 30, 40, 50 or more amino acids that flank the carboxy or amino terminus of the motif in the naturally occurring amino acid sequence of the peptide. TSP1 derived peptides include, for example, those derived from proteins WISP-1
    • (SPWSPCSTSCGLGVSTRI), NOVH(TEWTACSKSCGMGFSTRV) and UNC5C (TEWSVCNSRCGRGYQKRTR).
  • By “CXC derived peptide” is meant a peptide comprising a CXC Motif: G-X(3)-C-L. Exemplary CXC derived peptides are shown in Table 3. If desired, the peptide includes at least about 5, 10, 20, 30, 40, 50 or more amino acids that flank the carboxy or amino terminus of the motif in the naturally occurring amino acid sequence. CXC derived peptides include, for example, those derived from proteins GRO-α/CXCL1 (NGRKACLNPASPIVKKIIEKMLNS), GRO-γ/MIP-21β/CXCL3 (NGKKACLNPASPMVQKEEKIL), and ENA-78/CXCL5 (NGKEICLDPEAPFLKKVIQKILD).
  • By “Collagen IV derived peptide” is meant a peptide comprising a C—N—X(3)-V-C or P—F-X(2)-C collagen motif. Exemplary collagen IV derived peptides are shown in Table 5. If desired, the peptide includes at least about 5, 10, 20, 30, 40, 50 or more amino acids that flank the carboxy or amino terminus of the motif in the naturally occurring amino acid sequence. Type IV collagen derived peptides include, for example, LRRFSTMPFMFCNINNVCNF and FCNINNVCNFASRNDYSYWL, and LPRFSTMPFIYCNINEVCHY.
  • By “Somatotropin derived peptide” is meant a peptide comprising a Somatotropin Motif: L-X(3)-L-L-X(3)-S—X-L. Exemplary somatotropin derived peptides are shown in Table 8. If desired, the peptide includes at least about 5, 10, 20, 30, 40, 50 or more amino acids that flank the carboxy or amino terminus of the motif in the naturally occurring amino acid sequence.
  • By “Serpin derived peptide” is meant a peptide comprising a Serpin Motif: L-X(2)-E-E-X—P. Exemplary serpin derived peptides are shown in Table 9. If desired, the peptide includes at least about 5, 10, 20, 30, 40, 50 or more amino acids that flank the carboxy or amino terminus of the motif in the naturally occurring amino acid sequence.
  • By “Beta 1 integrin” is meant a polypeptide that binds a collagen IV derived peptide or that has at least about 85% identity to NP_596867 or a fragment thereof.
  • By “Beta 3 integrin” is meant a polypeptide that binds a collagen IV derived peptide or that has at least about 85% identity to P05106 or a fragment thereof.
  • By “CD36” is meant a CD36 glycoprotein that binds to a thrombospondin-derived peptide or that has at least about 85% identity to NP_001001548 or a fragment thereof. CD36 is described, for example, by Oquendo et al., “CD36 directly mediates cytoadherence of Plasmodium falciparum parasitized erythrocytes,” Cell 58: 95-101, 1989.
  • By “CD47” is meant a CD47 glycoprotein that binds to a thrombospondin-derived peptides or that has at least about 85% identity to NP_000315 or a fragment thereof. CD47 is described, for example, by Han et al., “CD47, a ligand for the macrophage fusion receptor, participates in macrophage multinucleation.” J. Biol. Chem. 275: 37984-37992, 2000.
  • By “CXCR3” is meant a G protein coupled receptor or fragment thereof having at least about 85% identity to NP_001495. CXCR3 is described, for example, by Trentin et al., “The chemokine receptor CXCR3 is expressed on malignant B cells and mediates chemotaxis.” J. Clin. Invest. 104: 115-121, 1999.
  • By “blood vessel formation” is meant the dynamic process that includes one or more steps of blood vessel development and/or maturation, such as angiogenesis, vasculogenesis, formation of an immature blood vessel network, blood vessel remodeling, blood vessel stabilization, blood vessel maturation, blood vessel differentiation, or establishment of a functional blood vessel network.
  • By “angiogenesis” is meant the growth of new blood vessels originating from existing blood vessels. Angiogenesis can be assayed by measuring the total length of blood vessel segments per unit area, the functional vascular density (total length of perfused blood vessel per unit area), or the vessel volume density (total of blood vessel volume per unit volume of tissue).
  • By “vasculogenesis” is meant the development of new blood vessels originating from stem cells, angioblasts, or other precursor cells.
  • By “blood vessel stability” is meant the maintenance of a blood vessel network.
  • By “alteration” is meant a change in the sequence or in a modification (e.g., a post-translational modification) of a gene or polypeptide relative to an endogeneous wild-type reference sequence.
  • By “ameliorate” is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.
  • By “antibody” is meant any immunoglobulin polypeptide, or fragment thereof, having immunogen binding ability.
  • In this disclosure, “comprises,” “comprising,” “containing” and “having” and the like can have the meaning ascribed to them in U.S. Patent law and can mean “includes,” “including,” and the like; “consisting essentially of or “consists essentially” likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.
  • A “cancer” in an animal refers to the presence of cells possessing characteristics typical of cancer-causing cells, for example, uncontrolled proliferation, loss of specialized functions, immortality, significant metastatic potential, significant increase in anti-apoptotic activity, rapid growth and proliferation rate, and certain characteristic morphology and cellular markers. In some circumstances, cancer cells will be in the form of a tumor; such cells may exist locally within an animal, or circulate in the blood stream as independent cells, for example, leukemic cells.
  • By “disease” is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ.
  • By “fragment” is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.
  • By “isolated nucleic acid molecule” is meant a nucleic acid (e.g., a DNA) that is free of the genes, which, in the naturally occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule which is transcribed from a DNA molecule, as well as a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.
  • By an “isolated polypeptide” is meant a polypeptide of the invention that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the invention. An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.
  • By “marker” is meant any protein or polynucleotide having an alteration in expression level or activity that is associated with a disease or disorder.
  • “By “neoplasia” is meant a disease that is caused by or results in inappropriately high levels of cell division, inappropriately low levels of apoptosis, or both. Solid tumors, hematological disorders, and cancers are examples of neoplasias.
  • By “operably linked” is meant that a first polynucleotide is positioned adjacent to a second polynucleotide that directs transcription of the first polynucleotide when appropriate molecules (e.g., transcriptional activator proteins) are bound to the second polynucleotide.
  • By “peptide” is meant any fragment of a polypeptide. Typically peptide lengths vary between 5 and 1000 amino acids (e.g., 5, 10, 15, 20, 25, 50, 100, 200, 250, 500, 750, and 1000).
  • By “polypeptide” is meant any chain of amino acids, regardless of length or post-translational modification.
  • By “promoter” is meant a polynucleotide sufficient to direct transcription. By “reduce” is meant a decrease in a parameter (e.g., blood vessel formation) as detected by standard art known methods, such as those described herein. As used herein, reduce includes a 10% change, preferably a 25% change, more preferably a 40% change, and even more preferably a 50% or greater change.
  • By “reference” is meant a standard or control condition.
  • By “substantially identical” is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and even more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.
  • Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e−3 and e−100 indicating a closely related sequence.
  • “Sequence identity” or “identity” in the context of two nucleic acid or polypeptide sequences includes reference to the residues in the two sequences which are the same when aligned for maximum correspondence over a specified comparison window, and can take into consideration additions, deletions and substitutions. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (for example, charge or hydrophobicity) and therefore do not deleteriously change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences which differ by such conservative substitutions are said to have sequence similarity. Approaches for making this adjustment are well-known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, for example, according to the algorithm of Meyers and Miller, Computer Applic. Biol. Sci., 4: 11-17, 1988, for example, as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif., USA).
  • “Percentage of sequence identity” means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions, substitutions, or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions, substitutions, or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • The term “substantial identity” or “homologous” in their various grammatical forms in the context of polynucleotides means that a polynucleotide comprises a sequence that has a desired identity, for example, at least 60% identity, preferably at least 70% sequence identity, more preferably at least 80%, still more preferably at least 90% and even more preferably at least 95%, compared to a reference sequence using one of the alignment programs described using standard parameters. One of skill will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like. Substantial identity of amino acid sequences for these purposes normally means sequence identity of at least 60%, more preferably at least 70%, 80%, 85%, 90%, and even more preferably at least 95%.
  • Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions. However, nucleic acids which do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This may occur, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. One indication that two nucleic acid sequences are substantially identical is that the polypeptide which the first nucleic acid encodes is immunologically cross reactive with the polypeptide encoded by the second nucleic acid, although such cross-reactivity is not required for two polypeptides to be deemed substantially identical.
  • An “expression vector” is a nucleic acid construct, generated recombinantly or synthetically, bearing a series of specified nucleic acid elements that enable transcription of a particular gene in a host cell. Typically, gene expression is placed under the control of certain regulatory elements, including constitutive or inducible promoters, tissue-preferred regulatory elements, and enhancers.
  • A “recombinant host” may be any prokaryotic or eukaryotic cell that contains either a cloning vector or expression vector. This term also includes those prokaryotic or eukaryotic cells that have been genetically engineered to contain the cloned gene(s) in the chromosome or genome of the host cell.
  • The term “operably linked” is used to describe the connection between regulatory elements and a gene or its coding region. That is, gene expression is typically placed under the control of certain regulatory elements, including constitutive or inducible promoters, tissue-specific regulatory elements, and enhancers. Such a gene or coding region is said to be “operably linked to” or “operatively linked to” or “operably associated with” the regulatory elements, meaning that the gene or coding region is controlled or influenced by the regulatory element.
  • A “reference sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least about 5, 10, or 15 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, about 100 amino acids, or about 150 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides about 300 nucleotides or about 450 nucleotides or any integer thereabout or therebetween.
  • Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math., 2: 482, 1981; by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol., 48: 443, 1970; by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 8: 2444, 1988; by computerized implementations of these algorithms, including, but not limited to: CLUSTAL in the PC/Gene program by Intelligenetics, Mountain View, Calif., GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 7 Science Dr., Madison, Wis., USA; the CLUSTAL program is well described by Higgins and Sharp, Gene, 73: 237-244, 1988; Corpet, et al., Nucleic Acids Research, 16:10881-10890, 1988; Huang, et al., Computer Applications in the Biosciences, 8:1-6, 1992; and Pearson, et al., Methods in Molecular Biology, 24:7-331, 1994. The BLAST family of programs which can be used for database similarity searches includes: BLASTN for nucleotide query sequences against nucleotide database sequences; BLASTX for nucleotide query sequences against protein database sequences; BLASTP for protein query sequences against protein database sequences; TBLASTN for protein query sequences against nucleotide database sequences; and TBLASTX for nucleotide query sequences against nucleotide database sequences. See, Current Protocols in Molecular Biology, Chapter 19, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York, 1995. New versions of the above programs or new programs altogether will undoubtedly become available in the future, and can be used with the present invention.
  • Unless otherwise stated, sequence identity/similarity values provided herein refer to the value obtained using the BLAST 2.0 suite of programs, or their successors, using default parameters (Altschul et al., Nucleic Acids Res, 2:3389-3402, 1997). It is to be understood that default settings of these parameters can be readily changed as needed in the future.
  • As those ordinary skilled in the art will understand, BLAST searches assume that proteins can be modeled as random sequences. However, many real proteins comprise regions of nonrandom sequences which may be homopolymeric tracts, short-period repeats, or regions enriched in one or more amino acids. Such low-complexity regions may be aligned between unrelated proteins even though other regions of the protein are entirely dissimilar. A number of low-complexity filter programs can be employed to reduce such low-complexity alignments. For example, the SEG (Wooten and Federhen, Comput. Chem., 17:149-163, 1993) and XNU (Clayerie and States, Comput. Chem., 17:191-1, 1993) low-complexity filters can be employed alone or in combination.
  • As used herein, the terms “treat,” “treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.
  • A “tumor,” as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues.
  • As used herein, the terms “prevent,” “preventing,” “prevention,” “prophylactic treatment” and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.
  • Following long-standing patent law convention, the terms “a,” “an,” and “the” refer to “one or more” when used in this application, including the claims. Thus, for example, reference to “a subject” includes a plurality of subjects, unless the context clearly is to the contrary (e.g., a plurality of subjects), and so forth.
  • Throughout this specification and the claims, the terms “comprise,” “comprises,” and “comprising” are used in a non-exclusive sense, except where the context requires otherwise. Likewise, the term “include” and its grammatical variants are intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items.
  • For the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing amounts, sizes, dimensions, proportions, shapes, formulations, parameters, percentages, parameters, quantities, characteristics, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about” even though the term “about” may not expressly appear with the value, amount or range. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are not and need not be exact, but may be approximate and/or larger or smaller as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art depending on the desired properties sought to be obtained by the presently disclosed subject matter. For example, the term “about,” when referring to a value can be meant to encompass variations of, in some embodiments, ±100% in some embodiments ±50%, in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods or employ the disclosed compositions.
  • Further, the term “about” when used in connection with one or more numbers or numerical ranges, should be understood to refer to all such numbers, including all numbers in a range and modifies that range by extending the boundaries above and below the numerical values set forth. The recitation of numerical ranges by endpoints includes all numbers, e.g., whole integers, including fractions thereof, subsumed within that range (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5, as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like) and any range within that range.
    • EXAMPLES
  • The following Examples have been included to provide guidance to one of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill can appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter. The following Examples are offered by way of illustration and not by way of limitation.
    • Example 1 Methods Synthesis of BR6
  • All chemicals were purchased from Sigma-Aldrich Chemical Co. (St. Louis, Mo., USA) and used without further purification. Bis(2-hydroxyethyl)disulfide (15.4 g, 10 mmol) and triethylamine (TEA, 37.5 mL, 300 mmol) were dissolved in 450 mL of tetrahydrofuran previously dried with NaSO4 in a 1000 ml round bottom flask. The flask was flushed with N2 for 10 min and then maintained under a N2 environment. Acryloyl chloride (24.4 mL, 300 mmol) was dissolved in 50 mL tetrahydrofuran then added to the flask dropwise over 2 hrs while stirring. The reaction was carried out for 24 hrs, then the TEA HCl precipitate was removed by filtration, and the solvent was removed by rotary evaporation. The product was dissolved in 100 mL dichloromethane and washed five times with 200 mL of an aqueous solution of 0.2 M Na2CO3 and three times with distilled water. The solution was dried with NaSO4 and the solvent was removed by rotary evaporation.
    • Polymer Synthesis
  • Base monomer BR6 was polymerized with side chain monomers S3, S4, and S5 at a base:side chain ratio of 1.2:1 by weight without solvent at 90° C. for 24 hrs while stirring. For end-capping with E10, base polymer was dissolved in anhydrous dimethyl sulfoxide at 100 mg/mL with 0.2 mM end-cap. The reaction was allowed to proceed for 1 hr at room temperature while shaking.
    • Example 2 Characteristics of Representative Polymer/Peptide Nanoparticles
  • Referring now to FIG. 5 are shown representative formation and sizing of polymer/peptide nanoparticles (by nanoparticle tracking analysis on a Nanosight LM10). Selected peptides and PBAEs were diluted in 25 mM sodium acetate buffer and then together in different weight-to-weight ratios. In some embodiments w/w is unity, 1:1, in other embodiments there is an excess of polymer to peptide. In some embodiments this ratio is 5:1, in other embodiments between 1:1-10:1, in other embodiments it is 10:1 to 20:1. In FIG. 6, both a 5:1 and 1:1 ratio is shown. The mixtures were incubated at room temperature for up to 10 minutes to allow for self-assembly and then loaded into the NanoSight laser cell. Using NanoSight nanoparticle tracking software and analysis, individual particles were tracked in order to determine the average size distribution of the particles. In the case of more hydrophilic peptides (DEAH Box poly8) and PBAEs (336), there were very few background particles, that is very few particles of peptide or PBAE only. However, when self-assembled, a noticeable nanoparticle distribution was observed, with an average size ranging from 100-150 nm. In the case of hydrophobic peptides and PBAEs, they have the possibility of aggregating with themselves. In the case of the peptide-PBAE mixture, a shift in the mean can be observed as a way to detect difference in nanoparticle formation.
  • Referring now to FIG. 6, is shown DEAH peptide release by 336 nanoparticles at 4° C. (above) and 37° C. (below). Changing polymer to peptide formulation ratios and concentrations are key to tune release. Slowing the reaction rate of degradation of the liable polymer bonds extends release from nanoparticles. This is shown by change in temperature, but could also be accomplished by increasing hydrophobicity of the polymer, increasing the molecular weight between liable ester groups, or other modifications known by someone in the art; FITC labeled DEAH peptide and 336 polymer were mixed and incubated for up to 10 minutes in sodium acetate buffer. Mixtures at different peptide concentrations, but constant polymer to peptide ratios, as well as peptide only were added to a 96-well plate. Fluorescence measurements were obtained using a plate reader and measured over time. The plates were kept either at 4° C. or 37° C.
  • Referring now to FIG. 7, is shown HUVEC viability/proliferation assays with polymer/SP6001/DEAH peptide; the CellTiter 96® AQueous One Solution Cell Proliferation assay was used to see the effect of both peptide and polymer on cell proliferation and viability. Polymers at the right concentrations have minimal cytotoxic effect on the cells, such as 336 below 100 uM. The individual peptides and Polymers were diluted in sodium acetate buffer and added to HUVECs in a 96-well plate. After incubating for a few days, the assay substrate was added and then incubated for a few hours at 37° C. Absorbance measurements were performed using a plate reader.
  • Referring now to FIG. 8 is shown HUVEC migration assays with 336 polymer/DEAH peptide. These nanoparticles inhibit endothelial migration in addition to proliferation and viability. Peptide-polymer nanoparticles were made as described previously. Samples were added to HUVEC cells and migration was measured using the ACEA time course cell migration system. Nanoparticle formulations at a total peptide concentration of 20 uM were able to inhibit migration more than any peptide only at 20 μM.
  • Referring now to FIG. 9 is shown in vivo 336 polymer nanoparticle/SP6001 DEAH peptide; Peptide-336 polymer nanoparticles were formulated as previously described and intravitreously injected to test in vivo efficacy. ACNV laser mouse model was used on C57 BL/6 female mice. The mice receive laser eye treatments on day zero, followed by the intravitreous injections. Mice are then perfused with fluorescein labeled dextran on day 14 and choroidal flat mounts (bottom) were analyzed via fluorescence microscopy. On day 14, both the peptide only and nanoparticles formulations significantly reduced angiogenesis in the eye (top) and did so to a similar extent. This suggests that all peptide was released from nanoparticles by day 14.
  • Referring now to FIG. 10 is shown (top) Particle size and (bottom) cell viability effects of various polymer/SP2012 nanoparticles as compared to peptide only of non-cytotoxic polymers; a range of polymer structures were mixed with SP2000 series peptides, in a similar manner as described above. Similar sizing is found with peptides from the same class with similar structural properties. For example, SP2000, SP2012, SP2024, SP2034, and SP2036 can be encapsulated similarly to each other with the same polymers, but different from peptides from other classes such as SP6001. Sizing was performed using the Malzern Zetasizer. Size strongly depends on polymer choice. Using the same cell viability assay as described previously, effects of nanoparticle vs. peptide only on HUVECs in a 96-well plate. Non-cytotoxic polymers are shown here. Referring once again to FIG. 10, the (top) panel suggests that some of these peptide-polymer formulations have an increased effect on HUVEC cell proliferation and viability (y-axis ratio less than one) as compared to peptide only. (Data also are normalized to any polymer-only effects. Pep-pol/pol/SP2012 refers to the change in cell proliferation/viability due to the peptide/polymer nanoparticle formulation divided by any change in cell proliferation/viability from the same dose of polymer by itself and this quantity divided by the change in cell proliferation/viability by delivering the same amount of peptide SP2012 as a bolus);
  • FIG. 11 shows polymer/peptide formulations for alternative peptides. Peptide-polymer formulations made as described previously. Here two different classes of peptides are used. Experiments performed in a 96-well plate, with final results obtained using the same cell viability/proliferation assay as described previously. An increased effect (decreased metabolic activity) is observed for the nanoparticle formulations over the free peptide.
    • Example 3 Hydrogels for Protein/Peptide Release
