EP3125881A1 - Agents thérapeutiques combinatoires supramoléculaires - Google Patents

Agents thérapeutiques combinatoires supramoléculaires

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Publication number
EP3125881A1
EP3125881A1 EP15772801.5A EP15772801A EP3125881A1 EP 3125881 A1 EP3125881 A1 EP 3125881A1 EP 15772801 A EP15772801 A EP 15772801A EP 3125881 A1 EP3125881 A1 EP 3125881A1
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EP
European Patent Office
Prior art keywords
acid
lipid
alcohol
combinatorial therapeutic
supramolecular combinatorial
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.)
Withdrawn
Application number
EP15772801.5A
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German (de)
English (en)
Other versions
EP3125881A4 (fr
Inventor
Shiladitya Sengupta
Monideepa ROY
Samad HOSSAIN
Aniruddha SENGUPTA
Sanghamitra MYLAVARAPU
Anubhab MUKHERJEE
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.)
Invictus Oncology Pvt Ltd
Original Assignee
Invictus Oncology Pvt Ltd
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Application filed by Invictus Oncology Pvt Ltd filed Critical Invictus Oncology Pvt Ltd
Priority claimed from PCT/US2015/023009 external-priority patent/WO2015153345A1/fr
Publication of EP3125881A1 publication Critical patent/EP3125881A1/fr
Publication of EP3125881A4 publication Critical patent/EP3125881A4/fr
Withdrawn legal-status Critical Current

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    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/554—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being a steroid plant sterol, glycyrrhetic acid, enoxolone or bile acid
    • 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
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present disclosure relates generally to supramolecular combinatorial therapeutics, compositions comprising same, and uses thereof.
  • the present disclosure provides hydrophobic taxane-lipid covalent conjugates which create supramolecular assemblies within lipid bilayer and micelles, providing an extra stabilization resulting in increased intratumoral concentration and hence increased efficacy.
  • the present disclosure also provides supramolecular combinatorial therapeutics, wherein a taxane-lipid conjugate is combined with one or more of a platinum compound, a kinase inhibitor, and an immunoregulator, each of which is optionally conjugated with a lipid.
  • Taxanes are well known taxanes commonly used for the treatment of metastatic ovarian cancer and breast cancer. Their unique anti proliferative mechanism of action and broad range of activity were attracted by scientific community. Taxanes promote hyper stabilization of microtubules in dividing cell and thereby prevents the disassembly of microtubules necessary for cell division. Due to the high potency and non specificity towards cancer cell it shows serious undesired side effects like nausea, vomiting, diarrhea, dizziness, or drowsiness etc.
  • lipophilic taxane 5 prodrugs have been synthesized where enhance permeability and retention (EPR) phenomenon considered as effective drug accumulation method to tumor and hence better in vivo efficacy.
  • the lipids used in those prodrugs include phospholipids, cholesterol, fatty acids etc.
  • Platinum-based chemotherapeutic agents are used as first line of therapy in over 70% of all cancers. Cisplatin undergoes rapid formation of cis-[Pt(NH 3 ) 2 Cl(OH 2 )] + and cis- [Pt(NH 3 ) 2 (OH 2 )] 2+ resulting in nephrotoxicity. Further, aquation of both carboplatin and oxaliplatin are significantly slower, resulting in decreased potency.
  • the present invention describes prodrugs of taxane can assemble into supramolecular structure with improved pharmacokinetic profile such as long circulation time, enhance uptake and slow release of drug inside tumor.
  • the taxane uptake to tumor can be achieved in higher amount by making supramolecular assemblies in aqueous buffer along with addition of some co-lipid to form nanoparticles with average particle size below 300 nm. Degradation of the supramolecular assembly as well as the prodrug releases effective drug inside the cell.
  • Pharmaceutical compositions of prodrug of a taxane comprise a linker wherein taxane is coupled through ester, ether, amide, or other covalent conjugation with the linker.
  • the lipid molecule can be cholesterol, alpha tocopherol, fatty acid or other naturally occurring lipid molecule which is conjugated to drug molecule through a suitable linker/spacer.
  • the spacer can be composed of succinic acid, fumaric acid, propargylic acid, ethylene glycol, diethylene glycol, natural or unnatural amino acids individually or in any combinations.
  • the disclosure provides a supramolecular combinatorial therapeutic (SCT).
  • SCT supramolecular combinatorial therapeutic
  • compositions e.g., pharmaceutical compositions comprising the supramolecular combinatorial therapeutic.
  • SCT the term "supramolecular combinatorial therapeutic” or “SCT” refers to nano- or micro-sized strucutres in which, or on which, the active agents to be delivered are not covalently (or otherwise chemically) bound to the structure, but are instead physically or mechanically contained within or retained by the structure. These structures can be stabilized by van der Waals forces or other forms of noncovalent bonding.
  • the supramolecular combinatorial therapeutic can be, but are not limited to, in the form of particles, liposomes, micelles, emulsions, In some embodiments, the superamolecular combinatorial therapeutic is in the form of a nano- or micro-particle. In some embodiments, the supramolecular combinatorial therapeutic is in the form of a particle, wherein particle has a lipid layer forming a lumen, wherein a taxane conjugate is present in or on the outer surface of the lipid layer.
  • Certain exemplary embodiments provide supramolecular combinatorial therapeutics, wherein a taxane-lipid conjugate is combined with one or more of a platinum compound, a kinase inhibitor, and an immunoregulator, each of which is optionally conjugated with a lipid.
  • a method of treating cancer comprising, administering a supramolecular combinatorial therapeutic as described herein to a patient in need of treatment for cancer.
  • the cancer is selected from the group consisting of: breast cancer; ovarian cancer; glioma; gastrointestinal cancer; prostate cancer; carcinoma, lung carcinoma, hepatocellular carcinoma, testicular cancer; cervical cancer; endometrial cancer; bladder cancer; head and neck cancer; lung cancer; gastro-esophageal cancer, and gynecological cancer.
  • the method further comprises coadministering one or more additional anti-cancer therapy to the patient.
  • the additional therapy is selected from the group consisting of surgery, chemotherapy, radiation therapy, thermotherapy, immunotherapy, hormone therapy, laser therapy, anti-angiogenic therapy, and any combinations thereof.
  • the additional therapy comprises administering an anti-cancer agent to the patient.
  • the disclosure also provides a method for prediciting the likehood of a cancer patient exhibiting a better response to self-assembled supramolecular particles than platinates or taxanes.
  • the method comprises assaying the expression level of one or more CAVl, CAV2, CAV3, LDLR, SMAD7, SMURF2, NEDD4 and PRKCA, and wherein increased expression level of at least one of CAVl, CAV2, CAV3, LDLR, SMAD7, SMURF2, NEDD4, PRKCA is indicative of a positive response to treatment comprising self- assembled supramolecular particles.
  • the sample can be a tumor sample.
  • the sample can be a biopsy sample or cells from ascitic fluid or cells from pleural effusion.
  • the sample to be assayed can be a fixed, wax-embedded tissue sample.
  • Fig. 1 shows in vitro characterization of supramolecular new taxane 2 according to an embodiment of the disclosure.
  • Fig. 2 shows in vitro characterization of supramolecular new taxane 16 according to an embodiment of the disclosure.
  • Fig. 3 shows expression of CAVl mRNA by qPCR in responsive (unshaded) and non-responsive (shaded) cell-lines.
  • FIG. 4 shows in vitro characterization of a supramolecular combinatorial therapeutic (SCT) molecule, according to an embodiment of the disclosure, where the supramolecular new taxane is attached to an antibody which binds a protein, receptor, or marker expressed on the surface of a cancer cell.
  • SCT supramolecular combinatorial therapeutic
  • Fig. 5 shows therapeutic effects of 10-125 and anti-PD-Ll against 4T1 (TNBC) tumors.
  • the supramolecular combinatorial therapeutic comprises a taxane-lipid conjugate. It is to be recognized that the supramolecular combinatorial therapeutic can comprise only one type of taxane conjugate or two or more different types of taxane conjugates. Accordingly, in some embodiments, the supramolecular combinatorial therapeutic comprises only one type of taxane conjugate. In some other embodiments, the supramolecular combinatorial therapeutic comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten or more) different types of taxane conjugates. By different types of taxane conjugates is meant that at least one element in the conjugates differes from each other.
  • the different types of conjugates can differ by the specific taxanes in the conjugates, the specific lipids in the conjugates, or the way the taxane and the lipid are conjugated together, i.e., the linker.
  • the taxane-lipid conjugate is a cabazitaxel-cholesterol conjugate.
  • the supramolecular combinatorial therapeutic can further comprise a lipid conjugated kinase inhibitor.
  • the supramolecular combinatorial therapeutic comprises a taxane-lipid conjugate and a kinase inhibitor-conjugate.
  • the supramolecular combinatorial therapeutic comprising the taxane conjugate and the kinase inhibitor conjugate can have the conjugates in any desired combination or ratio.
  • the supramolecular combinatorial therapeutic can comprise two or more (e.g. two, three, four, five, six, seven, eight, nine, ten or more) different types of taxane conjugates and one type of kinase inhibitor conjugate.
  • the supramolecular combinatorial therapeutic can comprise one type of taxane conjugate and two or more (e.g. two, three, four, five, six, seven, eight, nine, ten or more) different types of kinase inhibitor conjugates.
  • the supramolecular combinatorial therapeutic can comprise two or more (e.g. two, three, four, five, six, seven, eight, nine, ten or more) different types of taxane conjugates and two or more (e.g. two, three, four, five, six, seven, eight, nine, ten or more) different types of kinase inhibitor conjugates.
  • the kinase inhibitor is a PI3K inhibitor.
  • the supramolecular combinatorial therapeutic can comprise two or more (e.g. two, three, four, five, six, seven, eight, nine, ten or more) different types of platinum conjugates, two or more (e.g. two, three, four, five, six, seven, eight, nine, ten or more) different types of taxane conjugates, and two or more (e.g. two, three, four, five, six, seven, eight, nine, ten or more) different types of kinase inhibitor conjugates.
  • the supramolecular combinatorial therapeutic further comprises an anti-PD-Ll antibody (or an antigen binding fragment thereof), optionally conjugated with a lipid.
  • the supramolecular combinatorial therapeutic can also comprise a platinum compound conjugated with a lipid.
  • the supramolecular combinatorial therapeutic comprises a taxane-lipid conjugate and a platinum conjugate.
  • the supramolecular combinatorial therapeutic comprising the taxane conjugate and the platinum conjugate can have the conjugates in any desired combination or ratio.
  • the supramolecular combinatorial therapeutic can comprise two or more (e.g. two, three, four, five, six, seven, eight, nine, ten or more) different types of taxane conjugates and one type of platinum conjugate.
  • the supramolecular combinatorial therapeutic can comprise one type of taxane conjugate and two or more (e.g.
  • the supramolecular combinatorial therapeutic can comprise two or more (e.g. two, three, four, five, six, seven, eight, nine, ten or more) different types of taxane conjugates and two or more (e.g. two, three, four, five, six, seven, eight, nine, ten or more) different types of platinum conjugates.
  • the supramolecular combinatorial therapeutic further comprises an anti-PD-Ll antibody (or an antigen binding fragment thereof), optionally conjugated with a lipid.
  • the supramolecular combinatorial therapeutic further comprises an antibody (or an antigen binding fragment thereof) conjugated with a lipid.
  • the antibody can be useful for thereaputic purposes (i.e., a therapeutic antibody) or for targeting the the supramolecular combinatorial therapeutic to a desired site (i.e., a targeting antibody).
  • the supramolecular combinatorial therapeutic further comprises an immunomodulator.
  • Immunomodulators are active agents of immunotherapy, and can either activate or suppress an immune response.
  • the immunomodulator activates and stimulates an immune response against cancer cells, non- limiting examples of which include immune cells (e.g., natural killer cells, lymphokine- activated killer cells, cytotoxic T cells and dendritic cells), antibodies (e.g., anti-PD-Ll and anti-PD-1 antibodies, anti-CD52, anti-VEGF-A, anti- CD30, anti-EGFR, anti-CD33, anti- CD20, anti-CTLA4, and anti-HER-2 antibodies), and cytokines (e.g., interferons and interleukins).
  • immune cells e.g., natural killer cells, lymphokine- activated killer cells, cytotoxic T cells and dendritic cells
  • antibodies e.g., anti-PD-Ll and anti-PD-1 antibodies, anti-CD52, anti-VEGF-A, anti- CD
  • the immunomodulator is conjugated with a lipid.
  • a lipid An important negative co-stimulatory signal regulating T cell activation is provided by programmed death- 1 receptor (PD-1 )(CD279), and its ligand binding partners PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273).
  • PD-1 programmed death- 1 receptor
  • B7-H1, CD274 PD-L2
  • B7-DC CD273
  • the negative regulatory role of PD-1 was revealed by PD-1 knock outs (Pdcdl'), which are prone to autoimmunity [Nishimura et al, Immunity JJ. : 141-51 (1999); Nishimura et al, Science 291 : 319-22 (2001)].
  • PD-1 can be expressed on T cells, B cells, natural killer T cells, activated monocytes and dendritic cells (DCs).
  • DCs dendritic cells
  • PD-1 is expressed by activated, but not by unstimulated human CD4 + and CD8 + T cells, B cells and myeloid cells. This stands in contrast to the more restricted expression of CD28 and CTLA-4 [Nishimura et al, Int. Immunol. 8: 773-80 (1996); Boettler et al, J. Virol. 80: 3532-40 (2006)].
  • PD-L1 is constitutively expressed on mouse T and B cells, CDs, macrophages, mesenchymal stem cells and bone marrow-derived mast cells [Yamazaki et al., J. Immunol.
  • PD-L1 is expressed on a wide range of nonhematopoietic cells (e.g., cornea, lung, vascular epithelium, liver nonparenchymal cells, mesenchymal stem cells, pancreatic islets, placental synctiotrophoblasts, keratinocytes, etc.) [Keir et al, Annu. Rev. Immunol. 26: 677-704 (2008)], and is upregulated on a number of cell types after activation. PD-L2 expression is more restricted than PD-L1. PD-L2 is inducibly expressed on DCs, macrophages, and bone marrow-derived mast cells. PD-L2 is also expressed on about half to two-thirds of resting peritoneal Bl cells, but not on conventional B2 B cells [Zhong et al, Eur. J. Immunol. 37: 2405-10 (2007)].
  • nonhematopoietic cells e.g., cornea, lung, vascular epithel
  • PD-1 signaling typically has a greater effect on cytokine production than on cellular proliferation, with significant effects on IFN- ⁇ , TNF-a and IL-2 production.
  • PD-1 mediated inhibitory signaling also depends on the strength of the TCR signaling, with greater inhibition delivered at low levels of TCR stimulation. This reduction can be overcome by co- stimulation through CD28 [Freeman et al., J. Exp. Med. 192: 1027-34 (2000)] or the presence of IL-2 [Carter et al., Eur. J. Immunol. 32: 634-43 (2002)].
  • Evidence is mounting that signaling through PD-L1 and PD-L2 may be bidirectional.
  • signaling may also be delivered back to the cells expressing PD-L1 and PD-L2. While treatment of dendritric cells with a naturally human anti-PD-L2 antibody isolated from a patient with Waldenstrom's macroglobulinemia was not found to upregulate MHC II or B7 costimulatory molecules, such cells did produce greater amount of proinflammatory cytokines, particularly TNF-a and IL-6, and stimulated T cell proliferation [Nguyen et al, J. Exp. Med. 196: 1393-98 (2002)].
  • mice with this antibody also (1) enhanced resistance to transplated B16 melanoma and rapidly induced tumor- specific CTL [Radhakrishnan et al., J. Immunol. 170: 1830-38 (2003); Radhakrishnan et al., Cancer Res. 64: 4965-72 (2004); Heckman et al, Eur. J. Immunol. 37: 1827-35 (2007)]; (2) blocked development of airway inflammatory disease in a mouse model of allergic asthma [Radhakrishnan et al., J. Immunol. 173: 1360-65 (2004); Radhakrishnan et al, J. Allergy Clin.Immunol. UJy. 668-74 (2005)].
  • Certain aspects of the present invention describe prodrugs of combinations of chemotherapeutic drugs with anti-PD-1 or anti-PD-Ll antibodies that can assemble into supramolecular structures with improved tumor loading and upregulated T cell-mediated immune responses, and the use of these supramolecules in the treatment of cancer.
  • the anti-PD-Ll antibodies according to the present invention which have antibody dependent cell-mediated cytotoxicity (ADCC) activity, directly act on PD-L1 bearing tumor cells by inducing their lysis without showing any significant toxicity. Moreover, the antibodies block the interaction between human PD-L1 and human PD-1 and enhance T-cell function to upregulate cell-mediated immune responses for the treatment of cancer.
  • ADCC antibody dependent cell-mediated cytotoxicity
  • cancer is selected from the group consisting of: breast cancer; ovarian cancer; glioma; gastrointestinal cancer; prostate cancer; carcinoma, lung carcinoma, hepatocellular carcinoma, testicular cancer; cervical cancer; endometrial cancer; bladder cancer; head and neck cancer; lung cancer; gastro-esophageal cancer, and gynecological cancer.
  • the method further comprises co-administering one or more additional anti-cancer therapy to the patient.
  • the additional therapy is selected from the group consisting of surgery, chemotherapy, radiation therapy, thermotherapy, immunotherapy, hormone therapy, laser therapy, anti-angiogenic therapy, and any combinations thereof.
  • the additional therapy comprises administering an anti-cancer agent to the patient.
  • the method further comprises co-administration of one or more immunomodulators to the subject.
  • the immunomodulator is an anti-PD-Ll or an anti-PD-1 antibody.
  • the platinum/ taxane/anti-PD-Ll uptake to tumor can be achieved in higher amounts by supramolecular assembly in aqueous buffer along with addition of some co-lipid to form nanoparticles with average particle size below 300 nm. Degradation of supramolecular assembly as well as the prodrug releases effective drug inside cells.
  • Pharmaceutical composition of prodrug of a platinum/taxane comprises a linker wherein platinum/taxane is coupled through ester, ether, amide or other covalent conjugation with the linker.
  • the lipid molecule can be cholesterol, alpha tocopherol, fatty acid or other naturally occurring lipid molecule which is conjugated to drug molecule through a suitable linker/spacer.
  • the spacer can be composed of succinic acid, fumaric acid, propargylic acid, ethylene glycol, diethylene glycol, natural or unnatural amino acids individually or in any combinations.
  • the supramolecular combinatorial therapeutic is in the form of a particle, wherein the particle has a lipid layer forming a lumen, wherein a platinum/taxane/anti-PD-Ll conjugate is present in or on the outer surface of the lipid layer.
  • the disclosure provides a supramolecular combinatorial therapeutic comprising a taxane conjugate, e.g., a hydrophobic taxane-lipid conjugate.
  • Amount of the conjugate in the supramolecular combinatorial therapeutic can range from about 1% to about 99% (w/w).
  • the amount of the conjugate in the supramolecular combinatorial therapeutic can be from about 5% to about 95%, from about 10% to about 90%, from about 15%) to about 85%), from about 20%> to about 75%, or from about 25% to about 50%>.
  • the composition can comprise two or more (e.g., two, three, four, five, six, seven, eight, nine, ten or more) different taxane conjugates.
  • the different conjugates can be present in any desired ratio.
  • the different conjugates can be in a ratio ranging from about 100: 1 to 1 : 100.
  • the different conjugates can be in a ratio ranging from about 50: 1 to 1 :50, 25: 1 to 1 :25, 10: 1 to 1 : 10, 5: 1 to 1 :5, or 2.5: 1 to 1 :2.5.
  • the different conjugates can be in a ratio of about 1 : 1.
  • the supramolecular combinatorial therapeutic can be in any shape, size or form.
  • the supramolecular combinatorial therapeutic can be in the form of a nano- or micro-structure.
  • Such structures can include, but are not limited to liposome, emulsions, and micelles.
  • the supramolecular combinatorial therapeutic can be in the form of a liposome.
  • liposome encompasses any compartment enclosed by a lipid layer. Liposomes can have one or more lipid membranes. Liposomes can be characterized by membrane type and by size.
  • Small unilamellar vesicles have a single membrane and typically range between 0.02 and 0.05 ⁇ in diameter; large unilamellar vesicles (LUVS) are typically larger than 0.05 ⁇ .
  • Oligolamellar large vesicles and multilamellar vesicles have multiple, usually concentric, membrane layers and are typically larger than 0.1 ⁇ .
  • Liposomes with several nonconcentric membranes, i.e., several smaller vesicles contained within a larger vesicle, are termed multivesicular vesicles.
  • the lipid molecules comprise elongated non-polar (hydrophobic) portions and polar (hydrophilic) portions.
  • the hydrophobic and hydrophilic portions of the molecule are preferably positioned at two ends of an elongated molecular structure.
  • the lamellae are composed of two mono layer sheets of lipid molecules with their non-polar (hydrophobic) surfaces facing each other and their polar (hydrophilic) surfaces facing the aqueous medium.
  • the membranes formed by the lipids enclose a portion of the aqueous phase in a manner similar to that of a cell membrane enclosing the contents of a cell.
  • the bilayer of a liposome has similarities to a cell membrane without the protein components present in a cell membrane.
  • a liposome composition can be prepared by a variety of methods that are known in the art. See e.g., US Patent No. 4,235,871, No. 4,897,355 and No. 5,171,678; published PCT applications WO 96/14057 and WO 96/37194; Feigner, P. L. et al, Proc. Natl. Acad. Sci., USA (1987) 8:7413-7417, Bangham, et al. M. Mol. Biol. (1965) 23:238, Olson, et al. Biochim. Biophys. Acta (1979) 557:9, Szoka, et al. Proc. Natl. Acad. Sci.
  • the liposomes can be prepared to have substantially homogeneous sizes in a selected size range.
  • One effective sizing method involves extruding an aqueous suspension of the liposomes through a series of polycarbonate membranes having a selected uniform pore size; the pore size of the membrane will correspond roughly with the largest sizes of liposomes produced by extrusion through that membrane. See e.g., U.S. Patent No. 4,737,323, content of which is incorporated herein by reference in its entirety.
  • a lipid conjugated component of the supramolecular combinatorial therapeutic is present in or on the surface of the lipid layer.
  • the supramolecular combinatorial therapeutic is in the form of a liposome, wherein the lipid non-lipid portion of a lipid conjugated component (e.g., taxane, PI3K inhibitor, platinum, antibody portion of the conjugate) is on the outer surface of the lipid layer.
  • a lipid conjugated component e.g., taxane, PI3K inhibitor, platinum, antibody portion of the conjugate
  • the supramolecular combinatorial therapeutic can also be in the form of an emulsion.
  • Emulsions are typically heterogenous systems of one liquid dispersed in another in the form of droplets (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1 , p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1 , p.
  • Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other.
  • emulsions may be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety.
  • Emulsions can contain additional components in addition to the dispersed phases, and the conjugate disclosed herein can be present as a solution in either the aqueous phase or the oily phase or itself as a separate phase.
  • compositions can also be present in emulsions as needed.
  • Pharmaceutical emulsions can also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water- in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.
  • Such complex formulations often provide certain advantages that simple binary emulsions do not.
  • Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion.
  • a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.
  • Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion may be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion.
  • Emulsifiers can broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).
  • Synthetic surfactants also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199).
  • Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion.
  • HLB hydrophile/lipophile balance
  • surfactants may be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).
  • Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia.
  • Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations.
  • non-emulsifying materials can also be included in emulsion formulations and contribute to the properties of emulsions.
  • Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.
  • polysaccharides for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth
  • cellulose derivatives for example, carboxymethylcellulose and carboxypropylcellulose
  • synthetic polymers for example, carbomers, cellulose ethers, and
  • emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that may readily support the growth of microbes, these formulations often incorporate preservatives.
  • preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid.
  • Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation.
  • Antioxidants used can be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.
  • free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite
  • antioxidant synergists such as citric acid, tartaric acid, and lecithin.
  • Emulsion formulations for oral delivery have been very widely used because of ease of formulation, as well as efficacy from an absorption and bioavailability standpoint (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.
  • the supramolecular combinatorial therapeutic can be in the form of a particle.
  • the term "particle” encompasses liposomes, emulsions, vesicles and lipid particles.
  • the particle can be of any shape or form, e.g., spherical, rod, elliptical, cylindrical, capsule, or disc; and these particles can be part of a network or an aggregate. Without limitations, the particle can have any size from nm to millimeters.
  • the particle is a microparticle or a nanoparticle.
  • microparticle refers to a particle having a particle size of about 1 ⁇ to about 1000 ⁇ .
  • nanoparticle refers to particle having a particle size of about 0.1 nm to about 1000 nm.
  • the particles disclosed herein are nanoparticles and have an average diameter of from about 5 nm to about 500 nm.
  • the particles have an average diameter of from about 75 nm to about 500 nm, from about 25 nm to about 250 nm, from about 50 nm to about 150 nm, from about 75 nm to about 125 nm, from about 50 nm to about 500 nm, from about 75 nm to about 200 nm, from about 100 to about 175 nm, from about 125 nm to about 175 nm, from about 40 nm to about 90 nm, or from about 50 nm to about 80 nm.
  • a nanoparticle can be less than about 1 um in diameter, e.g., about 1 um or less in diameter, about 500 nm or less in diameter, about 400 nm or less in diameter, about 300 nm or less in diameter, about 200 nm or less in diameter, about 100 nm or less in diameter, about 50 nm or less in diameter, or about 10 nm or less in diameter.
  • a nanoparticle can be less than 1 um in diameter, e.g., 1 um or less in diameter, 500 nm or less in diameter, 400 nm or less in diameter, 300 nm or less in diameter, 200 nm or less in diameter, 100 nm or less in diameter, 50 nm or less in diameter, or 10 nm or less in diameter.
  • the nanoparticles in a composition can be from about 1 nm to about 1 um in diameter, e.g.
  • the nanoparticles in a composition can be from 1 nm to 1 um in diameter, e.g. from 1 nm to 500 nm in diameter, from 1 nm to 200 nm in diameter, from 10 nm to 200 nm in diameter, from 100 nm to 200 nm in diameter, or from 10 nm to 100 nm in diameter.
