KR20040004642A - Conjugates activated by cell surface proteases and therapeutic uses thereof - Google Patents

Abstract

Provided are conjugates, compositions, and methods for treating, preventing, or alleviating one or more symptoms of cell surface protease related diseases, including MTSP related, urokinase type plasminogen activator (uPA) or endothelial related diseases. Conjugates for use in the compositions and methods are peptidic conjugates containing a therapeutic agent comprising a cytotoxic agent.

Description

Conjugates activated by cell surface proteases and therapeutic uses thereof

Related Applications

US Provisional Application No. 60 / 293,267, filed May 23, 2001, filed by Applicants Edwin L. Madison, Joseph Edward Semple and George P. Vlasuk under the heading "CONJUGATES ACTIVATED BY CELL SURFACE PROTEASES AND THERAPEUTIC USES THEREOF." Claim the benefit of priority over calls. If allowed, the entirety of the above US provisional application is incorporated herein by reference.

Field of invention

Conjugates, compositions and methods are provided for localized delivery of therapeutic agents for treating various diseases such as proliferative diseases, autoimmune diseases, infectious diseases and inflammatory diseases. Conjugates that act as prodrugs contain a peptidic substrate that is cleaved by the therapeutic and cell surface proteases to release the therapeutic in the vicinity of the targeted cell.

Background of the Invention

Effective treatment of cancer and other proliferative diseases involves the administration of chemotherapeutic agents, typically systemic administration. Typically, chemotherapeutic agents are cytotoxic agents that act by inhibiting proliferation or other metabolic processes so that actively proliferating and growing cells can be targeted by the agent. However, such targeting agents are not highly specific and often have side effects.

Thus, the goal in pharmacology is the design of specific agents that act with very specific activity against targeted cells or tissues. This goal is important in the design of formulations for which the ratio of toxic dose to therapeutic dose is generally close to one and should be limited in dosage, for example as an agent for the treatment of proliferative diseases, including neoplastic diseases, and diseases of viral origin. Do. Numerous approaches have been developed to achieve this goal. Including the use of conjugates containing targeting agents such as antibodies and / or growth factors and therapeutic agents that act on specific cells; The use of antisense techniques to target specific genes and / or proteins; For example, there is the use of gene therapy to provide precise copies of deletion genes or pharmaceutically active compounds and the use of relatively non-toxic toxins when not delivered intracellularly. Thus, great success has been limited. There are only a limited number and type of potential targeting agents and the specificity of these agents is optimal.

Thus, there is a need to develop means for delivering therapeutic agents to targeted cells and tissues. Accordingly, it is an object of the present invention, in particular, to provide methods and compounds for targeted delivery of therapeutic agents.

Summary of the Invention

Provided herein are compounds and methods for targeted delivery of a therapeutic agent. The compound contains a peptide substrate or a soluble, secreted or released form thereof against the cell surface protease, and an agent that becomes a therapeutic agent upon degradation by the protease or that can be activated by a targeted cell or tissue or its vicinity. It is a conjugate. Such formulations include therapeutic agents (eg cytotoxic agents), drugs, therapeutic nucleic acid molecules and diagnostic agents (eg labeled residues and imaging agents). Cell surface proteases are proteases located on the cell surface and include, but are not limited to, membrane-bound proteases such as membrane-bound serine proteases (SP), including, for example, MTSPs and proteases designated as endotheliases. Do not. Also contemplated are proteases located on the cell surface due to specific binding interactions with receptors. Among these proteases are the urokinase plasminogen activator (u-PA) bound to the urokinase plasminogen activator receptor (u-PAR; see Hung (1984) Adv. Exp. Med. Biol. 172: 281-293; Cheng et al. (1989) Gene 69: 357-363). Such conjugates contain one or more substrates for one or multiple cell surface proteases linked directly or via a linker to a targeted agent comprising a therapeutic agent such as a cytotoxic agent. The conjugates provided herein contain the following components: (peptide substrate) _s , (linker) _q and (targeted agent) _t [where one or more peptidic substrate residues are together with the linker (L) or with the linker (L) ) Is connected to one or more therapeutic agents, s is at least 1 and each bone is the same or different, typically 1 to 6, generally 1, 2 or 3, q is a cell surface protease (s) Is at least 0 when cleaving the sexual substrate (s) and releasing the agent in the form of releasing the active therapeutic agent or converting it into the active form by the cell, tissue, or surrounding environment, q is from 0 to t, generally from 1 to 4 , t is at least 1, generally 1 or 2, each targeted agent is the same or different, the linker means any linker, and the targeted agent is any agent, typically a cytotoxic agent Remedy, Diagnostic agents such as acids, imaging agents or labeled residues, or drugs including but not limited to antitumor agents, anticancer agents, anti-angiogenic agents, pro-apoptotic and antimitotic agents or treatments] .

Therapeutic agents include any biologically active molecule. These agents include toxins, cytokines and lymphokines, growth factors, nucleic acid molecules such as antisense nucleic acids, dsRNAs and DNA molecules. Therapeutic agents include agents that are intracellularly active, such as cytotoxins or extracellularly active, such as activity modulators of extracellular demand. The therapeutic agent, when in the conjugate, is substantially inert and, when cleaved, is released in the active form or in a form that can be activated by the targeted cell or tissue or its environment.

In an exemplary embodiment, the conjugate for use in the methods and compositions provided herein comprises a compound of formula (peptide ⁱ ) _s- (linker) _q- (therapeutic) _t [where peptide ⁱ is a substrate for cell surface protease and s is At least 1, 1 to 6 or 1 or 2 or 1, the linker is any linker, q is at least 0 or 0 to 4 or 0 or 1 and the therapeutic agent is for example an antitumor agent, an angiogenesis agent , Cytotoxic agents, including but not limited to, anticancer agents, apoptosis promoters and antimitotic agents, and t is one or more or 1 or 2.] or a derivative thereof. In such conjugates, the therapeutic agent is covalently linked to the C terminus or N terminus of the peptidic substrate, optionally via a linker L.

In certain embodiments, since peptide ⁱ is a substrate for cell surface proteases, substantially inactive conjugates are cleaved at one point of the peptide substrate chain in response to protease to exhibit therapeutic activity in vitro and / or in vivo ( Peptide ^a ) _s- (linker) _q- (therapeutic) _t of a compound or a derivative thereof. In such conjugates, the therapeutic agent is, for example, a cytotoxic agent, and peptide ^a is a truncated form of peptide ⁱ resulting from cleavage at the P1-P1 'bond.

Such conjugates can be used to target and deliver targeted agents to specific cells, and therefore can be used to treat any disease associated with cells or tissues expressing cell surface proteases, including cell-attached and cell-localized proteases. Can be. Cells in which such proteases are expressed or cells in the vicinity thereof are not necessarily associated with disease or disease progression, but may serve to present proteases that are present and cleavage of the targeted conjugate.

A method of treating a disease associated with a cell or tissue expressing a cell surface protease, including cell-attached and cell-localized proteases. Such diseases include, but are not limited to, proliferative diseases, autoimmune diseases, infectious diseases and inflammatory diseases. For example, diseases include rheumatoid arthritis, lupus, multiple sclerosis, psoriasis, diabetic retinopathy, pterygium recurrence, Scars after excimer laser surgery and glaucoma removal, other eye disorders including various disorders in the anterior eye, cardiovascular disorders, restenosis, chronic inflammatory diseases, wounds, circulatory disorders, crest syndrome, bacterial infections, viral infections including AIDS, skin Diseases and cancers, including solid neoplasms and vascular tumors, including but not limited to lung cancer, colon cancer, breast cancer, ovarian cancer and prostate cancer.

Also provided herein are methods of identifying proteases that target the conjugate for treating a disease. The method includes identifying cell surface protease related diseases by identifying cells involved in cell surface protease related disease progression or cells in the vicinity of cells involved in cell surface protease related disease progression and identifying cell surface proteases on the cells. . Subsequently, a conjugate can be prepared that targets such protease provided herein.

Description of the Drawings

1-5 provide in vitro CT ₅₀ (time to 50% cleavage) (minutes) for the exemplary conjugates provided herein: A = 0.1-25 minutes; B = 25 to 100 minutes; C = 100 to 250 minutes; D => 250 min.

1 shows an in vitro CT ₅₀ (min) for exemplary doxorubicin conjugates provided herein and cleavage of such conjugates by MTSP1.

FIG. 2 shows an in vitro CT ₅₀ (min) for cleavage of the exemplary doxorubicin conjugate provided herein and the conjugate by u-PA.

3 shows an in vitro CT ₅₀ (min) for cleavage of an exemplary Taxol conjugate provided herein and MTSP1 by the conjugate.

FIG. 4 shows an in vitro CT ₅₀ (min) for cleavage of the exemplary Taxol conjugate provided herein and the conjugate by u-PA.

FIG. 5 shows an in vitro CT ₅₀ (min) for cleavage of exemplary doxorubicin and taxol conjugates provided herein and ET1 (endoteliaase 1) conjugates.

Detailed description of the aspect

A. Definition

Unless specifically defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents, patent applications, published applications and publications and GenBank sequences, websites and publications cited herein are incorporated by reference in their entirety unless otherwise noted. Where there are several definitions for terms herein, the definitions in this section are widely used. When referring to URLs or other identifiers or addresses, such identifiers may change and certain information on the Internet may be changed to something else, but it should be understood that searching the Internet may search for equivalent information. Reference to this proves that this information is available and widely disseminated publicly.

As used herein, a targeted agent is any agent intended for targeted delivery and includes therapeutic and diagnostic agents and any other agent intended for targeted delivery.

As used herein, targeted delivery means delivery to selected cells or tissues expressing the protease to release the targeted agent. Such delivery need not be limited to such selected cells or tissues, but should include and generally deliver higher amounts to these selected cells or tissues. Delivery includes introduction into or binding to a cell or tissue or release in the vicinity of a cell or tissue. For example, in some cases a tumor includes the production of a substrate associated with a protease, receptor, cofactor or stroma, so that delivery is such induced stromal activity, eg, in invading cells or cells in a targeted tumor. , Targeting of proteases, receptors, and / or enzyme cofactors.

As used herein, the therapeutic index is the ratio of LD ₅₀ / ED ₅₀ .

As used herein, a therapeutic agent is any drug or other agent intended for delivery to a proliferating cell comprising a target cell or tissue, eg, a tumor cell and a cell associated with a proliferative, usually undesirable response. to be. Therapeutic agents include anticancer agents, antiangiogenic agents, apoptosis promoters, antimitotic growth factors, cytokines (such as tumor necrosis factor and interleukin) and cytotoxic agents and other agents described herein and known to those skilled in the art. . Therapeutic agents include agents that act extracellularly and agents that act extracellularly, such as agents that modulate the activity of certain cell surface receptors (eg, G proteins that deliver extracellular signals).

As used herein, an inert therapeutic agent is a therapeutic agent that, by conjugation to a peptide, exhibits no activity due to conformational changes or other factors such as size or steric hindrance or exhibits substantially reduced activity compared to the released active therapeutic agent. For example, conjugated doxorubicin is not toxic to cells until released in a form capable of entering the cells from the conjugate. When the agent is cleaved from the conjugate, it is the active form or a form that is further processed into the active form intracellularly or on the cell by one or multiple steps, including enzymatic or chemical steps.

As used herein, an active therapeutic agent is a therapeutic agent that is cleaved from the conjugate by cleavage of the peptidic substrate portion of the conjugate. Active therapeutic agents can exhibit their intended activity due to cleavage, usually by entering the cell. When conjugated, the therapeutic agent has no or a reduced activity as a therapeutic agent and upon cleavage is released around the cell.

As used herein, anticancer agent (used interchangeably with "antitumor or antineoplastic agent") means any agent used in the treatment of cancer. These anticancer agents, when used alone or in combination with other compounds, can alleviate, reduce, ameliorate or prevent clinical symptoms or diagnostic markers associated with neoplasms, tumors and cancers, or provide or maintain a remission of these symptoms. And any agent that can be used in the methods, combinations, and compositions provided herein. Non-limiting examples of anti-neoplastic agents include anti-angiogenic agents, alkylating agents, anti-metabolites, certain natural products, platinum coordination complexes, anthracendions, substituted ureas, methylhydrazine derivatives, adrenal cortex inhibitors, certain hormones, antagonists, and anticancer polys Saccharides are included.

As used herein, substantially inert with respect to a conjugated therapeutic agent is compared to a nonconjugated therapeutic agent in a standard or in the field recognized assays, eg, in vitro or in vivo assays, to evaluate the therapeutic activity of the agent. 1% or more, generally 10, 20, 30, 50, 60, 70, 80 or 90 or 100% inactive.

As used herein, a targeted cell or tissue refers to a cell or tissue comprising a cell surface protease that cleaves the conjugate. The cell or tissue may be associated with a disease or may be present at disease loci or loci by participating in disease progression or only by chance.

As used herein, angiogenesis is directly or indirectly involved in the establishment and maintenance of new vasculature (angiogenesis), including but not limited to neovascularization, which is broadly associated with tumors. Include the entire process.

As used herein, antiangiogenic therapy or antiangiogenic agents, when used alone or in combination with other therapies or compounds, are one or more clinical symptoms or diagnostics associated with undesirable and / or uncontrolled angiogenesis. By any therapeutic regimen and compound that can alleviate, reduce, ameliorate, prevent, or provide or maintain a state of relief of these symptoms. Thus, for the purposes of the present application, an antiangiogenic agent refers to an agent that inhibits the establishment or maintenance of the pulse system. Such agents include, but are not limited to, antitumor agents and agents for the treatment of other diseases associated with undesirable angiogenesis such as diabetic retinopathy, restenosis, hyperproliferative diseases and other diseases.

As used herein, an antitumor agent that is not antiangiogenic means an antitumor agent that does not act primarily by inhibiting angiogenesis. Whether the antitumor agent mainly acts by inhibiting angiogenesis can be determined using the assays provided herein or using assays well known to those skilled in the art.

As used herein, undesirable and / or unregulated angiogenesis refers to pathological angiogenesis in which the effects of angiogenesis stimulators outweigh the effects of angiogenesis inhibitors. As used herein, defective angiogenesis refers to pathological angiogenesis associated with a defect in normal angiogenesis resulting in abnormal angiogenesis or in the absence or substantial reduction of angiogenesis.

As used herein, cell surface proteases are proteases located on or near the cell surface or attached to the cell surface due to specific binding interactions with any protease and / or receptor located on or at the cell surface. . Exemplary proteases located on the cell surface due to specific binding interactions with the receptor include urokinase plasminogen activator (u-PA) bound to the urokinase plasminogen activator receptor (u-PAR). Thus, cell surface proteases contemplated herein include cell surface-attached proteases. It also includes all forms thereof that can circulate or be inside a cell. To be classified as a cell surface protease, it must exist as one or more forms thereof that are located on the surface of the cell at some point in the cell cycle (ie, located on the surface or bound to a receptor, such as a transmembrane protease). Cell surface proteases include serine proteases and urokinase, including but not limited to transmembrane serine protease (MTSP) and endotheliase.

As used herein, serine protease (SP) refers to a variety of protease families in which serine residues are involved in hydrolysis of proteins or peptides. The serine residue may be part of a catalytic triad mechanism comprising serine, histidine and aspartic acid on catalysis or hydroxyl / ε-amine or hydroxyl / α-amine involving serine and lysine on catalysis It may be part of a catalytic diad mechanism. Of particular interest are SPs of mammals including human origin. Those skilled in the art generally recognize that single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity. Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Bejacmin / Cummings Pub. co., p 224].

As used herein, secreted, soluble and released forms of cell surface proteases are contemplated. Such forms include splice variants that do not contain a transmembrane domain and, for example, those found upon proteolytic degradation or upon removal of the extracellular portion of the membrane bound protease.

As presented herein, cell surface proteases and proteases attached to cells can be used to provide a means for concentrating a therapeutic agent, such as a cytotoxic agent, near such cells by providing a conjugate that is activated upon cleavage by such enzymes. Such conjugates locally release to the site of action a therapeutic agent such as a cytotoxic agent or a derivative thereof that can be converted to a therapeutic agent, such as a cytotoxic agent, upon action of a cell surface protease or cell-attached protease. As noted above, the substrate is designed to be a substrate of a targeted protease that is expressed or active on the surface of a cell, such as tumor cells or endothelial cells, present at or associated with the site (s) or locus or loci of the disease. Due to the specific expression, localization or activation of the protease or the receptor, substrate or enzymatic cofactor for the protease, the administration of the conjugate provided herein can target the therapeutic agent to these cells. Upon contact with the protease, the active therapeutic agent is released immediately adjacent to the targeted cell. For example, the specific profiles of some MTSPs are as follows.

As used herein, “transmembrane serine protease (MTSP)” refers to a family of transmembrane serine proteases that share common structural features as described herein. Hooper et al. (2001) J. Biol. Chem. 276: 857-860. Thus, for example, a reference to "MTSP" refers to all proteins encoded by the MTSP gene, including but not limited to MTSP1, MTSP3, MTSP4, MTSP6, MTSP7, MTSP9, MTSP10, MTSP12, MTSP20, MTSP22, and MTSP25, Or equivalent molecules obtained from any other source, prepared synthetically, or exhibit the same activity. Other MTSPs include, but are not limited to, chorin, enteropeptidase, human airway trypsin-type protease (HAT), MTSP1, TMPRSS2 and TMPRSS4. The MTSPs described herein can be used to identify other MTSPs. MTSP from full-length molecules and splice variants and other animals of interest such as cattle, sheep, goats, pigs, horses, primates (including chimpanzees and gorillas), rodents, dogs, cats and pets, farm and zoo animals Methods for identifying nucleic acids encoding other MTSPs, including nucleic acid molecules encoding A, are known to those of skill in the art and described herein. Nucleic acid molecules described herein, including nucleic acids set forth in the Sequence Listing below, may be prepared using a full-length MTSP of human source or other species origin, for example, by screening the appropriate library using the nucleic acid molecule or selected primers or probes based thereon. It can be used to obtain nucleic acid molecules encoding polypeptides.

Encoding nucleic acid molecular sequences and encoded amino acid sequences of exemplary MTSPs and / or domains thereof are shown in SEQ ID NOs: 1-45, 269-170, and 272-276. The term also includes MTSPs having conservative amino acid substituents that do not substantially alter the activity of each member, and also include polypeptides encoded by splice variants thereof. Thus, the polypeptide obtained can be obtained in 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of the undenatured polypeptide. MTSPs with amino acid substitutions to retain are included, and MTSPs encoded by splice variants thereof and MTSPs encoded by allelic variants such as single nucleotide polymorphisms (SNPs). Although not necessarily, suitable substitutions, including conservative substitutions of amino acids, are known to those skilled in the art and can be made without removing biological activity such as the catalytic activity of the resulting molecules. MTSPs are MTSPs of animal origin, such as mammals, including human origin.

As used herein, “protease domain of MTSP” refers to the extracellular domain of MTSP, which exhibits proteolytic activity and shares homology and structural features with the chymotrypsin / trypsin family protease domain. Thus, it is at least the minimum portion of the domain exhibiting proteolytic activity when assessed by standard in vitro assays. Contemplated herein are such protease domains and catalytically active portions thereof.