  • As shown in FIG. 12, FITC-tagged bovine serum albumin (BSA) was mixed with a macromer solution containing 10% (w/v) PEGDA (Mn-270 Da) with various amounts of B4S4, dissolved in a 1:1 (v/v) mixture of DMSO and PBS. Irgacure 2959 was added at 0.05% (w/v), and the solution was briefly vortexed and immediately polymerized to form gels. The gels were incubated at 37° C. in 1×PBS with shaking. PBS was removed at each time point to measure fluorescence.
  • The observed slowed release is due to two factors: first, increased overall hydrophobicity can decrease the movement of water in and out of the gel, reducing degradation rate and protein release. Furthermore, this method of mixing relatively hydrophobic diacrylates with hydrophilic diacrylates in a co-solvent (mixture of water and DMSO) that can dissolve both types of polymer causes the spontaneous formation of micro-emulsions within the gel (see SEM in FIG. 13; increasing B4S4 from top [0.2% w/w] to bottom [5% w/w]). Similar to traditionally studied controlled-release microparticles, these microparticles within photopolymerized gels could serve as another way to tune the release of an encapsulated peptide, protein, or drug.
    • Example 4 Stable Formulations
  • In this formulation nanoparticles were formed by mixing PBAE and DNA in 25 mM sodium acetate buffer (pH 5) at a 30:1 polymer:DNA ratio (w/w). After 10 min of incubation, sucrose solution was added at various concentrations. The particles were mixed, then frozen at −80° C. for 1 hr and lyophilized for 48 hr. They then were used for transfection or sizing or were stored at either room temperature, 4° C. or −20° C. and tested at various timepoints.
  • Referring now to FIG. 14, the size distribution of appropriately freeze-dried particles (bottom left, right-most histogram) remains the same as freshly-prepared particles (bottom left, left-most histogram). Freeze-dried particles also remain more stable in serum-containing medium than freshly-prepared particles (upper left). Using DNA-loaded nanoparticles, transfection efficiency is comparable between fresh particles and particles lyophilized with sucrose (right) even after 3 months of storage. Modifying type of sugar and concentration of sugar modulates the stability of the degradable nanoparticles.
    • Example 5 Inclusion of Lyophilized Nanoparticles into Pellets/Scaffolds
  • For coating of natural or pre-made synthetic scaffolds, DNA nanoparticles were prepared by mixing DNA and polymer in a sodium acetate buffer. Sucrose was added for a final concentration of 15 mg/mL, and the solution was used to coat the surface of a trabecular bone construct. This construct was then lyophilized for 2 days before being seeded with primary human cells (−50% GFP+ for ease of visualization). Referring now to FIG. 15, DsRed expression was observed within 24 hr, indicating that the nanoparticles remained functional and able to transfect cells in this new system.
  • Lyophilized nanoparticles also can be mixed with PLGA microparticles to form a larger construct that can be more easily manipulated and also can tune controlled release properties. In this embodiment, DsRed DNA-containing nanoparticles were compressed into a pellet with PLGA microparticles. This pellet was then placed within a well containing primary human glioblastoma cells (−20% GFP+ for ease of visualization through the opaque pellet). Referring now to FIG. 16, DsRed expression was observed within 4 days and remained very robust even after 12 days. Referring once again to FIG. 16, top=1 day, middle=4 days, bottom=12 days after transfection.
  • Further, as demonstrated in FIG. 17, DNA-loaded nanoparticles have been incorporated into natural and synthetic scaffolds, disks, microparticles, and hydrogels.
    • Example 6 Bioreducible Polymeric Particle Formulations for Delivery of siRNA
  • Reducible functional groups mediate successful siRNA-delivery, including transfection. In this example, GFP+ primary human glioblastoma cells were seeded in 96-well plates at a density of 104 cells/well in complete culture medium (DMEM/F-12 with 10% FBS and 1% antibiotic-antimycotic) and allowed to adhere overnight. Just before transfection, the culture medium was changed to serum-free medium. Particles were prepared by diluting polymer and siRNA both in 25 mM sodium acetate buffer (pH 5), then mixing them at a 100:1 polymer:siRNA ratio (w/w). Nanoparticles formed spontaneously after 10 min of incubation and were added to the cells in medium at a 1:5 ratio (v/v) and a final concentration of 60 nM. Each polymer/siRNA treatment group was paired with a control group using a scrambled siRNA sequence (scrRNA). Cells were incubated with the particles for 4 hr. The medium and particles were then aspirated and replaced with complete medium. On each of the following days, GFP expression was measured using a Synergy 2 multiplate fluorescence reader (Biotek). Background fluorescence was measured from GFP cells in medium and was subtracted from all other readings. Knockdown was calculated by normalizing GFP fluorescence (excitation 485 nm, emission 528 nm) from the siRNA-treated cells to the scrRNA-treated cells. Medium was changed every 3 days.
  • The reducible disulfide bond in the endgroup E10 (cystamine dihydrochloride) drastically improves siRNA delivery and gene knockdown. Referring now to FIG. 18, GFP+ glioblastoma cells were transfected with scrambled (control) siRNA (top panels) or siRNA against GFP (bottom). The polymers used as transfection agents consisted of B3-S5 at a 1.1:1 molar ratio, endcapped with (from left to right) E10, E3 (1,3=diaminopentane), or E6 (2-(3-aminopropylamino)ethanol). With the endgroups tested, the base polymer B3-S5 was able to achieve up to 8% knockdown; with E10 as the endgroup, over 80% knockdown was observed.
  • Referring now to FIGS. 1A-1C, the activity of R6-series polymers at delivering siRNA to knockdown GFP signal is GB cells is further demonstrated. % Knockdown of GFP expression in GFP+ glioblastoma cells transfected with siRNA against GFP, normalized to cells transfected with scrambled siRNA, using various BR6 polymers as a transfection agent. (A) Transfection with acrylate-terminated BR6 polymers with either S3, S4 or S5 as the side chain; (B) Transfection with E10 end-capped versions of the polymers in Figure A; and (C) GFP fluorescence images of cells transfected with BR6-S4-Ac complexed scrambled RNA (top) vs. siRNA against GFP (bottom);
  • Without wishing to be bound to any one particular theory, it is likely that E10 facilitates siRNA delivery by augmenting intracellular release because it degrades in the reducing intracellular environment. Results from gel retardation assay supports this hypothesis. Gel retardation assays were carried out by adding polymer of varying concentrations in sodium acetate buffer to a constant concentration of siRNA in sodium acetate. After 10 min of incubation, a solution of 30% glycerol in water was added at a 1:5 volumetric ratio as a loading buffer. Bromophenol blue or other dyes were not added, as they were found to interfere with binding. Samples were loaded into a 1% agarose gel with 1 μg/mL ethidium bromide at 125 ng siRNA per well. Samples were run for 15 min under 100 V, then visualized using UV exposure.
  • Referring now to FIG. 20, a gel retardation assay of siRNA with BR6-S5-E10 at varying ratios of polymer to RNA is shown. The polymer effectively retards siRNA (top), but in the presence of 5 mM glutathione siRNA is released immediately (bottom). These data demonstrate the hypothesized intracellular release of siRNA and elucidates the mechanism by which nanoparticles formed using BR6 facilitate strong siRNA transfection and GFP knockdown.
  • Referring also to FIG. 21, an E10-endcapped polymer (top) retards siRNA efficiently, but upon addition of 5 mM glutathione, siRNA is immediately released (bottom). Numbers refer to the w/w ratio of polymer-to-siRNA in all cases.
  • Referring now to FIG. 22, the same polymer as in FIG. 21, but with a different endcap (E7, 1-(3-aminopropyl)-4-methylpiperazine) also retards siRNA (top), but is not affected by application of glutathione (bottom).
  • Referring now to FIG. 23, gel permeation chromatography data of BR6 polymerized with S4 at a BR6:S4 ratio of 1.2:1 at 90° C. for 24 hours, before and after end-capping with E7, are provided.
  • Referring now to FIG. 24, knockdown efficiency also is affected by molecular weight of the polymer. In FIG. 24, 1.2:1, 1.1:1, and 1.05:1 refer to the ratio of reactants in the base polymer step growth reaction, which affects the ultimate molecular weight. Top 4310 formulations were able to achieve greater knockdown over time compared to commercially available reagents like Lipofectamine 2000 (Lipo).
  • Referring now to FIG. 25, combined DNA (RFP) and siRNA delivery (against GFP) in GB; GFP+GB cells were treated with scrambled siRNA (top) or siRNA against GFP (bottom), causing visible knockdown. Interestingly, different polymer structures seem ideal for siRNA versus DNA delivery or for both. One polymer effective in both was used to deliver both siRNA against GFP and plasmid DsRed DNA to GFP+ hMSCs, resulting in the ability to turn green cells red.
  • Referring now to FIG. 26, siRNA knockdown is affected by the endcap (E), base polymer (increasing hydrophobicity from L to R within each E), and molecular weight (increasing L to R within each base polymer). One endcap that shows high knockdown even at lower molecular weights is E10, which is strikingly more effective than the other endcaps tested for the same base polymers. Other PBAEs were also highly effective when synthesized at high molecular weight.
  • Referring now to FIG. 27, is shown 4410, 200 w/w (blue line on above graph), 8 days after transfection: Left: hMSCs treated with scrambled control; Right: hMSCs treated with siRNA.
  • Referring now to FIG. 28, in some embodiments, polymer molecular weight is between 4.00-10.00 kDa for siRNA delivery.
    • Example 7 DNA Delivery
  • Referring now to FIG. 29, the presently disclosed biomaterial can be used for other forms of delivery, for example DNA delivery. DNA transfection shows some similar trends compared with siRNA, but with different optimal endcaps. Specific polymer structure is critical to determine which polymers are effective for DNA delivery or siRNA delivery or both. Both DNA and siRNA transfection depend less on MW with high polymer hydrophobicity. High GFP DNA delivery was achieved using PBAEs, with transfection in 10% serum and at 5 μg DNA/mL. Referring now to FIG. 30, several formulations with up to 90% transfection and high (>90%) viability are shown.
  • Referring now to FIG. 31, GB transfection is demonstrated. More particularly, 551 GB cells cultured as neurospheres (undifferentiated). They were plated in monolayer on laminin 24 hr before transfection with DsRed DNA using 447 LG (red). 48 hr after transfection, they were stained for nestin (blue). Red and blue overlaid (left) show that transfection occurred in nestin+ cells (nestin only: right).
  • Referring now to FIG. 32, for a DNA delivery application, in some embodiments, polymer molecular weight is between 3.00-10.0 kDa.
    • Example 8 In Vivo Activity for Selected Peptides
  • In some embodiments, the presently disclosed subject matter demonstrates in vivo activity for selected peptides in DIVAA angioreactors and a lung cancer xenograft model, Koskimaki J E, Karagiannis E D, Tang B C, Hammers H, Watkins D N, Pili R, et al. Pentastatin-1, a collagen IV derived 20-mer peptide, suppresses tumor growth in a small cell lung cancer xenograft model. BMC Cancer 2010; 10:29, and in a breast cancer xenograft model using MDA-MB-231 cells. Koskimaki J E, Karagiannis E D, Rosca E V, Vesuna F, Winnard P T, Jr., Raman V, et al. Peptides derived from type IV collagen, CXC chemokines, and thrombospondin-1 domain-containing proteins inhibit neovascularization and suppress tumor growth in MDA-MB-231 breast cancer xenografts. Neoplasia 2009; 11(12):1285-91.
  • Following orthotopic inoculation of SCID mice in the mammary fat pad area using 2×106 cells, tumors grew to approximately 100 mm3 in 2 weeks; at that time 100 μL of peptide solution was injected i.p. once a day at peptide doses 10-20 mg/kg. PBS solution was injected as control. Several peptides have been found to inhibit tumor growth. See FIG. 33A. The microvessel density was determined by screening the immunohistologically stained CD31 sections. Inhibition of LEC migration in the ACEA migration assay also was determined (see FIG. 33B).