  • nanoparticles can be selected to be of specific sizes, e.g. less than about 200 nm in diameter.
  • Methods of selecting nanoparticles of a particular size and/or range of sizes are known in the art and can include, by way of non-limiting example, filtration, sedimentation, centrifugation, and/or chromatographic methods, e.g. SEC.
  • particle size refers to the mode of a size distribution of particles, i.e., the value that occurs most frequently in the size distribution.
  • Methods for measuring the particle size are known to a skilled artisan, e.g., by dynamic light scattering (such as photocorrelation spectroscopy, laser diffraction, low-angle laser light scattering (LALLS), and medium-angle laser light scattering (MALLS)), light obscuration methods (such as Coulter analysis method), or other techniques (such as rheology, and light or electron microscopy).
  • the particles can be substantially spherical. What is meant by “substantially spherical” is that the ratio of the lengths of the longest to the shortest perpendicular axis of the particle cross section is less than or equal to about 1.5. Substantially spherical does not require a line of symmetry. Further, the particles can have surface texturing, such as lines or indentations or protuberances that are small in scale when compared to the overall size of the particle and still be substantially spherical.
  • the ratio of lengths between the longest and shortest axes of the particle is less than or equal to about 1.5, less than or equal to about 1.45, less than or equal to about 1.4, less than or equal to about 1.35, less than or equal to about 1.30 ⁇ less than or equal to about 1.25 ⁇ less than or equal to about 1.20 ⁇ less than or equal to about 1.15 less than or equal to about 1.1.
  • surface contact is minimized in particles that are substantially spherical, which minimizes the undesirable agglomeration of the particles upon storage. Many crystals or flakes have flat surfaces that can allow large surface contact areas where agglomeration can occur by ionic or non-ionic interactions. A sphere permits contact over a much smaller area.
  • the particles can be, e.g., monodispersed or polydispersed and the variation in diameter of the particles of a given dispersion can vary.
  • the particles have substantially the same particle size.
  • Particles having a broad size distribution where there are both relatively big and small particles allow for the smaller particles to fill in the gaps between the larger particles, thereby creating new contact surfaces.
  • a broad size distribution can result in larger spheres by creating many contact opportunities for binding agglomeration.
  • the particles described herein are within a narrow size distribution, thereby minimizing opportunities for contact agglomeration.
  • a “narrow size distribution” is a particle size distribution that has a ratio of the volume diameter of the 90th percentile of the small spherical particles to the volume diameter of the 10th percentile less than or equal to 5.
  • the volume diameter of the 90th percentile of the small spherical particles to the volume diameter of the 10th percentile is less than or equal to 4.5, less than or equal to 4, less than or equal to 3.5, less than or equal to 3, less than or equal to 2.5, less than or equal to 2, less than or equal to 1.5, less than or equal to 1.45, less than or equal to 1.40, less than or equal to 1.35, less than or equal to 1.3, less than or equal to 1.25, less than or equal to 1.20, less than or equal to 1.15, or less than or equal to 1.1.
  • GSD Geometric Standard Deviation
  • ECD effective cutoff diameter
  • GSD is equal to the square root of the ratio of the ECD less than 84.17% to ECD less than 15.9%.
  • the GSD has a narrow size distribution when GSD ⁇ 2.5. In some embodiments, GSD is less than 2, less than 1.75, or less than 1.5. In one embodiment, GSD is less than 1.8.
  • the supramolecular combinatorial therapeutic can further include one or more additional lipids and/or other components.
  • additional lipids and/or other components can be included in the supramolecular combinatorial therapeutic for a variety of purposes, such as to prevent lipid oxidation, to stabilize bilayer, to reduce aggregation during formation or to attach ligands onto the particle surface.
  • Any of a number of lipids can be present, including but not limited to, amphipathic, neutral, cationic, anionic lipids, sterols, and phospholipids. Further, such lipids can be used alone or in any combination with each other.
  • the supramolecular combinatorial therapeutic further comprises a lipoprotein particle, e.g., HDL or LDL.
  • the supramolecular combinatorial therapeutic can comprise from about 1% to about 99% (w/w) of the additional lipid or component. Further the additional lipid or component can be present in 10: 1 to 1 : 10 ratio with the conjugate. If two or more different additional lipids are present in the supramolecular combinatorial therapeutic, each lipid can be independently in 10: 1 to 1 : 10 ratio with the conjugate. Further, if two or more different additional lipids are present in the supramolecular combinatorial therapeutic, the two lipids can be in 10:1 to 1 : 10 ratio.
  • two different components (conjugate and lipid or two different lipids) of the supramolecular combinatorial therapeutic can be in ratio 10: 1 to 1 : 10, 5: 1 to 1 :5, or 2.5: 1 to 1 :2.5.
  • two different components in the supramolecular combinatorial therapeutic can be in ratio of about 1 : 1, about 1 : 1.2, about 1 : 1.5, about 1 : 1.7, about 1 :2, about 1 :2.5, about 1 :3, about 1 :3.5, about 1 :4, about 1 :4.5, about 1 :5, about 1 :5.5, about 1 :6, about 1 :6.5, about 1 :7, about 1 :7.5, about 1 :8, about 1 :8.5, about 1 :9, about 1 :9.5, or about 1 : 10.
  • the supramolecular combinatorial therapeutic comprises more than two components ratio between any two components can be independent of ratio between any other two components.
  • the composition further comprises a first lipid in addition to the conjugate.
  • lipid as used herein means a substance that is soluble in organic solvents and includes, but is not limited to, oils, fats, sterols, triglycerides, fatty acids, phospholipids, and the like.
  • the lipid can be selected from the group consisting of sterol lipids, fatty acids, fatty alcohols, glycerolipids (e.g., monoglycerides, diglycerides, and triglycerides), phospholipids, glycerophospholipids, sphingolipids, prenol lipids, saccharolipids, polyketides, and any combination thereof.
  • the lipid can be a polyunsaturated fatty acid or alcohol.
  • polyunsaturated fatty acid or “polyunsaturated fatty alcohol” as used herein means a fatty acid or alcohol with two or more carbon-carbon double bonds in its hydrocarbon chain.
  • the lipid can also be a highly unsaturated fatty acid or alcohol.
  • highly polyunsaturated fatty acid or “highly polyunsaturated fatty alcohol” as used herein means a fatty acid or alcohol having at least 18 carbon atoms and at least 3 double bonds.
  • the lipid can be an omega-3 fatty acid.
  • omega-3 fatty acid as used herein means a polyunsaturated fatty acid whose first double bond occurs at the third carbon-carbon bond from the end opposite the acid group.
  • the lipid can be selected from the group consisting of cholesterol; 1,3-Propanediol Dicaprylate/Dicaprate; 10-undecenoic acid; 1-dotriacontanol; 1- heptacosanol; 1-nonacosanol; 2-ethyl hexanol; Androstanes; Arachidic acid; Arachidonic acid; arachidyl alcohol; Behenic acid; behenyl alcohol; Capmul MCM CIO; Capric acid; capric alcohol; capryl alcohol; Caprylic acid; Caprylic/Capric Acid Ester of Saturated Fatty Alcohol C12-C18; Caprylic/Capric Triglyceride; Caprylic/Capric Triglyceride; Ceramide phosphorylcholine (Sphingomyelin, SPH); Ceramide phosphorylethanolamine (Sphingomyelin, Cer-PE); Ceramide phosphorylglycerol; Ceroplastic acid; Ceramide phosphorylcholine (S
  • the first lipid is a phospholipid.
  • the phospholipids can be of natural origin, such as egg yolk or soybean phospholipids, or synthetic or semisynthetic origin.
  • the phospholipids can be partially purified or fractionated to comprise pure fractions or mixtures of phosphatidyl cholines, phosphatidyl cholines with defined acyl groups having 6 to 22 carbon atoms, phosphatidyl ethanolamines, phosphatidyl inositols, phosphatidic acids, phosphatidyl serines, sphingomyelin or phosphatidyl glycerols.
  • Suitable phospholipids include, but are not limited to, phosphatidylcholine, phosphatidylglycerol, lecithin, ⁇ , ⁇ -dipalmitoyl-a-lecithin, sphingomyelin, phosphatidylserine, phosphatidic acid, N-(2,3-di(9-(Z)-octadecenyloxy))- prop-l-yl-N,N,N-trimethylammonium chloride, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamme, phosphatidylinositol, cephalin, cardiolipin, cerebrosides, dicetylphosphate, dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, dioleoylphosphatidylglycerol, palm
  • Non-phosphorus containing lipids can also be used. These include, e.g., stearylamine, docecylamine, acetyl palmitate, fatty acid amides, and the like. Other phosphorus-lacking compounds, such as sphingo lipids, glycosphingolipid families, diacylglycerols, and ⁇ -acyloxyacids, can also be used.
  • the phospholipid in the supramolecular combinatorial therapeutic is selected from the group consisting of l,2-Didecanoyl-s/?-glycero-3- phosphocholine; 1 ,2-Dierucoyl-s/?-glycero-3 -phosphate (Sodium Salt); 1 ,2-Dierucoyl-sn- glycero-3-phosphocholine; 1 ,2-Dierucoyl-5/?-glycero-3-phosphoethanolamine; 1 ,2-Dierucoyl- 5/?-glycero-3[Phospho-rac-(l -glycerol) (Sodium Salt); l,2-Dilinoleoyl-s/?-glycero-3- phosphocholine; 1 ,2-Dilauroyl-s/?-glycero-3 -phosphate (Sodium Salt); 1 ,2-Dilauroyl-sn- glycero-3-phosphocholine; 1 ,2-Dil
  • the phospholipid is SPOC, egg PC, or Hydrogenated Soy PC (HSPC).
  • the phospholipid in the composition is SOPC.
  • the supramolecular combinatorial therapeutic further comprises a second lipid in addition to the conjugate and the first lipid.
  • the second lipid is a phospholipid.
  • the supramolecular combinatorial therapeutic further comprises a polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the PEG can be included in the composition by itself or conjugated with a component of the supramolecular combinatorial therapeutic.
  • the PEG can be conjugated with the conjugate or a lipid component of the supramolecular combinatorial therapeutic.
  • the PEG is conjugated with a lipid component of the supramolecular combinatorial therapeutic.
  • the PEG can be conjugated with any lipid or phospholipids.
  • the PEG conjugated lipid can be selected from the group consisting of PEG conjugated diacylglycerols and dialkylglycerols, PEG- conjugated phosphatidylethanolamine, PEG conjugated to phosphatidic acid, PEG conjugated ceramides (see, U.S. Patent No. 5,885,613), , PEG conjugated dialkylamines, PEG conjugated l ,2-diacyloxypropan-3-amines, and PEG conjugated to l ,2-distearoyl-sn-glycem-3-phosphoethanolamine (DSPE), and any combinations thereof.
  • the PEG conjugated lipid is 1 ,2-distearoyl-sn- glycem-3-phosphoethanolamine-N-[amino(poly ethylene glycol)-2000] (DSPE-PEG2000).
  • the supramolecular combinatorial therapeutic further comprises a targeting ligand.
  • targeting moiety or “targeting ligand” refers to any molecule that provides an enhanced affinity for a selected target, e.g., a cell, cell type, tissue, organ, region of the body, or a compartment, e.g., a cellular, tissue or organ compartment.
  • the targeting moiety or ligand can comprise a wide variety of entities. Such ligands can include naturally occurring molecules, or recombinant or synthetic molecules.
  • Exemplary targeting ligands include, but are not limited to, antibodies (polyclonal or monoclonal), antigen binding fragments of antibodies, antigens, folates, EGFR, albumin, receptor ligands, carbohydrates, aptamers, integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL and HDL ligands.
  • antibodies polyclonal or monoclonal
  • antigen binding fragments of antibodies include, but are not limited to, antibodies (polyclonal or monoclonal), antigen binding fragments of antibodies, antigens, folates, EGFR, albumin, receptor ligands, carbohydrates, aptamers, integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin
  • Additional exemplary ligands include, but are not limited to, polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (e.g., PEG-2K, PEG-5K, PEG-10K, PEG-12K, PEG-15K, PEG-20K, PEG-40K), MPEG, [MPEG] 2 , polyvinyl alcohol (PVA), polyurethane, poly(2- ethylacryllic acid), N-isopropylacrylamide polymers, polyphosphazine, polyethylenimine, cationic groups, spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic poly
  • psoralen mitomycin C
  • porphyrins e.g., TPPC4, texaphyrin, Sapphyrin
  • polycyclic aromatic hydrocarbons e.g., phenazine, dihydrophenazine
  • artificial endonucleases e.g., EDTA
  • lipophilic molecules e.g, steroids, bile acids, cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, l ,3-Bis-0(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1 ,3 -propanediol, heptadecyl group, palmitic acid, myristic acid,03-(oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid
  • biotin transport/absorption facilitators
  • transport/absorption facilitators e.g., naproxen, aspirin, vitamin E, folic acid
  • synthetic ribonucleases e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine- imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, AP, antibodies, hormones and hormone receptors, lectins, carbohydrates, multivalent carbohydrates, vitamins (e.g., vitamin A, vitamin E, vitamin K, vitamin B, e.g., folic acid, B12, riboflavin, biotin and pyridoxal), vitamin co factors, lipopolysaccharide, an activator of p38 MAP kinase, an activator of NF- ⁇ , taxon, vincristine, vinblastine, cytochalasin, nocodazole, ja
  • Peptide and peptidomimetic ligands include those having naturally occurring or modified peptides, e.g., D or L peptides; ⁇ , ⁇ , or ⁇ peptides; N-methyl peptides; azapeptides; peptides having one or more amide, i.e., peptide, linkages replaced with one or more urea, thiourea, carbamate, or sulfonyl urea linkages; or cyclic peptides.
  • a peptidomimetic also referred to herein as an oligopeptidomimetic is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide.
  • the peptide or peptidomimetic ligand can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.
  • Carbohydrate based targeting ligands include, but are not limited to, D-galactose, multivalent galactose, N-acetyl-D-galactose (GalNAc), multivalent GalNAc, e.g. GalNAc2 and GalNAc3; D-mannose, multivalent mannose, multivalent lactose, N-acetyl- galactosamine, N-acetyl-gulucosamine, multivalent fucose, glycosylated polyaminoacids and lectins.
  • the term multivalent indicates that more than one monosaccharide unit is present.
  • Such monosaccharide subunits can be linked to each other through glycosidic linkages or linked to a scaffold molecule.
  • a number of folate and folate analogs amenable to the present invention as ligands are described in U.S. Pat. Nos. 2,816,110; 5,552,545; 6,335,434 and 7,128,893, contents of all of which are herein incorporated in their entireties by reference.
  • targeting moieties such as ligands, cell surface receptors, glycoproteins, vitamins ⁇ e.g., riboflavin), aptamers and monoclonal antibodies, can also be used.
  • the targeting moieties can include the entire protein or fragments thereof. Targeting mechanisms generally require that the targeting agents be positioned on the surface of the liposome in such a manner that the targeting moiety is available for interaction with the target, for example, a cell surface receptor.
  • the targeting ligand binds a protein, receptor, or marker expressed on the surface of a cancer cell.
  • Targeting ligands that preferentially bind to and/or cross the membrane of cancer cells are known in the art, e.g.
  • iRGD iRGD, RGD, Lyp-1 peptide (CGNKRTRGC), NGR peptide, iNGR, RGR peptide, CAR peptide, tCAR peptide (CARSKNK); FSH-33, Allatostatin 1 , the pentapeptide CREKA, Hepatocarcinoma targeting peptide, Peptide GFE, anti-EGFR antibodies and/or antibody fragments, in particular Cetuximab, CendR, iRGD peptide (RGD-CendR hybrid peptide), small molecules, antibodies and/or antibody fragments binding to cancer-specific epitopes like e.g.
  • a targeting agent can be iRGD, e.g. a peptide having the sequence CRGDKGPDC.
  • the targeting ligand binds EGFR.
  • the targeting ligand is a polyclonal or monoclonal antibody or a fragment thereof retaining epitope binding activity or an antibody-based binding moiety.
  • the targeting ligand is a polyclonal or monoclonal antibody, antibody fragments, a peptide, or a molecule that is capable of binding protein receptors expressed on the surface of cancer cells.
  • the targeting ligand is an antibody selected from the group consisting of C242 antibody (CanAg), Rituximab (CD20), Trastuzumab (Her2), Cetuximab (EGFR), Bevacizumab (VEGF), Panitumumab, Alemtuzumab, Ofatumumab, Gemtuzumab (CD33), Inotuzumab (CD22), Lorvotuzumab (CD56), Brentuximab (CD30), Glembatumumab (GPNMB), epitope bind fragments thereof and any combinations thereof.
  • C242 antibody CanAg
  • Rituximab CD20
  • Trastuzumab Her2
  • Cetuximab EGFR
  • Bevacizumab VEGF
  • Panitumumab Alemtuzumab
  • Ofatumumab Ofatumumab
  • Gemtuzumab CD33
  • Inotuzumab CD22
  • the targeting ligand can be present, e.g. on the surface of a supramolecular combinatorial therapeutic described herein and/or partially embedded in the membrane or lipid layer of a supramolecular combinatorial therapeutic described herein.
  • Methods of incorporating a targeting agent are known in the art and non-limiting examples are described elsewhere herein.
  • a supramolecular combinatorial therapeutic described herein can comprise a two or more targeting agents, e.g. a supramolecular combinatorial therapeutic can comprise a combination of nanoparticles, each comprising a different targeting agent and/or a composition can comprise nanoparticles which each comprise multiple targeting agents.
  • a supramolecular combinatorial therapeutic described herein can comprise one targeting agent, two targeting agents, three targeting agents, or more targeting agents.
  • the targeting ligand can be linked to a component (e.g., a lipid) of the supramolecular combinatorial therapeutic.
  • the targeting ligand can be conjugated with a lipid.
  • a component e.g., a lipid
  • the targeting ligand can be conjugated with a lipid.
  • a variety of different targeting ligands and methods are known and available in the art, including those described, e.g., in Sapra, P. and Allen, TM, Prog. Lipid Res. 42(5):439-62 (2003); and Abra, RM et al, J. Liposome Res. 12: 1-3, (2002).
  • Other lipids conjugated with targeting moieties are described in US Patent Application Publication No. US2009/0247608 and No. US2012/0046478, content of both of which is incorporated herein by reference in its entirety.
  • the compositon can further comprise a therapeutic agent in addition to the taxane conjugate.
  • a therapeutic agent when present in the supramolecular combinatorial therapeutic, can be encapsulated in the supramolecular combinatorial therapeutic; present in a lipid layer of the supramolecular combinatorial therapeutic; or present on the surface of the supramolecular combinatorial therapeutic.
  • therapeutic agent means a molecule, group of molecules, complex or substance administered to an organism for diagnostic, therapeutic, preventative medical, or veterinary purposes.
  • the term "therapeutic agent” includes a "drug” or a “vaccine.” This term include externally and internally administered topical, localized and systemic human and animal pharmaceuticals, treatments, remedies, nutraceuticals, cosmeceuticals, biologicals, devices, diagnostics and contraceptives, including preparations useful in clinical and veterinary screening, prevention, prophylaxis, healing, wellness, detection, imaging, diagnosis, therapy, surgery, monitoring, cosmetics, prosthetics, forensics and the like.
  • This term can also be used in reference to agriceutical, workplace, military, industrial and environmental therapeutics or remedies comprising selected molecules or selected nucleic acid sequences capable of recognizing cellular receptors, membrane receptors, hormone receptors, therapeutic receptors, microbes, viruses or selected targets comprising or capable of contacting plants, animals and/or humans.
  • This term can also specifically include nucleic acids and compounds comprising nucleic acids that produce a therapeutic effect, for example deoxyribonucleic acid (DNA), ribonucleic acid (R A), or mixtures or combinations thereof, including, for example, DNAnanoplexes.
  • DNA deoxyribonucleic acid
  • R A ribonucleic acid
  • the term "therapeutic agent” also includes an agent that is capable of providing a local or systemic biological, physiological, or therapeutic effect in the biological system to which it is applied.
  • the therapeutic agent can act to control infection or inflammation, enhance cell growth and tissue regeneration, control tumor growth, act as an analgesic, promote anti-cell attachment, and enhance bone growth, among other functions.
  • Other suitable therapeutic agents can include anti-viral agents, hormones, antibodies, or therapeutic proteins.
  • Other therapeutic agents include prodrugs, which are agents that are not biologically active when administered but, upon administration to a subject are converted to biologically active agents through metabolism or some other mechanism.
  • a silk- based drug delivery composition can contain combinations of two or more therapeutic agents.
  • a therapeutic agent can include a wide variety of different compounds, including chemical compounds and mixtures of chemical compounds, e.g., small organic or inorganic molecules; saccharines; oligosaccharides; polysaccharides; biological macromolecules, e.g., peptides, proteins, and peptide analogs and derivatives; peptidomimetics; antibodies (polyconal and monoclonal) and antigen binding fragments thereof; nucleic acids; nucleic acid analogs and derivatives; an extract made from biological materials such as bacteria, plants, fungi, or animal cells; animal tissues; naturally occurring or synthetic compositions; and any combinations thereof.
  • the therapeutic agent is a small molecule.
  • small molecule can refer to compounds that are "natural product- like," however, the term “small molecule” is not limited to "natural productlike” compounds. Rather, a small molecule is typically characterized in that it contains several carbon— carbon bonds, and has a molecular weight of less than 5000 Daltons (5 kDa), preferably less than 3 kDa, still more preferably less than 2 kDa, and most preferably less than 1 kDa. In some cases it is preferred that a small molecule have a molecular weight equal to or less than 700 Daltons.
  • Exemplary therapeutic agents include, but are not limited to, those found in Harrison 's Principles of Internal Medicine , 13th Edition, Eds. T.R. Harrison et al. McGraw- Hill N.Y., NY; Physicians' Desk Reference, 50th Edition, 1997, Oradell New Jersey, Medical Economics Co.; Pharmacological Basis of Therapeutics, 8th Edition, Goodman and Gilman, 1990; United States Pharmacopeia, The National Formulary, USP XII NF XVII, 1990; current edition of Goodman and Oilman's The Pharmacological Basis of Therapeutics ; and current edition of The Merck Index , the complete contents of all of which are incorporated herein by reference.
  • the therapeutic agent can be linked to a component of the supramolecular combinatorial theraputic.
  • the therapeutic agent can be linked to a lipid or phospholipid component of the supramolecular combinatorial theraputic.
  • the therapeutic agent and the component of the supramolecular combinatorial therapeutic can be linked together by a bond or via a linker.
  • This linker can be cleavable or non-cleavable, depending on the application.
  • a cleavable linker can be used to release the therapeutic agent after transport to the desired target.
  • the intended nature of the conjugation or coupling interaction, or the desired biological effect will determine the choice of linker group.
  • the lipid in the lipid conjugated therapeutic agent is cholesterol.
  • the supramolecular combinatorial therapeutic can comprise from about 1% to about 99% (w/w) of the therapeutic agent or a conjugate thereof. Further the therapeutioc agent or a conjugate thereof can be present in 10: 1 to 1 : 10 ratio with the taxane conjugate. Without limitations, taxane conjugate and thereaputic agent (or a conjugate thereof) in the supramolecular combinatorial therapeutic can be in ratio 10: 1 to 1 : 10, 5: 1 to 1 :5, or 2.5: 1 to 1 :2.5.
  • taxane conjugate and thereaputic agent (or a conjugate thereof) in the supramolecular combinatorial therapeutic can be in ratio of about 1 : 1, about 1 : 1.2, about 1 : 1.5, about 1 : 1.7, about 1 :2, about 1 :2.5, about 1 :3, about 1 :3.5, about 1 :4, about 1 :4.5, about 1 :5, about 1 :5.5, about 1 :6, about 1 :6.5, about 1 :7, about 1 :7.5, about 1 :8, about 1 :8.5, about 1 :9, about 1 :9.5, or about 1 : 10.
  • the therapeutic agent is an antibody (e.g., polyclonal or monoclonal antibody) or an antigen binding fragment thereof.
  • the therapeutic agent is an antibody (e.g., polyclonal or monoclonal antibody), or an antigen binding fragment thereof, conjugated with a lipid, e.g., cholesterol.
  • the antibody is an immunomodulator comprising an anti-PD-1 antibody, an anti-PD-Ll antibody and combinations thereof.
  • the immunomodulator is conjugated with lipid, e.g. cholesterol or other lipids disclosed herein.
  • the therapeutic agent is a chemotherapeutic or anti-cancer agent.
  • chemotherapeutic agent refers to any chemical or biological agent with therapeutic usefulness in the treatment of diseases characterized by abnormal cell growth. Such diseases include tumors, neoplasms and cancer as well as diseases characterized by hyperplastic growth. These agents can function to inhibit a cellular activity upon which the cancer cell depends for continued proliferation.
  • a chemotherapeutic agent is a cell cycle inhibitor or a cell division inhibitor. Categories of chemotherapeutic agents that are useful in the methods of the invention include alkylating/alkaloid agents, antimetabolites, hormones or hormone analogs, and miscellaneous antineoplastic drugs.
  • a chemotherapeutic agent is a radioactive molecule.
  • a chemotherapeutic agent of use e.g. see Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principles of Internal Medicine, 14th edition; Perry et al. , Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2nd ed. 2000 Churchill Livingstone, Inc; Baltzer L, Berkery R (eds): Oncology Pocket Guide to Chemotherapy, 2nd ed. St.
  • the chemotherapeutic agent can be a cytotoxic chemotherapeutic.
  • cytotoxic agent refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells.
  • the term is intended to include radioactive isotopes (e.g.
  • chemotherapeutic agents such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof.
  • chemotherapeutic agent is a broad one covering many chemotherapeutic agents having different mechanisms of action. Generally, chemotherapeutic agents are classified according to the mechanism of action. Many of the available agents are antimetabolites of development pathways of various tumors, or react with the DNA of the tumor cells. There are also agents which inhibit enzymes, such as topoisomerase I and topoisomerase II, or which are antimiotic agents.