Exemplary MTSP polypeptides having the specified protease domains are shown in SEQ ID NOS: 1-45, 269-270 and 272-276, including smaller portions thereof, which possess or exhibit protease activity. Protease domains vary in size and configuration, including insertion and deletion in surface loops. They have a conserved structure, including one or more of the active site triads, primary specific pockets, oxyanion holes and / or other properties of the serine protease domain. Thus, for purposes herein, the protease domain is part of the MTSP as defined herein and is homologous to the domain of another MTSP. MTSPs include MTSP1, MTSP3, MTSP4, MTSP6, MTSP7, MTSP9, MTSP10, MTSP12, MTSP20, MTSP22 and MTSP25 (SEQ ID NOS: 1-19, 42-45, 269-270, and 272-276; see also International PCT Application WO 02/00860 (See SEQ ID NOs: 38 and 97 providing the MTSP12 variant); Corin (SEQ ID NOs: 28 and 29), Enteropeptidase (SEQ ID NOs: 30 and 31) Human Airway Trypsin-type Protease (HAT) (SEQ ID NOs: 32 and 33), Hepsin (SEQ ID NO: 32) 34 and 35), TMPRSS2 (SEQ ID NOS: 36 and 37) and TMPRSS4 (SEQ ID NOs: 38 and 39) Regarding the larger class of enzymes of the chymotrypsin (S1) fold (see Internet accessible MEROPS database), MTSP protease domains share a high level of amino acid sequence identity The His, Asp and Ser residues required for activity are present in conserved motifs In the MTSP protease domain activated by cleavage, the second chain in two chain forms Activation sites that produce N-terminus have conserved motifs And easily identified. See, for example, amino acids 801 to 806, SEQ ID NO: 29; amino acids 406 to 410, SEQ ID NO: 31; amino acids 186 to 190, SEQ ID NO: 33; amino acids 161 to 166 SEQ ID NO: 35; amino acids 255 to 259, SEQ ID NO: 37; amino acid 190 to 194, SEQ ID NO: 39 and sequences as known and / or described herein in the art).

For example, with respect to MTSP10 (see SEQ ID NOs: 44 and 45), there are the following disulfide bonds: C ₄₈₈ -C ₅₀₄ , C ₅₈₇ -C ₆₅₃ ; C ₆₁₉ -C ₆₃₂ ; C ₆₄₃ -C ₆₇₃ (see SEQ ID NOs: 44 and 45) (42-58; 136-201; chymotrimsin comprising 168-182 and 191-220). Disulfide bonds are formed between Cys residues C ₅₇₃ -C ₂₉₆ linking the protease domain to other domains such that the polypeptide obtained at activation cleavage (between residues R ₄₆₂ and I ₄₆₃ of SEQ ID NO: 45) is a two chain molecule. do. C ₅₇₃ (SEQ ID NO: 45) is free Cys in the protease domain of the Japanese chain form. As noted, proteases may also be provided as double-stranded molecules. Single and double stranded forms are proteolytically active. The double stranded form is typically produced by a bond between C ₅₇₃ and Cys outside the protease domain, eg, Cys ₂₉₆ . Upon activation cleavage, disulfide bonds are maintained to produce a double stranded polypeptide. The size of chain “A” is a function of the starting length of the polypeptide before activating cleavage between R ₄₆₂ and I ₄₆₃ . Polypeptides of any length are contemplated herein, including the protease domain (residues 463-692 of SEQ ID NO: 45) or catalytically active fragments thereof. The double stranded form comprises at least the protease domain of a polypeptide comprising C ₂₉₆ up to C ₅₇₃ .

As used herein, a protease domain in double-stranded form connects the protease domain to the "A" chain, which is the remainder of the polypeptide, such as Cys outside a protease domain such as, for example, Cys ₅₇₃ (SEQ ID NO: 45 for MTSP). The double-stranded form formed from the single-stranded form of the protease in which Cys pairing was made between. Double stranded protease domain form refers to any form in which the "rest of the polypeptide", ie, the "A" chain is shorter and contains Cys from outside the protease domain.

As used herein, a catalytically active domain of MTSP means a protease domain. Citation to the protease domain of MTSP generally refers to the Japanese chain of protein. It is highly specified when a double stranded form or both forms are intended. The simogen form of each protein is a single stranded form, which can be converted to an active double stranded or multiple stranded form by activating cleavage. Active form refers to an active form in vivo or in vitro.

As used herein, activating cleavage refers to cleavage of the protease at the N-terminus of the protease domain (generally between R and I or V in a full length protein). When cleaved due to Cys-Cys pairing between the outer Cys of the protease domain and Cys in the protein domain (eg, Cys _{573 in} SEQ ID NO: 45), the resulting polypeptide is divided into two chains ("A", the protease domain of MTSP). Chain and "B" chain). Cleavage can be caused by another protease or autocatalytically. The conjugates provided herein advantageously contain a site recognized by an active cell surface protease (or cell attached protease), thereby cleaving by the protease to release the active or inactive prodrug form of the therapeutic agent.

As used herein, in all instances referred to herein, MTSP1 is

A polypeptide encoded by the nucleotide sequence set forth in SEQ ID NO: 1 or 40,

A polypeptide encoded by a nucleotide sequence that hybridizes to a nucleotide sequence set forth in SEQ ID NO: 1 or 40 under low, moderate or high stringency conditions,

A polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or 41,

About 40%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% of the amino acid sequence set forth in SEQ ID NO: 2 or 41; A polypeptide comprising an amino acid sequence having at least 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity and / or

One or more, or all, or any combination of the polypeptides encoded by the splice variant of MTSP1 set forth in SEQ ID NOS: 1 and 40.

MTSP1 may be of animal, especially mammalian origin, and include, but are not limited to, humans, rodents, chickens, ruminants and other animals. Any domain thereof is contemplated, including a protease domain, which may be a full length simogen or double strand activated form, or a double strand activated form or single chain form.

As used herein, in all instances cited herein, MTSP3 is

A polypeptide encoded by the nucleotide sequence set forth in SEQ ID NO: 3,

A polypeptide encoded by a nucleotide sequence that hybridizes to a nucleotide sequence set forth in SEQ ID NO: 3 under low, moderate or high stringency conditions,

A polypeptide comprising the amino acid sequence set forth as amino acids 205 to 437 of SEQ ID NO: 4,

About 40%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% A polypeptide comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity and / or

One or more, or all, or any combination of the polypeptides encoded by the splice variants of MTSP3 set forth in SEQ ID NOS: 3 and 4.

MTSP3 may be of animal, especially mammalian origin, and include, but are not limited to, humans, rodents, chickens, ruminants and other animals. Any domain thereof is contemplated, including a protease domain, which may be a full length simogen or double strand activated form, or a double strand activated form or single chain form.

As used herein, in all instances referred to herein, MTSP4 is

A polypeptide encoded by the nucleotide sequence set forth in SEQ ID NO: 5, 7 or 9,

A polypeptide encoded by a nucleotide sequence that hybridizes under low, medium or high stringency conditions to the nucleotide sequence set forth in SEQ ID NO: 5, 7 or 9,

A polypeptide comprising an amino acid sequence as set forth in any one of SEQ ID NO: 6, 8 or 10,

About 40%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88 with the amino acid sequence set forth in SEQ ID NO: 6, 8 or 10 A polypeptide comprising an amino acid sequence having at least%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity and / or

One or more or all, or any combination of the polypeptides encoded by the splice variants of MTSP4 set forth in SEQ ID NOs: 7-10.

MTSP4 may be of animal, especially mammalian origin, and include, but are not limited to, humans, rodents, chickens, ruminants and other animals. Any domain thereof is contemplated, including a protease domain, which may be a full length simogen or double strand activated form, or a double strand activated form or single chain form.

As used herein, in all cases referred to herein, MTSP6,

A polypeptide encoded by the nucleotide sequence set forth in SEQ ID NO: 11,

A polypeptide encoded by a nucleotide sequence that hybridizes to a nucleotide sequence set forth in SEQ ID NO: 11 under low, moderate or high stringency conditions,

A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 12,

About 40%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% as shown in SEQ ID NO: 12 A polypeptide comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity and / or

One or more, or all, or any combination of the polypeptides encoded by the splice variant of MTSP6 set forth in SEQ ID NO: 12.

MTSP6 may be of animal, in particular mammalian origin and include, but are not limited to, humans, rodents, chickens, ruminants and other animals. Any domain thereof is contemplated, including a protease domain, which may be a full length simogen or double strand activated form, or a double strand activated form or single chain form. Of particular interest here are MTSP6 of SEQ ID NO: 12.

As used herein, in all cases referred to herein, MTSP7 is

A polypeptide encoded by the nucleotide sequence set forth in SEQ ID NO: 13,

A polypeptide encoded by a nucleotide sequence that hybridizes under low, moderate or high stringency conditions to the nucleotide sequence set forth in SEQ ID NO: 13,

A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 13,

About 40%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% A polypeptide comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity and / or

One or more, or all, or any combination of the polypeptides encoded by the splice variant of MTSP7 set forth in SEQ ID NO: 13.

MTSP7 may be of animal, in particular mammalian origin and include, but are not limited to, humans, rodents, chickens, ruminants and other animals. Any domain thereof is contemplated, including a protease domain, which may be a full length simogen or double strand activated form, or a double strand activated form or single chain form.

As used herein, in all instances cited herein, MTSP9 is

A polypeptide encoded by the nucleotide sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 42,

A polypeptide encoded by a nucleotide sequence which hybridizes under low, moderate or high stringency conditions to the nucleotide sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 42,

A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 18 or 43,

About 40%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, and the amino acid sequence set forth in SEQ ID NO: 18 or 270; A polypeptide comprising an amino acid sequence having at least 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity and / or

One or more, or all, or any combination of the polypeptides encoded by the splice variant of MTSP9 set forth in SEQ ID NO: 17.

MTSP9 may be of animal, in particular mammalian origin and include, but are not limited to, humans, rodents, chickens, ruminants and other animals. Any domain thereof is contemplated, including a protease domain, which may be a full length simogen or double strand activated form, or a double strand activated form or single chain form.

As used herein, in all instances cited herein, MTSP10 is

A polypeptide encoded by the nucleotide sequence set forth in SEQ ID NO: 44,

A polypeptide encoded by a nucleotide sequence that hybridizes under low, medium or high stringency conditions to the nucleotide sequence set forth in SEQ ID NO: 44,

A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 45,

About 40%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% as shown in SEQ ID NO: 45 A polypeptide comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity and / or

One or more, or all, or any combination of the polypeptides encoded by the splice variant of MTSP7 set forth in SEQ ID NO: 44.

MTSP10 may be of animal, in particular mammalian origin and include, but are not limited to, humans, rodents, chickens, ruminants and other animals. Any domain thereof is contemplated, including a protease domain, which may be a full length simogen or double strand activated form, or a double strand activated form or single chain form.

Provided herein are MTSP10, including but not limited to splice variants and nucleic acids encoding MTSP and domains, derivatives and analogs thereof. Also provided is a single-chain protease domain having an N-terminus that is functionally equivalent to the N-terminus produced by activation of MTSP10 in the zymogen form. The cleavage site of the protease domain of MTSP10 is between amino acid R and amino acid I (R ↓ llGGT) (residues 462-467, SEQ ID NO: 45).

As used herein, in all instances cited herein, MTSP12 is

SEQ ID NOs: 19 and 20,

A polypeptide encoded by a nucleotide sequence comprising a nucleotide sequence as set forth in SEQ ID NO: 19 or a nucleotide encoding an amino acid sequence as set forth in SEQ ID NO: 20,

A polypeptide encoded by a nucleotide sequence that hybridizes to a nucleotide sequence set forth in SEQ ID NO: 19 under low, moderate or high stringency conditions,

A polypeptide or catalytically active portion thereof comprising the amino acid sequence set forth in SEQ ID NO: 20,

About 40%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% with amino acid sequence as set forth in SEQ ID NO: 20 A polypeptide comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity and / or

One or more or all, or any combination of polypeptides encoded by splice variants of MTSP12 comprising the amino acid sequence set forth in SEQ ID NO: 20.

In particular, MTSP12 polypeptides having the protease domains set forth in SEQ ID NOs: 19 and 20 are provided. The polypeptide is a single chain or multichain polypeptide. In all cases cited herein the protease domain of MTSP12 is

A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 20 or a catalytically active moiety not comprising the amino acid sequence set forth in SEQ ID NO: 271,

A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 272 or a catalytically active fragment thereof,

A polypeptide containing amino acids 237 to 456 of SEQ ID NO: 6, a polypeptide containing amino acids 538 to 765 of SEQ ID NO: 20 and amino acids 861 to 1087 of SEQ ID NO: 20 but not comprising the amino acid sequence of SEQ ID NO: 271 Does not have a polypeptide,

a polypeptide encoded by a nucleotide sequence that hybridizes under low, medium or high stringency conditions to the nucleotide sequence encoding any of the polypeptides of (a) to (c),

A polypeptide encoded by a nucleotide sequence which hybridizes to a nucleotide sequence set forth in SEQ ID NO: 20 under low, moderate or high stringency conditions but does not encode an amino acid sequence set forth in SEQ ID NO: 271,

About 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% with amino acid sequence shown in SEQ ID NO: 20 , A polypeptide comprising an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity,

amino acid sequence of the polypeptides of (a) to (e) with about 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% , A polypeptide comprising an amino acid sequence having at least 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity,

One or more or all, or any combination, or catalytically active portion of a polypeptide encoded by a splice variant of a nucleotide sequence encoding any one of the above MTSP12.

Smaller portions thereof are also provided that maintain protease activity. MTSP12 may be of animal, especially mammalian origin, and include, but are not limited to, humans, rodents, chickens, ruminants and other animals. Any domain thereof is contemplated, including a protease domain, which may be a full length simogen or double strand activated form, or a double strand activated form or single chain form. MTSP12 also includes International PCT Application WO 02/00860 (see SEQ ID NOs: 38 and 97).

As used herein, in all instances cited herein, MTSP20 is

A polypeptide encoded by the nucleotide sequence set forth in SEQ ID NO: 273,

A polypeptide encoded by a nucleotide sequence that hybridizes under low, medium or high stringency conditions to the nucleotide sequence set forth in SEQ ID NO: 273,

A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 273,

About 40%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% as shown in SEQ ID NO: 274 A polypeptide comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity and / or

One or more or all of the polypeptides encoded by a splice variant of MTSP20 encoded by a nucleotide sequence comprising the sequence set forth in SEQ ID NO: 273, or any combination.

MTSP20 may be of animal, especially mammalian origin, and include, but are not limited to, humans, rodents, chickens, ruminants and other animals. Any domain thereof is contemplated, including a protease domain, which may be a full length simogen or double strand activated form, or a double strand activated form or single chain form.

As used herein, in all instances cited herein, MTSP22 is

A polypeptide encoded by the nucleotide sequence set forth in SEQ ID NO: 275,

A polypeptide encoded by a nucleotide sequence that hybridizes to a nucleotide sequence set forth in SEQ ID NO: 275 under low, medium or high stringency conditions,

A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 276,

About 40%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% as shown in SEQ ID NO: 276 A polypeptide comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity and / or

One or more or all, or any combination of the polypeptides encoded by the splice variant of MTSP22 set forth in SEQ ID NO: 275.

MTSP22 may be of animal, especially mammalian origin, and include, but are not limited to, humans, rodents, chickens, ruminants and other animals. Any domain thereof is contemplated, including a protease domain, which may be a full length simogen or double strand activated form, or a double strand activated form or single chain form.

As used herein, in all instances cited herein, MTSP25 is

A polypeptide encoded by the nucleotide sequence set forth in SEQ ID NO: 269,

A polypeptide encoded by a nucleotide sequence that hybridizes under low, medium or high stringency conditions to the nucleotide sequence set forth in SEQ ID NO: 269,

A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 270,

About 40%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% as shown in SEQ ID NO: 270 A polypeptide comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity and / or

One or more or all, or any combination of the polypeptides encoded by the splice variant of MTSP25 set forth in SEQ ID NO: 269.

MTSP25 may be of animal, especially mammalian origin, and include, but are not limited to, humans, rodents, chickens, ruminants and other animals. Any domain thereof is contemplated, including a protease domain, which may be a full length simogen or double strand activated form, or a double strand activated form or single chain form.

As used herein, a human protein is a protein encoded by a nucleic acid present in the human genome, including all allelic and conservative variants, unless the variant is found in another mammal.

Substantially not cleaved by plasmin or prostate specific antigen (PSA) (or other protease other than cell surface-attached protease) means targeted cell surface membrane protease, or prostate specific antigen (PSA) for cleavage of the conjugate. ) Or relative activity when compared with the in vitro assay comparing the activity of the catalytically active portion of the protease domain (or catalytically active form thereof) of plasminogen (under optimal conditions for each enzyme) 2: 1, 3 : 1, 4: 1, 5: 1, 10: 1, 50: 1 or greater than 100: 1.

As used herein, activity means the ratio K _cat / K _m , where k _cat is the catalytic conversion of a particular enzyme and K _m is the Michaelis constant for binding of the substrate.

As used herein, "nucleic acid encoding a protease domain or catalytically active portion of MTSP" encodes only the cited single-chain protease domain or active portion thereof and does not encode another contiguous portion of MTSP as a contiguous sequence. It is to be understood as meaning nucleic acid.

As used herein, the CUB domain is a motif already identified in various proteins that mediate protein-protein interactions in the complement component C1r / C1s and are involved in the developmental process.

As used herein, a simogen is a catalytically inactive protein that is converted into a proteolytic catalyst by the action of an activator, including autoactivation (ie, typically, but not necessarily, less than 1% of the active form) )to be. Inactive means less active than those skilled in the art consider to be the active form of the enzyme. The ratio of activation of the simogen to the activated form varies from enzyme to enzyme.

As used herein, "disease or disorder" refers to a pathological condition in an organism that results from, for example, an infection or genetic defect and is characterized by identifiable symptoms. Diseases to be treated herein include any disease wherein cell surface proteases, including cell-localized or cell-attached proteases, are associated with the diseased or targeted cells or tissues involved in disease progression. This association may be because the protease is inadvertently associated with cells involved in or associated with the disease. These diseases are referred to herein as cell surface protease related diseases. Thus, to treat these diseases, cell surface proteases are expressed that are expressed in cells associated with diseases such as immune cells in the case of immune disease treatment and virus infected cells in the case of viral disease treatment. The conjugate is designed as described herein for cleavage by the selected protease.

As used herein, neoplasia (neoplastic) refers to abnormal new growth and therefore has the same meaning as a tumor that may be benign or malignant. Unlike hyperplasia, neoplasia continues in the absence of early stimulation.

As used herein, neoplastic disease refers to any disease associated with cancer, including tumor development, growth, metastasis and progression.

As used herein, cancer is a general term for a disease caused by any type of malignant tumor.

As used herein, malignant refers to a primary tumor that, when applied to a tumor, has the ability to lose and metastasize growth and positional control.