  • Representative data showing the activity of free peptide and peptide encapsulated in the presently disclosed polymeric particles are shown in FIG. 33D, which shows the metabolic activity of free peptides and peptides in polymeric particles.
    • Example 9 Non-Viral Gene Delivery for Treatment of Glioblastoma and Brain Cancer Stem Cells
  • Glioblastoma (GB) is a grade IV brain cancer as defined by the WHO and is the most common primary CNS tumor in the United States. Current treatment includes surgical resection, radiotherapy, and chemotherapy. The median survival with treatment is approximately 14 months.
  • Brain cancer stem cells (BCSCs) possess genetic and morphological features similar to neural stem cells. Small numbers of BCSCs can initiate gliomas. BCSCs are refactory to conventional anti-cancer treatments.
  • Gene delivery typically is accomplished by either vaccine-mediated or polymer mediates techniques. Virus-mediated gene delivery is highly efficient, insertional mutagenesis, and toxicity/immunogenicity. Polymer-mediated gene delivery is chemically versatile, potentially safer than vaccine-mediated gene delivery, but typically is less efficient. See Green et al., 2008. Acc. Chem. Res. 41(6):749-59; Putnam 2006. Nat. Mater. 5(6):439-51.
  • Non-viral, e.g., polymer-mediated gene delivery, can be accomplished, in some embodiments, by using poly(beta-amino esters) (PBAEs). In particular embodiments, PBAEs suitable for use in target delivery can be synthesized in a two-step reaction provided herein below in Scheme 6 and can form nanocomplexes with negatively-charged cargo (e.g., DNA, siRNA) via electrostatic interactions as disclosed, for example, in some embodiments described in International PCT Patent Application Publication No. WO/2010/132879 for “Multicomponent Degradable Cationic Polymers,” to Green et al., which is incorporated herein by reference in its entirety.
  • Figure US20160374949A9-20161229-C00038
  • In some embodiments, the presently disclosed subject matter demonstrates the delivery of DNA to GB cells, i.e., bulk tumor (non-stem cells; verifies the efficacy of the presently disclosed methods in BCSCs; demonstrates the delivery of apoptosis-inducing genes in BCSCs; provides practical considerations for translation of the presently disclosed methods; and discusses how the presently disclosed methods can be used in conjunction with other methods for treating GB.
  • The delivery of DNA to GB cells, bulk tumor (non-stem cells) and the efficacy of the presently disclosed methods to deliver DNA to BCSCs is demonstrated in FIGS. 36-39.
  • Referring now to FIG. 34, the delivery of DNA to GB bulk tumor cells is demonstrated for representative biomaterials. Referring now to FIG. 35, the transfection of genes to BCSC is demonstrated for representative presently disclosed biomaterials. FIG. 36 demonstrates the delivery of DNA to fetal (healthy) cells. FIG. 37 also demonstrates the delivery of DNA to BCSCs. The delivery of apoptosis-inducing genes in BCSCs is demonstrated in FIGS. 38 to 39.
  • These data demonstrate that PBAEs can be used for highly effective DNA delivery to GB cells, including tumor-initiating stem cells; transfection occurs even in 3D neurospheres in suspension; transfection is much less efficient in non-cancer cells (F34 fetal cells) as compared to GB cells; and transfection with secreted TRAIL causes more death in BCSCs with not significant effect on healthy cells.
  • In practical considerations for translation, for lyophilized nanoparticles, the presently disclosed methods provide an ease of preparation, e.g., only water needs to be added to the lyophilized nanoparticles, long-term storage, large, consistent batches, manipulation for uses in other devices, and stability in suspension. See scheme in FIG. 3.
  • As shown in FIG. 40, particles lyophilized with sucrose and used immediately are as effective in transfection as freshly prepared particles. Further, no loss in efficiency is observed within three months; and approximately 50% efficiency is retained after six months. The use of the presently disclosed materials and methods for long-term gene delivery is demonstrated in FIGS. 41 and 42. Other methods for treatment of GB include siRNA delivery to GB cells (FIG. 43).
  • A comparison of siRNA vs. DNA delivery in GB cells is shown in FIGS. 44 and 45. More particularly, as shown in FIG. 45, both 4410 and 447 can form complexes with DNA and siRNA; a higher weight ratio of polymer-to-nucleic acid is needed for siRNA than for DNA; E10 polymers release siRNA immediately, but not DNA, upon addition of glutathione (GSH).
  • In summary, PBAE/nucleic acid nanoparticles can be fabricated in a form that remains stable over time and allow flexibility for clinical use; PBAEs can be used for effective DNA or siRNA delivery to GB-derived BCSCs; and efficient release of cargo is necessary for effective nucleic acid delivery, especially with siRNA.
    • Example 10 Microparticles for Peptide Delivery In some embodiments, microparticles for controlled release of nanoparticles, which themselves encapsulate biological agents, are illustrated in FIGS. 14-17.
  • More particularly, FIG. 46 depicts a strategy of combining nanoparticles within microparticles to extend release further. PLGA or blends of PLGA can be combined with the presently disclosed polymers to form microparticles by double emulsion. FIG. 47 shows release of a representative peptide, DEAH-FITC, from a presently disclosed microparticle. FIG. 48 shows slow extended release from microparticles containing nanoparticles that contain peptides; FITC-DEAH peptide was first mixed with the 336 PBAE to allow for self-assembly into nanoparticles and was then mixed with BSA (middle) or not (bottom) to form an aqueous mixture. This mixture was added to a DCM-PLGA phase and sonicated to form a w/o suspension. This suspension was then added to a PVA solution and homogenized to form the final w/o/w suspension. This mixture was finally added to another PVA solution to allow for the DCM to evaporate and harden the formed microparticle. Different release profiles can potentially be obtained as seen above for the different microparticle formulations. In all cases, there is a long-term release of the peptide. Forming nanoparticles that encapsulate the peptide within the microparticles, extends the release compared to encapsulating peptide directly into microparticles (middle figure). The particles can be designed to have different release depending on the local environment (top figure). In some embodiments, release is constant over time and zero-order with respect to time (bottom figure).
  • Referring now to FIG. 49 in shown the in vivo effects of microparticle formulations in both the CNV and rhoNEGF model over time. DEAH (SP6001)-336 PBAE nanoparticle formulation made as described previously. (Top) Intravitreal injections into CNV model mice as described previously show comparable effects after 14 days, even though only small fraction of peptide is released over that time from microparticles. (Middle) and (Bottom) A genetic model of wet form of age-related macular degeneration in mice used to test long-term effect of microparticles. After 1 week (middle) comparable effects seen in reduction of angiogenesis. After 8 weeks (bottom), however, while peptide only no longer inhibits angiogenesis, the microparticle still does, as it is still releasing peptide over this time. While PLGA is used to form the microparticles used above, other polymers may be used including the synthetic polyesters and polyamides described above. In certain embodiments, blends of these polymer are combined with PLGA to form microparticles with differing environmental sensitivity and release properties; (a) the effect of microparticle (SP-6001) in CNV model mouse; (b) the effect of microparticle (SP-6001) in rhoNEGF (V6) mouse, 1 week after injection; and (c) the effect of microparticle (SP-6001) in rhoNEGF (V6) mouse, 8 weeks after injection.
    • REFERENCES
  • All publications, patent applications, patents, and other references mentioned in the specification are indicative of the level of those skilled in the art to which the presently disclosed subject matter pertains. All publications, patent applications, patents, and other references are herein incorporated by reference to the same extent as if each individual publication, patent application, patent, and other reference was specifically and individually indicated to be incorporated by reference. It will be understood that, although a number of patent applications, patents, and other references are referred to herein, such reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.
    • Chiang A C, Massague J. Molecular basis of metastasis. N Engl J Med 2008; 359(26):2814-23.
    • Gupta G P, Massague J. Cancer metastasis: building a framework. Cell 2006; 127(4):679-95.
    • Sawyers C L. Cancer: mixing cocktails. Nature 2007; 449(7165):993-6.
    • Dorrell M I, Aguilar E, Scheppke L, Barnett F H, Friedlander M. Combination angiostatic therapy completely inhibits ocular and tumor angiogenesis. Proc Natl Acad Sci USA 2007; 104(3):967-72.
    • Farokhzad O C. Nanotechnology for drug delivery: the perfect partnership. Expert Opin Drug Deliv 2008; 5(9):927-9.
    • Putnam D. Polymers for gene delivery across length scales. Nat Mater 2006; 5(6):439-51.
    • Brigger I, Dubernet C, Couvreur P. Nanoparticles in cancer therapy and diagnosis. Adv Drug Deliv Rev 2002; 54(5):631-51.
    • Green J J, Shi J, Chiu E, Leshchiner E S, Langer R, Anderson D G. Biodegradable polymeric vectors for gene delivery to human endothelial cells. Bioconjug Chem 2006; 17:1162-9.
    • Green J J, Chiu E, Leshchiner E S, Shi J, Langer R, Anderson D G. Electrostatic ligand coatings of nanoparticles enable ligand-specific gene delivery to human primary cells. Nano Lett 2007; 7(4):874-9.
    • Harris T J, Green J J, Fung P W, Langer R, Anderson D G, Bhatia S N. Tissue-specific gene delivery via nanoparticle coating. Biomaterials 2010; 31(5):998-1006.
    • Green J J, Zugates G T, Tedford N C, Huang Y, Griffith L G, Lauffenburger D A, et al. Combinatorial modification of degradable polymers enables transfection of human cells comparable to adenovirus. Adv Mater 2007; 19(19):2836-42.
    • Lee J S, Green J J, Love K T, Sunshine J, Langer R, Anderson D G. Gold, poly(beta-amino ester) nanoparticles for small interfering RNA delivery. Nano Lett 2009; 9(6):2402-6.
    • Reichert J. Development trends for peptide therapeutics. Tufts Center for the Study of Drug Development 2008 [cited 2010; Available from: http://www.peptidetherapeutics.org/PTF_Summary_2008.pdf
    • Rosca E V, Koskimaki J E, Rivera C G, Pandey N B, Tamiz A P, Popel A S. Anti-angiogenic peptides for cancer therapeutics. Curr Pharm Biotechnol 12(8):1101-1116 (2011).
    • Lucas R, Holmgren L, Garcia I, Jimenez B, Mandriota S J, Borlat F, et al. Multiple forms of angiostatin induce apoptosis in endothelial cells. Blood 1998; 92(12):4730-41.
    • Green J J, Langer R, Anderson D G. A combinatorial polymer library approach yields insight into nonviral gene delivery. Acc Chem Res 2008; 41(6):749-59.
    • Shmueli R B, Anderson D G, Green J J. Electrostatic surface modifications to improve gene delivery. Expert Opin Drug Deliv 7(4):535-50.
    • Zhang S, Uludag H. Nanoparticulate systems for growth factor delivery. Pharm Res 2009; 26(7):1561-80.
    • Jain R A. The manufacturing techniques of various drug loaded biodegradable poly(lactide-co-glycolide) (PLGA) devices. Biomaterials 2000; 21(23):2475-90.
    • Little S R, Lynn D M, Ge Q, Anderson D G, Puram S V, Chen J Z, et al. Poly-beta amino ester-containing microparticles enhance the activity of nonviral genetic vaccines. Proc Natl Acad Sci USA 2004; 101(26):9534-9.
    • Koskimaki J E, Karagiannis E D, Tang B C, Hammers H, Watkins D N, Pili R, et al. Pentastatin-1, a collagen IV derived 20-mer peptide, suppresses tumor growth in a small cell lung cancer xenograft model. BMC Cancer 2010; 10:29.
    • Koskimaki J E, Karagiannis E D, Rosca E V, Vesuna F, Winnard P T, Jr., Raman V, et al. Peptides derived from type IV collagen, CXC chemokines, and thrombospondin-1 domain-containing proteins inhibit neovascularization and suppress tumor growth in MDA-MB-231 breast cancer xenografts. Neoplasia 2009; 11(12):1285-91.
  • Although the foregoing subject matter has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be understood by those skilled in the art that certain changes and modifications can be practiced within the scope of the appended claims.