  • Chemotherapeutic agents include, but are not limited to, an aromatase inhibitor; an antiestrogen, an anti-androgen (especially in the case of prostate cancer) or a gonadorelin agonist; a topoisomerase I inhibitor or a topoisomerase II inhibitor; a microtubule active agent, an alkylating agent, an anti-neoplastic anti- metabolite or a platin compound; a compound targeting/decreasing a protein or lipid kinase activity or a protein or lipid phosphatase activity, a further anti-angiogenic compound or a compound which induces cell differentiation processes; a bradykinin 1 receptor or an angiotensin II antagonist; a cyclooxygenase inhibitor, a bisphosphonate, a heparanase inhibitor (prevents heparan sulphate degradation), e.g., PI-88, a biological response modifier, preferably a lymphok
  • interferon ⁇ an ubiquitination inhibitor or an inhibitor which blocks anti- apoptotic pathways; an inhibitor of Ras oncogenic isoforms or a farnesyl transferase inhibitor; a telomerase inhibitor, e.g., telomestatin; a protease inhibitor, a matrix metalloproteinase inhibitor, a methionine aminopeptidase inhibitor, e.g., bengamide or a derivative thereof; a proteasome inhibitor, e.g., PS-341 (bortezomib / Velcade); agents used in the treatment of hematologic malignancies or FMS-like tyrosine kinase inhibitors; an HSP90 inhibitors; histone deacetylase (HDAC) inhibitors; mTOR inhibitors; somatostatin receptor antagonists; integrin antagonists; anti-leukemic compounds; tumor cell damaging approaches, such as ionizing radiation; EDG binders; anthranilic acid
  • chemotherapeutic agents include, but are not limited to, plant alkaloids, hormonal agents and antagonists, biological response modifiers, preferably lymphokines or interferons, antisense oligonucleotides or oligonucleotide derivatives; or miscellaneous agents or agents with other or unknown mechanism of action.
  • the chemotherapeutic agent can be linked to a component of the supramolecular combinatorial therapeutic.
  • the chemotherapeutic agent can be linked to a lipid or phospholipid component of the supramolecular combinatorial therapeutic.
  • the chemotherapeutic agent and the component of the supramolecular combinatorial therapeutic can be linked together by a bond or via a linker.
  • This linker can be cleavable or non- cleavable, depending on the application.
  • a cleavable linker can be used to release the chemotherapeutic agent after transport to the desired target.
  • the intended nature of the conjugation or coupling interaction, or the desired biological effect, will determine the choice of linker group.
  • the lipid in the lipid conjugated chemotherapeutic agent is cholesterol.
  • the supramolecular combinatorial therapeutic can comprise from about 1% to about 99% (w/w) of the chemtherapeutic agent or a conjugate thereof. Further the chemotherapeutioc agent or a conjugate thereof can be present in 10: 1 to 1 : 10 ratio with the taxane conjugate. Without limitations, taxane conjugate and chemothereaputic agent (or a conjugate thereof) in the supramolecular combinatorial therapeutic can be in ratio 10: 1 to 1 : 10, 5: 1 to 1 :5, or 2.5: 1 to 1 :2.5.
  • taxane conjugate and chemothereaputic agent (or a conjugate thereof) in the supramolecular combinatorial therapeutic can be in ratio of about 1 : 1, about 1 : 1.2, about 1 : 1.5, about 1 : 1.7, about 1 :2, about 1 :2.5, about 1 :3, about 1 :3.5, about 1 :4, about 1 :4.5, about 1 :5, about 1 :5.5, about 1 :6, about 1 :6.5, about 1 :7, about 1 :7.5, about 1 :8, about 1 :8.5, about 1 :9, about 1 :9.5, or about 1 : 10.
  • the chemotherapeutic agent can be a kinase inhibitor, e.g. a Phosphoinositide 3-kinase (PI 3-kinase or PI3K) inhibitor.
  • Phosphoinositide 3-kinases are a family of related enzymes that are capable of phosphorylating the 3 position hydroxyl group of the inositol ring of phosphatidylinositol. They are also known as phosphatidylinositol-3- kinases.
  • PI3Ks interact with the IRS (Insulin receptor substrate) in order to regulate glucose uptake through a series of phosphorylation events.
  • the phosphoinositol- 3-kinase family is composed of Class I , II and Class III , with Class I the only ones able to convert PI(4,5)P2 to PI(3,4,5)P3 on the inner leaflet of the plasma membrane.
  • Class I PI3K are heterodimeric molecules composed of a regulatory and a catalytic subunit; they are further divided between IA and IB subsets on sequence similarity.
  • Class IA PI3K are composed of one of five regulatory p85a, p55a, p50a, ⁇ 85 ⁇ or ⁇ 55 ⁇ subunit attached to a pi 10a, ⁇ or ⁇ catalytic subunit.
  • the first three regulatory subunits are all splice variants of the same gene ( Pik3rl ), the other two being expressed by other genes (Pik3r2 and Pik3r3, ⁇ 85 ⁇ and ⁇ 55 ⁇ , respectively).
  • the most highly expressed regulatory subunit is p85a, all three catalytic subunits are expressed by separate genes (Pik3ca, Pik3cb and Pik3cd for pi 10a, pi 10 ⁇ and pi 105, respectively).
  • the first two pi 10 isoforms (a and ⁇ ) are expressed in all cells, but ⁇ ⁇ is primarily expressed in leukocytes and it has been suggested it evolved in parallel with the adaptive immune system.
  • the regulatory plOl and catalytic pi 10 ⁇ subunits comprise the type IB PI3K and are encoded by a single gene each.
  • Class II comprises three catalytic isoforms (C2a, C2p, and C2y), but unlike Classes I and III, no regulatory proteins. These enzymes catalyse the production of PI(3)P from PI (may also produce PI(3,4)P2 from PI(4)P). C2a and C2p are expressed throughout the body, however expression of C2y is limited to hepatocytes.
  • the distinct feature of Class II PI3Ks is the C-terminal C2 domain. This domain lacks critical Asp residues to coordinate binding of Ca 2+ , which suggests class II PI3Ks bind lipids in a Ca 2+ independent manner.
  • Class III are similar to II in that they bias the production of PI(3)P from PI, but are more similar to Class I in structure, as they exist as a heterodimers of a catalytic ( Vps34 ) and a regulatory (pi 50) subunits. Class III seems to be primarily involved in the trafficking of proteins and vesicles.
  • a "PI3K inhibitor” refers to an agent that inhibits the activity of PI3K, as measured by the level of phosphorylation of the 3 position hydroxyl group of the inositol ring of phosphatidylinositol, or as measured by the activity and/or phosphorylation (where increased phosphorylation indicates PI3K activity) of molecules downstream of PI3K.
  • downstream molecules are known in the art and can include, but are not limited to AKT, SGK, mTOR, GSK3P, PSD-95, S6, and 4EBP1.
  • Methods of measuring the activity of PI3K directly or indirectly are well known in the art, and include, by way of non- limiting example determining the level of phosphorylation of a molecule downstream of PI3K using phospho-isoform specific antibodies, which are commercially available (e.g. anti- phospho-AKT antibody, Cat No. ab66138 Abeam, Cambridge, MA).
  • a PI3K inhibitor can be LY294002, PI 103, and/or PI828.
  • Further non-limiting examples of PI3K inhibitors can include wortmannin, demethoxyviridin, IC486068, IC87114, GDC-0941, perifosine, CAL101, PX-866, IPI-145, BAY 80-6946, BEZ235, P6503, TGR1202, SF1126, INK1117, BKM120, IL147, XL765, Palomid 529, GSK1059615, ZSTK474, PWT33597, TG100-115, CAL263, GNE-447, CUDC-907, and AEZS-136.
  • the conjugate comprises a PI3K inhibitor covalently linked with a lipid.
  • the lipid conjugated PI3K inhibitor is
  • PI3K inhibitors covalently linked with a lipid are described, for example, in PCT Patent Publication No. WO2013188763, the content of which is incorporated herein by reference in its entirety.
  • the supramolecular combinatorial therapeutic comprises at least one (e.g., one two, three, four, five six, seven, eight, nine, ten or more different types of) taxane conjugate and at least one taxane conjugate (e.g., one two, three, four, five six, seven, eight, nine, ten or more different types of) lipid conjugated PI3K inhibitor).
  • the supramolecular combinatorial therapeutic can comprise from about 1% to about 99% (w/w) of the PI3K inhibitor conjugate. Further the PI3K inhibitor conjugatecan be present in 10: 1 to 1 : 10 ratio with the taxane conjugate.
  • each PI3K inhibitor conjugate can be independently in 10: 1 to 1 : 10 ratio with the conjugate. Further, if two or more different PI3K inhibitor conjugates are present in the composition, the two PI3K inhibitor conjugates can be in 10: 1 to 1 : 10 ratio.
  • two different components (conjugate and PI3K inhibitor conjugate) of the supramolecular combinatorial therapeutic can be in ratio 10: 1 to 1 : 10, 5: 1 to 1 :5, or 2.5: 1 to 1 :2.5.
  • two different components in the supramolecular combinatorial therapeutic can be in ratio of about 1 : 1, about 1 : 1.2, about 1 : 1.5, about 1 : 1.7, about 1 :2, about 1 :2.5, about 1 :3, about 1 :3.5, about 1 :4, about 1 :4.5, about 1 :5, about 1 :5.5, about 1 :6, about 1 :6.5, about 1 :7, about 1 :7.5, about 1 :8, about 1 :8.5, about 1 :9, about 1 :9.5, or about 1 : 10. If the supramolecular combinatorial therapeutic comprises more than two components ratio between any two components can be independent of ratio between any other two components.
  • the chemotherapeutic agent is a platinate. Any platinum compound can be used in the methods and compositions described herein.
  • the platinum compound is a platinum (II) or platinum (IV) compound.
  • the platinum (II) compound is selected from the group consisting of cisplatin, oxaliplatin, carboplatin, paraplatin, sartraplatin, and combinations thereof.
  • the platinate is cisplatin or oxalipaltin.
  • Cisplatin [cis- dichlorodiammineplatinum(II)] (CDDP) has emerged as an important class of antitumor agents, and is widely used for the treatment of many malignancies including testicular, ovarian, cervical, head and neck, and non-small cell lung cancer (Jamieson, et al, Chem. Rev. (1999), 99(9): 2467-2498). It was also shown to be active in triple negative breast cancer (Leong, et al, J. Clin. Invest. (2007), 117(5): 1370-80). Its use is however dose-limited mainly because of nephrotoxicity or toxicity to the kidney (Madias, NE and Harrington, JT, Am. J. (1978), 65(2): 307-14).
  • the platinum can be dissociably linked to a lipid via at least one coordination bond.
  • the coordination bond is Pt->0. In some other embodiments, the coordination bond is Pt->N.
  • the conjugate comprises a platinum dissociably linked with a cholesterol via at least one coordination bond. In some embodiments, the lipid conjugated platinum compound is
  • the supramolecular combinatorial therapeutic comprises at least one (e.g., one two, three, four, five six, seven, eight, nine, ten or more different types of) taxane conjugate and at least one platinum conjugate (e.g., one two, three, four, five six, seven, eight, nine, ten or more different types of) lipid conjugated platinum).
  • the supramolecular combinatorial therapeutic can comprise from about 1% to about 99% (w/w) of the platinum conjugate. Further the platinum conjugatecan be present in 10: 1 to 1 : 10 ratio with the taxane conjugate.
  • each platinum conjugate can be independently in 10: 1 to 1 : 10 ratio with the taxane conjugate. Further, if two or more different platinum conjugates are present in the composition, the two platinum conjugates can be in 10: 1 to 1 : 10 ratio.
  • two different components (taxane conjugate and platinum conjugate) of the supramolecular combinatorial therapeutic can be in ratio 10: 1 to 1 : 10, 5: 1 to 1 :5, or 2.5:1 to 1 :2.5.
  • two different components in the supramolecular combinatorial therapeutic can be in ratio of about 1 :1, about 1 : 1.2, about 1 : 1.5, about 1 : 1.7, about 1 :2, about 1 :2.5, about 1 :3, about 1 :3.5, about 1 :4, about 1 :4.5, about 1 :5, about 1 :5.5, about 1 :6, about 1 :6.5, about 1 :7, about 1 :7.5, about 1 :8, about 1 :8.5, about 1 :9, about 1 :9.5, or about 1 : 10. If the supramolecular combinatorial therapeutic comprises more than two components ratio between any two components can be independent of ratio between any other two components.
  • the supramolecular combinatorial therapeutic can also include components selected to reduce aggregation of particles during formation, which can result from steric stabilization of particles which prevents charge-induced aggregation during formation.
  • Suitable components that reduce aggregation include, but are not limited to, polyethylene glycol (PEG)-modified lipids (i.e., PEG conjugated lipids), monosialoganglioside Gml, and polyamide oligomers (“PAO”) such as (described in US Pat. No. 6,320,017).
  • Exemplary suitable PEG-modified lipids include, but are not limited to, PEG-modified diacylglycerols and dialkylglycerols, PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG- ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, PEG-modified 1 ,2-diacyloxypropan-3 -amines, and PEG conjugated DSPE (e.g., DSPE- PEG2000).
  • PEG-modified diacylglycerols and dialkylglycerols include, but are not limited to, PEG-modified diacylglycerols and dialkylglycerols, PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG- ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG
  • Other compounds with uncharged, hydrophilic, steric-barrier moieties, which prevent aggregation during formation can also be coupled to lipids to reduce aggregation during formation.
  • ATTA-lipids are described, e.g., in U.S. Patent No. 6,320,017
  • PEG-lipid conjugates are described, e.g., in U.S. Patent Nos. 5,820,873, 5,534,499 and 5,885,613.
  • the concentration of the lipid component selected to reduce aggregation is about 0.1 to 15% (by mole percent of lipids). It should be noted that aggregation preventing compounds do not necessarily require lipid conjugation to function properly. Free PEG or free ATTA in solution can be sufficient to prevent aggregation. If the liposomes are stable after formulation, the PEG or ATTA can be dialyzed away before administration to a subject.
  • Neutral lipids when present in the composition, can be any of a number of lipid species which exist either in an uncharged or neutral zwitterionic form at physiological pH.
  • Such lipids include, but are not limited to, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides.
  • the selection of neutral lipids for use in liposomes described herein is generally guided by consideration of, e.g., liposome size and stability of the liposomes in the bloodstream.
  • the neutral lipid component is a lipid having two acyl groups, (i.e., diacylphosphatidylcholine and diacylphosphatidylethanolamine).
  • Lipids having a variety of acyl chain groups of varying chain length and degree of saturation are available or can be isolated or synthesized by well-known techniques.
  • lipids containing saturated or unsaturated fatty acids with carbon chain lengths in the range of C 6 to C22 e.g., C 6 , C 8 , Cio, C12, Ci4, Ci6, Ci8, C22, or C22
  • C 6 to C22 e.g., C 6 , C 8 , Cio, C12, Ci4, Ci6, Ci8, C22, or C22
  • lipids having mixtures of saturated and unsaturated fatty acid chains can be used.
  • the neutral lipids can be phosphatidylcholine, DOPE, DSPC, POPC, DMPC, DPPC or any related phosphatidylcholine.
  • the neutral lipids useful in the present invention can also be composed of sphingomyelin, dihydrosphingomyeline, or phospholipids with other head groups, such as serine and inositol.
  • the sterol component can be any of those sterols conventionally used in the field of liposome, lipid vesicle or lipid particle preparation.
  • a preferred sterol is cholesterol.
  • the cationic lipids can be any of a number of lipid species which carry a net positive charge at about physiological pH.
  • lipids include, but are not limited to, N,N-dioleyl-N,N- dimethylammonium chloride (“DODAC”); N-(2,3-dioleyloxy)propyl-N,N-N- triethylammonium chloride (“DOTMA”); N,N-distearyl-N,N-dimethylammonium bromide (“DDAB”); N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (“DOTAP”); l,2-Dioleyloxy-3-trimethylaminopropane chloride salt (“DOTAP.C1"); 3 ⁇ -( ⁇ -( ⁇ ', ⁇ '- dimethylaminoethane)-carbamoyl)cholesterol (“DC)
  • DODAC N,N-dioleyl-
  • cationic lipids can be used, such as, e.g., LIPOFECTIN (including DOTMA and DOPE, available from GIBCO/BRL), and LIPOFECTAMINE (comprising DOSPA and DOPE, available from GIBCO/BRL).
  • LIPOFECTIN including DOTMA and DOPE, available from GIBCO/BRL
  • LIPOFECTAMINE comprising DOSPA and DOPE, available from GIBCO/BRL
  • Other cationic lipids suitable for lipid particle formation are described in W098/39359, W096/37194.
  • Other suitable cationic lipids are described, for example in US Patent Application Publication No. 2011/0997720 and PCT Patent Application Publication No. WO 2009/132131 and No. WO 2009/132131, content of all of which is incorporated herein by reference in its entirety.
  • the anionic lipid can be any of a number of lipid species which carry a net negative charge at about physiological pH.
  • lipids include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoyl phosphatidylethanoloamine, N-succinyl phosphatidylethanolamine, N-glutaryl phosphatidylethanolamine, lysylphosphatidylglycerol, and other anionic modifying groups joined to neutral lipids.
  • amphipathic lipids refer to any suitable material, wherein the hydrophobic portion of the lipid material orients into a hydrophobic phase, while the hydrophilic portion orients toward the aqueous phase.
  • Such compounds include, but are not limited to, phospholipids, aminolipids, and sphingolipids.
  • programmable fusion lipids are also suitable for inclusion in the supramolecular combinatorial therapeutic described herein.
  • Particles containing programmable fusion lipids have little tendency to fuse with cell membranes and deliver their payload until a given signal event occurs. This allows the supramolecular combinatorial therapeutic to distribute more evenly after administration into an organism or disease site before it starts fusing with cells.
  • the signal event can be, for example, a change in pH, temperature, ionic environment, or time.
  • a fusion delaying or "cloaking" component such as an ATTA- lipid conjugate or a PEG-lipid conjugate, can simply exchange out of the particle membrane over time.
  • the particle By the time the particle is suitably distributed in the body, it has lost sufficient cloaking agent so as to be fusogenic. With other signal events, it is desirable to choose a signal that is associated with the disease site or target cell, such as lower pH at a site of tumor.
  • One or more complementary surface active agent can be added to the supramolecular combinatorial therapeutics, for example as complements to the characteristics of an amphiphilic agent or to improve particle stabilizing capacity or enable an improved solubilization.
  • Such complementary agents can be pharmaceutically acceptable non-ionic surfactants which preferably are alkylene oxide derivatives of an organic compound which contains one or more hydroxylic groups.
  • non-ionic surfactants which preferably are alkylene oxide derivatives of an organic compound which contains one or more hydroxylic groups.
  • ethoxylated and/or propoxylated alcohol or ester compounds or mixtures thereof are commonly available and are well known as such complements to those skilled in the art.
  • esters of sorbitol and fatty acids such as sorbitan monopalmitate, oily sucrose esters, polyoxyethylene sorbitane fatty acid esters, polyoxyethylene sorbitol fatty acid esters, polyoxyethylene fatty acid esters, polyoxyethylene alkyl ethers, polyoxyethylene sterol ethers, polyoxyethylene- polypropoxy alkyl ethers, block polymers and cethyl ether, as well as polyoxyethylene castor oil or hydrogenated castor oil derivatives and polyglycerine fatty acid esters.
  • esters of sorbitol and fatty acids such as sorbitan monopalmitate, oily sucrose esters, polyoxyethylene sorbitane fatty acid esters, polyoxyethylene sorbitol fatty acid esters, polyoxyethylene fatty acid esters, polyoxyethylene alkyl ethers, polyoxyethylene sterol ethers, polyoxyethylene- polypropoxy alkyl ethers, block polymers and cethyl ether
  • Suitable non- ionic surfactants include, but are not limited to various grades of PLURONIC®, POLOXAMER®, SPAN®, TWEEN®, POLYSORBATE®, TYLOXAPOL®, EMULPHOR® or CREMOPHOR® and the like.
  • the complementary surface active agents can also be of an ionic nature, such as bile duct agents, cholic acid or deoxycholic their salts and derivatives or free fatty acids, such as oleic acid, linoleic acid and others.
  • Other ionic surface active agents are found among cationic lipids like C6-C24 alkylamines or alkanolamine and cationic cholesterol esters.
  • the supramolecular combinatorial therapeutic comprises a PEG conjugated lipid and a phospholipid.
  • the supramolecular combinatorial therapeutic comprises a taxane-lipid conjugate and a PI3K inhibitor-lipid conjugate.
  • the supramolecular combinatorial therapeutic comprises a taxane-lipid conjugate and a platinum- lipid conjugate.
  • the supramolecular combinatorial therapeutic comprises a taxane-lipid conjugate and an antibody (or an antigen binding fragment thereof) lipid conjugate.
  • the antibody, or the antigen binding fragment thereof can be a therepautic agent or a targeting ligand.
  • the disclosure provides a conjugate comprising a taxane conjugated to with a lipid.
  • Taxus is generally referred to diterpene-containing compounds produced by the plants of the genus Taxus (e.g., yews, such as, but not limited to, Taxus baccata, Taxus brevifolia, Taxus canadensis, Taxus chinensis, Taxus cuspidata, Taxus floridana, Taxus globosa, Taxus sumatrana, Taxus walUchiana), and synthetic and semisynthetic forms thereof.
  • yews such as, but not limited to, Taxus baccata, Taxus brevifolia, Taxus canadensis, Taxus chinensis, Taxus cuspidata, Taxus floridana, Taxus globosa, Taxus sumatrana, Taxus walUchiana
  • the term denotes a compound containing the core
  • the basic taxane core structure may further be substituted or may contain unsaturations in the ring to yield a number of compounds, generically known as taxanes. Generally, such compounds may block cell growth by stopping mitosis by interfering with microtubules.
  • the term "diterpene,” as used herein, means chemical compounds having a carbon skeleton derived from four isoprene units.
  • the taxane group of compounds includes paclitaxel, docetaxel, and cabazitaxel.
  • Taxanes can be isolated from natural sources, and can also be prepared synthetically from naturally occumng precursors.
  • Paclitaxel (TAXOL®, Bnstol-Myers Squibb), for example, can be prepared from baccatin by attachment of protecting groups to the hydroxyl groups of baccatin that are to become the hydroxyl groups of paclitaxel, converting the precursor baccatin to paclitaxel, and then removing the protecting groups from the hydroxyl groups to obtain paclitaxel (see, e.g., WO93/10076. int. pub. date 05/27/93; K. V. Rao, U.S. Patent No. 5,200.534; R.A. Holton, U.S.
  • Non-limiting examples of taxanes can include paclitaxel and docetaxel, derivatives thereof, and mixtures thereof.
  • Taxanes can be used effectively to treat a variety of cancers.
  • Paclitaxel for example, has been found to have activity against ovarain and breast cancers, as well as against malignant melanoma, colon cancer, leukemias and lung cancer (see, e.g., Borman, Chemical & Engineering News, September 2, 1991 , pp. 11-18; The Pharmacological Basis of Therapeutics (Goodman Gilman et al, eds.), Pergamon Press, New York (1990), p. 1239; Suffness, Antrtumor Alkaloids, in: "The Alkaloids, Vol. XXV,” Academic Press, Inc. (1985), Chapter 1 , pp. 6-18; Rizzo et al, J. Pharm.
  • Paclitaxel acts against cancer cells by binding to tubulin in the cells nuclei, thereby blocking the disassembly of microtubules and consequently, inhibiting cell division (Schiff et al, Nature 277:665 (1979) [00114]
  • taxane also include bactins and bactin derivatives.
  • baccatin or "baccatin derivatives” refers to the taxane derivatives in which the side chain at the 13- position of the taxane skeleton is a hydroxy group.
  • taxane also include abeo-taxanes.
  • Abeo-taxanes have a 5-membered A ring.
  • the taxanes can be derived from natural sources, can be prepared synthetically or can be obtained from semisynthetic methods or a combination thereof.
  • Exemplary taxanes include, but are not limited to paclitaxel (TAXOLTM), docetaxel (taxotere), cabazitaxel, abretaxane, taxoprexin, xyotax, cephalomannine, 10-deacetylcephalomannine, baccatin, taxine, brevifoliol, 10- deacetylbaccatin, hongdoushan A, hongdoushan B, hongdoushan C, 7-epitaxol, 7-epibaccatin III, and lO-desacetyl-7-epitaxol.
  • TAXOLTM docetaxel
  • taxotere docetaxel
  • cabazitaxel abretaxane
  • taxoprexin xyotax
  • cephalomannine 10-deacetylcephalomannine
  • baccatin taxine
  • brevifoliol 10- deacetylbaccatin
  • the taxane in the conjugate is cabazitaxel.
  • the taxane and the lipid can be covalently conjugated via a linker.
  • linker means an organic moiety that connects two parts of a compound.
  • Linkers typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NR 1 , C(O), C(0)0, SO, S0 2 , S0 2 NH or a chain of atoms, such as substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl,
  • the linker can be a branched linker.
  • the branch-point of the branched linker can be at least trivalent, but can be a tetravalent, pentavalent or hexavalent atom, or a group presenting such multiple valencies.
  • the branch-point can be , -N, -N(Q)-C, -O-C, -S-C, -SS-C, -C(0)N(Q)-C, -OC(0)N(Q)-C, - N(Q)C(0)-C, or -N(Q)C(0)0-C; wherein Q is independently for each occurrence H or optionally substituted alkyl.
  • the branch-point can be glycerol or a glycerol derivative.
  • the linker comprises at least one cleavable linking group.
  • a cleavable linking group is one which is sufficiently stable outside the cell, but which upon entry into a target cell is cleaved to release the two parts the linker is holding together.
  • the cleavable linking group is cleaved at least 10 times or more, preferably at least 100 times faster in the target cell or under a first reference condition (which can, e.g., be selected to mimic or represent intracellular conditions) than in the blood or serum of a subject, or under a second reference condition (which can, e.g., be selected to mimic or represent conditions found in the blood or serum).
  • Cleavable linking groups are susceptible to cleavage agents, e.g., pH, redox potential or the presence of degradative molecules. Generally, cleavage agents are more prevalent or found at higher levels or activities inside cells than in serum or blood.