As used herein, an endothelia is a mammal comprising a human protein, which has a transmembrane domain, is expressed on the surface of endothelial cells and is active and has a protease domain, particularly an extracellular protease domain, which is generally a serine protease. Animal protein (see US Application No. 09 / 717,473 and International PCT Application WO 01/36604). Thus, for example, a reference to an endothelial agent includes all proteins encoded by the endothelial gene family, or equivalent molecules obtained from any other source, prepared synthetically, or exhibiting the same activity. The endothelial gene family is a transmembrane protease that is expressed or active in endothelial cells. These proteases include serine proteases. These include protease domains exhibiting sequence homology with endotheliases 1 and 2. Endotheliases 1 and 2, for example, exhibit about 40% or 45% identity. Sequence homology is at least about 25%, 40%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85 depending on the length of the sequence when aligned to maximize identity %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more of the number of residues Refers to sequence identity. Sequence homology also encodes a nucleic acid molecule or the same protein provided herein under at least moderate stringency conditions or more closely related proteins in the case of more closely related proteins, but the degeneracy of the genetic code Is assessed by determining whether the sequences hybridize to different nucleic acid molecules. In addition, "endoteliase" refers to 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, of the proteolytic activity of a polypeptide in which the polypeptide obtained is not modified. Endothelial agents having amino acid substitutions, such as those shown in Table 1, that retain 80%, 90%. Suitable conservative substitutions of amino acids are known to those skilled in the art and can generally be performed without changing the biological activity of the resulting molecules. As is well known, those of ordinary skill in the art generally recognize that single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity. Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Bejacmin / Cummings Pub. Co., p. 224]. In addition, the definition of "endoteliase" includes catalytically active fragments or secreted forms of endoteliase.

As used herein, in all instances recited herein, endotelia 1

A polypeptide encoded by the nucleotide sequence set forth in SEQ ID NO: 21,

A polypeptide encoded by a nucleotide sequence which hybridizes under low, moderate or high stringency conditions to the nucleotide sequence set forth in SEQ ID NO: 21,

A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 22,

About 40%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, and the amino acid sequence set forth in SEQ ID NO: 22 A polypeptide comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity and or

One or more, or all, or any combination of polypeptides encoded by splice variants of a nucleic acid molecule encoding a protein containing the polypeptide set forth in SEQ ID NO: 22.

Endotheliase 1 may be of animal, especially mammalian origin, and includes, but is not limited to, humans, rodents, chickens, ruminants and other animals. Any domain thereof is contemplated, including a protease domain, which may be a full length simogen or double strand activated form, or a double strand activated form or single chain form.

As used herein, in all instances recited herein, endotelia 2 is

A polypeptide encoded by the nucleotide sequence set forth in SEQ ID NO: 23 or 25,

A polypeptide encoded by a nucleotide sequence that hybridizes under low, moderate or high stringency conditions to the nucleotide sequence set forth in SEQ ID NO: 23 or 25,

A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 24 or 26,

About 40%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89 with the amino acid sequence set forth in SEQ ID NO: 24 or 26 A polypeptide comprising an amino acid sequence having at least%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity and or

One or more or all or any combination of polypeptides encoded by splice variants of the nucleic acid molecule set forth in SEQ ID NO: 23 or 25.

Endotheliase 2 may be of animal, especially mammalian origin, and includes, but is not limited to, humans, rodents, chickens, ruminants and other animals. Any domain thereof is contemplated, including a protease domain, which may be a full length simogen or double strand activated form, or a double strand activated form or single chain form.

As used herein, the protease domain of an endotelase refers to the polypeptide portion of an endotelase, an extracellular portion that exhibits protease activity. Protease domains generally contain at least a minimum of 50 or 100 amino acid numbers required for protease activity. Protease activity can be assessed experimentally, for example, by testing the ability of a polypeptide to act as a protease. Protease activity can be assessed using assays such as those described in the Examples, except that known substrates are used in place of test compounds. In addition, since proteases, particularly serine proteases, have specific structures and sequences or motifs, the protease domains can be readily identified by the structures and sequences or motifs.

As used herein, part of the protease domain of an endoteliase refers to the portion of the protease domain of an endoteliase that is located inside or is an extracellular domain of endoteliase and exhibits serine proteolytic activity. Thus, at least a portion of the extracellular domain exhibiting proteolytic activity as assessed by standard assays. Exemplary protease domains of endotelase are shown in SEQ ID NO: 22 and shown as amino acids 321-688 and 321-562 of SEQ ID NOs: 24 and 26, respectively. Smaller portions thereof that retain protease activity are contemplated. Protease domains vary in size and configuration, including insertion and deletion in surface loops. The domain exhibits a conserved structure, including one or more structural features such as active site triads, primary specific pockets, oxyanion holes, and / or other properties of the serine protease domain of the protease. Thus, for the purposes of this application, the protease domain is homologous in terms of similarity or homology of the sequence and features to the protease domain of a endotryase or chymotrypsin or trypsin as defined herein.

As used herein, homology means at least about 25%, 40%, 60%, 80%, 90%, 95% or 98% nucleic acid sequence identity. By sequence identity, the number of conserved amino acids is determined by the standard alignment algorithm program and used with default gap penalties established by each supplier. Homology can also be assessed by conserved nucleic acid sequences that include any nucleic acid that encodes a domain and hybridizes under at least low stringency conditions. Similarly, nucleic acid sequence alignment programs are commercially available (DNAStar "MegAlign" program (Madison, WI) and the "Gap" program (Madison, WI) of the University of Wisconsin Genetics Computer Group (UWG). Hybridization along the entire length of the nucleic acid of interest under moderate stringency or high stringency conditions, and nucleic acid molecules containing degenerate codons instead of codons are contemplated when hybridizing nucleic acid molecules.

As used herein, the description that a polypeptide consists essentially of a protease domain means that only the endothelial portion of the polypeptide is the protease domain or catalytically active portion thereof. The polypeptide may optionally include amino acid sequences from additional non-endotelase.

As used herein, a domain is functionally and / or functionally distinct from other portions of the molecule as part of a molecule, eg, a protein or nucleic acid.

As used herein, an active form of protease means an enzyme that catalyzes the hydrolysis of a protein or peptide. Citations to proteases include active and simogen or other less active forms.

As used herein, nucleic acids include analogs and mixtures thereof including DNA, RNA and peptide nucleic acids (PNA). The nucleic acid may be single stranded or double stranded. When referring to probes or primers optionally labeled with a detectable label, such as a fluorescent or radiolabel, single-chain molecules are considered. The molecule is typically a molecule whose length is such that its target is statistically unique and low copy number (typically less than 5, generally less than 3) for probing or priming a library. In general, a probe or primer contains at least 14, 16 or 30 consecutive sequences that are complementary or identical to the gene of interest. Probes and primers can be 10, 20, 30, 50, 100 or more nucleic acids long.

As used herein, a nucleic acid encoding a fragment or portion of an endothelial agent refers to a nucleic acid that encodes only the aforementioned fragment or portion of an endothelial protein and other consecutive portions of the endothelial agent do not encode as a continuous sequence. do.

As used herein, heterologous nucleic acid is not normally produced in vivo by the cells expressing it or affects the expression of endogenous nucleic acid (eg, DNA) by affecting transcription, translation or other controllable biochemical processes. A nucleic acid that encodes or encodes an RNA (if DNA) or protein (if RNA) that encodes or mediates a changing mediator or is located at a locus different from its normal locus. Heterologous nucleic acids are generally not endogenous to the cells to which they are to be introduced, either obtained from another cell or produced synthetically. Generally, but not necessarily, the nucleic acid encodes proteins and RNA that are not normally produced by the cell expressing it.

Heterologous nucleic acid (eg DNA) may also be referred to as foreign nucleic acid (eg DNA). Any nucleic acid (eg, DNA) that is heterologous to, or foreign to, a cell skilled in the art is recognized herein or is considered to be foreign to it, including heterologous nucleic acids that are also expressed inherently. It includes. Examples of heterologous nucleic acids include nucleic acids encoding traceable marker proteins, eg, proteins that confer drug resistance; Nucleic acids encoding therapeutically effective substances such as anticancer agents, enzymes and hormones; And nucleic acids and RNAs such as DNA encoding other types of proteins, eg, antibodies, such as nucleic acids such as RNA interference (RNAi) or other double stranded RNA and antisense RNA. Antibodies encoded by heterologous nucleic acid can be secreted or expressed on the surface of the cell into which the heterologous nucleic acid has been introduced.

For example, the nucleic acid may be a targeted agent such as a therapeutic or diagnostic agent in the conjugate. Nucleic acids can be used to inhibit the expression of targeted genes by creating loss of function (RNAi) [Chuang et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97: 4985 for the use of dsRNA. Mammal, seed. Organisms, including C. elegans, Drosophila, and plants, and methods involving the use of RNAi to silence genes in humans are known. Fire et al. (1998) Nature 391: 806-811 Fire (1999) Trends Genet. 15: 358-363; Sharp (2001) Genes Dev. 15: 485-490; Hammond, et al. (2001) Nature Rev. Genet. 2: 110-1119; Tuschl (2001) Chem. Biochem. 2: 239-245; Hamilton et al. (1999) Science 286: 950-952; Hammond et al. (2000) Nature 404: 293-296; Zamore et al. (2000) Cell 101: 25-33; Bernstein et al. (2001) Nature 409: 363-366; Elbashir et al. (2001) Genes Dev. 15: 188-200; Elbashir et al. (2001) Nature 411: 494-498; International PCT Application WO 01/29058; International PCT Application WO 99/32619. By selecting the appropriate sequence, the expression of dsRNA can interfere with the accumulation of endogenous mRNA encoding the targeted gene product. RNAi is prepared using a region that contains at least about 21 nucleotides and which is selective for a nucleic acid encoding a targeted gene product (ie, whose target is unique).

As used herein, gene therapy involves delivering heterologous nucleic acids (eg, DNA) into specific cells, ie target cells, of a mammal, particularly a human, having a disease or disorder for which such therapy is desired. The nucleic acid molecule is included in a conjugate linked through a cell surface protein cleavage site. Nucleic acid (eg DNA) is introduced into a selected target cell in such a way that heterologous nucleic acid (eg DNA) is expressed and thereby produced a therapeutic product encoded. Alternatively, the heterologous nucleic acid (eg, DNA) may mediate the expression of the DNA encoding the therapeutic product in some way, or the product (eg peptide) that directly or indirectly mediates the expression of the therapeutic product in some way. Or RNA). Gene therapy may also be used to deliver a nucleic acid encoding a gene product that replaces a defective gene or supplements a gene product produced by a cell or mammal into which a nucleic acid is introduced. The introduced nucleic acid is a therapeutic compound, such as a growth factor inhibitor, or a tumor necrosis factor or inhibitor thereof, that is not normally produced in a mammalian host or is not produced in a therapeutically effective amount or at a therapeutically useful time. For example, the receptor for this can be encoded. Said heterologous nucleic acid (eg, DNA) encoding a therapeutic product may be modified prior to introduction into the cell of a diseased host to enhance or alter the product or its expression. Gene therapy may also include the delivery of inhibitors or inhibitors or other modulators of gene expression. The conjugates herein may be used to deliver products such as nucleic acids for gene therapy.

As used herein, a therapeutically effective product for gene therapy is a product encoded by said heterologous nucleic acid, typically DNA, upon introduction of the heterologous nucleic acid into a host, thereby expressing symptoms of congenital or acquired disease. A product that ameliorates or eliminates or cures a disease. Also included are biologically active nucleic acid molecules such as RNAi and antisense.

As used herein, with respect to antisense oligonucleotides, a sequence complementary to at least a portion of RNA is generally complementary enough to hybridize with RNA under moderate or high stringency conditions to form a stable duplex. In the case of a double-stranded SP antisense nucleic acid, the single strand of the duplex DNA (dsDNA) can be tested or triplex formation can be assessed. The ability to hybridize depends on the degree of complementarity and the length of the antisense nucleic acid. In general, the longer the nucleic acid length to hybridize, the more base mismatch with the SP encoding RNA, which may still form and contain stable duplexes (or triplets). One skilled in the art can ascertain the extent of discrepancies that can be tolerated using standard procedures for measuring the melting point of hybridized complexes.

Amino acid substitutions can be made or occur in any SP and its protease domain. Amino acid substitutions include conservative substitutions as set forth in Table 1 below that do not eliminate proteolytic activity. As described herein, substitutions that change the properties of the protein are also contemplated, such as the removal of cleavage sites or other sites, which substitutions are generally non-conservative, but are readily available to those skilled in the art. Can be achieved.

Suitable conservative substitutions of amino acids are known to those skilled in the art and generally can be carried out without changing the biological activity of the resulting molecule, eg, enzyme activity. Also included within the definition are catalytically active fragments of SP, in particular single chain protease moieties. Conservative amino acid substitutions are made, for example, as set forth in Table 1 below:

Ala (A) Gly; Ser Arg (R) Lys, Orn Asn (N) Gln; His Asp (D) Glu Cys (C) Ser Gln (Q) Asn Glu (E) Asp Gly (G) Ala; Pro HIs (H) Asn; Gln Ile (I) Leu; Val; Nle; Met Leu (L) Ile; Val; Nle; Met; Lys (K) Arg; Gln; Glu Met (M) Leu; Tyr; Ile; Nle Phe (F) Met; Leu; Tyr; Trp Ser (S) Thr Thr (T) Ser Trp (W) Tyr; Phe Tyr (Y) Trp; Phe Val (V) Ile; Leu; Nle; Met

Other substitutions may also be acceptable and may be determined empirically or according to known conservative substitutions. For example, one or more amino acid residues in the sequence may be substituted with another amino acid of similar polarity which acts as a functional equivalent resulting in a silent alteration. Substitution for an amino acid in the sequence may be selected from other members of the class to which the amino acid belongs. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, prolyl, phenylalanine, tryptophan and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. Positively charged (basic) amino acids include arginine, lysine and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

As used herein, amino acids that occur in the various amino acid sequences shown herein are identified according to their well known three letter or one letter abbreviations. The nucleotides generated from the various DNA fragments are named according to standard single-letter nomenclature commonly used in the art. Other abbreviations include hR or hArg for homoarginine; HY or hTyr for homotyrosine; Cha for cyclohexylamine; Amf for 4-aminomethylphenylalanine; DPL for 2- (4,6-dimethylpyrimidinyl) lysine; (Imidazolyl) K for N '-(2-imidazolyl) lysine; Me2PO3-Y for 0-dimethylphosphotyrosine; O-Me-Y for O-methyltyrosine; TIC for tetrahydro-3-isoquinoline carboxylic acid; MeL for 2-keto-3-amino-5-methyl-hexane; DAP for 1,3-diaminopropane; TFA for trifluoroacetic acid; AA for acetic acid is included.

As used herein, splice variants refer to variants produced by different processing of primary transcripts of genomic DNA resulting in one or more mRNA types.

As used herein, a probe or primer based on the nucleotide sequence described herein comprises at least 10, 14, generally 16 or 30 or more, or 100 contiguous nucleotide sequences.

As used herein, antisense polynucleotide means a synthetic sequence of nucleotide bases that are complementary to the sense strand of an mRNA or double stranded DNA. Under suitable conditions, mixtures of sense and antisense polynucleotides result in the binding or hybridization of two molecules. When such polynucleotides bind to (hybridize with) mRNA, inhibition of protein synthesis (detoxification) occurs. When such polynucleotides bind to double stranded DNA, inhibition of RNA synthesis (transcription) occurs. The resulting inhibition of translation and / or transcription results in the inhibition of the synthesis of the protein encoded by the sense chain. Antisense nucleic acid molecules are typically five or more contiguous nucleotides, often 14, 16, or complementary to the coding portion of a nucleic acid encoding a gene of interest, e.g., a nucleic acid encoding the single stranded protease domain of an SP. It contains a sufficient number of nucleotides to specifically bind to a target nucleic acid that is at least 30 consecutive nucleotides or modified nucleotides.

As used herein, an array refers to a collection of elements, such as antibodies, containing three or more members. An addressable array is typically an array in which members of the array are identifiable by placing on a solid phase support. Thus, in general, the members of the array will be anchored to a discontinuous identifiable locus on the surface of the solid phase.

As used herein, an antibody refers to an immunoglobulin, whether produced naturally or in part or in whole, including any derivative thereof that retains the specific binding capacity of the antibody. Thus, an antibody includes any protein having a binding domain that is homologous or substantially homologous to an immunoglobulin binding domain. Antibodies include members of any immunoglobulin claim, including IgG, IgM, IgA, IgD, and IgE.

As used herein, antibody fragment refers to any derivative of an antibody shorter than the full length that retains at least a portion of the specific binding capacity of the full length antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab ', F (ab) ₂ , single chain Fvs (scFV), FV, dsFV diabody, and Fd fragments. The fragment may comprise multiple chains joined together by disulfide bridges. Antibody fragments generally contain at least about 50 amino acids and typically at least 200 amino acids.

As used herein, an Fv antibody fragment consists of one variable heavy chain (V _H ) and one variable light chain that are linked in a non-covalent interaction.

As used herein, dsFV means Fv with genetically engineered intramolecular disulfide bonds that stabilize the V _H -V _L pair.

As used herein, F (ab) ₂ fragments are antibody fragments resulting from cleavage of immunoglobulins with pepsin at pH 4.0 to 4.5, which can be expressed recombinantly to produce equivalent fragments.

As used herein, Fab fragments are antibody fragments resulting from cleavage of immunoglobulins using papain, which can be expressed recombinantly to produce equivalent fragments.

As used herein, scFV refers to antibody fragments containing the variable light (V _L ) and variable heavy (V _H ) covalently linked by polypeptide linkers in any order. The linker is such that the two variable domains are crosslinked without substantial interference. Exemplary linkers include, but are not limited to, (Gly-Ser) _n residues in which certain Glu or Lys residues are dispersed throughout to increase solubility.

As used herein, humanized antibodies are antibodies that have been modified to include the amino acid sequence of a human such that administration to humans does not cause an immune response. Methods for preparing such antibodies are known. For example, to prepare such antibodies, hybridomas or other prokaryotic or eukaryotic cells expressing monoclonal antibodies, such as E. coli. E. coli or CHO cells are modified with recombinant nucleic acid techniques to express antibodies in which the amino acid composition of the non-variable regions is based on human antibodies. A computer program is designed to identify the non-variable regions.

As used herein, diabodies are dimeric scFVs, which typically have shorter peptide linkers than scFV and they are generally dimerized.

As used herein, preparation by recombinant means using recombinant DNA methods means using well known methods of molecular biology to express proteins encoded by cloned DNA.

As used herein, the term evaluation is used to obtain an absolute value for the activity of an SP or a domain thereof present in a sample and to obtain an index, ratio, percentage, visible value or other value, which is also an indicator of this activity level. In this respect, it means quantitatively and qualitatively determining. The assessment may be direct or indirect, and the chemical species actually detected need not be of course the proteolytic product itself, but may be, for example, a derivative thereof or some additional substance.

As used herein, biological activity refers to the physiological response or in vivo activity of a compound that results from in vivo administration of a compound, composition or other mixture. Thus, biological activity includes therapeutic and pharmaceutical activity of the compounds, compositions and mixtures. Biological activity can be observed in in vitro systems designed to test or use such activity.

As used herein, a combination refers to any association between two or more items.

As used herein, fluid means any composition that can flow. Thus, fluid means a composition in the form of a semi-solid, paste, solution, aqueous mixture, gel, lotion, cream and other such compositions.

As used herein, an effective amount of a compound for treating a particular disease means an amount sufficient to ameliorate, or reduce in a particular way, the symptoms associated with the disease. The amount may be administered in a single dose or may be administered depending on the regimen for which the amount is effective. The amount can cure the disease, but is typically administered to ameliorate the symptoms of the disease. Repeated administration may be necessary to achieve the desired improvement of symptoms.