Claims (70)

That which is claimed:
1. A bioreducible, hydrolytically degradable polymer of formula (Ia):
Figure US20160374949A9-20161229-C00039
wherein:
n is an integer from 1 to 10,000;
R1, R2, R3, R4, R5, R6, R7, R8, and R9 are each independently selected from the group consisting of hydrogen, branched and unbranched alkyl, branched and unbranched alkenyl, branched and unbranched alkynyl, aryl, halogen, hydroxyl, alkoxy, carbamoyl, carboxyl ester, carbonyldioxyl, amide, thiohydroxyl, alkylthioether, amino, alkylamino, dialkylamino, trialkylamino, cyano, ureido, a substituted alkanoyl group, cyclic, cyclic aromatic, heterocyclic, and aromatic heterocyclic groups, each of which may be substituted with at least one substituent selected from the group consisting of branched or unbranched alkyl, branched and unbranched alkenyl, branched and unbranched alkynyl, amino, alkylamino, dialkylamino, trialkylamino, aryl, ureido, heterocyclic, aromatic heterocyclic, cyclic, aromatic cyclic, halogen, hydroxyl, alkoxy, cyano, amide, carbamoyl, carboxylic acid, ester, carbonyl, carbonyldioxyl, alkylthioether, and thiohydroxyl groups;
wherein R1 can be present or absent and when present the compound of formula (I) further comprises a counter ion selected from the group consisting of chloride, fluoride, bromide, iodide, sulfate, nitrate, fumarate, acetate, carbonate, stearate, laurate, and oleate; and
wherein at least one R comprises a backbone of a diacrylate having the following structure:
Figure US20160374949A9-20161229-C00040
wherein X1 and X2 are each independently substituted or unsubstituted C2-C20 alkylene, and wherein each X1 and X2 can be the same or different.
2. The bioreducible, hydrolytically degradable polymer of claim 1, wherein at least one R comprises a backbone of a diacrylate selected from the group consisting of:
Figure US20160374949A9-20161229-C00041
or co-oligomers comprising combinations thereof, wherein the diacrylate can be the same or different.
3. The bioreducible, hydrolytically degradable polymer of claim 1, wherein R′ comprises a side chain derived from compound selected from the group consisting of:
Figure US20160374949A9-20161229-C00042
4. The bioreducible, hydrolytically degradable polymer of claim 1, wherein R″ comprises an end group comprising an acrylate functional group or an end group derived from a compound selected from the group consisting of:
Figure US20160374949A9-20161229-C00043
5. The bioreducible, hydrolytically degradable polymer of claim 1, wherein n is an integer from 1 to 100.
6. The bioreducible, hydrolytically degradable polymer of claim 1, wherein n is an integer from 1 to 30.
7. The bioreducible, hydrolytically degradable polymer of claim 1, wherein n is an integer from 5 to 20.
8. The bioreducible, hydrolytically degradable polymer of claim 1, wherein n is an integer from 10 to 15.
9. The bioreducible, hydrolytically degradable polymer of claim 1, wherein n is an integer from 1 to 10.
10. The bioreducible, hydrolytically degradable polymer of claim 1, wherein at least one R″ comprises a biomolecule selected from the group consisting of poly(ethyleneglycol) (PEG), a targetinaligand, and a labeling molecule.
11. A nanoparticle, microparticle, or gel comprising the bioreducible, hydrolytically degradable polymer of claim 1.
12. The nanoparticle, microparticle, or gel of claim 11 further comprising a therapeutic agent.
13. The nanoparticle, microparticle, or gel of claim 12, wherein the therapeutic agent is a peptide or a combination of peptides.
14. The nanoparticle, microparticle, or gel of claim 12, wherein the therapeutic agent is an siRNA or a combination of siRNA.
15. The nanoparticle, microparticle, or gel of claim 12, wherein the therapeutic agent is selected from the group consisting of a gene, DNA, RNA, siRNA, miRNA, is RNA, agRNA, smRNA, a nucleic acid, a peptide, a protein, a chemotherapeutic agent, a hydrophobic drug, a small molecule drug, and combinations thereof.
16. A nanoparticle, microparticle, or gel comprising a compound of formula (I):
Figure US20160374949A9-20161229-C00044
wherein:
n is an integer from 1 to 10,000;
R1, R2, R3, R4, R5, R6, R7, R8, and R9 are each independently selected from the group consisting of hydrogen, branched and unbranched alkyl, branched and unbranched alkenyl, branched and unbranched alkynyl, aryl, halogen, hydroxyl, alkoxy, carbamoyl, carboxyl ester, carbonyldioxyl, amide, thiohydroxyl, alkylthioether, amino, alkylamino, dialkylamino, trialkylamino, cyano, ureido, a substituted alkanoyl group, cyclic, cyclic aromatic, heterocyclic, and aromatic heterocyclic groups, each of which may be substituted with at least one substituent selected from the group consisting of branched or unbranched alkyl, branched and unbranched alkenyl, branched and unbranched alkynyl, amino, alkylamino, dialkylamino, trialkylamino, aryl, ureido, heterocyclic, aromatic heterocyclic, cyclic, aromatic cyclic, halogen, hydroxyl, alkoxy, cyano, amide, carbamoyl, carboxylic acid, ester, carbonyl, carbonyldioxyl, alkylthioether, and thiohydroxyl groups;
wherein R1 can be present or absent and when present the compound of formula (I) further comprises a counter ion selected from the group consisting of chloride, fluoride, bromide, iodide, sulfate, nitrate, fumarate, acetate, carbonate, stearate, laurate, and oleate; and
at least one of R, R′, and R″ comprise a reducible or degradable linkage, and wherein each R, R′, or R″ can independently be the same or different;
under the proviso that when at least one R group comprises an ester linkage of the formula —C(═O)—O— and the compound of formula (I) comprises a poly(beta-amino ester), then the compound of formula (I) must also comprise one or more of the following characteristics:
(a) each R group is different;
(b) each R″ group is different;
(c) each R″ group is not the same as any of R′, R1, R2, R3, R4, R5, R6, R7, R8, and R9;
(d) the R″ groups degrade through a different mechanism than the ester-containing R groups, wherein the degradation of the R″ group is selected from the group consisting of a bioreducible mechanism or an enzymatically degradable mechanism; and/or
(e) the compound of formula (I) comprises a substructure of a larger cross-linked polymer, wherein the larger cross-linked polymer comprises different properties from compound of formula (I);
and one or more peptides selected from the group consisting of an anti-angiogenic peptide, an anti-lymphangiogenic peptide, an anti-tumorigenic peptide, and an anti-permeability peptide.
17. The nanoparticle, microparticle, or gel of claim 16, wherein n is an integer from 1 to 1,000.
18. The nanoparticle, microparticle, or gel of claim 16, wherein n is an integer from 1 to 100.
19. The nanoparticle, microparticle, or gel of claim 16, wherein n is an integer from 1 to 30.
20. The nanoparticle, microparticle, or gel of claim 16, wherein n is an integer from 5 to 20.
21. The nanoparticle, microparticle, or gel of claim 16, wherein n is an integer from 10 to 15.
22. The nanoparticle, microparticle, or gel of claim 16, wherein n is an integer from 1 to 10.
23. The nanoparticle, microparticle, or gel of claim 16, wherein the reducible or degradable linkage comprising R, R′, and R″ is selected from the group consisting of an ester, a disulfide, an amide, an anhydride or a linkage susceptible to enzymatic degradation, subject to the proviso of claim 16.
24. The nanoparticle, microparticle, or gel of claim 16, wherein R comprises a backbone of a diacrylate selected from the group consisting of:
Figure US20160374949A9-20161229-C00045
Figure US20160374949A9-20161229-C00046
25. The nanoparticle, microparticle, or gel of claim 16, wherein R′ comprises a side chain derived from compound selected from the group consisting of:
Figure US20160374949A9-20161229-C00047
26. The nanoparticle, microparticle, or gel of claim 16, wherein R″ comprises an end group derived from a compound selected from the group consisting of
Figure US20160374949A9-20161229-C00048
27. The nanoparticle, microparticle, or gel of claim 16, wherein the one or more peptide is a collagen IV peptide.