  • degradative agents include: redox agents which are selected for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, present in cells, that can degrade a redox cleavable linking group by reduction; esterases; amidases; endosomes or agents that can create an acidic environment, e.g., those that result in a pH of five or lower; enzymes that can hydrolyze or degrade an acid cleavable linking group by acting as a general acid, peptidases (which can be substrate specific) and proteases, and phosphatases.
  • redox agents which are selected for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, present in cells, that can degrade a redox cleavable linking group by reduction; esterases; amidases; endosomes or
  • a linker can include a cleavable linking group that is cleavable by a particular enzyme.
  • the type of cleavable linking group incorporated into a linker can depend on the cell to be targeted.
  • cleavable linking group is cleaved at least 1.25, 1.5, 1.75, 2, 3, 4, 5, 10, 25, 50, or 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).
  • the cleavable linking group is cleaved by less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or 1% in the blood (or in vitro conditions selected to mimic extracellular conditions) as compared to in the cell (or under in vitro conditions selected to mimic intracellular conditions).
  • Exemplary cleavable linking groups include, but are not limited to, redox cleavable linking groups (e.g., -S-S- and -C(R) 2 -S-S-, wherein R is H or Ci-C 6 alkyl and at least one R is Ci-C 6 alkyl such as CH 3 or CH 2 CH 3 ); phosphate -based cleavable linking groups (e.g., -0-P(0)(OR)-0-, -0-P(S)(OR)-0-, -0-P(S)(SR)-0-, -S-P(0)(OR)-0-, -O- P(0)(OR)-S-, -S-P(0)(OR)-S-, -0-P(S)(ORk)-S-, -S-P(S)(OR)-0-, -0-P(0)(R)-0-, -O- P(S)(R)-0-, -S-P(0)(R)-0-, -S
  • a peptide based cleavable linking group comprises two or more amino acids.
  • the peptide-based cleavage linkage comprises the amino acid sequence that is the substrate for a peptidase or a protease found in cells.
  • the linker comprises an acid labile group.
  • an acid cleavable linking group is cleaveable in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.5, 6.0, 5.5, 5.0, 4.5, 4.0, 3.5, 3.0, or lower), or by agents such as enzymes that can act as a general acid.
  • the linker comprises one or more of succinic acid, fumaric acid, propargylic acid, ethylene glycol, diethylene glycol, and natural or unnatural amino acids.
  • the linker comprises oxalic acid, malonic acid, succinic acid, glutaric acid, succinic acid, ethylene diamine, natural or unnatural amino acid, ethylene glycol, diethylene glycol, acetic acid, propionic acid, butyric acid, valeric acid, acrylic acid, but-2-enoic acid, pent-2-enoic acid, hex-2-enoic acid, 2-propynoic acid, but-2-ynoic acid, pent-2-ynoic acid, hex-2-ynoic acid, ethylene, propylene, 1-butene, 1-pentene, 1-hexene, acetylene, propyne, but-l-yne, pent-l-yne, or any combinations thereof [00125] In some embodiments, the linker comprises succinic acid and ethylene diamine; succinic acid and natural or unnatural amino acid; ethylene glycol and acetic acid; ethylene glycol and acrylic acid; diethylene
  • the linker is selected from the group consisting of - C(0)CH 2 CH 2 C(0)NHCH 2 CH 2 NHC(0)-; -C(0)CH 2 CH 2 C(0)NHCH 2 NHC(0)-;
  • the lipid in the taxane-lipid conjugate is cholesterol, alpha- tocopherol, or a fatty acid. In one embodiment, the lipid in the taxane-lipid conjugate is cholesterol.
  • the taxane and the lipid can be covalently conjugated with each other (or the linker) using a reactive functional group present in their respective structures.
  • the term "reactive functional group” refers to a functional group that is capable of reacting with another functional group.
  • exemplary reactive functional groups include, but are not limited to, hydroxyls, amines, thiols, thials, sulfmos, carboxylic acids, amides, and the like.
  • the reactive functional group on the lipid, the taxane, and the linker can be the same or different.
  • the reactive group on the lipid is a hydroxyl, an amine, a thiol, or a carboxylic acid.
  • the reactive group on the taxane is a hydroxyl, an amine, a thiol, or a carboxylic acid.
  • the taxane-lipid conjugate can be selected from the group consisting of taxane conjugates 1-33, strucutres of which are shown in the Examples section.
  • the methods described herein relate to treating a subject having or diagnosed as having cancer.
  • Subjects having cancer can be identified by a physician using current methods of diagnosing cancer.
  • Symptoms and/or complications of cancer which characterize these conditions and aid in diagnosis are well known in the art and include but are not limited to, growth of a tumor, impaired function of the organ or tissue harboring cancer cells, etc.
  • Tests that may aid in a diagnosis of, e.g. cancer include, but are not limited to, tissue biopsies and histological examination.
  • a family history of cancer, or exposure to risk factors for cancer e.g. tobacco products, radiation, etc.
  • Cancer can include, but is not limited to, carcinoma, including adenocarcinoma, lymphoma, blastoma, melanoma, sarcoma, leukemia, squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, Hodgkin's and non Hodgkin's lymphoma, pancreatic cancer, glioblastoma, basal cell carcinoma, biliary tract cancer, bladder cancer, brain cancer including glioblastomas and medulloblastomas; breast cancer, cervical cancer, choriocarcinoma; colon cancer, colorectal cancer, endometrial carcinoma, endometrial cancer; esophageal cancer, gastric cancer; various types of head and neck cancers, intraepithelial neoplasms including Bowen's disease and Paget's disease; hematological neoplasms including acute lymphocytic and myelogenous leukemia; Kaposi's
  • compositions and methods described herein can be administered to a subject having or diagnosed as having cancer.
  • the methods described herein comprise administering an effective amount of compositions described herein to a subject in order to alleviate a symptom of a cancer.
  • "alleviating a symptom of a cancer” is ameliorating any condition or symptom associated with the cancer. As compared with an equivalent untreated control, such reduction is by at least 5%, 10%, 20%, 40%>, 50%>, 60%>, 80%), 90%o, 95%), 99%o or more as measured by any standard technique.
  • a variety of means for administering the compositions described herein to subjects are known to those of skill in the art.
  • Such methods can include, but are not limited to oral, parenteral, intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, cutaneous, topical, injection, or intratumoral administration. Administration can be local or systemic.
  • the term "effective amount” as used herein refers to the amount of a composition described herein needed to alleviate at least one or more symptom of the disease or disorder, and relates to a sufficient amount of pharmacological composition to provide the desired effect.
  • the term "therapeutically effective amount” therefore refers to an amount of a composition described herein that is sufficient to provide a particular anti-tumor effect when administered to a typical subject.
  • An effective amount as used herein, in various contexts, would also include an amount sufficient to delay the development of a symptom of the disease, alter the course of a symptom disease (for example but not limited to, slowing the progression of a symptom of the disease), or reverse a symptom of the disease. Thus, it is not generally practicable to specify an exact "effective amount”. However, for any given case, an appropriate "effective amount” can be determined by one of ordinary skill in the art using only routine experimentation.
  • Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50%> of the population).
  • the dosage can vary depending upon the dosage form employed and the route of administration utilized.
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50.
  • Compositions and methods that exhibit large therapeutic indices are preferred.
  • a therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of a composition described herein, which achieves a half-maximal inhibition of symptoms) as determined in cell culture, or in an appropriate animal model.
  • IC50 i.e., the concentration of a composition described herein, which achieves a half-maximal inhibition of symptoms
  • Levels in plasma can be measured, for example, by high performance liquid chromatography.
  • the effects of any particular dosage can be monitored by a suitable bioassay, e.g., assay for tumor size and/or growth, among others.
  • the dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.
  • the technology described herein relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a supramolecular combinatorial therapeutic and a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents and/or dispersion media. The use of such carriers and diluents is well known in the art.
  • materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as e
  • wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation.
  • the terms such as “excipient”, “carrier”, “pharmaceutically acceptable carrier” or the like are used interchangeably herein.
  • the carrier inhibits the degradation of the active agent, e.g. a composition as described herein.
  • the pharmaceutical composition comprising a supramolecular combinatorial therapeutic can be a parenteral dose form. Since administration of parenteral dosage forms typically bypasses the patient's natural defenses against contaminants, parenteral dosage forms are preferably sterile or capable of being sterilized prior to administration to a patient. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions. In addition, contra lled-release parenteral dosage forms can be prepared for administration of a patient, including, but not limited to, DUROS ® -type dosage forms and dose-dumping.
  • Suitable vehicles that can be used to provide parenteral dosage forms of a composition as described herein are well known to those skilled in the art. Examples include, without limitation: sterile water; water for injection USP; saline solution; glucose solution; aqueous vehicles such as but not limited to, sodium chloride injection, Ringer's injection, dextrose Injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.
  • Compounds that alter or modify the solubility of a pharmaceutically acceptable salt can also be incorporated into the parenteral dosage forms of the disclosure, including conventional and controlled-release parenteral dosage forms.
  • compositions can also be formulated to be suitable for oral administration, for example as discrete dosage forms, such as, but not limited to, tablets (including without limitation scored or coated tablets), pills, caplets, capsules, chewable tablets, powder packets, cachets, troches, wafers, aerosol sprays, or liquids, such as but not limited to, syrups, elixirs, solutions or suspensions in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion, or a water-in-oil emulsion.
  • Such compositions contain a predetermined amount of the pharmaceutically acceptable salt of the disclosed compounds, and may be prepared by methods of pharmacy well known to those skilled in the art. See generally, Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams, and Wilkins, Philadelphia PA. (2005).
  • Conventional dosage forms generally provide rapid or immediate drug release from the formulation. Depending on the pharmacology and pharmacokinetics of the drug, use of conventional dosage forms can lead to wide fluctuations in the concentrations of the drug in a patient's blood and other tissues. These fluctuations can impact a number of parameters, such as dose frequency, onset of action, duration of efficacy, maintenance of therapeutic blood levels, toxicity, side effects, and the like.
  • controlled-release formulations can be used to control a drug's onset of action, duration of action, plasma levels within the therapeutic window, and peak blood levels.
  • controlled- or extended- release dosage forms or formulations can be used to ensure that the maximum effectiveness of a drug is achieved while minimizing potential adverse effects and safety concerns, which can occur both from under-dosing a drug (i.e., going below the minimum therapeutic levels) as well as exceeding the toxicity level for the drug.
  • a composition as described herein can be administered in a sustained release formulation.
  • Controlled-release pharmaceutical products have a common goal of improving drug therapy over that achieved by their non-controlled release counterparts.
  • the use of an optimally designed controlled-release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition in a minimum amount of time.
  • Advantages of controlled-release formulations include: 1) extended activity of the drug; 2) reduced dosage frequency; 3) increased patient compliance; 4) usage of less total drug; 5) reduction in local or systemic side effects; 6) minimization of drug accumulation; 7) reduction in blood level fluctuations; 8) improvement in efficacy of treatment; 9) reduction of potentiation or loss of drug activity; and 10) improvement in speed of control of diseases or conditions.
  • Controlled-release formulations are designed to initially release an amount of drug (active ingredient) that promptly produces the desired therapeutic effect, and gradually and continually release other amounts of drug to maintain this level of therapeutic or prophylactic effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body. Controlled-release of an active ingredient can be stimulated by various conditions including, but not limited to, pH, ionic strength, osmotic pressure, temperature, enzymes, water, and other physiological conditions or compounds.
  • a variety of known controlled- or extended-release dosage forms, formulations, and devices can be adapted for use with the salts and compositions of the disclosure. Examples include, but are not limited to, those described in U.S. Pat. Nos.: 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5674,533; 5,059,595; 5,591 ,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365,185 Bl ; each of which is incorporated herein by reference.
  • dosage forms can be used to provide slow or controlled-release of one or more active ingredients using, for example, hydroxypropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems (such as OROS (Alza Corporation, Mountain View, Calif. USA)), or a combination thereof to provide the desired release profile in varying proportions.
  • active ingredients for example, hydroxypropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems (such as OROS (Alza Corporation, Mountain View, Calif. USA)), or a combination thereof to provide the desired release profile in varying proportions.
  • OROS Alza Corporation, Mountain View, Calif. USA
  • the method of treatment disclosed herein comprises coadministering one or more additional anti-cancer therapies to the patient in addition to administering the conjugate or composition comprising the conjugate.
  • additional anti-cancer therapies include, but are not limited to, surgery, chemotherapy, radiation therapy, thermotherapy, immunotherapy, hormone therapy, laser therapy, anti-angiogenic therapy, and any combinations thereof.
  • the method comprises co-administering the conjugate and an anti-cancer agent or chemotherapeutic agent to the subject.
  • coadministering refers to the administration of two or more therapeutic agents to a subject, wherein the two or more therapeutic agents may be administered in the same or different pharmaceutical compositions, using the same or different dosage forms (e.g., oral, parenteral, intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, cutaneous, topical, injection, or intratumoral administration), and wherein the two or more therapeutic agents may be administered at the same time or different times over the course of treatment (e.g., different times within the same hour, different times within the same day, different times within the same week, different times within the same month).
  • anti-cancer agent refers to any compound (including its analogs, derivatives, prodrugs and pharmaceutically salts) or composition which can be used to treat cancer.
  • Anti-cancer compounds for use in the present invention include, but are not limited to, inhibitors of topoisomerase I and II, alkylating agents, microtubule inhibitors (e.g., taxol), and angiogenesis inhibitors.
  • anti-cancer compounds include, but are not limited to, paclitaxel (taxol); docetaxel; germicitibine; Aldesleukin; Alemtuzumab; alitretinoin; allopurinol; altretamine; amifostine; anastrozole; arsenic trioxide; Asparaginase; BCG Live; bexarotene capsules; bexarotene gel; bleomycin; busulfan intravenous; busulfanoral; calusterone; capecitabine; platinate; carmustine; carmustine with Polifeprosan Implant; celecoxib; chlorambucil; cladribine; cyclophosphamide; cytarabine; cytarabine liposomal; dacarbazine; dactinomycin; actinomycin D; Darbepoetin alfa; daunorubicin liposomal; daunorubicin, dauno
  • the anti-cancer agent is a paclitaxel-carbohydrate conjugate, e.g., a paclitaxel- glucose conjugate, as described in U.S. Pat. No. 6,218,367, content of which is herein incorporated by reference in its entirety.
  • the anti-cancer agent is a platinate selected from the group consisting of cisplatin, oxaliplatin, carboplatin, paraplatin, sartraplatin, and any combinations thereof.
  • the anti-cancer agent is an immunomodulator.
  • the methods of these embodiments comprise co-administering the conjugate and the immunomodulator to the subject.
  • the conjugate and the immunomodulator are co-administered in separate pharmaceutical compositions and at different times.
  • the conjugate and the immunomodulator are coadministered at the same time in the same pharmaceutical composition.
  • the immunomodulator activates and stimulates an immune response against cancer cells.
  • An immunomodulator may increase immune response by greater than 5%, 10%, 25%, 50%), 75%o, 90%), 100% or more.
  • An immunomodulator may reduce cancer cell numbers and/or growth by greater than 5%, 10%, 25%, 50%, 75%, 90%, 100% or more.
  • immunomodulators include, but are not limited to, immune cells (e.g., natural killer cells, lymphokine-activated killer cells, cytotoxic T cells and dendritic cells), antibodies (e.g., anti-PD-Ll and anti-PD-1 antibodies, anti-CD52, anti-VEGF-A, anti- CD30, anti- EGFR, anti-CD33, anti-CD20, anti-CTLA4, and anti-HER-2 antibodies), and cytokines (e.g., interferons and interleukins).
  • the immunomodulator is an anti-PD- LI, an anti-PD-1 antibody, or a mixture thereof.
  • the immunomodulator is conjugated with a lipid.
  • the methods of treatment can further include the use of radiation or radiation therapy. Further, the methods of treatment can further include the use of surgical treatments.
  • an effective dose of a composition as described herein can be administered to a patient once. In certain embodiments, an effective dose of a composition as described herein can be administered to a patient repeatedly.
  • subjects can be administered a therapeutic amount of a composition as described herein, such as, e.g. 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, or more.
  • the treatments can be administered on a less frequent basis. For example, after treatment biweekly for three months, treatment can be repeated once per month, for six months or a year or longer.
  • Treatment according to the methods described herein can reduce levels of a marker or symptom of a condition, e.g. tumor size and/or growth by at least 10%, at least 15%, at least 20%>, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80 % or at least 90% or more.
  • the dosage of a composition as described herein can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment. With respect to duration and frequency of treatment, it is typical for skilled clinicians to monitor subjects in order to determine when the treatment is providing therapeutic benefit, and to determine whether to increase or decrease dosage, increase or decrease administration frequency, discontinue treatment, resume treatment, or make other alterations to the treatment regimen.
  • the dosing schedule can vary from once a week to daily depending on a number of clinical factors, such as the subject's sensitivity a composition as described herein.
  • the desired dose or amount of activation can be administered at one time or divided into subdoses, e.g., 2-4 subdoses and administered over a period of time, e.g., at appropriate intervals through the day or other appropriate schedule.
  • administration can be chronic, e.g., one or more doses and/or treatments daily over a period of weeks or months.
  • dosing and/or treatment schedules are administration daily, twice daily, three times daily or four or more times daily over a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months, or more.
  • a composition as described herein can be administered over a period of time, such as over a 5 minute, 10 minute, 15 minute, 20 minute, or 25 minute period.
  • the dosage ranges for the administration of a composition as described herein, according to the methods described herein depend upon, for example, the form of a composition as described herein, its potency, and the extent to which symptoms, markers, or indicators of a condition described herein are desired to be reduced, for example the percentage reduction desired for tumor growth.
  • the dosage should not be so large as to cause adverse side effects.
  • the dosage will vary with the age, condition, and sex of the patient and can be determined by one of skill in the art.
  • the dosage can also be adjusted by the individual physician in the event of any complication.
  • compositions as described herein in, e.g. the treatment of a condition described herein, or to induce a response as described herein can be determined by the skilled clinician.
  • a treatment is considered "effective treatment," as the term is used herein, if one or more of the signs or symptoms of a condition described herein are altered in a beneficial manner, other clinically accepted symptoms are improved, or even ameliorated, or a desired response is induced e.g., by at least 10% following treatment according to the methods described herein.
  • Efficacy can be assessed, for example, by measuring a marker, indicator, symptom, and/or the incidence of a condition treated according to the methods described herein or any other measurable parameter appropriate, e.g.
  • Treatment includes any treatment of a disease in an individual or an animal (some non-limiting examples include a human or an animal) and includes: (1) inhibiting the disease, e.g., preventing a worsening of symptoms (e.g. pain or inflammation); or (2) relieving the severity of the disease, e.g., causing regression of symptoms.
  • An effective amount for the treatment of a disease means that amount which, when administered to a subject in need thereof, is sufficient to result in effective treatment as that term is defined herein, for that disease.
  • Efficacy of an agent can be determined by assessing physical indicators of a condition or desired response, (e.g. tumor size and/or growth). It is well within the ability of one skilled in the art to monitor efficacy of administration and/or treatment by measuring any one of such parameters, or any combination of parameters. Efficacy can be assessed in animal models of a condition described herein, for example treatment of cancer. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant change in a marker is observed, e.g. a decreased in tumor size and/or growth.
  • in vitro and animal model assays are provided herein which allow the assessment of a given dose of a composition as described herein.
  • the effects of a dose of a composition can be assessed by an in vitro cell viability assay.
  • a non- limiting example of a protocol for such an assay is as follows: cells, e.g. cancer cell lines, are contacted with compositions described herein and viability determined at one or more timepoints using a cell viability reagent, e.g. CellTiter 96 Aqueous One Solution reagents (PROMEGA, WI).
  • a cell viability reagent e.g. CellTiter 96 Aqueous One Solution reagents (PROMEGA, WI).
  • ovarian adenocarcinomas can be induced in K-Ras LSL/+ /Pten fl/fl mice via intrabursal delivery of adenovirus-carrying Cre recombinase. Once mice develop medium to large tumors, they can be administered a composition as described herein, e.g. via tail vein injection. Tumor imaging can be performed and/or mice can be sacrificed.
  • Caveolae are specialized invaginations of the plasma membrane, consisting primarily of proteins called caveolins, with key roles in numerous biochemical and cellular processes. Caveolin-1 (CAVl) is an important constituent of caveolae, involved in endocytotic trafficking. Caveolae have also been associated with uptake of drugs into cells. Association of self-assembled supramolecular particles with caveolae may assist in their uptake into cells, facilitating therapeutic intervention in cancer cells.
  • the current invention provides methods for predicting the likelihood of a cancer patient exhibiting a better response to self-assembled supramolecular particles than platinates or taxanes.
  • the methods involve determining the expression levels of a gene product that correlates with responsiveness to treatment with self-assembled supramolecular particles, predictive of higher internalization of the payload drug and better therapeutic efficacy for self-assembled supramolecular particles.
  • the disclosure also provides a method of predicting the likelihood of a cancer patient exhibiting a better response to self-assembled supramolecular particles than platinates or taxanes.
  • the method comprises: (a) assaying the expression level of EEA1, SRSF5, SMAD2, SNX3, PLCD1, OSBP, DNM1, DNM2, DNM3, SGK3, FAPP1, SMAD7, SMURF2, NEDD4, PRKCA, CDH-1, LDLR, CP, CD36, LYN, FLOT-1, FLOT-2, CA4, APOE, CAVl, CAV2, CAV3, LMAN2, LAT and STOM in tumor samples obtained from patients; and (b) predicting a likelihood that the patient will exhibit a positive response, wherein: increased expression level of the one or more genes selected from CAVl , CAV2, CAV3, LDLR, SMAD7, SMURF2, NEDD4, PRKCA is positively correlated with a likelihood of a positive response
  • test or "assaying” as used herein refer to performing a quantitative or qualitative analysis of a component in a sample.
  • the terms include laboratory or clinical observations, and/or measuring the level of the component in the sample.
  • expression level refers to qualitative or quantitative determination of an expression product or gene product. Expression level can be determined for the RNA expression level of a gene or for the polypeptide expression level of a gene.
  • normalized expression level refers to an expression level of a response indicator gene relative to the level of an expression product of a reference gene(s), which might be all measured expression products in the sample, a single reference expression product, or a particular set of expression products.
  • a gene exhibits an "increased expression level” when the expression level of an expression product is higher in a first sample, such as in a tumor cell, than in a second sample, such as a normal cell or non-tumor cell.
  • a gene exhibits an "increased normalized expression level” when the normalized expression level of an expression product is higher in a first sample than in a second sample.
  • reference gene refers to a gene whose expression level can be used to compare the expression level of a gene product in a test sample.
  • reference genes include housekeeping genes, such as beta- globin, alcohol dehydrogenase, or any other gene, the expression of which does not vary depending on the disease status of the cell containing the gene.
  • all of the assayed genes or a large subset thereof may serve as reference genes.
  • the term "expression product” or “gene product” are used herein to refer to the RNA transcription products (transcripts) of a gene, including mRNA, and the polypeptide translation products of such RNA transcripts.
  • An expression product can be, for example, an unspliced RNA, an mRNA, a splice variant mRNA, a microRNA, a fragmented RNA, a polypeptide, a post-translationally modified polypeptide, a splice variant polypeptide, etc.
  • the term “measuring” as used herein refers to performing a physical act of determining the dimension, quantity, or capacity of a component in a sample.
  • microarray refers to an ordered arrangement of hybridizable array elements, e.g., oligonucleotide or polynucleetide probes, on a substrate.
  • polynucleotide generally refers to any polyribonucleotide or polydeoxribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA.
  • polynucleotides as used herein include, without limitation, single- and double-stranded DNA, DNA including single- and double-stranded regions, single- and double-stranded RNA, and RNA including single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double- stranded or include single- and double-stranded regions.
  • polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • the strands in such regions may be from the same molecule or from different molecules.
  • the regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules.
  • One of the molecules of a triple- helical region often is an oligonucleotide.
  • polynucleotide also includes DNAs (including cDNAs) and RNAs and those that contain one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons, are "polynucleotides" as that term is used herein.
  • DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritiated bases are included within the term “polynucleotides” as used herein.
  • polynucleotide embraces all chemically, enzymatically and/or metabolically modified forms of unmodified polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells.
  • oligonucleotide refers to a relatively short polynucleotide, including, without limitation, single-stranded deoxyribonucleotides, single- or double-stranded ribonucleotides, RNA/DNA hybrids and double-stranded DNAs. Oligonucleotides, such as single-stranded DNA probe oligonucleotides, are often synthesized by chemical methods, for example using automated oligonucleotide synthesizers that are commercially available. However, oligonucleotides can be made by a variety of other methods, including in vitro recombinant DNA-mediated techniques and by expression of DNAs in cells and organisms.
  • primer refers to an oligonucleotide that acts to initiate synthesis of a complementary nucleic acid strand when placed under conditions in which synthesis of a primer extension product is induced, e.g., in the presence of nucleotides and a polymerization-inducing agent such as a DNA or RNA polymerase and at suitable temperature, pH, metal ion concentration, and salt concentration.
  • a polymerization-inducing agent such as a DNA or RNA polymerase and at suitable temperature, pH, metal ion concentration, and salt concentration.
  • Primers are generally of a length compatible with their use in synthesis of primer extension products, and can be in the range of between about 8 nucleotides and about 100 nucleotides (nt) in length, such as about 10 nt to about 75 nt, about 15 nt to about 60 nt, about 15 nt to about 40 nt, about 18 nt to about 30 nt, about 20 nt to about 40 nt, about 21 nt to about 50 nt, about 22 nt to about 45 nt, about 25 nt to about 40 nt, and so on, e.g., in the range of between about 18 nt and about 40 nt, between about 20 nt and about 35 nt, between about 21 and about 30 nt in length, inclusive, and any length between the stated ranges.
  • nt nucleotides
  • Primers can be in the range of between about 10-50 nucleotides long, such as about 15-45, about 18-40, about 20-30, about 21-25 nt and so on, and any length between the stated ranges. In some embodiments, the primers are not more than about 10, 12, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, or 70 nucleotides in length. In this context, the term "about” may be construed to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 more nucleotides either 5 Or 3' from either termini or from both termini.