As used herein, when quoting two nucleic acid sequences, equivalent means that the two corresponding sequences encode the same amino acid sequence or equivalent protein. When an equivalent is used to refer to two proteins or peptides, it is conservative that the two proteins or peptides do not substantially change the activity or function of the protein or peptide (ie, maintain at least about 1% activity). It means having a substantially identical amino acid sequence with only amino acid substitutions (see Table 1 above). Where an equivalent refers to a property, the property does not have to be present in the same range (eg, two peptides may exhibit different degrees of enzymatic activity of the same type), but the activity is generally substantially same. Complementarity means that when quoting two nucleotide sequences, the two nucleotide sequences can hybridize, typically with less than 25%, often less than 15% or even less than 5%, of inconsistency or no inconsistency between the corresponding nucleotides. . In general, the two molecules hybridize under high stringency conditions.

As used herein, a method of treating or preventing a disease or disorder associated with undesirable and / or uncontrolled angiogenesis improves or reduces symptoms of a disease or disorder associated with undesirable and / or uncontrolled angiogenesis. It is meant to alleviate or prevent, or to provide or maintain a relief state of these symptoms. This also means that features of pathological angiogenesis are eliminated, reduced or prevented by treatment. Non-limiting examples of features of pathological angiogenesis include uncontrolled degradation of the proximal extracellular matrix of the basement membrane and endothelial cells; Migration, division and organization of endothelial cells into new functional capillaries and maintenance of the functional capillaries.

As used herein, operably linked or operatively linked refers to the functional correlation of regulatory and effector sequences such as DNA and promoter, thereby transcriptional and translational termination sites, and other signal sequences. For example, an operative linkage of a DNA to a promoter may be achieved by physically and interfering between the DNA and the promoter such that transcription of the DNA is initiated from the promoter by RNA polymerase that specifically recognizes, binds to and transcribs the DNA. It means functional correlation. To optimize expression and / or in vitro transcription, at the level of transcription and translation, the cloning of clones to remove extra potentially inappropriate alternative translational initiation (ie, starting) codons or other sequences that may interfere with or reduce expression. It may be necessary to remove, add or modify the 5 'non-toxic portion. Alternatively, consensus ribosomal binding sites can be found in Kozak J. Biol. Chem. 266: 19867-19870 (1991) can be inserted closely to the 5 'of the starting codon and can enhance expression. The desirability (or necessity) of such modifications can be determined experimentally.

As used herein, a promoter region or promoter element refers to a segment of DNA or RNA that regulates the transcription of the DNA or RNA to which they are operatively linked. The promoter region contains specific sequences sufficient for RNA polymerase recognition, binding, and transcription initiation. This portion of the promoter region is referred to as the promoter. The promoter region also includes sequences that regulate the recognition, binding and transcription initiation activity of RNA polymerase. The sequences may be cis functional or reactive with trans functional factors. Depending on the regulatory characteristics, the promoter may be either constitutive or regulatory. Exemplary promoters contemplated for use in prokaryotes include bacteriophage T7 and T3 promoters.

As used herein, sample means anything that can contain an analyte for which analyte analysis is desired. The sample can be a biological sample such as a biological fluid or biological tissue. Examples of biological fluids include urine, blood, plasma, serum, saliva, semen, feces, sputum, cerebrospinal fluid, tears, mucus or amniotic fluid, and the like. Biological tissue is an aggregate of certain types of cells, usually with intracellular substances that form one of the structural substances of human, animal, plant, bacterial, fungal or viral structure, connective tissue, epithelial tissue, muscle tissue and neural tissue. It includes. Examples of biological tissues also include organs, tumors, lymph nodes, arteries and individual cell (s).

As used herein, hybridization under the specified stringency conditions is used to describe the stability of a hybrid formed between two single-stranded DNA fragments, and after the hybrid is washed, it is less stringent than the stringency conditions of the wash step. It means conditions of ionic strength and temperature annealed under equivalent conditions. Typically, high, medium and low stringency includes the following or equivalent conditions:

1) High stringency: 0.1 x SSPE, 0.1% SDS, 65 ℃

2) Medium stringency: 0.2 x SSPE or SSC, 0.1% SDS, 50 ℃

3) Low stringency: 1.0 x SSPE or SSC, 0.1% SDS, 50 ° C.

Equivalent conditions means conditions that are selected for substantially the same percentage of disagreement in the resulting hybrid. The addition of components such as formamide, Ficoll and Denhardt solutions affects parameters such as the conditions under which hybridization is performed and the reaction rate. Thus, hybridization at 42 ° C. in 5 × SSC, 20% formamide is substantially the same as described above, which hybridizes under low stringency conditions. Secrets for the preparation of SSPE, SSC and Denhardt solutions and deionized formamides are described, for example, in Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Chapter 3; Sambrook et al., Vol. 3, p. B. 13; It is also described in a number of catalogs describing commonly used laboratory solutions. It will be appreciated that equivalent stringency can be achieved using other buffers, salts, and temperatures.

The term substantially the same or similar depends on the content as understood by one skilled in the art and generally means at least 40%, 80%, 90% 95% or 95% identity.

As used herein, substantially identical to a product means that the properties of interest are not sufficiently changed so that substantially the same product can be used in place of the product.

As used herein, target cell refers to a cell that expresses a cell surface protease.

As used herein, a test substance comprising a compound provided herein is defined chemically, in which the interaction with or effect on the cell surface protein or cell surface-attached protein or domain thereof is measured by the methods herein. Compounds (eg organic molecules, inorganic molecules, organic / inorganic molecules, proteins, peptides, nucleic acids, oligonucleotides, lipids, polysaccharides, saccharides or hybrids, especially molecules such as glycoproteins) or mixtures of compounds (eg : Library of test compound, natural extract or culture supernatant).

As used herein, the terms therapeutic, therapeutic regimen, radioprotectant, chemotherapeutic agent means conventional drugs and drug therapies, including vaccines known to those skilled in the art. Radiotherapy agents are known in the art.

As used herein, vectors (or plasmids) are discrete elements used to introduce heterologous DNA into cells for expression and / or replication thereof. The vector is typically maintained as an episome, but can be designed to integrate a gene or part thereof into the chromosome of the genome. Also contemplated are vectors that are artificial chromosomes, such as yeast artificial chromosomes or mammalian artificial chromosomes. Selection and use of such vehicles are known to those skilled in the art. Expression vectors include vectors capable of expressing such DNA operably linked to regulatory sequences, such as promoter regions, which may affect the expression of DNA fragments. Thus, expression vectors refer to recombinant DNA or RNA constructs such as plasmids, phages, recombinant viruses or other vectors which, when introduced into a suitable host cell, result in expression of the cloned DNA. Suitable expression vectors are well known to those skilled in the art and include vectors that integrate into a vector or host cell genome that are maintained as episomal and vector replicable in eukaryotic and / or prokaryotic cells.

As used herein, chemically stable means that the compound is sufficiently stable to be formulated for pharmaceutical use. Such chemical stability is well known to those skilled in the art and can be measured using conventional methods well known. Whether a given compound is sufficient to formulate for pharmaceutical use depends on a number of factors, including but not limited to the type and route of administration desired, the disease to be treated and the method of preparing the pharmaceutical formulation. do.

As used herein, a “functional equivalent” of a side chain of an amino acid is a group or moiety that functions in substantially the same way as a natural side chain, resulting in substantially the same result (eg, a substrate for cell surface proteases). For example, functional equivalents of the side chains of arginine include homoarginine, guanidinoaminopropyl, guanidinoaminoethyl, (Me) ₂ arginine side chain, (Et) ₂ arginine side chain, (4-aminomethyl) phenylmethyl, 4 -Amidinophenylmethyl, 4-guanidinophenylmethyl or conformationally bound arginine side chain analogs, eg (Where x is 0 or 1) [Webb et al. (1991) J. Org. Chem. 56: 3009], or three-dimensionally bound arginine side chain analogs, eg, , , , or (Where d is an integer from 0 to 5 or 1 to 3, W is N or CH or a mono- or polysubstituted N-alkyl derivative of the above groups, and alkyl is in certain embodiments a lower alkyl such as methyl) Including but not limited to.

As used herein, pharmaceutically acceptable derivatives of a compound include salts, esters, enol ethers, enol esters, acids, bases, solvates, hydrates or prodrugs of the compound. Such derivatives can be readily prepared by those skilled in the art using methods known for such derivatization. The resulting compounds can be administered to animals or humans without substantial toxic effects and are pharmaceutically active or prodrugs. Pharmaceutically acceptable salts include N, N'-dibenzylethylenediamine, chloroprocaine, chlorine, ammonia, diethanolamine and other hydroxyalkylamines, ethylenediamine, N-methylglucamine, procaine, N-benzyl Phenethylamine, 1-para-chlorobenzyl-2-pyrrolidin-1'-ylmethyl-benzimidazole, diethylamine and other alkylamines, piperazine and tris (hydroxy-methyl) aminomethane Amine salts not limited thereto; Alkali metal salts including but not limited to lithium, potassium and sodium; Alkaline earth metal salts, including but not limited to barium, calcium and magnesium; Inorganic acids, including but not limited to transition metal salts including but not limited to zinc and other metal salts including but not limited to sodium hydrogen phosphate and disodium phosphate, and also including, but not limited to, hydrochlorides and sulfates Salts and acetates, lactates, maleates, tartrates, citrates, ascorbates, succinates, butyrates, valerates and fumarates. Pharmaceutically acceptable esters include, but are not limited to, acidic alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, hetero, including carboxylic acid, phosphoric acid, phosphinic acid, sulfonic acid, sulfinic acid and boronic acid Salts of organic acids, including but not limited to aralkyl, cycloalkyl and heterocyclyl esters. Pharmaceutically acceptable enol ethers are of the formula C = C (OR), wherein R is hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl or heterocyclyl Including but not limited to derivatives of Pharmaceutically acceptable enol esters have the general formula C = C (OC (O) R) wherein R is hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl or hetero Derivatives of cyclyl). Pharmaceutically acceptable solvates and hydrates are compounds and one or more solvent or water molecules, generally 1 to about 100, typically 1 to about 10, for example 1 to about 2, 3 Or a complex with four solvents or water molecules.

As used herein, treatment means any means by which the symptoms of a condition, disease or disorder are ameliorated or otherwise advantageously changed. Treatment also includes any pharmaceutical use of the compositions herein, eg, for treating cancer.

As used herein, amelioration of a symptom of a particular disease by administration of a particular pharmaceutical composition, whether permanent or temporary, persistent or temporary, may be attributed to or may be associated with the administration of the composition. It means a decrease in symptoms.

As used herein, a prodrug is a compound that is metabolized or converted into a biologically, pharmaceutically or therapeutically active compound in vivo administration. To produce prodrugs, pharmaceutically active compounds are modified such that the active compounds are regenerated by metabolic processes. The prodrug may be designed to alter the metabolic stability or transport properties of the drug, mask side effects or toxicity, improve the flavor of the drug, or change other characteristics or properties of the drug. Knowledge of pharmacokinetic processes and drug metabolism in vivo allows a person skilled in the art to design prodrugs of such compounds once known pharmaceutically active compounds are known. Nogrady (1985) Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, pages 388-392.

It will be appreciated that the conjugates provided herein may contain chiral centers. The chiral center can be in the (R) or (S) configuration or a mixture thereof. Thus, the compounds provided herein can be enantiomerically pure or stereoisomeric or diastereomeric mixtures. In the case of amino acid residues, the residues may be L-type or D-type. Coordination of natural amino acid residues is generally L-type. Unless otherwise specified, the residue is L-shaped. It will be appreciated that the chiral centers of the compounds provided herein can be epimerized in vivo. Thus, those skilled in the art will recognize that for compounds to be epimerized in vivo, administration of the compound in the (R) form is equivalent to administration of the compound in the (S) form.

The conjugates provided herein are prodrugs because they include a therapeutic agent in an inactive form that is finally converted to the active form in the targeted cell or tissue or environment thereof. Upon exposure to the targeted protease, a biologically, pharmaceutically or therapeutically active form of the compound or a derivative that can be further metabolized into a biologically, pharmaceutically or therapeutically active form of the compound is released.

As used herein, "substantially pure" refers to standard analytical methods such as thin layer chromatography (TLC), gel electrophoresis and high performance liquid chromatography (HPLC) used by those skilled in the art to assess this purity. As homogenous enough to be homogeneous enough to appear to be free of detectable impurities or pure enough to be able to detectably change the physical and chemical properties such as enzymatic and biological activity of the material for the intended purpose. Means that. Methods for purifying compounds to produce substantially chemically pure compounds are known in the art. However, substantially chemically pure compounds may be mixtures of stereoisomers. In such cases, further purification may increase the specific activity of the compound.

As used herein, peptidic substrates include peptides and molecules such as peptides including peptide mimetics and peptide binding surrogate.

As used herein, specific subsites of the protease substrate: Pn ... P3-P2-P1 ↓ P1'-P2'-P3 '... Pn' to refer to conventional terms [see Schecter et. al. (1967) Biochem. Biophys. Res. Commun. 27: 157-162 is used. Cleavable bonds (ie cleavage sites) of the substrate are indicated by arrows. The N-terminal position of the bond means the unprimed position. Subregions are then numbered based on their distance from the cleavable bond. Amino acids (or amino acid surrogates) forming a cleavable bond are designated as 1 and adjacent residues are designated as 2 and the like and numbered from the cleavable bond. Thus, each specific subsite of the substrate is uniquely identified by the same number and meaning as primed or unprimed.

As used herein, a substitute for peptide bonds is a divalent group having steric and / or electronic characteristics similar to -C (O) NH-. Peptide binding substitutes are alkene isomeric systems (-CR = CR-), in particular (E) -alkene isomeric systems of the formula -CH = CH-, hydroxyethylene isomeric systems (-CH (OH) CH _2- ) , Enamine isomeric system (-C (= CRR) NH-), amino alcohol isomeric system (-CH (OH) CH ₂ NH-), difluoroketone isomeric system (-C (O) CF _2- ), Retroinverso compounds (-NHC (O)-), divalent heterocyclyl or heteroaryl groups and cyclopropyl isomeric systems, for example, Including but not limited to.

As used herein, alkyl, alkenyl and alkynyl carbon chains contain 1 to 20 carbons, generally 1 to 16 carbons, and are straight or branched, unless otherwise specified. Alkenyl carbon chains of 2 to 20 carbon atoms typically contain 1 to 8 double bonds and alkenyl carbon chains of 2 to 16 carbon atoms typically contain 1 to 5 double bonds. Alkynyl carbon chains of 2 to 20 carbon atoms typically contain 1 to 8 triple bonds, and alkynyl carbon chains of 2 to 16 carbon atoms generally contain 1 to 5 triple bonds. Exemplary alkyl, alkenyl and alkynyl groups herein include methyl, ethyl, propyl, isopropyl, isobutyl, n-butyl, secondary-butyl, tert-butyl, isopentyl, neopentyl, tert-pentyl and Including but not limited to isohexyl. Alkyl, alkenyl and alkynyl groups may be optionally substituted by one or more groups, generally alkyl groups, which are the same or different, unless otherwise specified. As used herein, lower alkyl, lower alkenyl and lower alkynyl refer to carbon chains having less than about 6 carbons. As used herein, "alk (en) (yn) yl" means an alkyl group containing one or more double bonds and one or more triple bonds.

As used herein, “cycloalkyl” typically refers to a saturated monocyclic or polycyclic ring system having 3 to 10 carbon atoms, eg, 3 to 6, wherein cycloalkenyl and cycloalkynyl are each one or more double bonds. And monocyclic or polycyclic ring systems comprising one or more triple bonds. Cycloalkenyl and cycloalkynyl groups contain, for example, 3 to 10 carbon atoms, wherein the cycloalkenyl group generally contains 4 to 7 carbon atoms and the cycloalkynyl group is, for example, 8 Contains from 10 to 10 carbon atoms. Ring systems of cycloalkyl, cycloalkenyl and cycloalkynyl groups may be combined into one ring or two or more rings which may be bonded together in a fusion, bridge or spiro-linked manner and optionally substituted by one or more alkyl group substituents. Can be done. "Cycloalk (en) (yn) yl" means a cycloalkyl group containing one or more double bonds and one or more triple bonds.

As used herein, "substituted alkyl," "substituted alkenyl," "substituted alkynyl," "substituted cycloalkyl," "substituted cycloalkenyl" and "substituted cycloalkynyl" Each one or more substituents, in certain embodiments alkyl, halo, haloalkyl (eg halo lower alkyl), pseudohalo, aryl, amino, dialkylamino, nitro, cyano, azido, alkylsulfinyl, alkylsulfonyl, Alkylcarbonylamino, alkoxycarbonylamino, aminoimino, hydroxy, alkoxy, aryloxy, alkyloxy, alkylthio, arylthio, aralkyloxy, aralkylthio, carboxy, alkylcarbonyl, alkoxycarbonyl, oxo and Alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl and cycloalkynyl groups substituted with one to three substituents independently selected from cycloalkyl.

As used herein, "aryl" means a cyclic group containing 6 to 19 carbon atoms. Aryl groups include, but are not limited to, groups such as fluorenyl, substituted fluorenyl, phenyl, substituted phenyl, naphthyl and substituted naphthyl. As used herein, “aryl” also means aryl-containing groups, including but not limited to aryloxy, arylthio, arylcarbonyl and arylamino.

As used herein, “heteroaryl” is generally one or more, for example, one to three atoms in the ring system, which are heteroatoms, ie, elements other than carbon, such as nitrogen, oxygen and sulfur atoms. By about 5 to about 15 membered means a monocyclic or polycyclic aromatic ring system. Heteroaryl groups may optionally be fused to benzene rings. Exemplary heteroaryl groups include, for example, furyl, imidazolyl, pyrrolidinyl, pyrimidinyl, tetrazolyl, thienyl, pyridyl, pyrrolyl, N-methylpyrrolyl, quinolinyl and isoquinolin Examples include, but are not limited to, nil, pyridyl, thienyl and quinolinyl.

As used herein, “heteroaryl” also means heteroaryl-containing groups including but not limited to heteroaryloxy, heteroarylthio, heteroarylcarbonyl and heteroarylamino.

As used herein, “heterocyclyl” refers to one or more, eg, one to three, valent heteroatoms in the ring system, ie, elements other than carbon, such as nitrogen, oxygen, and / or ring atoms. Phosphorus, 3- to 10-membered, for example 4- to 7-membered or 5- to 6-membered monocyclic or polycyclic unopened sulfur ring systems.

As used herein, "substituted aryl," "substituted heteroaryl" and "substituted heterocyclyl" are each selected from alkyl, cyclo-alkyl, cycloalkylalkyl, aryl, and halo, halo alkyl and alkyl. Heteroaryl, aralkyl, heteroaralkyl, alkenyl containing 1 to 2 double bonds, optionally substituted by 1 to 3 substituents, 1 to 2 triple bonds Containing alkynyl, alk (en) (yn) yl groups, halo, pseudohalo, cyano, hydroxy, haloalkyl and polyhaloalkyl (e.g. halo lower alkyl, in particular trifluoromethyl), formyl , Alkylcarbonyl, and arylcarbonyl, heteroarylcarbonyl, carboxy, alkoxycarbonyl, aryloxycarbon optionally substituted by one or more, generally one to three substituents selected from halo, halo alkyl and alkyl Neyl, aminoimino, alkoxycar Carbonylamino, aryloxycarbonylamino, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, arylaminocarbonyl, diarylaminocarbonyl, aralkylaminocarbonyl, alkoxy, aryloxy, perfluoroalkoxy , Alkenyloxy, alkynyloxy, arylalkoxy, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, arylaminoalkyl, amino, alkylamino, dialkylamino, arylamino, alkylarylamino, alkylcarbonylamino, aryl Carbonylamino, azido, nitro, mercapto, alkylthio, arylthio, perfluoroalkylthio, thiocyano, isothiocyano, alkylsulfinyl, alkylsulfonyl, arylsulfinyl, arylsulfonyl, amino One or more substituents independently selected from sulfonyl, alkylaminosulfonyl, dialkylaminosulfonyl and arylaminosulfonyl, in certain embodiments aryl, heteroaryl substituted with 1 to 3 substituents It means a heterocyclyl group.