28. The nanoparticle, microparticle, or gel of claim 27, wherein the collagen IV peptide is selected from the group consisting of:
(SEQ ID NO: 2443) LRRFSTMPFMFCNINNVCNF (SEQ ID NO: 2444) LRRFSTMPFMFGNINNVGNF (SEQ ID NO: 2445) LRRFSTMPFMF (SEQ ID NO: 2446) LRRFSTMPFMF-Abu-NINV (SEQ ID NO: 2447) LRRFSTMPFMF-Abu (SEQ ID NO: 2448) LRRFSTMP (SEQ ID NO: 2449) NINNV-Abu-NF (SEQ ID NO: 2450) FMF-Abu-NINNV-Abu-NF (SEQ ID NO: 2451) STMPFMF-Abu-NINNV-Abu-NF (SEQ ID NO: 2452) LRRFSTMPFMF-Abu-NINNV-Abu-NF (SEQ ID NO: 2453) LNRFSTMPF (SEQ ID NO: 2454) LRRFST-Nle-PF-Nle-F (SEQ ID NO: 2455) LRRFSTMPAMF-Abu-NINNV-Abu-NF (SEQ ID NO: 2456) LRRFSTMPFAF-Abu-NINNV-Abu-NF (SEQ ID NO: 2457) LRRFSTMPFMA-Abu-NINNV-Abu-NF (SEQ ID NO: 2458) LRRFSTMPF-Nle-F-Abu-NINNV-Abu-NF (SEQ ID NO: 2459) LRRFSTMPFM(4-ClPhen)-Abu-CNINNV-Abu-NF (SEQ ID NO: 2460) F-Abu-NINNV-Abu-N (SEQ ID NO: 2461) F-Abu-NIN (SEQ ID NO: 2462) LRRFSTMPFMFSNINNVSNF (SEQ ID NO: 2463) LRRFSTMPFMFANINNVANF (SEQ ID NO: 2464) LRRFSTMPFMFININNVINF (SEQ ID NO: 2465) LRRFSTMPFMFTNINNVTNF (SEQ ID NO: 2466) LRRFSTMPFMFC(AllyGly)NINNV(AllyGly)NF (SEQ ID NO: 2467) LRRFSTMPFMFVNINNVVNF (SEQ ID NO: 2468) LRRFSTMPFMF-Abu-NINN (SEQ ID NO: 2469) LRRFSTMPFMFTNINV (SEQ ID NO: 2470) F-Abu-NINV (SEQ ID NO: 2471) FTNINNVTN (SEQ ID NO: 2472) LRRFSTMPFMFTNINN (SEQ ID NO: 2473) LRRFSTMPFMFININN (SEQ ID NO: 2474) LRRFSTMPF-dA-FININNVINF (SEQ ID NO: 2475) LRRFSTAPFAFININNVINF (SEQ ID NO: 2476) LRRFSTMPFAFININNVINF;
wherein Abu is 2-aminobutyric acid; Nle is Norleucine; and AllyGly is allyglycine.
29. The nanoparticle, microparticle, or gel of claim 16, wherein the one or more peptide is selected from the group consisting of:
RLRLLTLQSWLL (SEQ ID NO: 2477) LMRKSQILISSWF (SEQ ID NO: 2478) LLIVALLFILSWL (SEQ ID NO: 2479) LLRLLLLIESWLE (SEQ ID NO: 2480) LLRSSLILLQGSWF (SEQ ID NO: 2481) LLHISLLLIESRLE (SEQ ID NO: 2482) LLRISLLLIESWLE (SEQ ID NO: 2483)
30. The nanoparticle, microparticle, or gel of claim 16, wherein the one or more peptide is selected from the group consisting of an isolated peptide or analog thereof comprising the amino acid sequence W-X2-C-X3-C-X2-G (SEQ ID NO: 2486), wherein X denotes a variable amino acid; W is tryptophan; C is cysteine, G is glycine; and wherein the peptide reduces blood vessel formation in a cell, tissue or organ.
31. The nanoparticle, microparticle, or gel of claim 16, wherein the one or more peptide is selected from the group consisting of an isolated peptide or analog thereof comprising or consisting essentially of a sequence having at least 85% amino acid sequence identity to an amino acid sequence selected from the group consisting of:
THSD-1: QPWSQCSATCGDGVRERRR; (SEQ ID NO: 2350) THSD-3: SPWSPCSGNCSTGKQQRTR; (SEQ ID NO: 2351) THSD-6: WTRCSSSCGRGVSVRSR; (SEQ ID NO: 2352) CILP: SPWSKCSAACGQTGVQTRTR; (SEQ ID NO: 2325) WISP-1: SPWSPCSTSCGLGVSTRI; (SEQ ID NO: 2360) WISP-2: TAWGPCSTTCGLGMATRV; (SEQ ID NO: 2361) WISP-3: TKWTPCSRTCGMGISNRV; (SEQ ID NO: 2362) F-spondin: SEWSDCSVTCGKGMRTRQR; (SEQ ID NO: 2359) F-spondin: WDECSATCGMGMKKRHR; (SEQ ID NO: 2358) CTGF: TEWSACSKTCGMGISTRV; (SEQ ID NO: 2327) fibulin-6: ASWSACSVSCGGGARQRTR; (SEQ ID NO: 2331) fibulin-6: QPWGTCSESCGKGTQTRAR; (SEQ ID NO: 2330) fibulin-6: SAWRACSVTCGKGIQKRSR; (SEQ ID NO: 2329) CYR61: TSWSQCSKTCGTGISTRV; (SEQ ID NO: 2328) NOVH: TEWTACSKSCGMGFSTRV; (SEQ ID NO: 2332) UNC5-C: TEWSVCNSRCGRGYQKRTR; (SEQ ID NO: 2356) UNC5-D: TEWSACNVRCGRGWQKRSR; (SEQ ID NO: 2357) SCO-spondin: GPWEDCSVSCGGGEQLRSR; (SEQ ID NO: 2349) Properdin: GPWEPCSVTCSKGTRTRRR; (SEQ ID NO: 2335) C6: TQWTSCSKTCNSGTQSRHR; (SEQ ID NO: 2324) ADAMTS-like-4: SPWSQCSVRCGRGQRSRQVR; (SEQ ID NO: 2355) ADAMTS-4: GPWGDCSRTCGGGVQFSSR; (SEQ ID NO: 2293) ADAMTS-8: GPWGECSRTCGGGVQFSHR; ADAMTS-16: SPWSQCTASCGGGVQTR; (SEQ ID NO: 2310) ADAMTS-18: SKWSECSRTCGGGVKFQER; (SEQ ID NO: 2315) semaphorin 5A: GPWERCTAQCGGGIQARRR; (SEQ ID NO: 2346) semaphorin 5A: SPWTKCSATCGGGHYMRTR; (SEQ ID NO: 2347) semaphoring 5B: TSWSPCSASCGGGHYQRTR; (SEQ ID NO: 2348) papilin: GPWAPCSASCGGGSQSRS; (SEQ ID NO: 2334) papilin: SQWSPCSRTCGGGVSFRER; (SEQ ID NO: 2333) ADAM-9: KCHGHGVCNS; (SEQ ID NO: 2497) and ADAM-12: MQCHGRGVCNNRKN, (SEQ ID NO: 2288)
wherein A is alanine; I is isoleucine; M is methionine; H is histidine; Y is tyrosine; K is lysine; W is tryptophan; C is cysteine, T is threonine, S is serine; N is asparagine; G is glycine; R is arginine; V is valine, P is proline, and Q is glutamine wherein the peptide reduces blood vessel formation in a cell, tissue or organ.
32. The nanoparticle, microparticle, or gel of claim 16, wherein the one or more peptide is selected from the group consisting of an isolated peptide or analog thereof having at least 85% identity to an amino acid sequence selected from the group consisting of:
ENA-78: NGKEICLDPEAPFLKKVIQKILD; (SEQ ID NO: 2381) CXCL6: NGKQVCLDPEAPFLKKVIQKILDS; (SEQ ID NO: 2384) CXCL1: NGRKACLNPASPIVKKIIEKMLNS; (SEQ ID NO: 2388) Gro-γ: NGKKACLNPASPMVQKIIEKIL; (SEQ ID NO: 2392) Beta thromboglobulin/CXCL7: DGRKICLDPDAPRIKKIVQKKL, (SEQ ID NO: 2400) Interleukin 8 (IL-8)/CXCL8: DGRELCLDPKENWVQRVVEKFLK, (SEQ ID NO: 2396) GCP-2: NGKQVCLDPEAPFLKKVIQKILDS, (SEQ ID NO: 2384)
wherein A is alanine; I is isoleucine; F is phenylalanine; D is aspartic acid; M is methionine; H is histidine; Y is tyrosine; K is lysine; W is tryptophan; C is cysteine, T is threonine, S is serine; N is asparagine; G is glycine; R is arginine; V is valine, P is proline, and Q is glutamine; and wherein the peptide reduces blood vessel formation in a cell, tissue or organ.
33. The nanoparticle, microparticle, or gel of claim 16, wherein the one or more peptide is selected from the group consisting of an isolated peptide or analog thereof having at least 85% amino acid sequence identity to an amino acid sequence selected from the group consisting of
Alpha 6 fibril of type 4 collagen: YCNINEVCHYARRNDKSYWL; (SEQ ID NO: 2379) Alpha 5 fibril of type 4 collagen: LRRFSTMPFMFCNINNVCNF; (SEQ ID NO: 2443) Alpha 4 fibril of type 4 collagen: AAPFLECQGRQGTCHFFAN; (SEQ ID NO: 2373) Alpha 4 fibril of type 4 collagen: LPVFSTLPFAYCNIHQVCHY; (SEQ ID NO: 2371) Alpha 4 fibril of type 4 collagen: YCNIHQVCHYAQRNDRSYWL, (SEQ ID NO: 2372) and Collagen type IV, alpha6 fibril LPRFSTMPFIYCNINEVCHY (SEQ ID NO: 2494)
wherein A is alanine; I is isoleucine; F is phenylalanine; D is aspartic acid; M is methionine; H is histidine; Y is tyrosine; K is lysine; W is tryptophan; C is cysteine, T is threonine, S is serine; N is asparagine; G is glycine; R is arginine; V is valine, P is proline, and Q is glutamine wherein the peptide reduces blood vessel formation in a cell, tissue or organ.
34. The nanoparticle, microparticle, or gel of claim 16, wherein the one or more peptide is selected from the group consisting of an isolated peptide or analog thereof comprising one of the following amino acid sequences:
TSP Motif: W-X(2)-C-X(3)-C-X(2)-G, (SEQ ID NO: 2486) CXC Motif: G-X(3)-C-L Collagen Motif: C-N-X(3)-V-C (SEQ ID NO: 2487) Collagen Motif: P-F-X(2)-C Somatotropin Motif: L-X(3)-L-L-X(3)-S-X-L (SEQ ID NO: 2488) Serpin Motif: L-X(2)-E-E-X-P (SEQ ID NO: 2489)
wherein X denotes a variable amino acid and the number in parentheses denotes the number of variable amino acids; W denotes tryptophan; C denotes cysteine, G denotes glycine, V denotes valine; L denotes leucine, P is proline, and wherein the peptide reduces blood vessel formation in a cell, tissue or organ.
35. The nanoparticle, microparticle, or gel of claim 16, wherein the one or more peptide comprises an amino acid sequence shown in Table 1-6, 8 and 9.
36. The nanoparticle, microparticle, or gel of claim 16, wherein the one or more peptide comprises an isolated peptide or analog thereof having at least 85% identity to an amino acid sequence shown in Table 1-10.
37. The nanoparticle, microparticle, or gel of claim 16, wherein the one or more peptide comprises an amino acid sequence shown in Table 1-10.
38. The nanoparticle, microparticle, or gel of claim 16, wherein the one or more peptide consists essentially of an amino acid sequence shown in Table 1-10.