  • Primers are in many embodiments single-stranded for maximum efficiency in amplification, but can alternatively be double-stranded. If double-stranded, the primer is in many embodiments first treated to separate its strands before being used to prepare extension products. This denaturation step is typically effected by heat, but may alternatively be carried out using alkali, followed by neutralization. Thus, a "primer" is complementary to a template, and complexes by hydrogen bonding or hybridization with the template to give a primer/template complex for initiation of synthesis by a polymerase, which is extended by the covalent addition of bases at its 3' end.
  • a “primer pair” as used herein refers to first and second primers having nucleic acid sequence suitable for nucleic acid-based amplification of a target nucleic acid.
  • Such primer pairs generally include a first primer having a sequence that is the same or similar to that of a first portion of a target nucleic acid, and a second primer having a sequence that is complementary to a second portion of a target nucleic acid to provide for amplification of the target nucleic acid or a fragment thereof.
  • Reference to “first” and “second” primers herein is arbitrary, unless specifically indicated otherwise.
  • the first primer can be designed as a "forward primer” (which initiates nucleic acid synthesis from a 5' end of the target nucleic acid) or as a "reverse primer” (which initiates nucleic acid synthesis from a 5' end of the extension product produced from synthesis initiated from the forward primer).
  • the second primer can be designed as a forward primer or a reverse primer.
  • probe or "oligonucleotide probe”, used interchangeably herein, refers to a structure comprised of a polynucleotide, as defined above, that contains a nucleic acid sequence complementary to a nucleic acid sequence present in the target nucleic acid analyte (e.g., a nucleic acid amplification product).
  • the polynucleotide regions of probes may be composed of DNA, and/or R A, and/or synthetic nucleotide analogs.
  • Probes are generally of a length compatible with their use in specific detection of all or a portion of a target sequence of a target nucleic acid, and are in many embodiments in the range of between about 8 nt and about 100 nt in length, such as about 8 to about 75 nt, about 10 to about 74 nt, about 12 to about 72 nt, about 15 to about 60 nt, about 15 to about 40 nt, about 18 to about 30 nt, about 20 to about 40 nt, about 21 to about 50 nt, about 22 to about 45 nt, about 25 to about 40 nt in length, and so on, e.g., in the range of between about 18-40 nt, about 20-35 nt, or about 21-30 nt in length, and any length between the stated ranges.
  • a probe is in the range of between about 10-50 nucleotides long, such as about 15-45, about 18-40, about 20-30, about 21-28, about 22-25 and so on, and any length between the stated ranges. In some embodiments, the probes are not more than about 10, 12, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, or 70 nucleotides in length. In this context, the term "about” may be construed to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 more nucleotides either 5' or 3' from either termini or from both termini.
  • RNA transcript refers to the RNA transcription product of a gene, including, for example, mRNA, an unspliced RNA, a splice variant mRNA, a microRNA, and a fragmented RNA.
  • the sample for assaying the gene expression level can be any desired biological sample from a subject.
  • biological sample as used herein denotes a sample taken or isolated from a biological organism, e.g., cell lysate, a homogenate of a tissue sample from a subject.
  • biological sample also includes untreated or pre-treated (or pre- processed) biological samples.
  • the biological sample can be a tissue sample, a biological fluid, including, but not limited to, blood (including whole blood, plasma, cord blood and serum), lactation products (e.g., milk), amniotic fluids, sputum, saliva, urine, semen, cerebrospinal fluid, bronchial aspirate, perspiration, mucus, liquefied feces, synovial fluid, lymphatic fluid, tears, tracheal aspirate, and fractions thereof.
  • the biological sample can include cell lysate and fractions thereof. For example, cells can be harvested and lysed to obtain a cell lysate.
  • the sample is from a resection, biopsy, or core needle biopsy.
  • fine needle aspirate samples can be used. Samples can be either paraffin-embedded or frozen tissue. In some embodiments, the sample is a fixed, wax-embedded tissue sample.
  • the sample can be a tumor sample.
  • the tumor sample can be a biopsy sample or cells from ascitic fluid or cells from pleural effusion.
  • tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • tumor cell refers to a cancerous cell obtained from a cancer cell line or a cancer patient.
  • the term encompasses a tumor cell obtained from tumor tissue samples, for example, tissue obtained by surgical resection and tissue obtained by biopsy, such as for example, a core biopsy or a fine needle biopsy.
  • tumor cell also encompasses tumor cells obtained from sites other than the primary tumor, e.g., circulating tumor cells.
  • the term further encompasses cells that are the progeny of the patient's tumor cells, e.g.
  • cell culture samples derived from primary tumor cells or circulating tumor cells The term further encompasses samples that may comprise protein or nucleic acid material shed from tumor cells in vivo, e.g., bone marrow, blood, plasma, serum, and the like.
  • the biological sample can be a clinical sample.
  • a "clinical sample” is a sample derived from a human subject.
  • a biological sample can also be referred to as a "subject sample.”
  • a test biological sample is the biological sample that has been the object of analysis, monitoring, or observation.
  • a control biological sample can be either a positive or a negative control for the test biological sample.
  • the control biological sample contains the same types of tissues, cells and biological fluids as that of the test biological sample.
  • the sample can be obtained by removing a sample of cells from a subject, but can also be accomplished by using previously isolated cells.
  • the biological sample can be freshly collected or a previously collected sample.
  • the test sample or the biological sample can be a frozen biological sample, e.g., a frozen tissue or cells.
  • the frozen sample can be thawed before employing methods, assays and systems of the invention. After thawing, a frozen sample can be centrifuged before being subjected to the assays disclosed herein.
  • a biological sample can be a nucleic acid product amplified after polymerase chain reaction (PCR).
  • the nucleic acid product such as DNA, R A and mRNA
  • PCR polymerase chain reaction
  • the nucleic acid product can be isolated from a particular biological sample using any of a number of procedures, which are well-known in the art, the particular isolation procedure chosen being appropriate for the particular biological sample.
  • Methods of isolating and analyzing nucleic acid variants as described above are well known to one skilled in the art and can be found, for example in the Molecular Cloning: A Laboratory Manual, 3rd Ed., Sambrook and Russel, Cold Spring Harbor Laboratory Press, 2001.
  • the test sample or the biological sample can be treated with a chemical and/or biological reagent.
  • Chemical and/or biological reagents can be employed to protect and/or maintain the stability of the sample, including biomolecules (e.g., nucleic acid and protein) therein, during processing.
  • biomolecules e.g., nucleic acid and protein
  • One exemplary reagent is a protease inhibitor, which is generally used to protect or maintain the stability of protein during processing.
  • chemical and/or biological reagents can be employed to release nucleic acid or protein from the sample.
  • tumor cells or a tumor sample is assayed or measured for an expression level of an indicator gene product(s).
  • the tumor sample can be obtained from a solid tumor, e.g., via biopsy, or from a surgical procedure carried out to remove a tumor; or from a tissue or bodily fluid that contains cancer cells.
  • the tumor cell or tumor sample is obtained from a tumor of epithelial origin.
  • the tumor cell is a breast, colon, non-small cell lung, renal, ovarian, prostate, or melanoma tumor cell.
  • the tumor sample is obtained from a patient with breast cancer, colon cancer, non-small cell lung cancer, renal cancer, ovarian cancer, prostate cancer, or melanoma.
  • Methods of gene expression profiling include methods based on hybridization analysis of polynucleotides, methods based on sequencing of polynucleotides, and proteomics-based methods.
  • Exemplary methods known in the art for the quantification of mRNA expression in a sample include northern blotting and in situ hybridization (Parker & Barnes, Methods in Molecular Biology 106:247-283 (1999)); RNAse protection assays (Hod, Biotechniques 13:852-854 (1992)); and PCR-based methods, such as reverse transcription PCT (RT-PCR) (Weis et al, Trends in Genetics 8:263-264 (1992)).
  • RT-PCR reverse transcription PCT
  • Antibodies may be employed that can recognize sequence-specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes.
  • Representative methods for sequencing-based gene expression analysis include Serial Analysis of Gene Expression (SAGE), and gene expression analysis by massively parallel signature sequencing (MPSS).
  • the level of RNA transcript of one or more genes is assayed using a quantitative reverse transcription polymerase chain reaction (RT-PCR or qPCR).
  • RT-PCR quantitative reverse transcription polymerase chain reaction
  • qPCR quantitative reverse transcription polymerase chain reaction
  • the first step is the isolation of mRNA from a target sample.
  • the starting material is typically total RNA isolated from tumors or tumor cell lines, and corresponding normal tissues or cell lines, respectively.
  • RNA can be isolated from a variety of primary tumors, including breast, lung, colorectal, prostate, brain, liver, kidney, pancreas, spleen, thymus, testis, ovary, uterus, etc., tumor, or tumor cell lines, with pooled DNA from healthy donors.
  • mRNA can be extracted, for example, from frozen or archived paraffin-embedded and fixed (e.g. formalin-fixed) tissue samples.
  • RNA isolation can be performed using purification kit, buffer set and protease from commercial manufacturers, such as Qiagen, according to the manufacturer's instructions. For example, total RNA from cells in culture can be isolated using Qiagen RNeasy mini-columns.
  • RNA isolation kits include MasterPureTM Complete DNA and RNA Purification Kit (EPICENTRE®, Madison, WT), and Paraffin Block RNA Isolation Kit (Ambion, Inc.). Total RNA from tissue samples can be isolated using RNA Stat-60 (Tel- Test). RNA prepared from tumor can be isolated, for example, by cesium chloride density gradient centrifugation. [00182] As RNA cannot serve as a template for PCR, the first step in gene expression profiling by RT-PCR is the reverse transcription of the RNA template into cDNA, followed by its exponential amplification in a PCR reaction.
  • the two most commonly used reverse transcriptases are avilo myeloblastosis virus reverse transcriptase (AMV- RT) and Moloney murine leukemia virus reverse transcriptase (MMLV-RT).
  • AMV- RT avilo myeloblastosis virus reverse transcriptase
  • MMLV-RT Moloney murine leukemia virus reverse transcriptase
  • the reverse transcription step is typically primed using specific primers, random hexamers, or oligo-dT primers, depending on the circumstances and the goal of expression profiling.
  • extracted RNA can be reverse-transcribed using a GeneAmp RNA PCR kit (Perkin Elmer, CA, USA), following the manufacturer's instructions.
  • the derived cDNA can then be used as a template in the subsequent PCR reaction.
  • the PCR step can use a variety of thermostable DNA-dependent DNA polymerases, it typically employs the Taq DNA polymerase, which has a 5 '-3' nuclease activity but lacks a 3 '-5' proofreading endonuclease activity.
  • TaqMan® PCR typically utilizes the 5 '-nuclease activity of Taq or Tth polymerase to hydrolyze a hybridization probe bound to its target amplicon, but any enzyme with equivalent 5' nuclease activity can be used.
  • Two oligonucleotide primers are used to generate an amplicon typical of a PCR reaction.
  • a third oligonucleotide, or probe is designed to detect nucleotide sequence located between the two PCR primers.
  • the probe is non-extendible by Taq DNA polymerase enzyme, and is labeled with a reporter fluorescent dye and a quencher fluorescent dye. Any laser-induced emission from the reporter dye is quenched by the quenching dye when the two dyes are located close together as they are on the probe.
  • the Taq DNA polymerase enzyme cleaves the probe in a template-dependent manner.
  • the resultant probe fragments disassociate in solution, and signal from the released reporter dye is free from the quenching effect of the second fluorophore.
  • One molecule of reporter dye is liberated for each new molecule synthesized, and detection of the unquenched reporter dye provides the basis for quantitative interpretation of the data.
  • TaqMan® RT-PCR can be performed using commercially available equipment, such as, for example, ABI PRISM 7700TM Sequence Detection SystemTM (Perkin-Elmer- Applied Biosystems, Foster City, CA, USA), or Lightcycler (Roche Molecular Biochemicals, Mannheim, Germany).
  • the 5' nuclease procedure is run on a realtime quantitative PCR device such as the ABI PRISM 7700TM Sequence Detection SystemTM.
  • the system consists of a thermocycler, laser, charge-coupled device (CCD), camera and computer.
  • the system amplifies samples in a 96-well format on a thermocycler.
  • laser-induced fluorescent signal is collected in real-time through fiber optics cables for all 96 wells, and detected at the CCD.
  • the system includes software for running the instrument and for analyzing the data.
  • 5'-Nuclease assay data are initially expressed as Ct, or the threshold cycle.
  • Ct fluorescence values are recorded during every cycle and represent the amount of product amplified to that point in the amplification reaction. The point when the fluorescent signal is first recorded as statistically significant is the threshold cycle (C t ).
  • C t The point when the fluorescent signal is first recorded as statistically significant is the threshold cycle (C t ).
  • RT-PCR is usually performed using an internal standard. The ideal internal standard is expressed at a constant level among different tissues, and is unaffected by the experimental treatment.
  • RNAs most frequently used to normalize patterns of gene expression are mRNAs for the housekeeping genes glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and ⁇ -actin.
  • GPDH glyceraldehyde-3-phosphate-dehydrogenase
  • a more recent variation of the RT-PCR technique is the real time quantitative PCR, which measures PCR product accumulation through a dual-labeled f uorigenic probe (i.e., TaqMan® probe).
  • Real time PCR is compatible both with quantitative competitive PCR, where internal competitor for each target sequence is used for normalization, and with quantitative comparative PCR using a normalization gene contained within the sample, or a housekeeping gene for RT-PCR.
  • quantitative competitive PCR where internal competitor for each target sequence is used for normalization
  • quantitative comparative PCR using a normalization gene contained within the sample, or a housekeeping gene for RT-PCR.
  • the obtained cDNA is spiked with a synthetic DNA molecule (competitor), which matches the targeted cDNA region in all positions, except a single base, and serves as an internal standard.
  • the cDNA/competitor mixture is PCR amplified and is subjected to a post-PCR shrimp alkaline phosphatase (SAP) enzyme treatment, which results in the dephosphorylation of the remaining nucleotides.
  • SAP shrimp alkaline phosphatase
  • the PCR products from the competitor and cDNA are subjected to primer extension, which generates distinct mass signals for the competitor- and cDNA- derives PCR products. After purification, these products are dispensed on a chip array, which is pre-loaded with components needed for analysis with matrix-assisted laser desorption ionization tinie-of- flight mass spectrometry (MALDI-TOF MS) analysis.
  • MALDI-TOF MS matrix-assisted laser desorption ionization tinie-of- flight mass spectrometry
  • the cDNA present in the reaction is then quantified by analyzing the ratios of the peak areas in the mass spectrum generated. For further details see, e.g. Ding and Cantor, Proc. Natl. Acad. Sd. USA 100:3059-3064 (2003). c.
  • PCR-based techniques include, for example, differential display (Liang and Pardee, Science 257:967-971 (1992)); amplified fragment length polymorphism (iAFLP) (Kawamoto et al., Genome Res.
  • BeadArrayTM technology (Illumina, San Diego, CA; Oliphant et al., Discovery of Markers for Disease (Supplement to Biotechniques), June 2002; Ferguson et al, Analytical Chemistry 72:5618 (2000)); BeadsArray for Detection of Gene Expression (BADGE), using the commercially available Luminex 100 LabMAP system and multiple color-coded microspheres (Luminex Corp., Austin, TX) in a rapid assay for gene expression (Yang et al, Genome Res. 11 : 1888-1898 (2001)); and high coverage expression profiling (HiCEP) analysis (Fukumura et al, Nucl Acids. Res. 31(16) e94 (2003)).
  • PCR amplified inserts of cDNA clones are applied to a substrate in a dense array.
  • Preferably at least 10,000 nucleotide sequences are applied to the substrate.
  • the microarrayed genes, immobilized on the microchip at 10,000 elements each, are suitable for hybridization under stringent conditions.
  • Fluorescently labeled cDNA probes may be generated through incorporation of fluorescent nucleotides by reverse transcription of RNA extracted from tissues of interest. Labeled cDNA probes applied to the chip hybridize with specificity to each spot of DNA on the array.
  • the chip After stringent washing to remove non-specifically bound probes, the chip is scanned by confocal laser microscopy or by another detection method, such as a CCD camera. Quantitation of hybridization of each arrayed element allows for assessment of corresponding mRNA abundance. With dual color fluorescence, separately labeled cDNA probes generated from two sources of R A are hybridized pairwise to the array. The relative abundance of the transcripts from the two sources corresponding to each specified gene is thus determined simultaneously. The miniaturized scale of the hybridization affords a convenient and rapid evaluation of the expression pattern for large numbers of genes.
  • Microarray analysis can be performed by commercially available equipment, following manufacturer's protocols, such as by using the Affymetrix GenChip technology, or Incyte's microarray technology.
  • Serial analysis of gene expression is a method that allows the simultaneous and quantitative analysis of a large number of gene transcripts, without the need of providing an individual hybridization probe for each transcript.
  • a short sequence tag (about 10-14 bp) is generated that contains sufficient information to uniquely identify a transcript, provided that the tag is obtained from a unique position within each transcript.
  • many transcripts are linked together to form long serial molecules, that can be sequenced, revealing the identity of the multiple tags simultaneously.
  • the expression pattern of any population of transcripts can be quantitatively evaluated by determining the abundance of individual tags, and identifying the gene corresponding to each tag. For more details see, e.g. Velculescu et al, Science 270:484-487 (1995); and Velculescu et al, Cell 88:243-51 (1997).
  • MPSS Massively Parallel Signature Sequencing
  • the free ends of the cloned templates on each microbead are analyzed simultaneously, using a fluorescence- based signature sequencing method that does not require DNA fragment separation. This method has been shown to simultaneously and accurately provide, in a single operation, hundreds of thousands of gene signature sequences from a yeast cDNA library.
  • Immunohistochemistry methods are also suitable for detecting the expression levels of genes.
  • antibodies or antisera preferably polyclonal antisera, and most preferably monoclonal antibodies specific for each marker are used to detect expression.
  • the antibodies can be detected by direct labeling of the antibodies themselves, for example, with radioactive labels, fluorescent labels, hapten labels such as, biotin, or an enzyme such as horse radish peroxidase or alkaline phosphatase.
  • unlabeled primary antibody is used in conjunction with a labeled secondary antibody, comprising antisera, polyclonal antisera or a monoclonal antibody specific for the primary antibody. Immunohistochemistry protocols and kits are well known in the art and are commercially available.
  • raw expression level data measured for a gene product can be normalized relative to the mean expression level data obtained for one or more reference genes.
  • reference genes include housekeeping genes, such as GAPDH.
  • all of the assayed genes or a large subset thereof may also concurrently serve as reference genes and normalization can be based on the mean or median signal (Ct) of all of the assayed genes or a subset thereof (often referred to as "global normalization" approach).
  • measured normalized amount of a patient tumor mRNA can be compared to the amount found in a cancer tissue reference set. See e.g., Cronin, M. et al, Am. Soc. Investigative Pathology 164:35-42 (2004).
  • the normalization can be carried out such that a one unit increase in normalized expression level of a gene product generally reflects a 2-fold increase in quantity of expression product present in the sample.
  • normalization techniques applicable to qRT-PCR data from tumor tissue see e.g., Silva, S. et al. (2006) BMC Cancer 6, 200; deKok, J. et al. (2005) Laboratory Investigation 85, 154-159.
  • kits comprising agents, which can include gene-specific or gene-selective probes and/or primers, for quantitating the expression of the disclosed genes.
  • agents can include gene-specific or gene-selective probes and/or primers, for quantitating the expression of the disclosed genes.
  • kits can optionally contain reagents for the extraction of RNA from tumor samples, in particular, fixed paraffm- embedded tissue samples and/or reagents for RNA amplification.
  • the kits can optionally comprise the reagent(s) with an identifying description or label or instructions relating to their use in the methods of the present invention.
  • kits can comprise containers (including microliter plates suitable for use in an automated implementation of the method), each with one or more of the various reagents (typically in concentrated form) utilized in the methods, including, for example, pre-fabricated microarrays, buffers, the appropriate nucleotide triphosphates (e.g., dATP, dCTP, dGTP and dTTP; or rATP, rCTP, rGTP and UTP), reverse transcriptase, DNA polymerase, RNA polymerase, and one or more probes and primers of the present invention (e.g., appropriate length poly(T) or random primers linked to a promoter reactive with the RNA polymerase).
  • the appropriate nucleotide triphosphates e.g., dATP, dCTP, dGTP and dTTP; or rATP, rCTP, rGTP and UTP
  • reverse transcriptase DNA polymerase
  • RNA polymerase e.g.,
  • the methods and systems described herein can be implemented in numerous ways. In one embodiment, the methods involve use of a communications infrastructure, for example, the internet. Several embodiments of the invention are discussed below.
  • the present invention may also be implemented in various forms of hardware, software, firmware, processors, or a combination thereof.
  • the methods and systems described herein can be implemented as a combination of hardware and software.
  • the software can be implemented as an application program tangibly embodied on a program storage device, or different portions of the software implemented in the user's computing environment (e.g., as an applet) and on the reviewer's computing environment, where the reviewer may be located at a remote site (e.g., at a service provider's facility).
  • portions of the data processing can be performed in the user-side computing environment.
  • the user-side computing environment can be programmed to provide for defined test codes to denote a likelihood "score," where the score is transmitted as processed or partially processed responses to the reviewer's computing environment in the form of test code for subsequent execution of one or more algorithms to provide a result and/or generate a report in the reviewer's computing environment.
  • the score can be a numerical score (representative of a numerical value) or a non-numerical score representative of a numerical value or range of numerical values (e.g., "A”: representative of a 90-95% likelihood of sensitivity to a PI3K inhibitor; "High”: representative of a greater than 50% chance of sensitivity to (or some other selected threshold of likelihood); “Low”: representative of a less than 50% chance of sensitivity (or some other selected threshold of likelihood), and the like.
  • A representative of a 90-95% likelihood of sensitivity to a PI3K inhibitor
  • “High” representative of a greater than 50% chance of sensitivity to (or some other selected threshold of likelihood)
  • “Low” representative of a less than 50% chance of sensitivity (or some other selected threshold of likelihood)
  • the system generally includes a processor unit.
  • the processor unit operates to receive information, which can include test data (e.g., level of a predictive gene product(s); level of a reference gene product(s); normalized level of a predictive gene product(s); and may also include other data such as patient data.
  • This information received can be stored at least temporarily in a database, and data analyzed to generate a report as described above.
  • Part or all of the input and output data can also be sent electronically.
  • Certain output data e.g., reports
  • Exemplary output receiving devices can include a display element, a printer, a facsimile device and the like.
  • Electronic forms of transmission and/or display can include email, interactive television, and the like.
  • all or a portion of the input data and/or output data e.g., usually at least the final report
  • the data may be accessed or sent to health professionals as desired.
  • the input and output data, including all or a portion of the final report can be used to populate a patient's medical record that may exist in a confidential database as the healthcare facility.
  • the disclosure also provides a computer-readable storage medium (e.g., CD- ROM, memory key, flash memory card, diskette, etc.) having stored thereon a program which, when executed in a computing environment, provides for implementation of algorithms to carry out all or a portion of the results of a response likelihood assessment as described herein.
  • a computer-readable storage medium e.g., CD- ROM, memory key, flash memory card, diskette, etc.
  • the program includes program instructions for collecting, analyzing and generating output, and generally includes computer readable code devices for interacting with a user as described herein, processing that data in conjunction with analytical information, and generating unique printed or electronic media for that user.
  • the storage medium includes a program that provides for implementation of a portion of the methods described herein (e.g., the user-side aspect of the methods (e.g., data input, report receipt capabilities, etc.))
  • the program provides for transmission of data input by the user (e.g., via the internet, via an intranet, etc.) to a computing environment at a remote site. Processing or completion of processing of the data is carried out at the remote site to generate a report. After review of the report, and completion of any needed manual intervention, to provide a complete report, the complete report is then transmitted back to the user as an electronic document or printed document (e.g., fax or mailed paper report).
  • the storage medium containing a program according to the invention can be packaged with instructions (e.g., for program installation, use, etc.) recorded on a suitable substrate or a web address where such instructions may be obtained.
  • the computer-readable storage medium can also be provided in combination with one or more reagents for carrying out a response likelihood assessment (e.g., primers, probes, arrays, or such other kit components).
  • “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level.
  • “Complete inhibition” is a 100% inhibition as compared to a reference level.
  • a decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.
  • the terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statically significant amount.
  • the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%>, or at least about 50%>, or at least about 60%>, or at least about 70%), or at least about 80%>, or at least about 90%> or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4-fold, or at least about a 5 -fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.
  • an "increase" is a
  • a "subject” means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters.
  • Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon.
  • the subject is a mammal, e.g., a primate, e.g., a human.
  • the terms, "individual,” “patient” and “subject” are used interchangeably herein.
  • the subject is a mammal.
  • the mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of cancer.
  • a subject can be male or female.
  • a subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g. cancer) or one or more complications related to such a condition, and optionally, have already undergone treatment for cancer or the one or more complications related to cancer.
  • a subject can also be one who has not been previously diagnosed as having cancer or one or more complications related to cancer.
  • a subject can be one who exhibits one or more risk factors for cancer or one or more complications related to cancer or a subject who does not exhibit risk factors.
  • a "subject in need" of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition.
  • protein and “polypeptide” are used interchangeably herein to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues.
  • protein and “polypeptide” refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function.
  • modified amino acids e.g., phosphorylated, glycated, glycosylated, etc.
  • amino acid analogs regardless of its size or function.
  • Protein and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term “peptide” is often used in reference to small polypeptides, but usage of these terms in the art overlaps.
  • polypeptide proteins and “polypeptide” are used interchangeably herein when referring to a gene product and fragments thereof.
  • exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.
  • nucleic acid or “nucleic acid sequence” refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or an analog thereof.
  • the nucleic acid can be either single-stranded or double-stranded.
  • a single-stranded nucleic acid can be one nucleic acid strand of a denatured double- stranded DNA. Alternatively, it can be a single-stranded nucleic acid not derived from any double-stranded DNA.
  • the nucleic acid can be DNA.
  • nucleic acid can be RNA.