As used herein, "aralkyl" refers to an alkyl group in which one hydrogen atom of alkyl is replaced with an aryl group.

As used herein, “heteroaralkyl” refers to an alkyl group in which one hydrogen atom of alkyl is replaced with a heteroaryl group.

As used herein, the terms alkyl, alkoxy, carbonyl and the like are used as generally understood by those skilled in the art. For example, as used herein, alkyl refers to a saturated carbon chain containing one or more carbons, wherein the chain may be straight or branched and may comprise a cyclic moiety or be cyclic.

If any number of optional substituents is not specified (eg, "haloalkyl"), one or more substituents may be present. For example, "haloalkyl" can include one or more identical or different halogens. As another example, “C _1-3 alkoxyphenyl” may include one or more of the same or different alkoxy groups containing one, two or three carbons.

As used herein, "halo", "halogen" or "halide" means F, Cl, Br or I.

As used herein, pseudohalides are compounds that behave substantially similarly to halides. Such compounds include halides ^may be used and treated in the same manner as (X, wherein X is a halogen such as Cl or Br). Pseudohalides include, but are not limited to, cyanide, cyanate, thiocyanate, selenocyanate, trifluoromethoxy, difluoromethoxy, dichloromethoxy and azide.

As used herein, "haloalkyl" refers to lower alkyl radicals in which one or more hydrogen atoms have been replaced by halogen. Such groups include, but are not limited to, chloromethyl, trifluoromethyl and 1-chloro-2-fluoroethyl and the like.

As used herein, "haloalkoxy" means RO-, where R is a haloalkyl group.

As used herein, "sulfinyl" or "thionyl" means -S (O)-. As used herein, "sulfonyl" or "sulfuryl" means -S (O) _2- . As used herein, “sulfo” means —S (O) ₂ O—.

As used herein, “carboxy” means a divalent radical, —C (O) O—.

As used herein, "aminocarbonyl" means -C (O) NH ₂ .

As used herein, "alkylaminocarbonyl" means -C (O) NHR, where alkyl is like R hydrogen or lower alkyl.

As used herein, “dialkylaminocarbonyl” as used herein is —C (O) NR′R wherein R ′ and R are independently selected from hydrogen or alkyl, eg, lower alkyl. ) And "carboxamide" means a group of the formula -NR'COR.

As used herein, “diarylaminocarbonyl” refers to —C (O) NRR ′ where R and R ′ are independently selected from aryl, such as hydrogen or lower aryl, eg, phenyl. it means.

As used herein, “aralkylaminocarbonyl” refers to C (O) NRR ′ wherein one of R and R ′ is aryl, such as lower aryl, eg phenyl and the other of R and R ′. Is alkyl, for example lower alkyl).

As used herein, "arylaminocarbonyl" means -C (O) NHR, where R is aryl, such as lower aryl, for example phenyl.

As used herein, "hydroxycarbonyl" means -COOH.

As used herein, "alkoxycarbonyl" means -C (O) OR, where R is alkyl, such as lower alkyl.

As used herein, "aryloxycarbonyl" means -C (O) OR, where R is aryl, such as lower aryl, for example phenyl.

As used herein, "alkoxy" and "alkylthio" means RO- and RS-, where R is alkyl, eg, lower alkyl.

As used herein, "aryloxy" and "arylthio" mean RO- and RS-, where R is aryl, such as lower aryl, for example phenyl.

As used herein, “alkylene” is, for example, a straight, branched or cyclic, eg straight or branched divalent hydrocarbon group having 1 to about 20 carbon atoms, eg, 1 to 12 carbon atoms, An example is lower alkylene. One or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms may optionally be inserted along the alkylene group, where the nitrogen substituents are alkyl as described above. Exemplary alkylene groups include methylene (-CH _2- ), ethylene (-CH ₂ CH _2- ), propylene (-(CH ₂ ) _3- ), cyclohexylene (-C ₆ H _10- ), methylenedi Oxy (-O-CH ₂ -O-) and ethylenedioxy (-O- (CH ₂ ) ₂ -O-). The term "lower alkylene" means an alkylene group having 1 to 6 carbon atoms. Exemplary alkylene groups include lower alkylenes such as alkylene having 1 to 3 carbon atoms.

As used herein, "alkenylene" is, for example, straight, branched or cyclic, typically having 2 to about 20 carbon atoms, generally 1 to 12 carbon atoms and having one or more double bonds. As a straight or branched divalent aliphatic hydrocarbon group, for example lower alkenylene. One or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms may optionally be inserted along the alkenylene group, where the nitrogen substituents are alkyl as described above. Exemplary alkenylene groups include —CH═CH—CH═CH— and —CH═CH—CH ₂ —. The term "lower alkenylene" refers to alkenylene groups having 2 to 6 carbon atoms. Exemplary alkenylene groups are lower alkenylene, for example alkenylene having 3 to 4 carbon atoms.

As used herein, "alkynylene" is, for example, straight, branched or cyclic, generally straight chain having 2 to 20, generally 1 to 12 carbon atoms and one or more triple bonds. Or branched divalent aliphatic hydrocarbon group, for example lower alkynylene. One or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms may optionally be inserted along the alkynylene group, where the nitrogen substituents are alkyl as described above. Exemplary alkynylene groups include -C≡CC≡C-, -C≡C-, and -C≡C-CH _2- . The term "lower alkynylene" means an alkynylene group having 2 to 6 carbon atoms. Exemplary alkynylene groups refer to alkynylene having 3 to 4 carbon atoms, eg, alkynylene groups having 3 to 4 carbon atoms. Exemplary alk (en) (phosphorus) ylenes are lower alk (en) (phosphorus) yls such as, for example, 4 carbon atoms.

As used herein, "alk (en) (in) ylene" has from 2 to about 20 carbon atoms and at least one triple bond and at least one double bond, typically, for example, lower alk (en A straight, branched or cyclic, generally straight or branched divalent aliphatic hydrocarbon group having 1 to 12 carbon atoms, such as (in) ylene. One or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms may optionally be inserted along the alkynylene group, where the nitrogen substituents are alkyl as described above. Exemplary alk (en) (in) ylene groups include -C = C- (CH ₂ ) _n -C≡C, where n is 1 or 2. The term "lower alk (en) (phosphorus) ylene" refers to alk (en) (phosphorus) ylenes having 6 or less carbon atoms.

As used herein, "cycloalkylene" generally refers to a divalent saturated monocyclic or polycyclic ring system having 3 to 10 carbon atoms, for example 3 to 6 carbon atoms, wherein cycloalkenylene and cycloalkynylene are each one It means a bivalent monocyclic or polycyclic ring system containing at least a double bond and at least one triple bond. Cycloalkenylene and cycloalkynylene groups may contain 3 to 10 carbon atoms, for example, cycloalkenylene groups contain 4 to 7 carbon atoms and cycloalkynylene groups may contain 8 to 10 carbon atoms Contains sweets The ring system of cycloalkylene, cycloalkenylene and cycloalkynylene groups can consist of one ring or two or more rings which can be bonded together in a fusion, bridge or spiro-linked manner. "Cycloalk (en) (in) ylene" means a cycloalkylene group containing at least one double bond and at least one triple bond.

As used herein, "substituted alkylene," "substituted alkenylene," "substituted alkynylene," "substituted cycloalkylene," "substituted cycloalkenylene," and "substituted cycloalkyi Nylene "means halo, haloalkyl (e.g., halo lower alkyl), aryl, hydroxy, alkoxy, aryloxy, alkyloxy, alkylthio, arylthio, aralkyloxy, aralkylthio, carboxy alkoxycarbonyl, oxo and One or more substituents independently selected from cycloalkyl, in certain embodiments alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene and cycloalkynylene groups substituted with one to three substituents.

As used herein, “arylene” is monocyclic or polycyclic, eg monocyclic, having, for example, 5 to about 20, eg, 5 to 12 carbon atoms and one or more aromatic rings. Divalent aromatic groups, for example lower arylene. One or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms can optionally be inserted around the arylene group, where the nitrogen substituents are alkyl as described above. Exemplary arylene groups include 1,2-, 1,3- and 1,4-phenylene. The term "lower arylene" means an arylene group having 5 to 6 carbon atoms. Exemplary arylene groups are lower arylene.

As used herein, “heteroarylene” refers to one or more, typically, for example, one to three valent heteroatoms in the ring system, ie, elements other than carbon, such as nitrogen, oxygen, and / Or a sulfur 5-membered to 15-membered divalent monocyclic or polycyclic aromatic ring system.

As used herein, “heterocyclylene” refers to one or more, eg, one to three, valent heteroatoms in the ring system, ie, elements other than carbon, such as nitrogen, oxygen, and / or sulfur atoms. (S), which are generally 3- to 10-membered, for example 4- to 7- or 5- to 6-membered bivalent monocyclic or polycyclic nonaromatic ring systems.

As used herein, "substituted arylene," "substituted heteroarylene" and "substituted heterocyclylene" are each selected from alkyl, cycloalkyl, cycloalkylalkyl, aryl, and halo, halo alkyl and alkyl. Heteroaryl, aralkyl, heteroaralkyl, alkenyl containing 1 to 2 double bonds, optionally substituted by 1 to 3 substituents, 1 to 2 triple bonds selected Alkynyl, alk (en) (yn) yl groups, halo, pseudohalo, cyano, hydroxy, haloalkyl and polyhaloalkyl, for example halo lower alkyl (e.g. trifluoromethyl) ), Formyl, alkylcarbonyl, and arylcarbonyl, heteroarylcarbonyl, carboxy, alkoxycarbono optionally substituted by one or more, for example, one to three substituents selected from halo, halo alkyl and alkyl. Aryl, aryloxycarbonyl, aminocarbonyl, Kylaminocarbonyl, dialkylaminocarbonyl, arylaminocarbonyl, diarylaminocarbonyl, aralkylaminocarbonyl, alkoxy, aryloxy, perfluoroalkoxy, alkenyloxy, alkynyloxy, arylalkoxy, amino Alkyl, alkylaminoalkyl, dialkylaminoalkyl, arylaminoalkyl, amino, alkylamino, dialkylamino, arylamino, alkylarylamino, alkylcarbonylamino, arylcarbonylamino, azido, nitro, mercapto, alkyl Thio, arylthio, perfluoroalkylthio, thiocyano, isothiocyano, alkylsulfinyl, alkylsulfonyl, arylsulfinyl, arylsulfonyl, aminosulfonyl, alkylaminosulfonyl, dialkylaminosulfonyl And one or more substituents independently selected from arylaminosulfonyl, in certain embodiments arylene, heteroarylene and heterocyclylene groups substituted with one to three substituents.

As used herein, "alkylidene" means a divalent group, such as = CR'R ", which is bonded to one atom of another group to form a double bond. Exemplary alkylidene groups are methylidene (= CH ₂ ) and ethylidene (= CHCH ₃ ) As used herein, “aralkylidene” refers to an alkylidene group wherein R ′ or R ”is an aryl group. A "cycloalkylidene" group is a group in which R 'and R "are joined to form a carbocyclic ring. A" heterocyclidene "group contains one or more of R' and R" in the chain and contains R atoms 'And R' are connected to form a heterocyclic ring.

As used herein, "amido" refers to the divalent group -C (O) NH-. "Tioamido" means the divalent group -C (S) NH-. "Oxiamido" means the divalent group -OC (O) NH-. "Tiaamido" means the divalent group -SC (O) NH-. "Dithiaamido" means the divalent group -SC (S) NH-. "Ureido" means the divalent group -HNC (O) NH-. "Tioureido" means the divalent group -HNC (S) NH-.

As used herein, “semicarbazide” means —NHC (O) NHNH—. "Carbazate" refers to the divalent group -OC (O) NHNH-. "Isothiocarbezate" means the divalent group -SC (O) NHNH-. "Tiocarbazate" means a divalent group -OC (S) NHNH-. "Sulfonylhydrazide" refers to the group -SO ₂ NHNH-. "Hydrazide" means the divalent group -C (O) NHNH-. "Azo" means a divalent group -N = N-. "Hydrazinyl" means a divalent group -NH-NH-.

As used herein, the term “amino acid” is an α-amino acid that is either racemate or in either D- or L-configuration. The name "d" before the amino acid name (eg dAla, dSer, dVal, etc.) refers to the D-isomer of the amino acid. The name "dl" before the amino acid name (eg dlPip) refers to a mixture of L- and D-isomers of an amino acid.

As used herein, when any particular group, such as phenyl or pyridyl, is specified, it means that the group is unsubstituted or substituted. Exemplary substituents are halo, halo lower alkyl and lower alkyl unless otherwise specified.

As used herein, abbreviations for any protective group, amino acid, and other compound, unless stated otherwise, refer to the IUPAC-IUB Committee on its general usage, approved abbreviations, or biochemical nomenclature (see (1972) Biochem . 11: 942-944.

As used herein, HHT and CHT mean hexahydrotyrosyl (also known as cyclohexyltyrosyl or p-hydroxycyclohexylalanyl), CHA is cyclohexylalanyl, Pyr and PyroGlu means pyroglutamic acid, Pip is pipecolinic acid, Sar is sarcosine, nLeu and Nle are norleucine, nVal is norvaline, Aib is 2-aminoisobutyric acid, Quat is (R) -Glu (α- (3-amidinobenzyl)) and Abu and But are 2-aminobutyric acid.

As used herein, PEG is a polyethylene glycol containing substituent having a specified number of ethyleneoxy subunits. Thus, the term PEG (2) The term PEG (6) Indicates.

When R ¹ and R ² are combined to form-(CH ₂ ) _h- , the cyclic residues and heteroatom-containing cyclic residues thus defined are , , , , , , , , , Including but not limited to. As used herein, the term “hydroxylation” refers to the substitution by hydroxyl moieties on the substitutable carbons of the ring systems described as such. As used herein, the term “polyhydroxylation” refers to substitution by two, three or four hydroxyl residues on two or more substitutable carbons of the ring system described as such.

As used herein, the term “(d) (2,3-dihydroxypropionyl)” refers to the structure Indicates.

As used herein, the term "(2R, 3S) -2,3,4-trihydroxybutanoyl" refers to the structure Indicates.

As used herein, the term "quinyl" refers to a structure Or diastereomers thereof.

As used herein, the term "gluronyl" refers to a structure Or diastereomers thereof.

As used herein, the term "cotininyl" refers to a structure Or diastereomers thereof.

As used herein, the term "galyl" refers to a structure Indicates.

As used herein, the term "4-ethoxyscuaryl" refers to the structure Indicates.

As used herein, 1-methylHis or (1Me) H is a structure Indicates.

As used herein, 3-methylHis or (3Me) H structure Exit

As used herein, Quat ² is a structure Quat ³ represents the structure Quat ⁴ Quat ⁵ represents the structure Indicates.

Other abbreviations used herein are as follows:

Abbreviation meaning Aib 2-aminoisobutyryl 4,4-dimethylThr 2-amino-3-hydroxy-4-methylpentanoyl Met (O ₂ ) Methioninyl-S, S-Dioxide Ser (OMe) O-methyl ether of serinyl, known as 2-amino-3-methoxypropanoyl hSer Also known as homoserinyl, 2-amino-4-hydroxybutanoyl (hS) Gly N- (2-hydroxyethyl) glycyl N, N-dimethylGly N, N-dimethylglycyl β-Ala 3-aminopropanoyl Cys (Me) S-methylcysteinyl t-Butyl Gly 2-amino-3,3-dimethylbutanoyl F (Gn) 4-guanidinylphenylalanyl hCHA Homocyclohexylalanyl or 2-amino-4-cyclohexylbutanoyl Hexyl Gly 2-aminooctanoyl Gly 2-amino-4-pentenoyl Inact. Inert NT Not tested MeOEtCO 3-methoxypropanoyl 3,4-methyldioxyPhAc 3,4-methylenedioxyphenylacetyl L-3-Ph Lactyl L-2-hydroxy-3-phenylpropanoyl MeOEtOCO 2-methoxyethoxycarbonyl MeOCO Methoxycarbonyl MeO (EtO) 2Ac 2- (2-methoxyethoxy) ethoxyacetyl 2-pyridylAc 2-pyridylacetyl PhOAc Phenoxyacetyl MeOAc Methoxyacetyl PhAc Phenylacetyl MeOEtOAc 2-methoxyethoxyacetyl HOOCButa Glutaryl Z Benzyloxycarbonyl EtOCO Ethoxycarbonyl βA Beta-alanyl or 3-aminopropanoyl NapAc 1-naphthylacetyl iBoc Isobutoxycarbonyl HOAc Hydroxyacetyl MeSucc 3-methoxycarbonylpropanoyl Succ Succinyl HCO Formyl 4- (Guan) Phg 4-guanidinylphenylglycosyl Dox Doxorubicin Tax Taxol dA (Chx) or dCha d-cyclohexylalanyl dhF d-homophenylalanyl P (OH) 4-hydroxyprolyl

B. Protease Targets

The conjugate herein is designed to target proteases located on the cell surface, particularly tumor cells and cells involved in oncogenic processes and angiogenesis and other proliferative processes. The conjugates described in detail below contain a peptidic substrate for a selected targeted cell surface protease, linked directly or via a linker, to a therapeutic agent, typically a cytotoxic agent, which is substantially inert. The therapeutic agent is released in active form or in a form that can be activated in the vicinity of the targeted cell or tissue to which the therapeutic agent is delivered. As a result, the active therapeutic agent accumulates in or accumulates in the targeted cell or tissue.

Targeted proteases are located in cells or tissues involved in the disease process or by chance in the vicinity of cells or tissues involved in the disease or disease process and are generally, if not all, never in many other cells or tissues or Selection is made by identifying proteases that are absent or that exhibit activity or altered activity or specificity at lower levels, generally substantially lower, in such cells or tissues. The type and number of untargeted cells or tissues expressing an active protease will depend on the particular protease and the disease to be treated. Those skilled in the art will select targets based on acceptable levels of disease, targeted agents and side effects. The goal is to achieve enhanced therapeutic indices compared to when the targeted agent is administered by itself.

Targeted proteases may or may not be involved in the disease process, the expression of which may be incidental, and for this purpose, their specific role or lack thereof is not critical and is substantially targeted at the site of the targeted tissue or cell. It is important whether the protease is active. For example, many cell surface proteins of interest herein are expressed or active in tumor cells or cells involved in oncogenic processes. Any method known to those skilled in the art for measuring or detecting tissue or cell expression profiles can be used. For example, RNA blots composed of RNA from numerous tissues (eg, multiple crude expression (MTE) arrays (CLONTECH, Palo Alto, CA)) can be screened with probes based on the nucleic acid sequence of the protease of interest, Cells expressing proteases can be identified. In addition, Northern blot analysis can be used to test expression.