39. The nanoparticle, microparticle, or gel of claim 16, wherein the one or more peptide comprises an isolated peptide or analog thereof comprising or consisting essentially of a sequence having at least 85% amino acid sequence identity to an amino acid sequence selected from the group consisting of:
Placental Lactogen LLRISLLLIESWLE (SEQ ID NO: 2483) hGH-V LLRISLLLTQSWLE (SEQ ID NO: 2490) GH2 LLHISLLLIQSWLE (SEQ ID NO: 2491) Chorionic LLRLLLLIESWLE (SEQ ID NO: 2480) somatomammotropin Chorionic LLHISLLLIESRLE (SEQ ID NO: 2482) somatomammotropin hormone-like 1 Transmembrane LLRSSLILLQGSWF (SEQ ID NO: 2481) protein 45A IL-17 receptor C RLRLLTLQSWLL (SEQ ID NO: 2477) Neuropeptide FF LLIVALLFILSWL (SEQ ID NO: 2479) receptor 2 Brush border LMRKSQILISSWF (SEQ ID NO: 2478) myosin-I
wherein the peptide reduces blood vessel formation in a cell, tissue or organ.
40. The nanoparticle, microparticle, or gel of claim 16, wherein the one or more peptide comprises an isolated peptide or analog thereof comprising or consisting essentially of a sequence having at least 85% amino acid sequence identity to an amino acid sequence selected from the group consisting of:
DEAH box polypeptide EIELVEEEPPF (SEQ ID NO: 2485) 8 (“DEAH” disclosed as SEQ ID NO: 2484) Caspase 10 AEDLLSEEDPF (SEQ ID NO: 2492) CKIP-1 TLDLIQEEDPS (SEQ ID NO: 2493)
wherein the peptide reduces blood vessel formation in a cell, tissue or organ.
41. The nanoparticle, microparticle, or gel of claim 16, wherein the one or more peptide comprises an isolated peptide or analog thereof comprising or consisting essentially of a sequence having at least 85% amino acid sequence identity to an amino acid sequence selected from the group consisting of:
Collagen LPRFSTMPFIYCNINEVCHY (SEQ ID NO: 2494) type IV, alpha6 fibril
wherein the peptide reduces blood vessel formation in a cell, tissue or organ.
42. The nanoparticle, microparticle, or gel of claim 16, wherein the one or more peptide comprises a collagen peptide in combination with a somatotropin peptide.
43. The nanoparticle, microparticle, or gel of claim 16, wherein the one or more peptide has the sequence EIELVEEEPPF (SEQ ID NO: 2485) and the polymer has the following structure:
Figure US20160374949A9-20161229-C00049
wherein:
R is selected from the group consisting of:
Figure US20160374949A9-20161229-C00050
R′ is selected from the group consisting of:
Figure US20160374949A9-20161229-C00051
R″ is selected from the group consisting of:
Figure US20160374949A9-20161229-C00052
44. The nanoparticle, microparticle, or gel of claim 16, wherein the one or more peptide has the sequence NGRKACLNPASPIVKKIIEKMLNS (SEQ ID NO: 2388) and the polymer has the following structure:
Figure US20160374949A9-20161229-C00053
wherein:
R is selected from the group consisting of:
Figure US20160374949A9-20161229-C00054
R′ is selected from the group consisting of:
Figure US20160374949A9-20161229-C00055
R″ is selected from the group consisting of:
Figure US20160374949A9-20161229-C00056
45. The nanoparticle, microparticle, or gel of claim 16, wherein the one or more peptide has the sequence LLRISLLLIESWLE (SEQ ID NO: 2483) and the polymer has the following structure:
Figure US20160374949A9-20161229-C00057
wherein:
R is selected from the group consisting of:
Figure US20160374949A9-20161229-C00058
R′ is selected from the group consisting of:
Figure US20160374949A9-20161229-C00059
R″ is selected from the group consisting of:
Figure US20160374949A9-20161229-C00060
46. The nanoparticle, microparticle, or gel of claim 16, wherein the one or more peptide has the sequence LRRFSTMPFMF-Abu-NINNV-Abu-NF (SEQ ID NO: 2452) and the polymer has the following structure:
Figure US20160374949A9-20161229-C00061
wherein:
R is selected from the group consisting of:
Figure US20160374949A9-20161229-C00062
R′ is selected from the group consisting of:
Figure US20160374949A9-20161229-C00063
and
R″ is selected from the group consisting of:
Figure US20160374949A9-20161229-C00064
47. The nanoparticle, microparticle, or gel of claim 16, wherein the polymer has the following structure:
Figure US20160374949A9-20161229-C00065
and the one or more peptide is selected from the group consisting of:
EIELVEEEPPF; (SEQ ID NO: 2485) SPWSPCSTSCGLGVSTRI; (SEQ ID NO: 2360) LRRFSTMPFMFCNINNVCNF; (SEQ ID NO: 2375) and NGRKACLNPASPIVKKIIEKMLNS. (SEQ ID NO: 2388)
48. The nanoparticle, microparticle, or gel of claim 16, further comprising one or more peptides encapsulated therein.
49. A multilayer particle comprising a core and one or more layers, wherein the core comprises a material selected from the group consisting of a compound of formula (I), a gold nanoparticle, an inorganic nanoparticle, an organic polymer, and the one or more layers comprise a material selected from the group consisting of a compound of formula (I), an organic polymer, one or more peptides, and one or more additional biological agents.
50. The multilayer particle of claim 49, further comprising alternating cationic and anionic layers.
51. A microparticle comprising a compound of formula (I), poly(lactide-co-glycolide) (PLGA), or combinations thereof.
52. The microparticle of claim 51, further comprising a nanoparticle comprising a compound of formula (I).
53. The microparticle of claim 51, further comprising one or more peptides encapsulated therein.
54. A method for stabilizing a suspension of nanoparticles and/or microparticles of formula (I), the method comprising:
(a) providing a suspension of nanoparticles and/or microparticles of formula (I);
(b) admixing a lyroprotectant with the suspension;
(c) freezing the suspension for a period of time; and
(d) lyophilizing the suspension for a period of time.
55. The method of claim 54, wherein the lyroprotectant is sucrose.
56. A pellet or scaffold comprising one or more lyophilized particle, wherein the one or more lyophilized particle comprises a compound of formula (I).
57. The pellet or scaffold of claim 56, wherein the one or more lyophilized particle comprises cargo.
58. The pellet or scaffold of claim 57, wherein the cargo is selected from the group consisting of a peptide, a protein, an siRNA, DNA, an imaging agent, and combinations thereof.
59. The pellet or scaffold of claim 56, wherein the scaffold comprises a bone construct.
60. The pellet or scaffold of claim 56, further comprising one or more microparticle, which has the same or different composition as the one or more lyophilized particle.
61. The pellet or scaffold of claim 56, wherein the one or more lyophilized particle has a size of about 20 nm to about 100 nm.
62. The pellet or scaffold of claim 56, wherein the one or more lyophilized particle has a size of about 100 nm to about 300 nm.
63. The pellet or scaffold of claim 56, wherein the one or more lyophilized particle has a size of about 300 nm to about 1000 nm.
64. The pellet or scaffold of claim 56, wherein the one or more lyophilized particle has a size of about 1 micron to about 10 microns.
65. The pellet or scaffold of claim 56, wherein the one or more lyophilized particle has a size of about 10 microns to about 30 microns.
66. A method of treating a disease or condition, the method comprising administering to a subject in need of treatment thereof a therapeutically effective amount of a nanoparticle, microparticle, gel, or multilayer particle comprising a compound of formula (I), wherein the nanoparticle, microparticle, gel, or multilayer particle further comprises a therapeutic agent specific for the disease or condition to be treated.
67. The method of claim 66, wherein the disease or condition comprises an angiogenesis-dependent disease or condition.
68. The method of claim 67, wherein the angiogenesis disease or condition is selected from the group consisting of a cancer and age-related macular degeneration.
69. The method of claim 66, wherein the disease or condition is a non-angiogenic disease or condition.
70. The method of claim 66, wherein the therapeutic agent is selected from the group consisting of gene, DNA, RNA, siRNA, miRNA, is RNA, agRNA, smRNA, a nucleic acid, a peptide, a protein, a chemotherapeutic agent, a hydrophobic drug, a small molecule drug, and combinations thereof.
US13/272,042 2009-05-15 2011-10-12 Peptide/particle delivery systems Active 2031-04-12 US9717694B2 (en)