  • Suitable nucleic acid molecules are DNA, including genomic DNA or cDNA. Other suitable nucleic acid molecules are RNA, including mRNA.
  • agent refers generally to any entity which is normally not present or not present at the levels being administered to a cell, tissue or subject.
  • An agent can be selected from a group including but not limited to: polynucleotides; polypeptides; small molecules; and antibodies or antigen-binding fragments thereof.
  • a polynucleotide can be RNA or DNA, and can be single or double stranded, and can be selected from a group including, for example, nucleic acids and nucleic acid analogues that encode a polypeptide.
  • a polypeptide can be, but is not limited to, a naturally-occurring polypeptide, a mutated polypeptide or a fragment thereof that retains the function of interest.
  • agents include, but are not limited to a nucleic acid aptamer, peptide-nucleic acid (PNA), locked nucleic acid (LNA), small organic or inorganic molecules; saccharide; oligosaccharides; polysaccharides; biological macromolecules, peptidomimetics; nucleic acid analogs and derivatives; extracts made from biological materials such as bacteria, plants, fungi, or mammalian cells or tissues and naturally occurring or synthetic compositions.
  • PNA peptide-nucleic acid
  • LNA locked nucleic acid
  • An agent can be applied to the media, where it contacts the cell and induces its effects.
  • an agent can be intracellular as a result of introduction of a nucleic acid sequence encoding the agent into the cell and its transcription resulting in the production of the nucleic acid and/or protein environmental stimuli within the cell.
  • the agent is any chemical, entity or moiety, including without limitation synthetic and naturally-occurring non-proteinaceous entities.
  • the agent is a small molecule having a chemical moiety selected, for example, from unsubstituted or substituted alkyl, aromatic, or heterocyclyl moieties including macrolides, leptomycins and related natural products or analogues thereof. Agents can be known to have a desired activity and/or property, or can be selected from a library of diverse compounds.
  • small molecule can refer to compounds that are "natural product-like,” however, the term “small molecule” is not limited to "natural product-like” compounds. Rather, a small molecule is typically characterized in that it contains several carbon— carbon bonds, and has a molecular weight more than about 50, but less than about 5000 Daltons (5 kD). Preferably the small molecule has a molecular weight of less than 3 kD, still more preferably less than 2 kD, and most preferably less than 1 kD. In some cases it is preferred that a small molecule have a molecular mass equal to or less than 700 Daltons.
  • the term "inhibitor” refers to an agent which can decrease the expression and/or activity of the targeted expression product (e.g. mRNA encoding the target or a target polypeptide), e.g. by at least 10% or more, e.g. by 10% or more, 50%> or more, 70% or more, 80% or more, 90% or more, 95% or more, or 98 % or more.
  • the efficacy of an inhibitor of, for example, PI3K e.g. its ability to decrease the level and/or activity of PI3K can be determined, e.g.
  • PI3K polypeptide by measuring the level of a PI3K polypeptide (and/or mRNA encoding such a polypeptide) and/or the activity of PI3K.
  • Methods for measuring the level of a given mRNA and/or polypeptide are known to one of skill in the art, e.g. RTPCR with primers can be used to determine the level of RNA and Western blotting with an antibody can be used to determine the level of a polypeptide.
  • the activity of, e.g. PI3K can be determined using methods known in the art and described above herein. .
  • the terms “treat,” “treatment,” “treating,” or “amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder, e.g. cancer.
  • the term “treating” includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder associated with a cancer.
  • Treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective” if the progression of a disease is reduced or halted.
  • treatment includes not just the improvement of symptoms or markers, but also a cessation of, or at least slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment.
  • Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and/or decreased mortality, whether detectable or undetectable.
  • treatment also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).
  • the term "pharmaceutical composition” refers to the active agent in combination with a pharmaceutically acceptable carrier e.g. a carrier commonly used in the pharmaceutical industry.
  • a pharmaceutically acceptable carrier e.g. a carrier commonly used in the pharmaceutical industry.
  • pharmaceutically acceptable is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • administering refers to the placement of a compound as disclosed herein into a subject by a method or route which results in at least partial delivery of the agent at a desired site.
  • Pharmaceutical compositions comprising the compounds disclosed herein can be administered by any appropriate route which results in an effective treatment in the subject.
  • amphiphilic refers to a molecule that has both a hydrophobic portion and a lipophobic portion, i.e. at least one a polar, water-soluble group and at least one a nonpolar, water- insoluble group.
  • an amphiphilic molecule will partition to the interface of the two phases.
  • an amphiphile is a molecule that is soluble in both an aqueous environment and a non-aqueous environment.
  • amphiphile refers to an amphiphilic molecule.
  • compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
  • the term "consisting essentially of” refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment.
  • aromatase inhibitor relates to a compound which inhibits the estrogen production, i.e., the conversion of the substrates androstenedione and testosterone to estrone and estradiol, respectively.
  • the term includes, but is not limited to, steroids, especially atamestane, exemestane and formestane; and, in particular, non- steroids, especially aminoglutethimide, roglethimide, pyridoglutethimide, trilostane, testolactone, ketokonazole, vorozole, fadrozole, anastrozole and letrozole.
  • anti-estrogen relates to a compound which antagonizes the effect of estrogens at the estrogen receptor level.
  • the term includes, but is not limited to, tamoxifen, fulvestrant, raloxifene and raloxifene hydrochloride.
  • anti-androgen relates to any substance which is capable of inhibiting the biological effects of androgenic hormones and includes, but is not limited to, bicalutamide.
  • gonadorelin agonist includes, but is not limited to, abarelix, goserelin and goserelin acetate. Goserelin is disclosed in U.S. Patent No. 4,100,274 and is marketed as ZOLADEX. Abarelix can be formulated, e.g., as disclosed in U.S. Patent No. 5,843,901.
  • topoisomerase I inhibitor includes, but is not limited to, topotecan, irinotecan, gimatecan, camptothecin and its analogues, 9-nitrocamptot ecin and the macromolecular camptothecin conjugate PNU-166148 (compound Al in WO 99/17804).
  • topoisomerase II inhibitor includes, but is not limited to, the anthracyclines, such as doxorubicin, daunorubicin, epirubicin, idarubicin and nemorubicin; the anthraquinones mitoxantrone and losoxantrone; and the podophillotoxines etoposide and teniposide.
  • anthracyclines such as doxorubicin, daunorubicin, epirubicin, idarubicin and nemorubicin
  • the anthraquinones mitoxantrone and losoxantrone include the podophillotoxines etoposide and teniposide.
  • microtubule active agent relates to microtubule stabilizing, microtubule destabilizing agents and microtublin polymerization inhibitors including, but not limited to, taxanes, e.g., paclitaxel and docetaxel; vinca alkaloids, e.g., vinblastine, especially vinblastine sulfate; vincristine, especially vincristine sulfate and vinorelbine; discodermolides; colchicines; and epothilones and derivatives thereof, e.g., epothilone B or D or a derivative thereof. Also included are Epotholine derivatives which are disclosed in U.S. Patent No.
  • Alkylating agents are polyfunctional compounds that have the ability to substitute alkyl groups for hydrogen ions.
  • alkylating agents include, but are not limited to, bischloroethylamines (nitrogen mustards, e.g. chlorambucil, cyclophosphamide, ifosfamide, mechlorethamine, melphalan, uracil mustard), aziridines (e.g. thiotepa), alkyl alkone sulfonates (e.g. busulfan), nitrosoureas (e.g.
  • anti-neoplastic and anti-metabolite agents refers to the group of compounds that interfere with metabolic processes vital to the physiology and proliferation of cancer cells. Actively proliferating cancer cells require continuous synthesis of large quantities of nucleic acids, proteins, lipids, and other vital cellular constituents. Many of the antimetabolites inhibit the synthesis of purine or pyrimidine nucleosides or inhibit the enzymes of DNA replication. Some antimetabolites also interfere with the synthesis of ribonucleosides and RNA and/or amino acid metabolism and protein synthesis as well. By interfering with the synthesis of vital cellular constituents, antimetabolites can delay or arrest the growth of cancer cells.
  • antimetabolic agents include, but are not limited to, 5-fluorouracil (5-FU); asparaginase; capecitabine; cladribine (2-CDA); cytarabine; DNA de- methylating agents, such as 5-azacytidine and decitabine; edatrexate; floxuridine (5-FUdR); fludarabine phosphate; folic acid antagonists such as pemetrexed; gemcitabine; hydroxyurea; leucovorin; mercaptopurine (6-MP); methotrexate; pentostatin; and thioguanine (6-TG).
  • 5-fluorouracil 5-FU
  • asparaginase capecitabine
  • cytarabine DNA de- methylating agents, such as 5-azacytidine and decitabine
  • edatrexate such as 5-azacytidine and decitabine
  • floxuridine 5-FUdR
  • Patent No. 5,093,330 e.g., midostaurin
  • examples of further compounds include, e.g., UCN-01 ; safmgol; BAY 43-9006; Bryostatin 1; Perifosine; llmofosine; RO 318220 and RO 320432; GO 6976; Isis 3521; LY333531/LY379196; isochinoline compounds, such as those disclosed in WO 00/09495; FTIs; PD184352 or QAN697 (a PI3K inhibitor); x) compounds targeting, decreasing or inhibiting the activity of protein tyrosine kinase inhibitors include imatinib mesylate (GLEEVEC/GLIVEC) or a tyrphostin.
  • a tyrphostin is preferably a low molecular weight (M r ⁇ 1500) compound, or a pharmaceutically acceptable salt thereof, especially a compound selected from the benzylidenemalonitrile class or the S-arylbenzenemalonirile or bisubstrate quinoline class of compounds, more especially any compound selected from the group consisting of Tyrphostin A23/RG-50810, AG 99, Tyrphostin AG 213, Tyrphostin AG 1748, Tyrphostin AG 490, Tyrphostin B44, Tyrphostin B44 (+) enantiomer, Tyrphostin AG 555, AG 494, Tyrphostin AG 556 and AG957 and adaphostin (4- ⁇ [(2,5- dihydroxyphenyl)methyl] amino ⁇ -benzoic acid adamantyl ester, NSC 680410); and xi) compounds targeting, decreasing or inhibiting the activity of the epidermal growth factor family of receptor tyrosine kin
  • Further anti-angiogenic compounds include compounds having another mechanism for their activity, e.g., unrelated to protein or lipid kinase inhibition, e.g., thalidomide (THALOMID) and TNP-470.
  • TAALOMID thalidomide
  • TNP-470 TNP-470.
  • cyclooxygenase inhibitor includes, but is not limited to, e.g., Cox-2 inhibitors, 5-alkyl substituted 2-arylaminophenylacetic acid and derivatives, such as celecoxib (CELEBREX), rofecoxib (VIOXX), etoricoxib, valdecoxib (BEXTRA) or a 5- alkyl- 2-arylaminophenylacetic acid, e.g., 5-methyl-2-(2'-chloro-6'-fluoroanilino)phenyl acetic acid (lumiracoxib, PREXIGE).
  • Cox-2 inhibitors such as celecoxib (CELEBREX), rofecoxib (VIOXX), etoricoxib, valdecoxib (BEXTRA) or a 5- alkyl- 2-arylaminophenylacetic acid, e.g., 5-methyl-2-(2'-chloro-6'-fluoroanilino
  • bisphosphonate includes, but is not limited to, etridonic, clodronic, tiludronic, pamidronic, alendronic, ibandronic, risedronic and zoledronic acid.
  • heparanase inhibitor refers to compounds which target, decrease or inhibit heparin sulphate degradation.
  • the term includes, but is not limited to, PI-88.
  • telomerase inhibitor refers to compounds which target, decrease or inhibit the activity of telomerase.
  • Compounds which target, decrease or inhibit the activity of telomerase are especially compounds which inhibit the telomerase receptor, e.g., telomestatin.
  • methionine aminopeptidase inhibitor refers to compounds which target, decrease or inhibit the activity of methionine aminopeptidase.
  • Compounds which target, decrease or inhibit the activity of methionine aminopeptidase are, e.g., bengamide or a derivative thereof.
  • proteasome inhibitor refers to compounds which target, decrease or inhibit the activity of the proteasome.
  • Compounds which target, decrease or inhibit the activity of the proteasome include, e.g., PS-341 and MLN 341.
  • matrix metalloproteinase inhibitor or "MMP inhibitor”, as used herein, includes, but is not limited to, collagen peptidomimetic and non-peptidomimetic inhibitors; tetracycline derivatives, e.g., hydroxamate peptidomimetic inhibitor batimastat; and its orally- bioavailable analogue marimastat (BB-2516), prinomastat (AG3340), metastat (NSC 683551) BMS-279251 , BAY 12-9566, TAA211 , MMI270B or AAJ996.
  • MMP inhibitor matrix metalloproteinase inhibitor
  • agents used in the treatment of hematologic malignancies includes, but is not limited to, FMS-like tyrosine kinase inhibitors, e.g., compounds targeting, decreasing or inhibiting the activity of Flt-3; interferons; cytosine arabinoside (Ara- C); bisulfan; and ALK inhibitors, i.e. compounds which target, decrease or especially inhibit anaplastic lymphoma kinase (ALK).
  • FMS-like tyrosine kinase inhibitors e.g., compounds targeting, decreasing or inhibiting the activity of Flt-3
  • interferons cytosine arabinoside (Ara- C); bisulfan
  • ALK inhibitors i.e. compounds which target, decrease or especially inhibit anaplastic lymphoma kinase (ALK).
  • FMS-like tyrosine kinase inhibitors includes, but is not limited to, compounds which target, decrease or inhibit the activity of FMS-like tyrosine kinase receptors such as especially compounds, proteins or antibodies which inhibit Flt-3, e.g., PKC412, midostaurin, a staurosporine derivative, SU 11248 and MLN518.
  • HSP90 inhibitors includes, but is not limited to, compounds targeting, decreasing or inhibiting the intrinsic ATPase activity of HSP90; degrading, targeting, decreasing or inhibiting the HSP90 client proteins via the ubiquitin proteasome pathway.
  • Compounds targeting, decreasing or inhibiting the intrinsic ATPase activity of HSP90 are especially compounds, proteins or antibodies which inhibit the ATPase activity of HSP90, e.g., 17-allylamino,17-demethoxygeldanamycin (17-AAG), a geldanamycin derivative; other geldanamycin-related compounds; radicicol and HDAC inhibitors.
  • histone deacetylase inhibitors or "HDAC inhibitors” relates to compounds which target, decrease or especially inhibit the activity of histone deacetylase (HDAC), such as sodium butyrate and suberoylanilide hydroxamic acid (SAHA).
  • HDAC histone deacetylase
  • SAHA sodium butyrate and suberoylanilide hydroxamic acid
  • Specific HDAC inhibitors include MS275, SAHA, FK228 (formerly FR901228), Trichostatin A and compounds disclosed in U.S. Patent No.
  • mTOR inhibitors relates to compounds which target, decrease or inhibit the activity/function of the serine/threonine mTOR kinase family and are especially compounds, proteins or antibodies which inhibit members of the mTOR kinase family, e.g., CCI-779, ABT578, SAR543, rapamycin and derivatives/analogs thereof, AP23573 and AP23841 from Ariad, everolimus (CERTICAN, RAD001 ) and sirolimus (RAPAMUNE).
  • Somatostatin receptor antagonists refers to agents which target, treat or inhibit the somatostatin receptor, such as octreotide and SOM230.
  • integratedin antagonists includes, but is not limited to, e.g. ⁇ 3 antagonists and ⁇ 5 antagonists.
  • Tumor cell damaging approaches refers to approaches, such as ionizing radiation.
  • ionizing radiation means ionizing radiation that occurs as either electromagnetic rays, such as X-rays and gamma rays; or particles, such as alpha and beta particles. Ionizing radiation is provided in, but not limited to, radiation therapy and is known in the art. See Hellman, Cancer, 4 th Edition, Vol. 1 , Devita et al, Eds., pp. 248-275 (1993).
  • anti-leukemic compounds includes, e.g., Ara-C, a pyrimidine analog, which is the 2'-a-hydroxy ribose (arabinoside) derivative of deoxycytidine. Also included is the purine analog of hypoxanthine, 6-mercaptopurine (6-MP) and fludarabine phosphate.
  • EDG binders refers to a class of immunosuppressants that modulates lymphocyte recirculation, such as FTY720.
  • ribonucleotide reductase inhibitors refers to pyrimidine or purine nucleoside analogs including, but not limited to, fludarabine and/or Ara-C; 6-thioguanine; 5- FU; cladribine; 6-mercaptopurine, especially in combination with Ara-C against ALL; and/or pentostatin.
  • Ribonucleotide reductase inhibitors are especially hydroxyurea or 2-hydroxy- 7H-isoindole-l,3-dione derivatives, such as PL-1 , PL-2, PL-3, PL-4, PL-5, PL-6, PL-7 or PL-8. See Nandy et al, Ada Oncologica, Vol. 33, No. 8, pp. 953-961 (1994).
  • S-adenosylmethionine decarboxylase inhibitors includes, but is not limited to, the compounds disclosed in U.S. Patent No. 5,461,076.
  • ACE inhibitors include benazepril (CIBACEN), enazepril (LOTENSIN), captopril, enalapril, fosinopril, lisinopril, moexipril, quinapril, ramipril, perindopril and trandolapril.
  • a supramolecular combinatorial therapeutic comprising a taxane-lipid conjugate.
  • taxane conjugate is a cabazitaxel-lipid conjugate, a paclitaxel-lipid conjugate or a docetaxel-lipid conjugate.
  • the supramolecular combinatorial therapeutic of paragraph 9 wherein the supramolecular combinatorial therapeutic comprises from about 1% to about 99% (w/w) of the platinum conjugate.
  • the supramolecular combinatorial therapeutic of paragraph 9 or 10 wherein the supramolecular combinatorial therapeutic comprises the taxane conjugate and the platinum conjugate in about 10: 1 to about 1 : 10 ratio.
  • the supramolecular combinatorial therapeutic of any of paragraphs 12-15 wherein the supramolecular combinatorial therapeutic comprises the taxane conjugate and the antibody conjugate in about 10: 1 to about 1 : 10 ratio.
  • lipid is selected from the group consisting of cholesterol; 1,3 -Propanediol Dicaprylate/Dicaprate; 10-undecenoic acid; 1-dotriacontanol; 1-heptacosanol; 1- nonacosanol; 2-ethyl hexanol; Androstanes; Arachidic acid; Arachidonic acid; arachidyl alcohol; Behenic acid; behenyl alcohol; Capmul MCM CIO; Capric acid; capric alcohol; capryl alcohol; Caprylic acid; Caprylic/Capric Acid Ester of Saturated Fatty Alcohol C12-C18; Caprylic/Capric Triglyceride; Caprylic/Capric Triglyceride; Ceramide phosphorylcholine (Sphingomyelin, SPH); Ceramide phosphorylethanolamine (Sphingomyelin, Cer-PE); Ceramide phosphorylglyce
  • Phosphatidylethanolamine (cephalin, PE); Phosphatidylinositol (PI); Phosphatidylinositol bisphosphate (PIP2); Phosphatidylinositol phosphate (PIP); Phosphatidylinositol triphosphate (PIP3); Phosphatidylserine (PS); polyglyceryl- 6-distearate; Pregnanes; Propylene Glycol Dicaprate; Propylene Glycol Dicaprylocaprate; Propylene Glycol Dicaprylocaprate; Psyllic acid; recinoleaic acid; recinoleyl alcohol; Sapienic acid; soy lecithin; Stearic acid; Stearidonic; stearyl alcohol; Tricosylic acid; Tridecyl alcohol; Tridecylic acid; Triolein; Undecyl alcohol; undecylenic acid; Undecylic
  • lipid is cholesterol, alpha-tocopherol, or a fatty acid.
  • the phospholipid is selected from the group consisting of phosphatidyl cholines, phosphatidyl cholines with acyl groups having 6 to 22 carbon atoms, phosphatidyl ethanolamines, phosphatidyl inositols, phosphatidic acids, phosphatidyl serines, sphingomyelin, phosphatidyl glycerols, and any combinations thereof.
  • the phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylglycerol, lecithin, ⁇ , ⁇ -dipalmitoyl-a-lecithin, sphingomyelin, phosphatidylserine, phosphatidic acid, N-(2,3-di(9-(Z)-octadecenyloxy))-prop-l- yl-N,N,N-trimethylammonium chloride, phosphatidylethanolamme, lysolecithin, lysophosphatidylethanolamine, phosphatidylinositol, cephalin, cardiolipin, cerebrosides, dicetylphosphate, dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, dioleoy
  • the supramolecular combinatorial therapeutic of paragraph 26 wherein the phospholipid is SOPC.
  • PEG polyethylene glycol
  • the supramolecular combinatorial therapeutic of paragraph 31 wherein the PEG is conjugated with a component of the supramolecular combinatorial therapeutic.
  • the PEG conjugated lipid is selected from the group consisting of PEG conjugated diacylglycerols and dialkylglycerols, PEG- conjugated phosphatidylethanolamine and phosphatidic acid, PEG conjugated ceramides, PEG conjugated dialkylamines, PEG conjugated 1 ,2-diacyloxypropan-3 -amines, and any combinations thereof.
  • the targeting ligand is selected from the group consisting of peptides, polypeptides, proteins, enzymes, peptidomimetics, glycoproteins, antibodies (monoclonal or polyclonal) and portions and fragments thereof, lectins, nucleosides, nucleotides, nucleoside and nucleotide analogues, nucleic acids, monosaccharides, disaccharides, trisaccharides, oligosaccharides, polysaccharides, lipopolysaccharides, vitamins, steroids, hormones, cofactors, receptors, receptor ligands, and analogs and derivatives thereof.
  • the supramolecular combinatorial therapeutic of paragraph 37 wherein the targeting ligand binds a protein, receptor, or marker expressed on the surface of a cancer cell.
  • the supramolecular combinatorial therapeutic of paragraph 41 wherein the supramolecular combinatorial therapeutic comprises about 1% to about 99% (w/w) of the chemotherapeutic agent.
  • chemotherapeutic agent is selected from the group consisting of PI3K inhibitors; platinum compounds; inhibitors of topoisomerase I and II; alkylating agents; microtubule inhibitors; angiogenesis inhibitors; and any combinations thereof.
  • chemotherapeutic agent is selected from the group consisting of PI3K inhibitors, platinum compounds, germicitibine; Aldesleukin; Alemtuzumab; alitretinoin; allopurinol; altretamine; amifostine; anastrozole; arsenic trioxide; Asparaginase; BCG Live; bexarotene capsules; bexarotene gel; bleomycin; busulfan intravenous; busulfanoral; calusterone; capecitabine; platinate; carmustine; carmustine with Polifeprosan Implant; celecoxib; chlorambucil; cladribine; cyclophosphamide; cytarabine; cytarabine liposomal; dacarbazine; dactinomycin; actinomycin D; Darbepoetin alfa; daunorubicin
  • PI3K inhibitor is selected from the group consisting of PI103; P1828; LY294002; wortmannin; demethoxyviridin; IC486068; IC87114; GDC-0941; perifosine; CAL101; PX-866; IPI-145; BAY 80-6946; BEZ235; P6503; TGR1202; SF1126; INK1117; BKM120; IL147; XL765; Palomid 529; GSK1059615; ZSTK474; PWT33597; TG100-115; CAL263; GNE-447; CUDC-907; and AEZS-136, and any combinations thereof.
  • the supramolecular combinatorial therapeutic comprises at least one (e.g., one, two, three, four, five, six, seven, eight, nine, ten or more different) taxane conjugate and at least one (e.g., one, two, three, four, five, six, seven, eight, nine, ten or more different) PI3K inhibitor-lipid conjugate.
  • the supramolecular combinatorial therapeutic comprises at least one (e.g., one, two, three, four, five, six, seven, eight, nine, ten or more different) taxane conjugate and at least one (e.g., one, two, three, four, five, six, seven, eight, nine, ten or more different) PI3K inhibitor-lipid conjugate.
  • the supramolecular combinatorial therapeutic of any of paragraphs 1-50 wherein the supramolecular combinatorial therapeutic comprises at least one (e.g., one, two, three, four, five, six, seven, eight, nine, ten or more different) taxane conjugate and at least one (e.g., one, two, three, four, five, six, seven, eight, nine, ten or more different) platinum-lipid conjugate.
  • the supramolecular combinatorial therapeutic comprises at least one (e.g., one, two, three, four, five, six, seven, eight, nine, ten or more different) taxane conjugate and at least one (e.g., one, two, three, four, five, six, seven, eight, nine, ten or more different) platinum-lipid conjugate.
  • the supramolecular combinatorial therapeutic comprises at least one (e.g., one, two, three, four, five, six, seven, eight, nine, ten or more different) taxane conjugate and at least one (e.g., one, two, three, four, five, six, seven, eight, nine, ten or more different) antibody-lipid conjugate.
  • the supramolecular combinatorial therapeutic comprises at least one (e.g., one, two, three, four, five, six, seven, eight, nine, ten or more different) taxane conjugate and at least one (e.g., one, two, three, four, five, six, seven, eight, nine, ten or more different) antibody-lipid conjugate.
  • composition comprises the conjugate, the phospholipid, and the PEG conjugated lipid in ratio from about 10-0.1 : 10-0.1 : 10-0.01.
  • phospatidylcholine is selected from the group consisting of SOPC, Egg PC, HSPC, and any combinations thereof.
  • the supramolecular combinatorial therapeutic of paragraph 59 wherein the nanoparticle about 50 nm to about 200 nm in diameter. 61.
  • a method of treating cancer comprising, administering a supramolecular combinatorial therapeutic of any of paragraphs 1-61 to a subject in need of treatment for cancer.
  • the cancer is selected from the group consisting of breast cancer; ovarian cancer; glioma; gastrointestinal cancer; prostate cancer; carcinoma, lung carcinoma, hepatocellular carcinoma, testicular cancer; cervical cancer; endometrial cancer; bladder cancer; head and neck cancer; lung cancer; gastro-esophageal cancer, and gynecological cancer.