Among the targeted proteases are the type II membrane-bound serine proteases (MTSP; see International PCT Application No. 09 / 776,191, filed February 2, 2001 and International PCT Application WO 01/57194). PCT / US01 / 03471; International PCT Application No. PCT / US02 / 07903; US Provisional Patent Application Nos. 60 / 275,592, 60 / 278,166, 60 / 279,228, 60 / 291,001, 60 / 291,501 60 / 316,818, 60 / 302,939, 60 / 316,818, 60 / 328,529, 60 / 328,530, 60 / 332,015, 60 / 328,939, and Agent Case No. 24745-P1624 Provisional Patent Application, filed May 20, 2002; filed May 14, 2002, under U.S. Application Nos. 10 / 099,700, 10 / 104,271, 10 / 112,221, Representative Event No. 24745-1616 Application) and proteases found in endothelial cells, designated as endothelial agents (see US Application No. 09 / 717,473, filed Nov. 20, 2000 and International PCT Application WO 01/36604). Published international PCT application PCT / US00 / 31803; Also included are SEQ ID NOs: 3 to 26, 269 to 270, and 272 to 276.

In addition, proteases located on the cell surface due to specific interactions with cell surface proteins are contemplated. An example of such a protease is the urokinase plasminogen activator (u-PA) bound to the urokinase plasminogen activator receptor (u-PAR). Nucleic acid sequence information and expression profiles of exemplary MTSPs and endotheliases are as follows (see also Example 6).

MTSP

Cell surface proteolysis is a mechanism for producing biologically active proteins that mediate various cellular functions. These membrane-attached proteins include disintegrin-type and metalloproteinases (ADAM) and membrane-type matrix metalloproteinases (MT-MMP). In addition to MMPs, serine proteases have been involved in neoplastic disease progression. Most serine proteases that are secreted enzymes or sequestered in intracellular storage organelles play a role in blood clotting, wound healing, digestion, immune responses, and tumor invasion and metastasis.

Transmembrane serine protease (MTSP) appears to be involved in the etiology and pathogenicity of tumors. These enzymes are expressed in cells associated with certain cancerous and tumor cells and other proliferative disorders and other disease states, for example inflammatory cells, and may be tissue specific or organ specific. In mammals, at least 20 members of the family are known. Hooper et al. (2001) J. Biol. Chem. 276: 857-860, and also U.S. Application Nos. 09 / 776,191 and PCT / US01 / 03471, filed Feb. 2, 2001; U.S. Provisional Patent Applications 60 / 275,592 and 60 / 278,166 number; And SEQ ID NOs: 1 to 37. These include Coryne (Approval Nos. AF133845 and AB013874; See Yan et al. (1999) J. Biol. Chem. 274: 14926-14938; Tomia et al. (1998) J. Biochem. 124: 784-789; Uan et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97: 8525-8529; Enterpeptidase [also named enterokinase; Accession number U09860 for human proteins; See Kitamoto et al. (1995) Biochem. 27: 4562-4568; Yahagi et al. (1996) Biochem. Biophys. Res. Commun. 219: 806-812; Kitamoto et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91: 7588-7592; Matsushima et al. (1994) J. Biol. Chem. 269: 19976-19982; Human airway trypsin-type protease [HAT; Authorization number AB002134; See Yamaoka et al. J. Biol. Chem. 273: 11894-11901; MTSP1 [also named TADG-15 and Matriptase; See SEQ ID NOs: 1 and 2; Authorization Nos. AF133086 / AF118224, AF4280022; Takeuchi et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96: 11054-1161; Lin et al. (1999) J. Biol. Chem. 274: 18231-18236; Takeuchi et al. (2000) J. Biol. Chem. 275: 26333-26342; and Kim et al. (1999) Immunogenetics 49: 420-429; Hepsin (see Accession Nos. M18930, AF030065, X70900); Leytus et al. (1988) Biochem. 27: 11895-11901; Vu et al. (1997) J. Biol. Chem. 272: 31315-31320; and Farley et al. (1993) Biochem. Biophys. Acta 1173: 350-352; And US Pat. No. 5,972,616; TMPRS2 (see Accession Nos. U75329 and AF113596); Paoloni-Giacobino et al. (1997) Genomics 44: 309-320; and Jacquinet et al. (2000) FEBS Lett. 468: 93-100; And TMPRSS4 (Accession No. NM 016425; Wallrapp et al. (2000) Cancer 60: 2602-2606. Also known MTSP3, MTSP4, MTSP6, MTSP7, MTSP9, MTSP10, MTSP12, MTSP20, MTSP22 and MTSP25 (see SEQ ID NOs: 3 to 26, 269 to 270 and 272 to 276; International PCT Application No. PCT / US01 / 03471, also published as US Application No. 09 / 776,191, filed February 2, 2001 and International PCT Application WO 01/57194; International PCT Application No. PCT / US02 / 07903; Also, U.S. Provisional Patent Application Nos. 60 / 275,592, 60 / 278,166, 60 / 279,228, 60 / 291,001, 60 / 291,501 60 / 316,818, 60 / 302,939, 60 / 316,818 , Provisional Patent Application, filed May 20, 2002 under Nos. 60 / 328,529, 60 / 328,530, 60 / 332,015, 60 / 328,939 and Representative Case No. 24745-P1624; US Application Nos. 10 / 099,700, 10 / 104,271, 10 / 112,221, applications filed May 14, 2002 under Agent Case No. 24745-1616).

Serine proteases, including transmembrane serine proteases, have been involved in the processes involved in neoplasia and progression. Although the exact role of the protease has not been described in detail, serine proteases and inhibitors thereof are numerous intracellular and extracellular physiological, including degradative activity, metastatic spread, and neovascularization of tumors in cancer cell invasion involved in tumor progression. It is involved in the regulation of the process. Proteases are involved in the degradation of extracellular matrix (ECM), contribute to tissue remodeling, and are thought to be necessary for cancer invasion and metastasis. The activity and / or expression of some proteases have been shown to correlate with tumor progression and development and have also been shown to be active in certain cell types.

For example, epithelial cancer and normal tissues [Takeucuhi et al. (1999) Proc. Natl. Acad. Sci. USA 96: 11054-61; membrane-type serine protease MTSP1 (also referred to as Matriptase: SEQ ID NOs: 1 and 2 in US Pat. No. 5,972,616; and Genbank Accession No. AF118224; (1999) J. Biol Chem. 274: 18231-18236; US Pat. No. 5,792,616; Takeuchi (1999) Proc. Natl. Acad. Sci. USA 96: 11054-1161, which has been suggested to play a role in metastasis of breast cancer. Its main cleavage specificity is Arg-Lys residues Matriptase is also expressed in various epithelial tissues with high levels of activity and / or expression in the human gastrointestinal tract and prostate.

Hepcine, a cell surface serine protease identified in liver cancer cells, is overexpressed in ovarian cancer. Tanimoto et al. (1997) Cancer Res., 57 (14): 2884-7]. Hepcine transcripts appear to be abundant in carcinoma tissues and are rarely expressed in normal adult tissues including normal ovaries. Heptin is often overexpressed in ovarian cancer and has been suggested to be a candidate protease in the invasive process and growth capacity of ovarian cancer.

A serine protease-type gene named Normal Epithelial Cell-Specific 1 (NES1) (Liu et al., Cancer Res., 56 (14): 3371-9 (1996)) has been identified. Although expression of NES1 mRNA is observed in all normal and immortalized non-tumoral epithelial cell lines, most human breast cancer cell lines appear to have sharply reduced or completely lacked its expression. Structural similarity of NES1 to polypeptides known to modulate growth factor activity and inverse correlation of NES1 expression with breast tumor development suggest a direct or indirect role for this protease-type gene product in the inhibition of tumor development. .

Example MTSP

Each MTSP has a characteristic tissue expression profile, which MTSP, in particular, is not only expressed or activated in the tumor, but exhibits a characteristic tumor tissue expression or activation profile. In some cases, MTSPs may have different activity than non-tumor cells in tumor cells due to changes in the substrate or cofactors thereof or other elements that may alter the functional activity of MTSP. Thus, each is a diagnostic marker for a particular tumor due to activity and / or expression level or function in a subject having a neoplastic disease (ie, a mammal, especially a human) as compared to a subject or subject not suffering from a neoplastic disease. Can act as In addition, detection of activity (and / or expression) in certain tissues may be an indicator of neoplastic disease. In addition, each activity and / or expression profile allows the MTSP to act as a therapeutic target, for example, by administering its activity modulator or by administering a prodrug that is specifically activated by one of the MTSPs. Each MTSP or either one may exhibit activity or expression levels or substrate specificities that differ in levels from tumor cells in tumor cells. Such tumor cells include, but are not limited to, colon, lung, prostate, breast, esophagus, pancreas, cervix, uterus, endometrial tumors and other solid tumors and blood and lymphatic tumors. Thus, the conjugates provided herein can be designed by selecting substrate specificities for the treatment of either of these tumors and neoplastic conditions.

Tissue expression profile

Exemplary tissue and gene (see Example 8) profiles of some exemplary MTSPs are as follows. These profiles are not intended to define the full range of expression or activation of these MTSPs, but because MTSPs are expressed in tumors, expression or activation or substrate specificity at the surface of tumor cells may be used in the methods herein and one of these MTSPs. A conjugate designed according to the methods herein and as exemplified by the above can be prepared and used to treat neoplastic or other diseases or conditions, or targeted to cells expressing these proteins on the surface. Prove that.

MTSP1 (Matriptase)

MTSP1 (also referred to as Matriptase) is a trypsin-type serine protease with a broad spectrum of cleavage activity and two potential regulatory modules. MTSP1 was named "Matriptase" based on its ability to degrade extracellular matrix and its trypsin-type activity. When isolated from breast cancer cells (or T-47D cell conditioned medium), MTSP1 has been reported to exist mainly in uncomplexed form. MTSP1 was isolated from human breast milk, and when isolated from human breast milk, it was reported to exist in one of two complexed forms, 95 kDa (main form) and 110 kDa, and uncomplexed MTSP1 was not detected. Liu, et al. (1999) J. Biol. Chem. 274: 18237-18242. MTSP1 has been proposed to exist as an uncomplexed protease when active. In breast milk, matriptase has been reported to exist in complex with fragments of hepatocyte growth factor inhibitor-1 (HAI-1), a Kuntz-type serine protease inhibitor that is active against trypsin-type serine protease. .

Nucleic acids encoding proteins termed Matriptase have been cloned from T-47D human breast cancer cell-conditioned media. Lin et al. (1999) J. Biol. Chem. 274: 18231-18236. In the analysis of cDNA, the full length protease has 683 amino acids and three main structural regions, the serine protease adjacent to the carboxyl-terminal region, four tandem low-density lipoprotein receptor domains and two tandem complement subcomponents C1r and C1s ( It was determined to contain SEQ ID NO: 1). Studies to identify additional serine proteases produced by cancer cells were performed using PC-3 cells. The serine protease, designated “MT-SP1” by the authors, reported to be a transmembrane protease, has been cloned. Takeuchi et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96: 11054-11061. It was later found that the translated sequence of the cDNA encoding MT-SP1 included the initially identified Matriptase sequence. The nucleic acid initially encoding the protein named metliptase is a partial MTSP1 clone lacking 516 of the coding nucleotides. Takeuchi et al., J. Biol. Chem. 275: 26333-26342 (2000). Since the reported Matriptase coding cDNA sequence encodes a possible starting methionine, alternating splicing has been suggested to be able to produce a protein lacking the N-terminal region of MTSP1. Thus, Matriptase is a variant of MTSP1 herein.

MTSP1 is a type II transmembrane protein that exhibits trypsin-type protease activity and has an extracellular protease domain. Studies on substrate specificity of MTSP1 include protease-activated receptor 2 (IPAR2), pro-hepatocyte growth factor (pre-HGF) and single-chain urokinase-type plasminogen activator (sc-uPA). Denotes the macromolecular substrate of MTSP1. PAR2 functions in inflammation, cytoprotection and / or cell adhesion, while sc-uPA functions in tumor cell invasion and metastasis. HGF acts as a growth and angiogenesis factor.

Exemplary nucleotide sequences encoding human MTSP1 are shown in SEQ ID NOs: 1 and 2. As noted above, SEQ ID NO: 1 presents the MTSP1 encoding nucleic acid sequence. Such sequences are longer and contain protease domains common to both variants.

MTSP1 is expressed in breast, prostate and colon tumors. Thus, conjugates with substrates for MTSP1 can be used for the treatment of these tumors.

MTSP3

MTSP3 transcripts were detected in lung carcinoma (LX-1), colon adenocarcinoma (CX-1), colon adenocarcinoma (GI-112) and ovarian carcinoma (GI-102). No obvious signals were detected in other forms of lung carcinoma (GI-117), breast carcinoma (GI-101), pancreatic adenocarcinoma (GI-103) and prostate adenocarcinoma (PC3).

MTSP4

The MTSP4 transcript, the DNA fragment encoding the portion of the LDL receptor domain, and the protease domain were used to probe RNA blots composed of 76 different human tissues (Cat. No. 7775-1; Human Multiple Tissue Expression (MTE) array; CLONTECH). Was used. As in the northern gel blot analysis, a very strong signal was observed in the liver. Signals from other tissues were observed (at decreasing signal levels) as follows: fetal liver> heart = kidney = adrenal = testes = fetal heart and kidney = skeletal muscle = bladder = placenta> brain = spinal cord = colon = stomach = spleen = Lymph node = bone marrow = organ = uterus = pancreas = salivary gland = mammary gland = lung. MTSP4 is also less abundantly expressed in several tumor cell lines, including: Hela S3 = leukemia K-562 = Burkitt's lymphoma (Raji and Daudi) = colorectal adenocarcinoma (SW480)> lung carcinoma (A549) = leukemia MOLT-4 = leukemia HL-60. PCR of MTSP4 transcripts from a cDNA library (human tumor multi-tissue cDNA panel, catalog number K1522-1, manufactured by CLONTECH) made from several human primary tumors transplanted into nude mice was carried out using MTSP4-specific primers. Was performed. MTSP4 transcripts were detected in breast carcinoma (GI-101), lung carcinoma (LX-1), colon adenocarcinoma (GI-112) and pancreatic adenocarcinoma (GI-103). No obvious signals were detected in other forms of lung carcinoma (GI-117), colon adenocarcinoma (CX-1), ovarian carcinoma (GI-102) and prostate adenocarcinoma (PC3). MTSP4 transcript was also detected in the LNCaP and PC-3 prostate cancer cell lines as well as the HT-1080 human fibrosarcoma cell line.

MTSP6

MTSP6 is expressed at high levels in the colon. It is also expressed in the stomach, airways, mammary glands, thyroid gland, salivary glands, pituitary gland and pancreas. It is expressed at low levels in other tissues (see Example 6).

MTSP6 is also expressed in several tumor cell lines, including: HeLa S3> Rectal Colorectal Adenocarcinoma (SW480)> Leukemia MOLT-4> Leukemia K-562. In mouse xenograft models, MTSP6 transcripts were strongly detected in lung carcinoma (LX-1), moderately detected in pancreatic adenocarcinoma (GI-103), weakly detected in ovarian carcinoma (GI-102) and colon adenocarcinoma (GI). -112 and CX-1), breast carcinoma (GI-101), lung carcinoma (GI-117) and prostate adenocarcinoma (PC3). MTSP6 transcript was also detected in breast cancer cell line MDA-MB-231, prostate cancer cell line PC-3, but not in HT-1080 human fibrosarcoma cell line. MTSP6 is also expressed in mammary carcinoma cDNA (Clontech). MTSP6 is also overexpressed in ovarian tumor cells.

MTSP7

MTSP7 transcript was detected in lung carcinoma (A549 cell line), leukemia (K-562 cell line) and cervical carcinoma (HeLaS3 cell line). MTSP7 is thought to be expressed in lung, colon, prostate, breast, cervical tumors and other tumors.

MTSP9

MTSP9 is expressed, for example, in esophageal tumor tissue, lung carcinoma, colorectal carcinoma, lymphoma, cervical carcinoma (HeLaS3) and leukemia cell lines, as well as certain carcinoma cells and tissues. MTSP9 may also be a marker for breast, prostate, cervical and rectal cancers.

MTSP9 is highly expressed in the esophagus and at low levels in many other tissues. MTSP9 transcripts include kidneys (adults and fetuses), spleen (adults and fetuses), placenta, liver (adults and fetuses), thymus, peripheral blood leukocytes, lungs (adults and fetuses), pancreas, lymph nodes, bone marrow, organs, uterus, It is found in the prostate, testes, ovaries, and glandular organs (mammary gland, adrenal gland, thyroid gland, pituitary gland and salivary gland). MTSP9 is also expressed in esophageal tumor tissue and lung carcinoma and at low levels in colorectal carcinoma, lymphoma, cervical carcinoma (HeLaS3) and leukemia cell lines.

MTSP10

MTSP10 is expressed, for example, in esophageal tumor tissue, lung carcinoma, prostate cancer, pancreatic and breast cancer and cell lines, as well as certain cells and tissues (eg see examples for tissue specific expression profiles). The level of activated MTSP10 may be a diagnostic indicator of prostate cancer, uterine cancer, lung esophageal cancer or rectal cancer or leukemia or other cancers. Expression and / or activation of MTSP10 on or near cells or in body fluids in a subject can be a marker of breast cancer, prostate cancer, lung cancer, colon cancer, esophageal cancer and other cancers.

MTSP10 transcript was detected in the pancreas, lung and kidney. MTSP10 transcript was also detected in small intestine Marathon-Ready cDNA (Clontech). MTSP10 transcripts were detected in breast carcinoma (GI-101), lung carcinoma (LX-1 and GI-117), ovarian carcinoma (GI-102), and pancreatic adenocarcinoma (GI-103). MTSP10 transcript was weakly detected in prostate adenocarcinoma (PC3). MTSP10 transcript was also detected in CWR22R prostate tumors grown in nude mice. No obvious signal was detected in two forms of rectal adenocarcinoma (GI-112 and CX-1).

MTSP12

MTSP12 transcript was detected in the pancreas, lung and kidney. MTSP12 transcript was also detected in small intestine marathon-ready cDNA (Clontech). MTSP12 transcripts were detected in breast carcinoma (GI-101), lung carcinoma (LX-1 and GI-117), ovarian carcinoma (GI-102) and pancreatic adenocarcinoma (GI-103). MTSP12 transcript was weakly detected in prostate adenocarcinoma (PC3). MTSP12 transcript was also detected in CWR22R prostate tumors grown in nude mice. No obvious signal was detected in two types of colon adenocarcinoma (GI-112 and CX-1).

MTSP20

MTSP20 is expressed in lung, colon, cervical tumor and leukemia cells. It can also be expressed in breast, ovary, pancreas, prostate tumors and other tumors. MTSP20 transcripts were detected in the liver, lymph nodes, cerebellum, pancreas, prostate, uterus, testes, glands (adrenal gland, thyroid and salivary glands), thymus, kidney and spleen. Low transcript levels have been found in the lungs, placenta, bladder, ovaries, digestive system, circulatory system and other parts of the brain. MTSP20 is also a specific tumor, including lung carcinoma (A519), colorectal carcinoma (SW480), lymphoma (Raji and Daudi), cervical carcinoma (HeLaS3) and leukemia (HL-60, K-562 and MOLT-4) cell lines. It is expressed in cell lines.

MTSP22

MTSP22 is expressed in uterine tissue, thymus, adipose tissue and lymph nodes. It can also be expressed in lung, stomach, uterus, breast, ovary, prostate tumors and other tumors. MTSP22 transcript was detected in some uterine tissue samples, but not in matching tumor samples thereof. In one of the 42 uterine samples, MTSP22 is expressed in tumors and metastatic tissues thereof, but not in normal tissue counterparts. MTSP22 is also expressed in some gastric and lung tumors but not in its normal tissue counterparts. MTSP22 is also expressed in normal tissue matched cDNA pairs of the pancreas. MTSP22-encoding cDNA was detected in thymus, adipose tissue and lymph nodes.