Priority Applications (4)

Application Number Priority Date Filing Date Title
US13/272,042 US9717694B2 (en) 2009-05-15 2011-10-12 Peptide/particle delivery systems
US14/438,353 US20190209690A9 (en) 2011-10-12 2013-10-25 Bioreducible Poly (Beta-Amino Ester)s For siRNA Delivery
US15/645,337 US10786463B2 (en) 2009-05-15 2017-07-10 Peptide/particle delivery systems
US17/149,583 US20210220287A1 (en) 2009-05-15 2021-01-14 Peptide/particle delivery systems

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US17861109P 2009-05-15 2009-05-15
PCT/US2010/035127 WO2010132879A2 (en) 2009-05-15 2010-05-17 Multicomponent degradable cationic polymers
US39222410P 2010-10-12 2010-10-12
US201161543046P 2011-10-04 2011-10-04
US201161542995P 2011-10-04 2011-10-04
US13/272,042 US9717694B2 (en) 2009-05-15 2011-10-12 Peptide/particle delivery systems

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2010/035127 Continuation-In-Part WO2010132879A2 (en) 2009-05-15 2010-05-17 Multicomponent degradable cationic polymers

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US14/438,353 Continuation-In-Part US20190209690A9 (en) 2011-10-12 2013-10-25 Bioreducible Poly (Beta-Amino Ester)s For siRNA Delivery
US15/645,337 Continuation US10786463B2 (en) 2009-05-15 2017-07-10 Peptide/particle delivery systems

Publications (3)

Publication Number Publication Date
US20120114759A1 US20120114759A1 (en) 2012-05-10
US20160374949A9 true US20160374949A9 (en) 2016-12-29
US9717694B2 US9717694B2 (en) 2017-08-01

Family

ID=46019847

Family Applications (3)

Application Number Title Priority Date Filing Date
US13/272,042 Active 2031-04-12 US9717694B2 (en) 2009-05-15 2011-10-12 Peptide/particle delivery systems
US15/645,337 Active US10786463B2 (en) 2009-05-15 2017-07-10 Peptide/particle delivery systems
US17/149,583 Pending US20210220287A1 (en) 2009-05-15 2021-01-14 Peptide/particle delivery systems

Family Applications After (2)

Application Number Title Priority Date Filing Date
US15/645,337 Active US10786463B2 (en) 2009-05-15 2017-07-10 Peptide/particle delivery systems
US17/149,583 Pending US20210220287A1 (en) 2009-05-15 2021-01-14 Peptide/particle delivery systems

Country Status (1)

Country Link
US (3) US9717694B2 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11697804B2 (en) 2018-10-01 2023-07-11 The Johns Hopkins University Transcriptionally targeted and CpG-free plasmid for theranostic gene therapy
US11883541B2 (en) 2017-10-02 2024-01-30 The Johns Hopkins University Nonviral gene transfer to the suprachoroidal space

Families Citing this family (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8507434B2 (en) * 2007-01-03 2013-08-13 The Johns Hopkins University Peptide modulators of angiogenesis and use thereof
US9717694B2 (en) * 2009-05-15 2017-08-01 The Johns Hopkins University Peptide/particle delivery systems
WO2014066811A1 (en) * 2012-10-25 2014-05-01 The Johns Hopkins University Bioreducible poly (b-amino ester)s for sirna delivery
WO2013056113A1 (en) * 2011-10-13 2013-04-18 The Johns Hopkins University Nanocomposites of gold and polymers
WO2014066898A1 (en) * 2012-10-26 2014-05-01 The Johns Hopkins University A layer-by-layer approach to co-deliver dna and sirna via aunps: a potential platform for modifying release kinetics
GB201304245D0 (en) * 2013-03-08 2013-04-24 Inst Quimic De Sarria Chemical compounds
CN105431162B (en) * 2013-06-07 2019-12-10 约翰霍普金斯大学 Biomimetic peptides and biodegradable delivery platforms for the treatment of angiogenesis-and lymphangiogenesis-dependent diseases
WO2015023775A1 (en) 2013-08-13 2015-02-19 Baylor College Of Medicine A novel plga-modified polyethylenimine self-assembly nanotechnology for nucleic acid and drug delivery
CA2924013A1 (en) * 2013-11-05 2015-05-14 Clontech Laboratories, Inc. Dry transfection compositions and methods for making and using the same
US20170216363A1 (en) 2014-08-07 2017-08-03 The Johns Hopkins University Nanoparticle modification of human adipose-derived mesenchymal stem cells for treating brain cancer and other neurological diseases
AU2018266690A1 (en) * 2017-05-08 2019-12-12 Asclepix Therapeutics, Inc. Biodegradable microparticles for sustained delivery of anti-angiogenic peptide
CN111886031B (en) * 2018-01-10 2022-10-14 G2G生物公司 Collagen peptide-containing polycaprolactone microsphere filler and preparation method thereof
WO2019204402A1 (en) * 2018-04-17 2019-10-24 Asclepix Therapeutics, Inc. Compositions and methods for treatment of cancer
EP3952913A4 (en) * 2019-04-12 2023-05-10 The Johns Hopkins University Tolerogenic artificial antigen-presenting cells
EP4048287A4 (en) * 2019-10-25 2023-12-06 The Johns Hopkins University Polymeric nanoparticles for intracellular protein delivery
CN112778520A (en) * 2019-11-08 2021-05-11 天津大学 Cationic polymer, preparation method and application
EP4163006B8 (en) * 2021-10-11 2024-03-20 leon-nanodrugs GmbH Methods for validating an apparatus for producing nanoparticles
WO2023077150A1 (en) * 2021-11-01 2023-05-04 The Johns Hopkins University Polymers and nanoparticles for intramuscular nucleic acid delivery
WO2024030661A1 (en) * 2022-08-05 2024-02-08 University Of Washington Systems and methods for selective shape transformation of 3d-printed materials

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6998115B2 (en) 2000-10-10 2006-02-14 Massachusetts Institute Of Technology Biodegradable poly(β-amino esters) and uses thereof
US7427394B2 (en) 2000-10-10 2008-09-23 Massachusetts Institute Of Technology Biodegradable poly(β-amino esters) and uses thereof
US20100144641A1 (en) 2005-09-12 2010-06-10 Popel Aleksander S Compositions Having Antiangiogenic Activity and Uses Thereof
US8507434B2 (en) 2007-01-03 2013-08-13 The Johns Hopkins University Peptide modulators of angiogenesis and use thereof
US9717694B2 (en) * 2009-05-15 2017-08-01 The Johns Hopkins University Peptide/particle delivery systems
US8992991B2 (en) * 2009-05-15 2015-03-31 The Johns Hopkins University Multicomponent degradable cationic polymers

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11883541B2 (en) 2017-10-02 2024-01-30 The Johns Hopkins University Nonviral gene transfer to the suprachoroidal space
US11697804B2 (en) 2018-10-01 2023-07-11 The Johns Hopkins University Transcriptionally targeted and CpG-free plasmid for theranostic gene therapy

Also Published As

Publication number Publication date
US9717694B2 (en) 2017-08-01
US10786463B2 (en) 2020-09-29
US20210220287A1 (en) 2021-07-22
US20120114759A1 (en) 2012-05-10
US20180028455A1 (en) 2018-02-01

Similar Documents

Publication Publication Date Title
US20210220287A1 (en) Peptide/particle delivery systems
US20210252154A1 (en) Multicomponent degradable cationic polymers
Song et al. Self-assembly of peptide amphiphiles for drug delivery: the role of peptide primary and secondary structures
Wang et al. The effect of fluorination on the transfection efficacy of surface-engineered dendrimers
US9187544B2 (en) Peptide modulators of angiogenesis and use thereof
US20230107757A1 (en) Poly(beta-amino ester)-co-polyethylene glycol (peg-pbae-peg) polymers for gene and drug delivery
US9943608B2 (en) Multi-arm biodegradable polymers for nucleic acid delivery
Hou et al. Partially PEGylated dendrimer-entrapped gold nanoparticles: A promising nanoplatform for highly efficient DNA and siRNA delivery
ES2635990T3 (en) Dual-derived chitosan nanoparticles and methods for manufacturing and using them for in vivo gene transfer
Li et al. Development of a reactive oxygen species (ROS)-responsive nanoplatform for targeted oral cancer therapy
Wang et al. Zwitterionic Janus Dendrimer with distinct functional disparity for enhanced protein delivery
US11649326B2 (en) Functional, segregated, charged telodendrimers and nanocarriers and methods of making and using same
Wang et al. Affinity-controlled protein encapsulation into sub-30 nm telodendrimer nanocarriers by multivalent and synergistic interactions
US20200009069A1 (en) Zwitterionic dendritic amphiphiles, zwitterionic dendrimers, zwitterionic telodendrimers, nanocarriers comprising same, and methods of making and using same
Seo et al. The biological efficiency and bioavailability of human growth hormone delivered using injectable, ionic, thermosensitive poly (organophosphazene)-polyethylenimine conjugate hydrogels
WO2022075459A1 (en) Oligonucleic acid conjugate
Li et al. Hypoxia-reinforced antitumor RNA interference mediated by micelleplexes with programmed disintegration
Liu et al. Zinc coordinated cationic polymers break up the paradox between low molecular weight and high transfection efficacy
JP2022524859A (en) Reversible coating of chitosan-nucleic acid nanoparticles and how to use them
US20170100484A1 (en) Drug delivery carrier for sustained release of medicinal proteins and method for production thereof
CN117355339A (en) Dendritic peptide conjugated polymers for efficient intracellular delivery of nucleic acids to immune cells
US8263133B2 (en) Multivalent clustering targeting strategy for drug carriers
WO2023149528A1 (en) High molecular micelles including novel block copolymer
Yokoo et al. Oligosarcosine Conjugation of Arginine-Rich Peptides Improves the Intracellular Delivery of Peptide/pDNA Complexes
EP4217365A1 (en) Polymers and nanoparticle formulations for systemic nucleic acid delivery

Legal Events

Date Code Title Description
AS Assignment

Owner name: THE JOHNS HOPKINS UNIVERSITY, MARYLAND

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:GREEN, JORDAN JAMIESON;POPEL, ALEKSANDER S.;SUNSHINE, JOEL CHAIM;AND OTHERS;SIGNING DATES FROM 20111106 TO 20111107;REEL/FRAME:027366/0029

STCF Information on status: patent grant

Free format text: PATENTED CASE

CC Certificate of correction
AS Assignment

Owner name: NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT, MARYLAND

Free format text: CONFIRMATORY LICENSE;ASSIGNOR:JOHNS HOPKINS UNIVERSITY;REEL/FRAME:052625/0033

Effective date: 20200507

MAFP Maintenance fee payment

Free format text: PAYMENT OF MAINTENANCE FEE, 4TH YR, SMALL ENTITY (ORIGINAL EVENT CODE: M2551); ENTITY STATUS OF PATENT OWNER: SMALL ENTITY

Year of fee payment: 4