  • the immunomodulator selected from the group consisting of natural killer cells, lymphokine-activated killer cells, cytotoxic T cells and dendritic cells, anti-PD-Ll antibodies, anti-PD-1 antibodies, anti- CD52 antibodies, anti-VEGF-A antibodies, anti-CD30 antibodies, anti-EGFR antibodies, anti-CD33 antibodies, anti-CD20 antibodies, anti-CTLA4 antibodies, anti-HER-2 antibodies, interferons and interleukins
  • a method for predicting the likelihood of a cancer patient exhibiting an enhanced response to self-assembled supramolecular particles than platinates or taxanes comprising assaying an expression level of one or more genes in a sample obtained from the patient; and predicting a likelihood that the patient will exhibit a positive response, wherein: increased expression level of the one or more genes selected from CAVl, CAV2, CAV3, LDLR, SMAD7, SMURF2, NEDD4, or PR CA, relative to a reference or control, is positively correlated with a likelihood of a positive response to treatment comprising self-assembled supramolecular particles.
  • the one or more genes are selected from the group consisting of EEA1, SRSF5, SMAD2, SNX3, PLCD1, OSBP, DNM1, DNM2, DNM3, SGK3, FAPP1, SMAD7, SMURF2, NEDD4, PRKCA, CDH-1, LDLR, CP, CD36, LYN, FLOT-1, FLOT-2, CA4, APOE, CAVl, CAV2, CAV3, LMAN2, LAT and STOM.
  • RNA transcript of the one or more genes is assayed using reverse transcription polymerase chain reaction (RT-PCR).
  • a conjugate comprising taxane conjugated to a lipid, preferably the taxane is cabazitaxel, paclitaxel or docetaxel, more preferably the taxane is cabazitaxel.
  • linker comprises one or more of succinic acid, fumaric acid, propargylic acid, ethylene glycol, diethylene glycol, and natural or unnatural amino acids.
  • the linker comprises at least one of oxalic acid, malonic acid, succinic acid, glutaric acid, succinic acid, ethylene diamine, natural or unnatural amino acid, ethylene glycol, diethylene glycol, acetic acid, propionic acid, butyric acid, valeric acid, acrylic acid, but-2-enoic acid, pent-2-enoic acid, hex-2-enoic acid, 2-propynoic acid, but-2-ynoic acid, pent-2-ynoic acid, hex-2-ynoic acid, ethylene, propylene, 1-butene, 1-pentene, 1- hexene, acetylene, propyne, but-l-yne, pent-l-yne, and any combinations thereof.
  • the linker is selected from the group consisting of -C(0)CH 2 CH 2 C(0)NHCH 2 CH 2 NHC(0)-;
  • lipid is selected from the group consisting of cholesterol; 1,3 -Propanediol Dicaprylate/Dicaprate; 10- undecenoic acid; 1-dotriacontanol; 1-heptacosanol; 1-nonacosanol; 2-ethyl hexanol; Androstanes; Arachidic acid; Arachidonic acid; arachidyl alcohol; Behenic acid; behenyl alcohol; Capmul MCM CIO; Capric acid; capric alcohol; capryl alcohol; Caprylic acid; Caprylic/Capric Acid Ester of Saturated Fatty Alcohol C12-C18; Caprylic/Capric Triglyceride; Caprylic/Capric Triglyceride; Ceramide phosphorylcholine (Sphingomyelin, SPH); Ceramide phosphorylethanolamine (Sphingomyelin, Cer-PE); Ceramide phosphorylglycerol; Ceroplastic acid
  • composition comprising a conjugate of any of paragraphs 1-10.
  • composition of paragraph 11 wherein the composition comprises from about 1% to about 99% (w/w) of the conjugate.
  • composition paragraph 1 1 or 12 wherein the composition further comprises a first lipid in addition to the conjugate.
  • the phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylglycerol, lecithin, ⁇ , ⁇ - dipalmitoyl-a-lecithin, sphingomyelin, phosphatidylserine, phosphatidic acid, N- (2,3-di(9-(Z)-octadecenyloxy))-prop- 1 -yl-N,N,N-trimethylammonium chloride, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylinositol, cephalin, cardiolipin, cerebrosides, dicetylphosphate, dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, dioleoylphosphatidylglycerol, diole
  • composition of paragraph 18, wherein the phospholipid is SOPC.
  • composition of any of paragraphs 13-20 wherein the composition comprises about 1% to about 99% of total lipid.
  • composition of any of paragraphs 13-21 wherein the composition comprises the conjugate and total lipid in about 10: 1 to about 1 : 10 ratio.
  • composition further comprises polyethylene glycol (PEG).
  • composition of paragraph 23, wherein the PEG is conjugated with a component of the composition wherein the PEG is conjugated with a component of the composition.
  • the PEG conjugated lipid is selected from the group consisting of PEG conjugated diacylglycerols and dialkylglycerols, PEG- conjugated phosphatidylethanolamine and phosphatidic acid, PEG conjugated ceramides, PEG conjugated dialkylamines, PEG conjugated l,2-diacyloxypropan-3-amines, and any combinations thereof.
  • composition of paragraph 26, wherein the PEG conjugated lipid is 1,2- distearoyl-sn-glycem-3-phosphoethanolamine-N-[amino(polyethylene glycol)- 2000] (DSPE-PEG2000).
  • composition of any of paragraphs 11-27, further comprising a targeting ligand comprising a targeting ligand
  • the targeting ligand is selected from the group consisting of peptides, polypeptides, proteins, enzymes, peptidomimetics, glycoproteins, antibodies (monoclonal or polyclonal) and portions and fragments thereof, lectins, nucleosides, nucleotides, nucleoside and nucleotide analogues, nucleic acids, monosaccharides, disaccharides, trisaccharides, oligosaccharides, polysaccharides, lipopolysaccharides, vitamins, steroids, hormones, cofactors, receptors, receptor ligands, and analogs and derivatives thereof.
  • composition of paragraph 29, wherein the targeting ligand binds a protein, receptor, or marker expressed on the surface of a cancer cell.
  • composition of paragraph 28 or 29, wherein the targeting ligand is conjugated with a component of the composition wherein the targeting ligand is conjugated with a component of the composition.
  • composition of paragraph 33 wherein the composition comprises about 1% to about 99% (w/w) of the chemotherapeutic agent.
  • chemotherapeutic agent is selected from the group consisting of PI3K inhibitors; platinum compounds; inhibitors of topoisomerase I and II; alkylating agents; microtubule inhibitors; angiogenesis inhibitors; and any combinations thereof.
  • chemotherapeutic agent is selected from the group consisting of PI3K inhibitors, platinum compounds, germicitibine; Aldesleukin; Alemtuzumab; alitretinoin; allopurinol; altretamine; amifostine; anastrozole; arsenic trioxide; Asparaginase; BCG Live; bexarotene capsules; bexarotene gel; bleomycin; busulfan intravenous; busulfanoral; calusterone; capecitabine; platinate; carmustine; carmustine with Polifeprosan Implant; celecoxib; chlorambucil; cladribine; cyclophosphamide; cytarabine; cytarabine liposomal; dacarbazine; dactinomycin; actinomycin D; Darbepoetin alfa; daunorubicin liposomal; daun
  • the PI3K inhibitor is selected from the group consisting of PI103; P1828; LY294002; wortmannin; demethoxyviridin; IC486068; IC87114; GDC-0941; perifosine; CAL101; PX- 866; IPI-145; BAY 80-6946; BEZ235; P6503; TGR1202; SF1126; INK1117; BKM120; IL147; XL765; Palomid 529; GSK1059615; ZSTK474; PWT33597; TG100-115; CAL263; GNE-447; CUDC-907; and AEZS-136, and any combinations thereof.
  • composition of any of paragraphs 33-37, wherein the chemotherapeutic agent is conjugated with a component of the composition wherein the chemotherapeutic agent is conjugated with a component of the composition.
  • composition of paragraph 38 wherein the chemotherapeutic agent is conjugated with a lipid or PEG.
  • composition of paragraph 39 wherein the chemotherapeutic agent is conjugated with cholesterol.
  • composition of paragraph 40 wherein the lipid conjugated chemotherapeutic
  • composition of paragraph 42, wherein the immunomodulator is conjugated with lipid is conjugated with lipid.
  • composition of paragraph 43, wherein the immunomodulator is conjugated with cholesterol.
  • composition of any of paragraphs 1 1-44 wherein the composition further comprises a neutral lipid, a cationic lipid, an anionic lipid, an amphiphilic lipid, a sterol, a programmable fusion lipid, or any combinations thereof.
  • composition of paragraph 46 wherein the composition comprises the conjugate, the phospholipid, and the PEG conjugated lipid in ratio from about 10- 0.1 : 10-0.1 : 10-0.1.
  • composition of any of paragraphs 46-48, wherein the phospholipid is phosphatidylcholine .
  • composition of paragraph 49 wherein the phospatidylcholine is selected from the group consisting of SOPC, POPC, Egg PC, HSPC, and any combinations thereof.
  • composition of paragraph 52 wherein the nanoparticle is about 5 nm to about 500 nm in diameter.
  • composition of paragraph 53 wherein the nanoparticle about 50 nm to about 200 nm in diameter.
  • a method of treating cancer comprising, administering a composition of any of paragraphs 11-55 to a subject in need of treatment for cancer.
  • the cancer is selected from the group consisting of breast cancer; ovarian cancer; glioma; gastrointestinal cancer; prostate cancer; carcinoma, lung carcinoma, hepatocellular carcinoma, testicular cancer; cervical cancer; endometrial cancer; bladder cancer; head and neck cancer; lung cancer; gastro-esophageal cancer, and gynecological cancer.
  • the additional therapy is selected from the group consisting of surgery, chemotherapy, radiation therapy, thermotherapy, immunotherapy, hormone therapy, laser therapy, anti-angiogenic therapy, and any combinations thereof.
  • the immunomodulator selected from the group consisting of natural killer cells, lymphokine-activated killer cells, cytotoxic T cells and dendritic cells, anti-PD-Ll antibodies, anti-PD-1 antibodies, anti- CD52 antibodies, anti-VEGF-A antibodies, anti-CD30 antibodies, anti-EGFR antibodies, anti-CD33 antibodies, anti-CD20 antibodies, anti-CTLA4 antibodies, anti-HER-2 antibodies, interferons and interleukins
  • taxane-lipid conjugate 1 the drug cabazitaxel is conjugated to cholesterol through a linker comprise of succinic acid and ethylene diamine. Cholesterol and one end of ethylene diamine are covalently bonded through a carbamate linkage where as another end is coupled to succinic acid through amide linkage. Succinic acid and C-2' hydroxyl group of cabazitaxel is coupled through an ester bond.
  • taxane-lipid conjugate 2 the drug cabazitaxel is conjugated to cholesterol through a linker comprise of succinic acid and natural or unnatural amino acid.
  • the acid group of amino acid is connected to cholesterol through an ester bond where as the amine group is connected to succinic acid through amide bond.
  • Cabazitaxel C-2' hydroxyl group is connected to succinic acid via an ester bond.
  • taxane-lipid conjugate 3 the drug cabazitaxel is conjugated to alpha tocopherol through a linker comprise of succinic acid and natural or unnatural amino acid.
  • the acid group of amino acid is connected to alpha tocopherol through an ester bond where as the amine group is connected to succinic acid through amide bond.
  • Cabazitaxel C-2' hydroxyl group is connected to succinic acid via an ester bond.
  • taxane-lipid conjugate 4 the drug cabazitaxel is conjugated to cholesterol through a linker comprise of ethylene glycol and acetic acid.
  • a linker comprise of ethylene glycol and acetic acid.
  • One end of ethylene glycol is connected to cholesterol via an ether bond and another hydroxyl group is connected to an acid group via a methylene spacer.
  • the acid group is connected to C-2' hydroxyl group of cabazitaxel is coupled through an ester bond.
  • taxane-lipid conjugate 5 the drug cabazitaxel is conjugated to cholesterol through a linker comprise of ethylene glycol and acrylic acid.
  • a linker comprise of ethylene glycol and acrylic acid.
  • One end of ethylene glycol is connected to cholesterol via an ether bond and another hydroxyl group is connected to an acid group via two methylene unit.
  • the acid group is connected to C-2' hydroxyl group of cabazitaxel is coupled through an ester bond.
  • taxane-lipid conjugate 6 the drug cabazitaxel is conjugated to cholesterol through a linker comprise of diethylene glycol and acetic acid.
  • a linker comprise of diethylene glycol and acetic acid.
  • One end of diethylene glycol is connected to cholesterol via an ether bond and another hydroxyl group is connected to an acid group via a methylene spacer.
  • the acid group is connected to C-2' hydroxyl group of cabazitaxel is coupled through an ester bond.
  • taxane-lipid conjugate 7 the drug cabazitaxel is conjugated to cholesterol through a linker comprise of hydrophobic amino acid and glycolic acid. Hydroxyl group of glycolic acid is connected with cholesterol via an ether bond and the acid end is connected via an amide bond with amine group of amino acid. The C-2' hydroxyl group of cabazitaxel is connected to the acid group of amino acid through an ester bond.
  • R H, CH3, CH(CH3)2, CH2CH(CH3)2, C(CH3)CH2CH3, CH2Ph
  • R H, CH3, CH(CH3)2, CH2CH(CH3)2, C(CH3)CH2CH3, CH2Ph
  • Step a To an ice cooled solution of ethylenediamine(22.2 mL) in dichliromethane (40 mL) was added a solution of cholesteryl chloroformate 1.01 (5g, 11.13 mmol) in dichliromethane (50 mL) drop wise over a period of 45 min and stirred at the same temperature for 1 h.
  • Step b To an ice cooled solution of intermediate 1.02 (1.0 g, 2.12 mmol) in dry dichliromethane (20 mL) was addedpyridine (3.41 mL, 42.3 mmol) and stirred for 20 minutes under nitrogen atmosphere. Succinic anhydride (1.06 g, 10.6mmol) was added to the reaction mixture and stirred for another 12 h at room temperature. After completion the reaction mixture was diluted with chloroform (50 mL), washed with 0.1N HC1 (3 x 100 mL) and brine (1 x 100 mL) successively.
  • Step c To a 10 mL single neck round bottom flask acid intermediate 1.03 (68 mg, 0.1196 mmol) was taken in anhydrous CH 2 C1 2 (3 mL) under nitrogen atmosphere at 0 °C. To this cooled solution DIPC (18 ⁇ , 0.1196 mmol) followed by DMAP (14.6 mg, 0.1196 mmol) and stirred at same temperature for 1 h. To this activated acid solution cabazitaxel (50 mg, 0.1196 mmol) was added and stirred for another 3h at room temperature and TLC was checked.
  • Step a To a 250 mL single neck round bottom flask BocHNCH2COOH (2g,
  • Step b To a 25 mL single neck round bottom flask intermediate 2.02 (1 g crude, 1.838mmol) was taken in anhydrous CH2CI2 (5 mL) under nitrogen atmosphere at 0 °C. To this reaction mixture TFA (2 mL) was added slowly over a period of 5 minute and stirred at room temperature for 3 h and TLC was checked. After completion the solvent was removed under reduced pressure and the crudeamine compound 2.03was utilized for the next reaction without further purification.
  • Step c The crude amine compound 2.03obtained from the previous reaction was diluted with dichloromethane (20 mL) and cooled to 0 °C. To this ice cooled solution DIPEA (3 mL, 17.12 mmol) was added slowly followed by succinic anhydride (856 mg, 8.56 mmol) and stirred at room temperature for 12h. After completion the reaction mixture was quenched with water (10 mL), washed with 1% HC1 solution, extracted with CH2CI2 (3 X 10 mL), dried over anhydrous Na 2 S0 4 and purified by silica gel to obtain pure acid intermediate2.04in 88% yield.
  • DIPEA 3 mL, 17.12 mmol
  • succinic anhydride 856 mg, 8.56 mmol
  • Step d To a 10 mL single neck round bottom flask acid intermediate 2.04 (137 mg, 0.2392 mmol) was taken in anhydrous CH2CI2 (10 mL) under nitrogen atmosphere at 0 °C. To this cooled solution DMAP (29 mg, 0.2392 mmol) followed by DIPC (37 ⁇ , 0.2392 mmol) was added and stirred at same temperature for 1 h. To this activated acid solution cabazitaxel (100 mg, 0.1196 mmol) was added and stirred for another 4 h at room temperature and TLC was checked.
  • reaction mixture was quenched with water, extracted with CHC13, dried over anhydrous sodium sulphate, concentrated and purified by silica gel chromatography to obtain conjugate 2 which was further precipitated in acetone -Hexane to obtain pure product in 76 % yield.
  • Step b To the solution of intermediate6.02 (crude 6 g, 0.011 mol) in dioxane (30 mL) was added diethylene glycol (20 mL) and allowed to reflux for 6 h. After completion the reaction mixture was quenched with water (20 mL) and extracted with ethyl acetate. The organic layer was washed with water (3 X 50 mL) and brine (20 mL) successively and dried over anhydrous Na 2 S0 4 . The organic layer was concentrated under reduced pressure and purified by silica gel chromatography utilizing methanol-chloroform as mobile phase to obtain intermediate 6.03 as viscous liquid (52% over two steps).
  • Step c To a 100 mL single neck round bottom flask NaH (594 mg, 14.84 mmol) was taken in THF (10 mL) under nitrogen atmosphere. The reaction was cooled to 0 °C under ice bath and a solution of intermediate 6.03 (2.35 g, 4.95 mmol) in THF (15 mL) was added slowly. The resulting solution was stirred for 1 h and ethyl bromoacetate was added slowly and stirred for 6 h at room temperature and TLC was checked. After completion the reaction mixture was cooled to 0 °C, quenched with water and extracted with ethyl acetate (3 X 15 mL).
  • Step d To a 100 mL single neck round bottom flask ester intermediate 6.04(1.272 g, 2.27 mmol) was taken in 20 mL of THF/H 2 0 (3 : 1) and cooled to 0 °C under ice bath. To this ice cooled solution LiOH (136 mg, 5.67 mmol) was added and was stirred at rt for 4 h and TLC was checked. After completion the reaction mixture was acidified by Na2HS04, extracted with ethyl acetate. The organic layer was dried over anhydrous Na 2 S0 4 and concentrated under vacuum and columnperformed to obtain pure acid intermediate 6.05 in 50% yield.
  • Step e To a 25 mL single neck round bottom flask acid 6.05 (25.46 mg, 0.047 mmol) was taken in anhydrous CH 2 CI 2 (5 mL) under nitrogen atmosphere at 0 °C. To this cooled solution DIPC (7 ⁇ ,, 0.0478 mmol) followed by DMAP (5.84 mg, 0.0478 mmol) and stirred at same temperature for 2h.
  • cabazitaxel (20 mg, 0.0239 mmol) was added and stirred for another 3h at room temperature and TLC was checked. After completion the reaction mixture was quenched with water, extracted with CHC13, dried over anhydrous sodium sulphate, concentrated and purified by silica gel chromatography to obtain conjugate 6 in 92 % yield.
  • Step a To an ice cooled solution of cholesterol 2.01(10 g, 0.026 mol) in dichloromethane (45 mL) was added pyridine (15 mL) and stirred for 15 minutes. To this solution p-toluene sulphonyl chloride (9.8 g, 0.052 mol) was added and stirred for 6 h at 0 °C and TLC was checked. After completion the reaction mixture was diluted with CHC1 3 (20 mL) and washed with IN HC1 (3 X 50 mL) and brine (20 mL) successively. The organic layer was dried over anhydrous Na 2 S0 4 and concentrated under vacuum to afford intermediate 7.01 and directly move for the next reaction without further purification.
  • Step b To the solution of crude intermediate 7.01 (10 g, 0.018 mol) in dioxane
  • Step c To solution of intermediate 7.02(1 g, 2.322 mmol) in acetone -THF mixture (30+5 mL) was added chromic acid (4 mL) drop wise under vigorous stirring at room temperature for 1 h and TLC was checked. After completion the reaction mixture was filtered through a thin pad of silica gel, washed with acetone, dried on Na2S04 and concentrated under reduced pressure and diluted with water (10 mL) and extracted with EtOAc (2 X 10 mL). The organic layer was dried over anhydrous Na 2 S0 4 , concentrated and column purified to afford intermediate 7.03 in 83% yield.
  • Step d To a 50 mL single neck round bottom flask acid intermediate 7.03 (500 mg, 1.124 mmol) was taken in anhydrous CH 2 C1 2 (15 mL) under nitrogen atmosphere at 0 °C. To this cooled solution EDCI (430 mg, 2.248 mmol) followed by HOBT (303 mg, 2.248 mmol) and stirred at same temperature for lh. To this activated acid solution DIPEA (0.8 mL, 4.497) followed by glycine methyl ester (282 mg, 2.248 mmol) was added and stirred for another 5 h at room temperature and TLC was checked.
  • DIPEA 0.8 mL, 4.497
  • Step e To a 50 mL single neck round bottom flask ester compound 7.04 (560 mg, 1.086 mmol) was taken in THF / water (12 mL, 3: 1) and cooled to 0 °C. To this ice cooled solution LiOH (136 mg, 3.258 mmol) was added and stirred at room temperature for 3 h and TLC was checked. After completion the reaction mixture was acidified with saturated NaHS0 4 up to P H 3 and extracted with Ethyl acetate (3 X 10 mL). The combined organic layer was dried over anhydrous Na 2 S0 4 and column purified to obtain pure acid 7.05 in good yield (390 mg, 65 %).
  • 1H NMR of 7.05 500 MHz, CDC1 3 ) ⁇ : 7.20 (s, 1H), 5.34 (s, 1H),
  • Step f To a 10 mL single neck round bottom flaskacid intermediate 7.05 (23.9 mg, 0.0478 mmol) was taken in anhydrous CH 2 C1 2 (2 mL) under nitrogen atmosphere at 0 °C. To this cooled solution DIPC (7 ⁇ , 0.0478 mmol) followed by DMAP (5.84 mg, 0.0478 mmol) and stirred at same temperature for 1 h. To this activated acid solution cabazitaxel (20 mg, 0.0239 mmol) was added and stirred for another 3h at room temperature and TLC was checked.
  • Step a To a 250 mL single neck round bottom flaskcholesterol (10 g, 25.862 mmol) was taken in THF (60 mL) under nitrogen atmosphere at 0 oC. Sodium hydride (2.068 g, 51.724 mmol) was added by pinch to the reaction mixture over a period of 10 minutes. The resulting solution was stirred for 20 minutes and methyl acrylate (11.6 mL, 129.31 mmol) in THF (20 mL) was added slowly and stirred for 2 h at 0 °C and TLC was checked.
  • reaction mixture was cooled to 0 °C and quenched with water and extracted with ethyl acetate, dried over anhydrous Na2S04, concentrated and column purified by silica gel chromatography to obtain product 8.01 in 20% yield.
  • Step b To a 50 mL single neck round bottom flask ester intermediate 8.01 (1 g, 4.231 mmol) was taken in THF / water (12 mL, 3: 1) and cooled to 0 °C. To this ice cooled solution LiOH (355 mg 8.462 mmol) was added and stirred at room temperature for 2 h and TLC was checked. After completion the reaction mixture was acidified with saturated NaHS0 4 up to P H 3 and extracted with Ethyl acetate (3 X 10 mL). The combined organic layer was dried over anhydrous Na 2 S0 4 and column purified to obtain pure compound 8.02 in good yield (80 %).
  • Step c To a 100 mL single neck round bottom flask acid intermediate 8.02 (1 g, 2.179 mmol) was taken in anhydrous dichloromethane (15 mL) under nitrogen atmosphere at 0 °C. To this cooled solution DIPC (0.67 ml, 4.359 mmol) followed by DMAP (133 mg, 1.089 mmol) were added and stirred at same temperature for 0.5 h. To this activated acid solution DIPEA (1.5 mL. 8.716 mmol) followed by glycine methyl ester (547 mg, 4.359 mmol) was added and stirred for another 6 h at room temperature and TLC was checked.
  • DIPC 0.67 ml, 4.359 mmol
  • DMAP 133 mg, 1.089 mmol
  • Step d To a 50 mL single neck round bottom flask ester intermediate 8.03 (1 g, 1.887 mmol) was taken in THF / water (21 mL, 3: 1) and cooled to 0 °C. To this ice cooled solution LiOH (230 mg, 5.662 mmol) was added and stirred at room temperature for 3 h and TLC was checked. After completion the reaction mixture was acidified with saturated NaHS0 4 up to P H 3 and extracted with Ethyl acetate (3 X 10 mL). The combined organic layer was dried over anhydrous Na 2 S0 4 and column purified to obtain pure compound 8.04 as white solid (800 mg, 82%).
  • Step e To a 10 mL single neck round bottom flask acidintermediate 8.04 (184 mg, 0.3588 mmol) was taken in anhydrous CH 2 C1 2 (10 mL) under nitrogen atmosphere at 0 °C. To this cooled solution DIPC (111 ⁇ , 0.7176 mmol) followed by DMAP (58.4 mg, 0.4758 mmol) and stirred at same temperature for 0.5 h. To this activated acid solution cabazitaxel (200 mg, 0.2392 mmol) was added and stirred for another 5 h at room temperature and TLC was checked.
  • Step a To an ice cooled solution of cholesterol (10 g, 0.026 mol) in CH 2 C1 2 (45 mL) was added pyridine (15 mL) and stirred for 15 minutes. To this solution p-toluene sulphonyl chloride(9.8 g, 0.052 mol) was added and stirred for 6 h at 0 °C and TLC was checked. After completion the reaction mixture was diluted with CHCI 3 (20 mL) and washed with IN HC1 (3 X 50 mL) and brine (20 mL) successively. The organic layer was dried over anhydrous Na 2 S0 4 and concentrated under vacuum to afford intermediate 14.01 and directly move for the next reaction without further purification.
  • Step b To the solution of crude intermediate 14.01 (0.026 mol) in dioxane (45 mL) was added ethylene glycol (15 mL) and refluxed for 4 h. The TLC was checked. After completion the reaction mixture was extracted with ethyl acetate and washed with water (3 X 50 mL) and brine (20 mL) successively. The organic layer was dried over anhydrous Na 2 S0 4 and concentrated under vacuum and column purified to afford intermediatel4.02as white solid (55% yield over two steps).