MTSP25

MTSP25 is expressed in breast, colon, uterus, ovary, kidney, prostate and testicular cancer tissues. It can also be expressed in lung, stomach, prostate tumors and other tumors. MTSP25 transcript was weakly expressed in lymph nodes. In cancer profiling array analysis, MTSP25 is highly expressed in prostate samples (normal and cancer samples). MTSP25 was highly expressed in kidney tumor samples but not in its normal tissue counterparts. MTSP25 was also expressed in breast cancer samples but not in its normal tissue counterparts. MTSP25 was expressed in normal uterine samples but not in their tumor sample counterparts. MTSP25 was also expressed in ovarian cancer samples. Of the three samples, expression of MTSP25 was also detected in one of the matched normal tissue counterparts. MTSP25 expression was also detected in colon cDNA pairs in tumor samples.

PCR analysis indicated that MTSP25 cDNA was strongly detected in testicular and mammary adenocarcinoma, weakly detected in brain, placenta, lung, spleen, prostate, small intestine, colon and leukocytes, and very weakly in heart, liver and pancreas.

2. Endotheliase

Endothelia refers to cells involved in various proliferative processes, including undesirable angiogenesis associated with tumor growth and proliferation and other hyperproliferative disorders (e.g. restenosis, scars, diabetic retinopathy, diseases and disorders in the anterior eye), In particular endothelial cells, in particular the cell surface protease family expressed in proliferating endothelial cells (see US Application No. 09 / 717,473 and International PCT Application No. PCT / US00 / 31803, filed November 20, 2000).

Proliferative disease

Endothelial agents are particularly useful targets for the delivery of therapeutic agents for the treatment of any disorder associated with abnormal angiogenesis. Endothelial cells play an important role in angiogenesis, which is the creation of new blood vessels from parental microvessels. Angiogenesis plays a large role in the metastasis of cancer and the pathology of various other disorders.

Regulated angiogenesis and unqualified angiogenesis proceed in a similar manner. Peripheral and endothelial cells surrounded by the basement membrane form capillaries. Angiogenesis begins with erosion of the basement membrane by enzymes released by endothelial cells and leukocytes. Subsequently, endothelial cells surrounding the lumen of the blood vessel protrude through this basement membrane. Angiogenic stimulators induce these endothelial cells to migrate through this eroded basement membrane. The migrating cells form "sprouts" that extend from the parent vessels, where endothelial cells proliferate and proliferate. These endothelial clouds form capillary loops that integrate with each other to create new blood vessels.

Angiogenesis, Regulators and Related Diseases

Angiogenesis is highly regulated by angiogenic stimulator and suppressor systems. Examples of known angiogenic stimulators include certain growth factors, cytokines, proteins, peptides, carbohydrates and lipids (Norby (1997) APMIS 105: 417-437); Polverini (1995) Crit. Rev. Oral. Biol. Med. 6: 230-247. Various endogenous and exogenous angiogenesis inhibitors are known in the art. See Jackson et al. (1997) FASEB 11: 457-465; Norrby (1997) APMIS 105: 417-437); and O'Reilly (1997) Investigational New Drugs, 15: 5-13.

Angiogenesis is essential for normal placenta, embryo, fetus, and postnatal development and growth, but most physiological events do not occur in adulthood except in very specific limited circumstances. For example, angiogenesis is commonly observed during wound healing, fetal and embryonic development and formation of the corpus luteum, endometrium and placenta. Angiogenesis in adults is often associated with disease states.

Unregulated angiogenesis occurs in many disease states, tumor metastases and abnormal growth of endothelial cells and supports the pathological damage seen in these conditions. A variety of pathological disease states that result in uncontrolled angiogenesis were classified as angiogenic dependent or angiogenic related diseases.

The regulation of angiogenesis is altered in certain disease states, and in many cases the pathological damage associated with such disease is associated with unregulated angiogenesis (generally, Norrby (1997) APMIS 105: 417-437); and O'Reilly (1997) Investigational New Drugs 15: 5-13. Thus, angiogenesis is a specific ocular disorder including various diseases, such as various inflammatory diseases such as rheumatoid arthritis, psoriasis, diabetic retinopathy, pterygium recurrence, scarring after excimer laser surgery and glaucoma removal, and various disorders of the anterior eye. Involved in obvious signs or progression of cancer, including chronic inflammatory diseases, wound repair, circulatory disorders, crest syndrome, skin diseases (US Pat. Nos. 5,593,990, 5,629,327 and 5,712,291) and solid neoplasms and vascular tumors do. Angiogenesis is essential for the growth and maintenance of solid tumors and their metastasis. Inhibition, elimination or regulation of this activity should affect the pathogenesis of the diseases and act as a therapeutic intervention point. In disease states, prevention of angiogenesis can avoid the damage caused by new microvascular invasion. Therapies aimed at regulating angiogenic processes can lead to the elimination or alleviation of these diseases. Thus, there is a need to develop therapeutic agents that target angiogenesis and regulate, particularly inhibit, abnormal or unregulated angiogenesis.

Thus, conjugates containing endothelial substrates may be used to treat rheumatoid arthritis, psoriasis, diabetic retinopathy, pterygium recurrence, scarring after excimer laser surgery and glaucoma removal, and certain eye disorders including various disorders in the anterior eye, cardiovascular disorders, autoimmunity. Diseases, chronic inflammatory diseases, wounds, circulatory disorders, crest syndrome, restenosis, psoriasis and other skin disorders (see US Pat. Nos. 5,593,990, 5,629,327 and 5,712,291) and solid neoplasms and vascular tumors It can be used to deliver therapeutic agents for the treatment of diseases including but not limited to cancer.

Endotheliase 1 and 2

Examples of endotheliases are two different endotheliases and variants called endotheliase 1 and endotheliase 2 (see SEQ ID NOs: 21-27). Other members of the family have at least 40%, 60%, 80%, 90%, 95%, or 98% sequence identity with the protease domain of the endothelial enzyme identified herein, or endothelial agents provided herein under high stringency conditions. This can be identified by hybridizing along the entire length of the nucleic acid encoding the protease domain of and probing genes expressed in endothelial cells or by examining libraries for these genes.

Alternatively and as a way of identifying endotheliase that may have low sequence identity, the endothelial agent identifies EST or other nucleic acid fragments having sequences similar to proteases, and then uses these fragments as probes to determine full length protease or The cDNA clones encoding their protease domains can be identified and selected using methods such as identifying and selecting those that are characteristic of transmembrane proteins, and then measuring gene expression profiles to identify those expressed on the surface of endothelial cells. Can be. Encoded proteins that have protease activity and that include transmembrane domains and extracellular domains and are expressed in endothelial cells are endothelial agents. Contemplated herein are proteins encoding a transmembrane protease expressed in endothelial cells or any method for identifying genes encoding such proteins.

Endotheliase 1

Examples of endotelases include endotelase 1 and endotelase 2. They are expressed in endothelial cells. An example of full-length endothelial agent 1 is an endothelial agent comprising the amino acid sequence set forth in SEQ ID NO: 42. See International PCT Application No. WO 00/5006, which describes a gene named DESC1 expressed in squamous cell carcinoma and prostate tumor. number]. As noted, endothelial agents are expressed in endothelial cells. Its protease domain is shown in SEQ ID NO: 22.

Expression Profile of Endotheliase 1

To obtain information related to the tissue distribution of endotheliase 1, the DNA insert of clone H117 was selected from 76 different human tissues (Cat. No. 7775-1; human multiple tissue expression (MTE) array; manufactured by CLONTECH, Palo Alto, RNA blot consisting of CA) was used for probing. Significant expression was observed in the esophagus, and low expression levels were observed in the stomach, salivary glands, pancreas, prostate, bladder, organs and uterus. Northern analysis using RNA blots (Catalog Nos. 7765-1 and 7782-1; Human Muscle and Digestive System Multiple Tissue Northern (MTN) blot; CLONTECH, Palo Alto, Calif.) Confirmed that expression was restricted to the esophagus. Two transcripts (about 1.7 and 2 kb) were detected in the esophagus. Endothelial 1 is also expressed in PC3 and LnCAP cells, which are cord blood endothelial cells.

Nucleic Acids Encoding Endotheliase 2 and Endotolia 2

Two splice variants of endothelial agent 2, designated as endoteliase 2-S and endothelial 2-L, are exemplified herein (see SEQ ID NOs: 23-26). The open reading frame of the nucleic acid encoding endothelial 2-S (SEQ ID NO: 23) consists of 1,689 bp transcribed into 562 amino acid proteins (SEQ ID NO: 24), and the ORF of endothelial 2-L is It consists of 2,067 bp (SEQ ID NO: 25) transcribed into 688 amino acid proteins (SEQ ID NO: 26).

The nucleic acid encoding the protease domain of endotelase 2-S consists of 729 bp transcribed into 242 amino acid proteins (amino acids 321 to 562 of SEQ ID NOs 23 and 24) and encodes the protease domain of endoteliase 2-L. The nucleic acid is composed of 1,107 bp transcribed to 368 amino acid proteins (amino acids 321 to 688 of SEQ ID NO: 25 and 26).

Endothelia-2 protein

Any and all of the above-mentioned endotheliases and / or protease domains thereof as encoded by nucleic acids comprising the amino acid sequences of SEQ ID NOs: 22, 24, 26 and 27 or hybridizing to such sequences under the conditions described above Contemplated for use in the methods herein. Also contemplated herein are proteins that contain amino acid sequence changes as shown in Table 1 above and retain protease activity.

Gene Expression Profile and Transcript Size of Endotheliase 2 in Normal and Tumor Tissues

In addition to expression in endothelial cells, endothelia 2 is expressed in placenta, pancreas, thyroid, liver and lung tissue. It is also expressed at low levels in the mammary gland, salivary glands, kidneys, organs, esophagus, caecum, heart and fetal lung. Endotheliase 2 is also expressed in certain tumor cell lines, including some tumor cell lines and lung tumors and colon tumors, and including breast carcinomas, lung carcinomas, colon adenoma, pancreatic adenocarcinoma (GI-103) and ovarian carcinoma. It has also been detected in prostate and fibrosarcoma cell lines.

C. Conjugate

Conjugates are provided that are substrates for proteases on the cell surface, in particular serine proteases, including type II membrane-bound serine proteases and endotheliases. Any cell surface protease, including cell-attached or cell-localized proteases, is contemplated herein. In general, proteases expressed as active forms at high levels in essential tissues are not ideal target candidates. Such proteases include proteases that are expressed in a relatively limited number of cells or appear at disease levels in the vicinity of cells that are expressed at high levels in cells such as endothelial cells and immune cells involved in tumor cells and disease states or are involved in such disease states. do. For example, endothelial cells are involved in numerous proliferative disorders due to their role in angiogenesis, and immune cells are involved in a number of disease processes including cancer and disease and inflammatory diseases. Other cell surface proteases are expressed at higher levels in certain tumors than normal cells. Regardless of whether such proteases play a role in a disease, their higher expression in cells involved in the disease state is sufficient to use the therapeutic agents in the conjugates provided herein for targeting.

Conjugates containing therapeutic agents such as cytotoxic agents are activated upon cleavage by cell surface proteases, including cell-attached and cell-localized proteases. Examples of such proteases include MTSPs including, but not limited to, MTSP1, MTSP3, MTSP4, MTSP6, MTSP7, MTSP9, MTSP10, MTSP12, MTSP20, MTSP22, MTSP25, urokinase, and endoteliase. Thus, conjugates targeted to these proteases are prodrugs that are inactive when the therapeutic agent is administered and finally activated in the vicinity of the targeted cell or tissue. Cell surface proteases, such as transmembrane proteases are intended targets, but any protease, such as released, secreted or soluble form, may be targeted.

Thus, therapeutic agents such as cytotoxic agents, conjugates containing peptide residues and optionally linkers, which are substrates (ie, peptidic substrates) for targeted cell surface proteases, are substantially inert before the cell surface protease acts. The therapeutic agent in the conjugate is activated when the peptide substrate of the conjugate is cleaved by the cell surface protease. A therapeutic agent, such as a cytotoxic agent, is released as a free agent, or a peptide substrate (P1-P2-P3-etc. (Ie, N-terminus) or P1'-P2 to which the agent was linked in the conjugate, optionally via a linker). Is released in the vicinity of the cell expressing the protease In certain embodiments, activation is achieved by a therapeutic agent such as a cytotoxic agent. This is achieved because after the action of the cell surface protease it can cross the cell membrane or interact with the cell or tissue to exhibit therapeutic activity In another embodiment, any residual peptidic residue or amino acid can be cleaved from the therapeutic agent to make the therapeutic agent active. The conjugate acts as a prodrug because it is substantially inactive when the therapeutic agent is conjugated. Upon cleavage, the therapeutic agent is released in the active form or in a form that is activated by the targeted cell or tissue or the surrounding environment.

In one exemplary embodiment, the targeted agent is a cytotoxic agent and the conjugate for use in the methods and compositions provided herein comprises a compound of formula (peptide ⁱ ) _s- (linker) _q- (cytotoxic agent) _t [where the peptide ⁱ is a peptidic substrate for cell cell surface proteases or released, secreted or other unbound proteases, s is at least 1, 1 to 6 or 1 or 2 or 1, the linker is any linker, q is at least 0 or 0-4 or 0 or 1, the cytotoxic agent is an anti-tumor, anticancer or antimitotic agent, including antiangiogenic agents, and t is at least 1 or 1 or 2], or Derivatives thereof. In such conjugates, the cytotoxic agent is covalently linked via the linker, optionally to the C-terminus or N-terminus of the peptidic substrate. In embodiments in which a therapeutic agent such as a cytotoxic agent is bound to the C-terminus of the peptide substrate, the N-terminus is optionally capped. N-terminal caps for use herein include, but are not limited to, acyl, sulfonyl and carbamoyl groups. In embodiments in which the therapeutic agent is bound to the N-terminus of the peptidic substrate, the C-terminus is a carboxamide derivative.

In certain embodiments, since peptide ⁱ is a peptide surface substrate for cell surface proteases or soluble MTSPs, substantially inactive conjugates cleave P1-P1 'bonds upon action of such proteases, such as cytotoxic activity in vitro and in vivo. It releases a compound of the formula (peptide ^a ) _s- (linker) _q- (therapeutic agent) _t or a derivative thereof that exhibits therapeutic activity. In such compounds, peptide ^a is a truncated form of peptide ⁱ resulting from cleavage at the P1-P1 'bond.

In another embodiment, the conjugate for use in the methods and compositions provided herein is the same, optionally linked via linker ¹ and linker ² , respectively, to the C-terminus and N-terminus of the peptidic substrate for cell surface protease or soluble MTSP. Or therapeutic agents such as two different cytotoxic agents. In this embodiment, the conjugate is a compound of formula (Therapeutic ¹ ) _x- (Linker ¹ ) _w- (Peptide ⁱ ) _s- (Linker ² ) _q- (Therapeutic ² ) _t [wherein peptide ⁱ is directed to a cell surface protease or soluble MTSP Is a peptidic substrate, s is at least 1 or 1 to 6 or 1 or 2 or 1, linker ¹ and linker ² are each independently any linker and are the same or different, and q and w are each independently at least 0 or 0 To 4 or 0 or 1, wherein the therapeutic agent is the same or differently an antitumor agent, an anticancer agent or an antimitotic agent, and t and x are each independently one or more or 1 or 2] or a derivative thereof.

In this embodiment, the peptide ⁱ is a peptide surface substrate for cell surface protease or soluble MTSP, so that a substantially inactive conjugate is cleaved at one point of the peptide chain upon action of such protease to yield the formula (Therapeutic ¹ ) _x- (Linker ¹ 2 compounds of) _w- (peptide ^a1 ) _s and (peptide ^a2 ) _s- (linker ² ) _q- (therapeutic agent ² ) _t or derivatives thereof. The released therapeutic agents are either active or further activated by cells, tissues or the surrounding environment. In these compounds, peptide ^a1 and peptide ^a2 are the N-terminal and C-terminal truncated portions, respectively, of peptide ⁱ resulting from cleavage at the P1-P1 'bond.

In one embodiment, the conjugate for use in the compositions and methods provided herein is a compound of Formula (I) or a derivative thereof.

X ^n- (P6) _m- (P5) _p- (P4) _i- (P3) _j- (P2) _l -P1- (P1 ') _u- (P2') _k- (P3 ') _r- (L ) _n -Z

In the above formula,

Z is a cure,

L is a linker,

l, j, i, p and m are chosen as follows:

l is 0 or 1, when l is 0, j, i, p and m are 0, when l is 1 j is 0 or 1 and when j is 0 i, p and m are 0 I is 0 or 1 when j is 1, p and m are 0 when i is 0, p is 0 or 1 when i is 1, and m is 0 when p is 0 When p is 1, m is 0 or 1,

u, k and r are selected as follows:

u is 0 or 1, when u is 0 k and r are 0, when u is 1 k is 0 or 1, when k is 0 r is 0 and k is 1 r is 0 or 1,

n is 0 or 1,

X ⁿ is hydrogen or acyl, sulfonyl or carbamoyl cap,

P6 to P3 'are amino acid residues as defined below.

In this embodiment, the P6 to P3 residues are linked by peptide bonds or peptide bond surrogate. Thus, the P6 to P3 'portions of the conjugates are peptidic substrates as defined herein.

In another embodiment, the conjugate for use in the compositions and methods provided herein is a compound of Formula II or a derivative thereof.

Z- (L) _n- (P6) _m- (P5) _p- (P4) _i- (P3) _j- (P2) _l -P1- (P1 ') _u- (P2') _k- (P3 ') _r -X ^c

In the above formula,

Z is a cure,

L is a linker,

l, j, i, p and m are chosen as follows:

l is 0 or 1, when l is 0 j, i, p and m are 0, when l is 1 j is 0 or 1 and when j is 0 i, p and m are 0 I is 0 or 1 when j is 1, p and m are 0 when i is 0, p is 0 or 1 when i is 1, and m is 0 when p is 0 When p is 1, m is 0 or 1,

u, k and r are selected as follows:

u is 0 or 1, k and r are 0 when u is 0, k is 0 or 1 when u is 1, r is 0 when k is 0, and k is 1 r is 0 or 1,

n is 0 or 1,

X ^c together with the carbonyl group of the bound amino acid residue form a carboxylic acid or carboxamide group,

P6 to P3 'are amino acid residues as defined below.

In this embodiment, the P6 to P3 'residues are linked by peptide bonds or peptide bond surrogate. Thus, the P6 to P3 'portions of the conjugates are peptidic substrates as defined herein.

In a further aspect, the conjugate for use in the compositions and methods provided herein is a compound of Formula III or a derivative thereof.

Z ^1- (L ¹ ) _n- (P6) _m- (P5) _p- (P4) _i- (P3) _j- (P2) _l -P1- (P1 ') _u- (P2') _k- (P3 ') _r- (L ² ) _v -Z ²

In the above formula,

Z ¹ and Z ² are each a therapeutic agent and are the same or different,

L ¹ and L ² are each a linker and are the same or different,

l, j, i, p and m are chosen as follows:

l is 0 or 1, when l is 0 j, i, p and m are 0, when l is 1 j is 0 or 1 and when j is 0 i, p and m are 0 I is 0 or 1 when j is 1, p and m are 0 when i is 0, p is 0 or 1 when i is 1, and m is 0 when p is 0 When p is 1, m is 0 or 1,

u, k and r are selected as follows:

u is 0 or 1, k and r are 0 when u is 0, k is 0 or 1 when u is 1, r is 0 when k is 0, and k is 1 r is 0 or 1,

n and v are each independently 0 or 1,

P6 to P3 'are amino acid residues as defined below.