  • Alcohol intermediate 14.02 (500 mg, 1.160 mmol) in dry THF (10 ml) was added to the reaction mixture and allowed to stir for another 1.5 hour at room temperature and TLC was checked. After Completion the reaction mixture was quenched with excess diethyl ether and filtered through a thin pad of celite. Combined organic layer was concentrated under vacuum and purified by column chromatography to obtain aldehyde intermediate 14.03 in 78% yield. As the aldehyde intermediate 14.03 is less stable over storage, was utilized for next reaction without further characterization.
  • Step d To 50 ml single neck round bottom flask Wittig salt (1 g, 2.733 mmol) was taken in Toluene (7 ml) and heated at 80 °C. To this solution aldehyde intermediate 14.03 (390 mg, 0.911 mmol) in toluene (5 mL) was added and allowed to stir for overnight at same temperature and TLC was checked. After completion, the reaction was quenched with Hexane to precipitate out unreacted Wittig reagent and Filtered off through a thin pad of celite and washed with hexane (15 mL). Combined Organic Layer was concentrated under vacuum and purified by column chromatography to obtain ester intermediate 14.04 in good yield (85%).
  • Step e To 50 ml single neck round bottom flask ester intermediatel4.04 (380 mg, 0.722 mmol) was taken in dry DCM (3 ml) and cooled to °C. To this solution TFA (1 mL) was added, stirred for 2.5 hour at room temperature and TLC was checked. After Completion the reaction mixture was concentrated under vacuum to remove TFA (using NaOH trap on rota vapour). Solid residue was purified by silica gel chromatography to obtain acid intermediate 14.05 in good yield.
  • Step f To a 25 mL single neck round bottom flask acid 14.05(112 mg, 0.2393 mmol) was taken in anhydrous CH 2 CI 2 (10 mL) under nitrogen atmosphere at 0 °C. To this cooled solution DMAP (14 mg, 0.1196 mmol) followed by DIPC (37 uL, 0.2393 mmol) was added and stirred at same temperature for 1 hour. To this activated acid solution cabazitaxel (100 mg, 0.1196 mmol) was added and stirred for another 4 hour at room temperature and TLC was checked.
  • Step a To a 250 mL single neck round bottom flask BocHNCH2COOH (2g, 11.417 mmol), cholesterol 2.01 (4.414g, 11.417 mmol) and DMAP (697 mg, 5.708 mmol) were taken in anhydrous dichloromethane (75 mL) under nitrogen atmosphere and stirred at 0 °C for 20 minutes. To this cooled solution DCC (2.591g, 12.558 mmol) was added and stirred for another 24 h at room temperature and TLC was checked. After completion the reaction mixture was quenched with water, extracted with CHC13, dried over anhydrous sodium sulphate, concentrated and purified by silica gel chromatography to obtain intermediate 2.02 in 60% yield.
  • Step b To a 25 mL single neck round bottom flask intermediate 2.02 (1 g crude, 1.838mmol) was taken in anhydrous CH 2 CI 2 (5 mL) under nitrogen atmosphere at 0 °C. To this reaction mixture TFA (2 mL) was added slowly over a period of 5 minute and stirred at room temperature for 3 h and TLC was checked. After completion the solvent was removed under reduced pressure and the crudeamine compound 2.03 was utilized for the next reaction without further purification.
  • Step c The crude amine intermediate 2.03 obtained from the previous reaction was diluted with dichloromethane (20 mL) and cooled to 0 °C. To this ice cooled solution DIPEA (3 mL, 17.12 mmol) was added slowly followed by succinic anhydride (856 mg, 8.56 mmol) and stirred at room temperature for 12h. After completion the reaction mixture was quenched with water (10 mL), washed with 1% HC1 solution, extracted with CH 2 CI 2 (3 X 10 mL), dried over anhydrous Na 2 S0 4 and purified by silica gel to obtain pure acid intermediate 2.04 in 88% yield.
  • Step d To a 10 mL single neck round bottom flask acid 2.04 (286 mg, 0.5269 mmol) was taken in anhydrous CH 2 CI 2 (10 mL) under nitrogen atmosphere at 0 °C. To this cooled solution DMAP (85 mg, 0.7026 mmol) followed by DIPC (109 ⁇ , 0.7026 mmol) was added and stirred at same temperature for lh. To this activated acid solution paclitaxel (300 mg, 0.3513 mmol) was added and stirred for another 4 h at room temperature and TLC was checked.
  • DMAP 85 mg, 0.7026 mmol
  • DIPC 109 ⁇ , 0.7026 mmol
  • Step a To a 250 mL single neck round bottom flask BocHNCH2COOH (2g, 11.417 mmol), cholesterol 2.01 (4.414g, 11.417 mmol) and DMAP (697 mg, 5.708 mmol) were taken in anhydrous dichloromethane (75 mL) under nitrogen atmosphere and stirred at 0 °C for 20 minutes.
  • Step b To a 25 mL single neck round bottom flask intermediate 2.02 (1 g crude, 1.838mmol) was taken in anhydrous CH 2 CI 2 (5 mL) under nitrogen atmosphere at 0 °C. To this reaction mixture TFA (2 mL) was added slowly over a period of 5 minute and stirred at room temperature for 3 h and TLC was checked. After completion the solvent was removed under reduced pressure and the crudeamine compound 2.03 was utilized for the next reaction without further purification.
  • Step c The crude amine compound 2.03obtained from the previous reaction was diluted with dichloromethane (20 mL) and cooled to 0 °C. To this ice cooled solution DIPEA (3 mL, 17.12 mmol) was added slowly followed by succinic anhydride (856 mg, 8.56 mmol) and stirred at room temperature for 12h. After completion the reaction mixture was quenched with water (10 mL), washed with 1% HC1 solution, extracted with CH 2 CI 2 (3 X 10 mL), dried over anhydrous Na 2 S0 4 and purified by silica gel to obtain pure acid intermediate 2.04 in 88% yield.
  • DIPEA 3 mL, 17.12 mmol
  • succinic anhydride 856 mg, 8.56 mmol
  • Step d To a 25 mL single neck round bottom flask acid intermediate 2.04 (137 mg, 0.2392 mmol) is taken in anhydrous CH 2 CI 2 (10 mL) under nitrogen atmosphere at 0 °C. To this cooled solution DMAP (29 mg, 0.2392 mmol) followed by DIPC (37 ⁇ , 0.2392 mmol) is added and stirred at same temperature for 1 h. GDC 0623 (100 mg, 0.1196 mmol) is added to the activated acid solution and stirred for another 4 h at room temperature and TLC is checked. After completion the reaction mixture is quenched with water, extracted with CHC13, dried over anhydrous sodium sulphate, concentrated and purified by silica gel chromatography to obtain novel MEK inhibitor 33.
  • Step a To a flame dried two necked round bottom flask phosphorus oxychloride (1.3 mL, 13.930 mmol) is taken in anhydrous THF (50 mL) at 0 oC. Cholesteryl alcohol 7.02 (5 g, 11.609 mmol) and triethylamine (1.78 mL, 24.18 mmol) in THF (100 mL) is dropped slowly to the reaction mixture while vigorous stirring. Triethylammonium chloride is slowly precipitated from thereaction mixture. The reaction is continued under the protection of argon for further 5 hours andthe intermediate 28.01 is used directly for the next step without any treatment.
  • Step b Triethylamine (3.88 mL, 34.825 mmol) and ethanolamine (0.834 mL. 13.93 mmol) in THF (50 mL) is added to the above reaction mixture slowly while vigorous stirring at 0 oC. Triethylammonium chloride is slowly precipitated from the solution and the reaction is continued under the protection of nitrogen for further 5 hours. After completiontriethylammonium chloride is removed by suction filtration and the solvent is evaporated to obtainintermediate 28.02 as white gum which is used directly for the next step without further purification.
  • Step c The intermediate 28.02 is dissolved in THF (50 mL) at room temperature.
  • Acetic acid- water solution (15g acetic acidin 40 mL water) is added in one portion and heated at 70 °C for 5 h under theprotection of nitrogen atmosphere. Then the mixture is condensed by azeotropic distillation withethanol and the ring-openproduct 28.03 is precipitated out by dripping the solutionslowly into acetone under vigorous stirring. The precipitation is collected by suctionfiltration, dried under high vacuum and used directly for the next step.
  • Step d The white gummy intermediate 28.03 is taken in dichloromethane (50 mL) and pyridine (10 mL) is added to the reaction mixture at room temperature. To this solution succinic hydride (5.5 g, 55.6 mmol) is added in one portion andstirred at room temperature for 12 h under the protection of nitrogen. After completion the reaction is diluted with CHC13 (20 mL) and washed with HC1 solution (1 N, 250 mL) and concentrated under reduced pressure. The slurry is precipitated in acetone to obtain intermediate 28.04 (1.5 g, 19.7% over four steps starting from 28.01).
  • Step e To a 25 mL single neck round bottom flask acid intermediate 28.04 (152 mg, 0.2392 mmol) is taken in anhydrous dichloromethane (5 mL) under nitrogen atmosphere at 0 °C. To this cooled solution DIPC (37 ⁇ , 0.2392 mmol) followed by DMAP (29 mg, 0.2392 mmol) is added and stirred at same temperature for lh. To this activated acid solution cabazitaxel (100 mg, 0.1196 mmol) is added and stirred for another 5 h at room temperature and TLC is checked. After completion the reaction mixture is diluted with chloroform (10 mL) and washed with 0.1N HC1 (10 mL) solution. The organic layer is evaporated under reduced pressure and the residue is purified on silica to obtain new taxane 28.
  • Step a To a 100 mL single neck round bottom flask octadecyl bromide 29.01 (31.122 g, 93.632 mmol) and mono NBoc ethylenediamine 29.02 (5 g, 31.211 mmol) is taken in acetonitrile (100 mL) and refluxed in presence of anhydrous K2C03 (17.253 g, 124.844 mmol) for 24 h under nitrogen atmosphere. After completion the reaction mixture is concentrated under reduced pressure and diluted with ethyl acetate (100 mL). The organic layer is washed with water (100 mL), dried over anhydrous sodium sulphate and concentrated. The residue is purified on silica gel to afford intermediate 29.03.
  • Step b To a 250 mL single neck round bottom flask intermediate 29.03 (5 g, 82.685 mmol) and methyl iodide (7.72 mL, 124.028 mmol) is taken in acetone (100 mL) and refluxed for 24 h under nitrogen atmosphere. After completion the reaction mixture is concentrated under reduced pressure and the solid residue is washed with hexane (10 mL X 2) to obtain intermediate 29.04 as white powder.
  • Scheme 10 Reagents and conditions: (a) K2C03, CH3CN, reflux, 24h (b) CH3I, (CH3)2CO, reflux, 24h
  • Step c To a 100 mL single neck round bottom flask intermediate 29.04 (2 g, 2.477 mmol) is taken in anhydrous dichloromethane (12 mL) at 0 oC under nitrogen atmosphere. To this solution TFA (3 mL) is added and stirred at same temperature for 4 h and TLC is checked. After completion the reaction mixture is concentrated under reduced pressure and the solid residue of 29.05 is utilized for next reaction without further purification.
  • Step d The crude amine intermediate29.05obtained from the previous reaction is diluted with dichloromethane (20 mL) and cooled to 0 °C. To this ice cooled solution DIPEA (2.21 mL, 12.385 mmol) is added slowly followed by succinic anhydride (495 mg, 4.954 mmol) and stirred at room temperature for 12h. After completion the reaction mixture is quenched with water (10 mL), washed with 1% HCl solution, extracted with CH 2 CI 2 (3 X 10 mL), dried over anhydrous Na 2 S0 4 and purified by silica gel to obtain pure acid intermediate29.06.
  • Step e To a 25 mL single neck round bottom flask acid intermediate 29.06 (193 mg, 0.2392 mmol) is taken in anhydrous dichloromethane (5 mL) under nitrogen atmosphere at 0 °C. To this cooled solution DIPC (37 ⁇ , 0.2392 mmol) followed by DMAP (29 mg, 0.2392 mmol) is added and stirred at same temperature for lh. To this activated acid solution cabazitaxel (100 mg, 0.1196 mmol) is added and stirred for another 5 h at room temperature and TLC is checked. After completion the reaction mixture is diluted with chloroform (10 mL) and washed with 0.1N HC1 (10 mL) solution. The organic layer is evaporated under reduced pressure and the residue is purified on silica to obtain new taxane 29.
  • Gemcitabine an anticancer agent which acts against wide range of tumors, is known to be administered in very high dose (1000 mg/m 2 ) due to its very short plasma half life. Gemcitabine itself is water soluble. Functionalization of this molecule by hydrophobic lipids through linkers would be given amphiphilic nature to this molecule. Formulation of these compounds to produce supramolecular assembly could provide a protection against its short plasma half life and also could contribute to an enhanced permeation and retention (EPR) effect.
  • EPR enhanced permeation and retention
  • Step a To a 100 niL single neck R.B cholesterol 2.01 (2 g, 5.172 mmol) was taken in anhydrous THF (20 mL) under nitrogen atmosphere at 0 oC. NaH (2.859 mg, 20.690 mmol) was added to the reaction mixture by pinch over a period of 5 minutes. The resulting solution was stirred for 20 minutes and ethyl bromo acetate (1.191 mL, 10.354 mmol) was added slowly and stirred for another 4 h at room temperature and TLC was checked. After completion the reaction mixture was quenched with water and the compound was extracted with ethyl acetate (2 X 20 mL). The combined organic layer was dried over anhydrous sodium sulphate, concentrated and purified by silica gel chromatography to obtain intermediate 7.01 in 10 % yield.
  • Step b To a 100 mL single neck round bottom flask ester intermediate 7.01 (200 mg, 0.432 mmol) was taken in 8 mL of THF/H 2 0 (3: 1) and cooled to 0 °C under ice bath. To this ice cooled solution LiOH (35 mg, 0.846 mmol) was added and was stirred at rt for 4 h and TLC was checked. After completion the reaction mixture was acidified by Na2HS04, extracted with ethyl acetate. The organic layer was dried over anhydrous Na 2 S0 4 and concentrated under vacuum and column performed to obtain pure acid intermediate 7.02 in 60% yield.
  • Reagents and conditions (a) NaH, THF,Ethyl bromoacetate, 0 oC to r.t, 4 h (b) LiOHJHF / H20 (3:1 ), 0 oC to r.t, 3 h (c) EDCI, HOBT, DIPEA, CH2CI2, 0 oC to r.t, 12 h (d) LiOHJHF / H20 (3:1 ), 0 oC to r.t, 3 h (e) DIPC, DMAP, CH2CI2, 3 h
  • Step c To a 25 mL single neck round bottom flask acid 7.02 (100 mg, 0.2248 mmol) was taken in anhydrous CH 2 CI 2 (5 mL) under nitrogen atmosphere at 0 °C. To this cooled solution EDCI (86.2 mg, 0.4497 mmol) followed by HOBT (60 mg, 0.4497 mmol) and stirred at same temperature for 1 h. To this activated acid solution DIPEA (0.15 mL,
  • Step d To a 50 mL single neck round bottom flask ester intermediate 7.03 (96 mg, 0.186 mmol) was taken in THF / water (4 mL, 3: 1) and cooled to 0 °C. To this ice cooled solution LiOH (23.4 mg, 0.558 mmol) was added and stirred at room temperature for 2 h and TLC was checked. After completion the reaction mixture was acidified with saturated NaHS0 4 up to P H 3 and extracted with Ethyl acetate (3 X 10 mL). The combined organic layer was dried over anhydrous Na 2 S0 4 and column purified to obtain pure compound 7.04 80% yield.
  • Step e To a 10 mL single neck round bottom flask acid intermediate 7.04 (23.9 mg, 0.0478 mmol) was taken in anhydrous CH 2 C1 2 (2 mL) under nitrogen atmosphere at 0 °C. To this cooled solution DIPC (7 microliter, 0.0478 mmol) followed by DMAP (5.84 mg, 0.0478 mmol) and stirred at same temperature for 1 h. To this activated acid solution cabazitaxel (20 mg, 0.0239 mmol) was added and stirred for another 3 h at room temperature and TLC was checked.
  • Step a To an ice cooled solution of cholesterol 2.01 (5 g, 0.013 mol) in CH 2 C1 2 (20 mL) is added pyridine (5 mL) and stirred for 15 minutes. To this solution paratoluene sulphonyl chloride (4.9 g, 0.026 mol) is added and stirred for another 4 h at 0 °C and TLC is checked. After completion the reaction mixture is diluted with CHCI 3 (20 mL), washed with IN HC1 (3 X 50 mL) and brine (20 mL) successively. The organic layer was dried anhydrous Na 2 S0 4 and concentrated under vacuum to afford intermediate 6.02 which is utilized for the next reaction without further purification.
  • Step b To a solution of intermediate 6.02 (crude 6 g, 0.011 mol) in dioxane (30 mL) is added ethylene glycol (20 mL) and allowed to reflux for 12 h. After completion dioxane is removed under vacuum and quenched with water (20 mL). The compound is extracted in ethyl acetate and washed with water (3 X 50 mL) and brine (20 mL) successively. The combined organic layer is dried over anhydrous Na 2 S0 4 and concentrated under reduced pressure. The residue is purified on silica gel utilizing methanol-chloroform as mobile phase to obtain intermediate 14.01.
  • Step c To 50 mL single neck round bottom flask IBX (1.301 g, 4.647 mmol) is taken in anhydrous dimethy sulphoxide (3 mL) and stirred for 30 minutes to get a clear solution. To this solution alcohol intermediate 14.01 (1 g, 2.323 mmol) is added in anhydrous THF (10 mL) over a period of 10 minutes. The resulting solution is stirred for another 3 h at room temperature and TLC is checked. After completion the reaction mixture is diluted with diethyl ether and the white solid is filtered through a thin pad of celite and washed with water (2 X 15 mL). The organic layer is dried over anhydrous Na2S04, concentrated under vacuum and purified by silica gel chromatography to obtain aldehyde intermediate 14.02.
  • Step e To a 25 mL single neck round bottom flask intermediate 14.03 (500 mg, 0.949 mmol) is taken in anhydrous CH 2 CI 2 (5 mL) under nitrogen atmosphere at 0 °C. To this reaction mixture TFA (2 mL) is added slowly over a period of 5 minute and stirred at room temperature for 3 h and TLC is checked. After completion the solvent is removed under reduced pressure and washed with hexane (5 mL). The residue is purified by silica gel chromatography to obtain acid intermediate 14.04.
  • Step f To a 25 mL single neck round bottom flask flask acid intermediate 14.04 (112 mg, 0.2392 mmol) is taken in anhydrous CH 2 CI 2 (7 mL) under nitrogen atmosphere at 0 °C. To this cooled solution DIPC (37 ⁇ , 0. 2392 mmol) followed by DMAP (29 mg, 0. 2392 mmol) is added and stirred at same temperature for 2 h. To this activated acid solution cabazitaxel (100 mg, 0.1196 mmol) is added and stirred for another 3 h at room temperature and TLC is checked. After completion the reaction mixture is quenched with water, extracted with CHC13, dried over anhydrous sodium sulphate, concentrated and purified by silica gel chromatography to obtain conjugate 14.
  • Supramolecular nanostructures were formulated using different mole ratios of phospholipids (such as POPC, SOPC, Egg PC or HSPC), taxane supramolecules and PEGylated-phospholipids (such as DSPE-PEG).
  • POPC, SOPC, Egg PC or HSPC phospholipids
  • DSPE-PEG PEGylated-phospholipids
  • This supramolecule was lyophilized (5% Lactose solution was used as cryo- protectant) over 16-20 hrs.
  • the white solid powder formed thereafter was reconstituted by adding required volume of water.
  • DLS study of this reconstituted supramolecular formulation reveals similar size, PDI, surface potential of supramolecules as it was before lyophilization.
  • UV measurement reveals % encapsulation efficiency of this formulation to be 14 mol% of taxane supramolecule.
  • the resulting solution was lyophilized and stored at -20°C.
  • the weight cut-off (the final weight ratio of total lipid to taxane supramolecule) of this system was determined to be -8.66:1.
  • the solution was lyophilized (5% Lactose solution was used as cryo-protectant) over 16-20 hrs and the white solid powder formed thereafter was reconstituted by adding required volume of water. DLS study of this reconstituted supramolecular formulation reveals similar size, PDI, surface potential of supramolecules as it was before lyophilization.
  • UV measurement reveals % encapsulation efficiency of this formulation to be 14 mol% of taxane supramolecule.
  • the resulting solution was lyophilized and stored at -20°C.
  • the weight cut-off (the final weight ratio of total lipid to taxane supramolecule) of this system was determined to be -8.5: 1.
  • the solution was lyophilized (5% Lactose solution was used as cryo-protectant) over 16-20 hrs and the white solid powder formed thereafter was reconstituted by adding required volume of water. DLS study of this reconstituted supramolecular formulation reveals similar size, PDI, surface potential of supramolecules as it was before lyophilization.
  • UV measurement reveals % encapsulation efficiency of this formulation to be 5.7 mol% of taxane supramolecule.
  • the resulting solution was lyophilized and stored at -20°C.
  • the final weight ratio of total lipid to taxane supramolecule of this system was determined to be ⁇ 9.1 :1.
  • Cell culture Mammalian cells were grown in specific culture media, supplemented with 10% fetal bovine serum (FBS) and antibiotics in a humidified environment containing 5% C0 2 at 37°C.
  • FBS fetal bovine serum
  • Cell viability assay The effects of supramolecular taxane conjugates on the viability of cancer cells were measured using MTT assay ( 6 ' 7 ' 8 ). Cells in 100 ⁇ culture-media were plated in 96-well plates (3000-5000 cells/well) and allowed to adhere overnight in a humidified environment containing 5% C0 2 at 37°C. Fresh media ( ⁇ ) containing different concentrations of compounds were added to cells and incubated for 48, 72 and 96 hrs. Following incubation, cell viability was determined using the MTT assay.
  • Example 16 Supramolecular combinatorial therapeutics including immunotherapeutics
  • Cell culture Mammalian cells were grown in specific culture media, supplemented with 10% fetal bovine serum (FBS) and antibiotics in a humidified environment containing 5% C02 at 37°C.
  • FBS fetal bovine serum
  • Combinatorial therapy Therapeutic effects of supramolecules and PD-Ll or PD-
  • mice were examined in melanoma, breast or lung carcinoma models. Mice (4-6 per group) were inoculated subcutaneous ly with cells (melanoma (B16F10): 3 x 10 5 cells; breast (4T1): 5 x 10 5 cells; lung (LLC): 5 x 1°5 cells).
  • drug therapy consisted of the following administration: saline (for control group), platinum drug (5mg/kg); platinum drug (5mg/kg) plus anti-PD-1 or PD- Llantibodies (2mg/kg); platinum drug (lOmg/kg); platinum drug (lOmg/kg) plus anti-PD-1 or PD-Ll antibodies (2mg/kg); anti-PD-1 or PD-Ll antibodies (2mg/kg); platinum- supramolecules (5mg/kg); platinum-supramolecules (5mg/kg) plus anti-PD-1 or PD- Ll antibodies (2mg/kg); platinum-supramolecules (lOmg/kg); platinum-supramolecules (lOmg/kg) plus anti-PD-1 or PD-Ll antibodies (2mg/kg); taxane (5mg/kg); taxane (5mg/kg) plus anti-PD-1 or PD-Ll antibodies (2mg/kg); taxane (5mg/kg); taxane (5mg/kg) plus
  • the compounds were administered via tail vein, with platinum/taxane compounds being administered at q2d dosing and anti-PD-1 or PD-L1 antibodies being dosed on subsequent days with the same dosing regimen.
  • Tumor volumes and body weights were recorded every alternate day for 2 weeks. Tumor volumes were determined in individual mouse by measuring two opposing diameters and represented as mm 3 . The animals were sacrificed when the average tumor volume exceeded 2000 mm 3 . The tumors were harvested immediately following sacrifice and stored in 10% formalin or flash frozen for further analysis.
  • Example 17 Supramolecular combinatorial therapeutics including immunotherapeutics
  • Combinatorial therapy Therapeutic effects of platinum supramolecules and PD- Ll were examined in TNBC models. Mice (6 per group) were inoculated subcutaneously with TNBC cells (4 x 10 5 cells). In tumor bearing mice, once the average tumor volume reaches between 60-80 mm 3 , drug therapy consisted of the following administration: saline (for control group), Carboplatin (5mg/kg); anti-PD-Ll antibodies (2mg/kg); 10-125 (5mg/kg); 10-125 (5mg/kg) plus anti-PD-Ll antibodies (2mg/kg).
  • the compounds were administered via tail vein, with platinum compounds being administered at q2d dosing and anti-PD-Ll antibodies being dosed on subsequent days with the same dosing regimen. Tumor volumes and body weights were recorded every alternate day for 1 week. Tumor volumes were determined in individual mouse by measuring two opposing diameters and represented as mm 3 . The tumors were harvested immediately following sacrifice and stored in 10% formalin or flash frozen for further analysis.
  • Fig. 5 shows the therapeutic effects of 10-125 and anti-PD-Ll against 4T1 (TNBC) tumors.
  • Tumor-bearing mice received platinum drugs (grey arrows) or immunization with anti-PD-Ll (orange arrows).
  • mg. 5 tumor sizes were reduced by 35% when a combination of 10-125 and anti-PD-Ll was used to treat TNBC.
  • the tumor volume was calculated by using the formula, (LxWxW)/2, where the longest diameter measured was considered 'L' and the shortest diameter was 'W.

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Abstract

La présente invention concerne d'une manière générale des agents thérapeutiques combinatoires supramoléculaires, des compositions les comprenant et des utilisations correspondantes. La présente invention concerne en particulier des conjugués covalents de lipides hydrophobes de taxane qui créent un ensemble supramoléculaire, dans une bicouche lipidique par exemple. Cela accroît la stabilisation et entraîne ainsi une augmentation de concentration intratumorale et une amélioration de l'efficacité. La présente invention concerne également des agents thérapeutiques combinatoires supramoléculaires, dans lesquels un conjugué de lipides hydrophobes de taxane est combiné à un ou plusieurs d'un composé de platine, d'un inhibiteur de kinase, et d'un immunorégulateur, qui peuvent chacun être éventuellement conjugués avec un lipide.
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