In this embodiment, the P6 to P3 'residues are linked by peptide bonds or peptide bond surrogate. Thus, the P6 to P3 'portions of the conjugates are peptidic substrates as defined herein.

In another embodiment, the conjugate for use in the compositions and methods provided herein is a compound of Formula IV or a derivative thereof.

X ^n- (P6) _m- (P5) _p- (P4) _i- (P3) _j- (P2) _l -P1- (P1 ') _u- (P2') _k- (P3 ') _r- (P4 ') _s- (L) _n -Z

In the above formula,

Z is a cure,

L is a linker,

l, j, i, p and m are chosen as follows:

l is 0 or 1, when l is 0 j, i, p and m are 0, when l is 1 j is 0 or 1 and when j is 0 i, p and m are 0 I is 0 or 1 when j is 1, p and m are 0 when i is 0, p is 0 or 1 when i is 1, and m is 0 when p is 0 When p is 1, m is 0 or 1,

u, k, r and s are selected as follows:

u is 0 or 1, when u is 0, k, r and s are 0, when u is 1, k is 0 or 1, when k is 0, r and s are 0 and k is R is 0 or 1 when 1, s is 0 when r is 0, s is 0 or 1 when r is 1,

n is 0 or 1,

X ⁿ is hydrogen or acyl, sulfonyl or carbamoyl cap,

P6 to P4 'are amino acid residues as defined below.

In this embodiment, the P6 to P4 'residues are linked by peptide bonds or peptide bond surrogate. Thus, the P6 to P4 ′ portions of the conjugates are peptidic substrates as defined herein. In another embodiment, the conjugate for use in the compositions and methods provided herein is a compound of Formula (V) or a derivative thereof.

Z- (L) _n- (P6) _m- (P5) _p- (P4) _i- (P3) _j- (P2) _l -P1- (P1 ') _u- (P2') _k- (P3 ') _r- (P4 ') _s -X ^c

In the above formula,

Z is a cure,

L is a linker,

l, j, i, p and m are chosen as follows:

l is 0 or 1, when l is 0 j, i, p and m are 0, when l is 1 j is 0 or 1 and when j is 0 i, p and m are 0 I is 0 or 1 when j is 1, p and m are 0 when i is 0, p is 0 or 1 when i is 1, and m is 0 when p is 0 When p is 1, m is 0 or 1,

u, k, r and s are selected as follows:

u is 0 or 1, when u is 0, k, r and s are 0, when u is 1, k is 0 or 1, when k is 0, r and s are 0 and k is R is 0 or 1 when 1, s is 1 when r is 0, s is 0 or 1 when r is 1,

n is 0 or 1,

X ^c together with the carbonyl group of the bound amino acid residue form a carboxylic acid or carboxamide group,

P6 to P4 'are amino acid residues as defined below.

In this embodiment, the P6 to P4 'residues are linked by peptide bonds or peptide bond surrogate. Thus, the P6 to P4 ′ portions of the conjugates are peptidic substrates as defined herein.

In a further aspect, the conjugate for use in the compositions and methods provided herein is a compound of Formula VI: or a derivative thereof.

Z ^1- (L ¹ ) _n- (P6) _m- (P5) _p- (P4) _i- (P3) _j- (P2) _l -P1- (P1 ') _u- (P2') _k- (P3 ') _r- (P4') _s- (L ² ) _v -Z ²

In the above formula,

Z ¹ and Z ² are each a therapeutic agent and are the same or different,

L ¹ and L ² are each a linker and are the same or different,

l, j, i, p and m are chosen as follows:

l is 0 or 1, when l is 0 j, i, p and m are 0, when l is 1 j is 0 or 1 and when j is 0 i, p and m are 0 I is 0 or 1 when j is 1, p and m are 0 when i is 0, p is 0 or 1 when i is 1, and m is 0 when p is 0 When p is 1, m is 0 or 1,

u, k, r and s are selected as follows:

u is 0 or 1, when u is 0, k, r and s are 0, when u is 1, k is 0 or 1, when k is 0, r and s are 0 and k is R is 0 or 1 when 1, s is 0 when r is 0, s is 0 or 1 when r is 1,

n and v are each independently 0 or 1,

P6 to P4 'are amino acid residues as defined below.

In this embodiment, the P6 to P4 'residues are linked by peptide bonds or peptide bond surrogate. Thus, the P6 to P3 'portions of the conjugates are peptidic substrates as defined herein.

Exemplary peptidic substrates, therapeutic agents, linkers and exemplary conjugates of Formulas I-VI are described in more detail below. It is intended herein to include within the scope of the invention conjugates resulting from all combinations and / or permutations of the groups mentioned below for the various formulas (I)-(VI).

1. Peptide Substrates

The peptidic substrate contemplated for use in the conjugate is a targeted cell surface protease or a soluble, secreted or released form thereof and contains a sufficient number of amino acid residues to allow any therapeutic agent in the conjugate to be substantially inactive. In an exemplary embodiment, where the therapeutic agent is for example doxorubicin, the conjugate is substantially inert since the conjugated therapeutic agent cannot cross the cell membrane. In certain embodiments, the peptidic substrate contains 1, 2, 3, 4 or 5 or more amino acid residues and may contain up to 9 or 10 residues. Longer peptidic substrates can be used in such conjugates where the therapeutic or therapeutic-amino acid or therapeutic-peptide residue conjugate obtained upon cleavage exhibits the desired therapeutic effect in vivo and in vitro.

Thus, exemplary peptide substrates for use in the conjugates provided herein have one or more amino acids (P1), two amino acids (P1-P1 '), three amino acids (P2-P1-P1') and are typically Contains 4, 5 or 6 amino acid residues (P3-P2-P1-P1 ', P4-P3-P2-P1-P1' or P4-P3-P2-P1-P1'-P2 '), Wherein P1-P1 ′ binding is, for example, cell surface protease, such as but not limited to uPA, MTSP, and endothelia bound to receptor, cell surface protease such as but not limited to cell surface protease or soluble thereof, Cleavage site in secreted or released form. The peptidic substrate optionally further has a P5, P6 or P3 'amino acid residue, and in certain embodiments has a P7, P8, P9, P10, P4', P5 ', P6' residue. Thus, the peptidic substrates for use in the conjugates provided herein are pepta-, hexa-, hepta-, octa- and nona-peptidic substrates, the therapeutic or therapeutic agent-amino acids or therapeutic agents obtained upon cleavage of the conjugate by proteases. When the peptidic residue conjugate exhibits the desired therapeutic effect in vivo and in vitro, it may contain 10, 11, 12, 13, 14, 15 or more residues.

The peptidic substrate may be linked to a therapeutic agent (or linker to which the therapeutic agent is linked) via a C-terminal residue (ie, P1 ', P2' or P3 ') or an N-terminal residue (ie, P6, P5 or P4) or optionally through an internal residue. Are bonded. The peptidic substrate can be, for example, straight chain but can be cyclized or can comprise cyclized sites.

In embodiments where the conjugation is through the C-terminus of the peptidic substrate, the peptidic substrate optionally has a cap such as an acyl or carbamoyl cap at the N-terminus. In embodiments where the conjugation is through the N-terminus of the peptidic substrate, the peptidic substrate further has a terminal group, such as a carboxamide group, at the C-terminus.

The conjugate may contain multiple peptide substrates and multiple therapeutic agents. For example, in a conjugate containing two therapeutic agents that are the same or different, conjugation to the therapeutic agent (s) or linkers linked thereto may be through the C-terminal and N-terminal residues of the peptidic substrate.

The methods described for the selection of such substrates can be used to design suitable substrates. In addition, the substrate can be designed based on the known specificity of other proteases. For example, trypsin-type and specificity of trypsin family members may be helpful in designing possible substrates. Substrate preferences for specific serine proteases are summarized as follows: Harris et al. (2000) PNAS 97 (14): 7754-7759.

Protease Exemplary P1 Residue (s) Exemplary P2 Residue (s) Exemplary P3 Residue (s) Chymotrypsin Tyr, Phe, Trp - - Trypsin Arg, Lys - - Thrombin Arg, Lys Phe Thr, Trp Plasmin Lys, Arg Trp, Tyr, Met Gln Granzyme B Asp - - Human neutrophil elastase Ala, Val, Ile - - Tissue Plasminogen Factor Arg Ser, Gly, Ala Met, Tyr Eurokinase Arg Ser, Ala Thr, Ser Factor Xa Arg Gly -

Conventional protocols for the preparation of conjugates include (1) identifying a targeted protease; (2) expression and assay development steps; (3) identifying, for example, substrates cleaved by the selected target protease by testing chromogenic or fluorescent substrates, and identifying peptide peptides cleaved by the targeted protease using substrate phage displays, Selecting the substrate by using a natural protein or peptide substrate or a natural inhibitor of the protease and using a combinatorial library to identify the substrate cleaved by the targeted protease; (4) synthesizing the conjugates containing the identified substrates, and (5) in vitro assays, cell culture assays, biological assays, and in vivo animal models (see, eg, Example 10). And, biologically assessing the conjugate.

The conjugate can be designed using any method known to those skilled in the art. The following provides an example protocol. First, a series of commercially available chromogenic and fluorescent peptide substrates can be tested for cleavage by proteases of interest (see the list of exemplary chromogenic and fluorescent substrates and examples in the table below). The peptidic portions of these substrates occupy the unprimed binding site of the protease and the reporter group is located on the primed side of the cleavable bond. The effective conjugate can then be designed based on the structure of the substrate that is effectively cleaved by the protease.

The peptidic portion of the substrate that is cleaved effectively can be used as the unprimed region of the conjugate and can be used as a Ser-therapeutic agent (eg, cytotoxic agent (eg doxorubicin)), Ser-Leu-therapeutic agent or Ser-Ser-Leu- The therapeutic agent can be used as the primed region of the conjugate. Cleavage of the conjugate prodrugs releases a Ser-, Ser-Leu-, or Ser-Ser-Leu-therapeutic compound. In another embodiment, the Ser in the released Ser-therapeutic may be replaced with other amino acids, including but not limited to Ala, hSer, Abu, Thr, Met, nLeu and Val. In another embodiment, for example, when the therapeutic agent is doxorubicin, the amino acid residues conjugated to the therapeutic agent have a hydrophilic side chain. Such amino acid residues include, but are not limited to, Leu, Abu, nLeu, nVal, CHA, hCHA, (hexyl) Gly, (allyl) Gly, (propargyl) Gly and (cyclopropyl) Ala. In another embodiment, for example, where the therapeutic agent is Taxol, the amino acid residues conjugated to the therapeutic agent have side chains that are not stericly large in size. Such amino acids include but are not limited to Gly and Ala. The P1'-therapeutic, P1'-P2'-therapeutic or P1'-P2'-P3'-therapeutic compounds obtained can be further treated with active therapeutic agents in vivo.

Another approach to design conjugate prodrugs for protease substrates is to use substrate phage notation to reveal the optimal subsite occupancy for the protease. The information obtained can then be used to design the unprimed peptidic portion of the conjugate. As described above, the primed region of the conjugate may be immobilized as a Ser-therapy, Ser-Leu-therapeutic or Ser-Ser-Leu-therapeutic.

A third approach to designing effective prodrug conjugates involves the use of combinatorial fluorescent substrate libraries to determine optimal residues for the unprimed regions of the protease substrate. This selected sequence can then be used as the unprimed portion of the conjugate prodrug and Ser-therapeutics (eg doxorubicin), Ser-Leu-therapeutics or Ser-Ser-Leu-therapeutics can be used as primed regions of the conjugate. have. These methods were used to design the peptidic substrate portion of the conjugates provided herein. For example, sequences comprising GSGR (and related sequences such as TGR, SGR and extended variants, etc.) are based on or derived from substrate phage notation experiments using u-PA as a target protease. Many Matriptase conjugates such as (R / K) -X-S-R and X- (R / K) -S-R and related sequences provided herein are based on data from combinatorial libraries. In another embodiment, sequences in natural substrates or natural inhibitors of protease targets such as VSPA, PGR (derived from P3-P1 of plasminogen) and related sequences are used in the design of u-PA-targeted conjugates. . In another embodiment, sequences from chromogenic substrates such as D-HHT-Gly-Arg and related sequences are used in the design of ET-1-targeted conjugates.

Chromic / Fluorescent Substrate

enzyme temperament rescue MTSP1 Spectrozyme t-PA CH ₃ SO ₂ -D-HHT-Gly-Arg-pNA.AcOH MTSP1 S 2765 N-α-Z-D-Arg-Gly-Arg-pNA.2HCl MTSP3 Spectrozyme fXIIa H-D-CHT-Gly-Arg-pNA.2AcOH MTSP4a Spec PL H-D-Nle-HHT-Lys-pNA.2AcOH MTSP5 S 2765 N-α-Z-D-Arg-Gly-Arg-pNA.2HCl MTSP6 Spectrozyme t-PA CH ₃ SO ₂ -D-HHT-Gly-Arg-pNA.AcOH MTSP7 S 2366 Fatigue Glu-Pro-Arg-pNA.HCl MTSP9 Pefachrome fVIIa CH ₃ SO ₂ -D-CHA-But-Arg-pNA MTSP10 Spectrozyme t-PA CH ₃ SO ₂ -D-HHT-Gly-Arg-pNA.AcOH MTSP22 S 2366 Fatigue Glu-Pro-Arg-pNA.HCl ET-1 Spectrozyme t-PA CH ₃ SO ₂ -D-HHT-Gly-Arg-pNA.AcOH ET-2 S 2765 N-α-Z-D-Arg-Gly-Arg-pNA.2HCl u-PA S-2444 Fatigue Glu-Gly-Arg-pNA.HCl ^a coupled assay, activation of plasminogen in the presence of Spec PL

Briefly stated, for coupled assays, the ability of proteases to activate enzymes such as plasminogen or trypsinogen is tested. To carry out this assay, the protease is described above in the presence of a known known substrate for a simogen, such as plasminogen or trypsinogen, for example lys-plasminogen or Spec PL (for plasmin). Incubate with Simogen. If the protease activates the simogen, activated enzymes such as plasmin and trypsin will degrade the substrate by changing the spectral properties of the substrate.

Exemplary Peptide Substrates

The description below provides a general discussion of the properties of exemplary peptide substrates and residues for cleavage by proteases such as MTSP1 (or Matriptase), endotheliase 1, and urokinase. In a similar manner, peptide substrates for cleavage by other cell surface proteases or soluble, secreted or released forms thereof, identify peptide substrates for the selected protease and then prepare conjugates containing such peptide substrates. This can be designed similarly.

a. P1 residue

Amino acid residues for use at the P1 position of the peptidic substrate for use in the conjugates provided herein include Arg, Arg surrogate and Lys. Arg surrogate is a P1 residue in a substrate for a non-natural amino acid (e.g., MTSP1, urokinase or endoteliase) that has a group or moiety that functions in substantially the same way as the natural side chain of arginine, resulting in substantially the same result. Function). Arg surrogates include side chains of homoarginine; Guanidinoaminopropyl; Guanidinoaminoethyl; (Me) ₂ arginine side chains; (Et) ₂ arginine side chains; (4-aminomethyl) phenylmethyl; 4-amidinophenylmethyl; Arg substitutes include, but are not limited to, α-amino acids having any of 4-guanidinophenylmethyl as side chains (Where z is 0 or 1) [Webb et al. (1991) J. Org. Chem. 56: 3009 or a conformationally bound arginine analogue such as , , , or Conformationally bound arginine side chains or mono- or di-substituted N-alkyl derivatives of the above groups such as d is an integer from 0 to 5 or from 1 to 3, and W is N or CH, wherein alkyl Is lower alkyl, for example methyl).

In certain embodiments herein, the P1 residue is Arg.

b. P2 residue

In the conjugates provided herein, the P2 residues are Phe, Ser, Gly, Ala, Ser (OMe), hSer, 1-methylHis, 3-methylHis, His, nVal, nLeu, Abu, (hS) Gly, Thr, Selected from Aib, CHA and Tyr. In another embodiment, the P2 residue is selected from Phe, Ser, Gly and Ala. In certain embodiments herein, the P2 residue is Ser or Ala. In another embodiment, the P2 residue is Gly or Ala.

c. P3 residue

Amino acid residues for use at the P3 position of the conjugates provided herein include Arg, Lys, Gln, Quat, Arg surrogate, Ser, Thr, hSer, dSer, Pro, (hS) Gly, Tyr, 4,4-dimethylThr , Asn, Met (O ₂ ), Quat ² , Quat ³ , Quat ⁴ and Quat ⁵ . In other embodiments, the P3 residue is selected from Arg, Lys, Gln, Quat and Arg surrogate. Arg surrogates include those described above for P1.

In certain embodiments, the P3 residue is Gln or Ser.

d. P4 residue

In the conjugates provided for use in the compositions and methods provided herein, the P4 residues are Pro, Arg, Ser, Ala, Lys, Gly, nLeu, Leu, Tyr, Glu, Phe, Val, N, N-dimethylGly, β- Ala, Cys (Me), Gln, t-butyl Gly and nVal. In other embodiments, the P4 residue is selected from Pro, Arg, Ser, Ala, Lys, Gly, nLeu, Leu, Tyr, Glu, Phe and Val. In further embodiments, the P4 residue is selected from Pro, Arg, Ser, Ala, Lys, Gly, nLeu, Phe or Val. In certain embodiments herein, the P4 residue is Arg or Gly.

e. P5 and P6 residues

In certain embodiments herein, the peptidic substrate used in the conjugate contains P5 and optionally P6 residues. P5 residues include Ile, Arg and Arg surrogate. In other embodiments, the P5 residues include Arg and Arg surrogate. Arg surrogates include those described above for the P1 residue. P6 residues include, for example, Leu, Val and Arg. In another embodiment, the P6 residues include, for example, Leu.

f. P1 'residue

P1 ′ residues of the conjugates provided herein are Gly, Ser, Ala, Leu, Ile, d-Ile, nLeu, Val, nVal, Aib, Abu, Met, 6-aminohexanoyl, Thr or hSer. In other embodiments, the PI 'residues of the conjugates provided herein are Gly, Ser, Ala, Leu, Ile, d-Ile, nLeu, Val, nVal, Aib, Abu, Met or 6-aminohexanoyl. In another embodiment, the PI 'residue is Ser, Ala, hSer, Abu, Thr, Met, nLeu or Val. In another embodiment, the PI 'residue is Gly or Ala. In another embodiment, the PI 'residue is Ser, Ala or Gly. In another embodiment, the PI 'residue is Leu, Abu, nLeu, nVal, CHA, hCHA, (hexyl) Gly, (allyl) Gly, (propargyl) Gly or (cyclopropyl) Ala. In certain embodiments herein, the PI 'residue is Ala, Ser, Gly, Ile or d-Ile.

g. P2 'residue

In certain embodiments herein, the conjugates provided herein have a P2 'residue. P2 ′ residues for use herein include Gly, Ser, Ala, Leu, Ile, d-Ile, nLeu, Val, nVal, Aib, Abu, Met, 6-aminohexanoyl, hCHA, CHA, hexylGly, allylGly And Phe. In other embodiments, P2 ′ residues for use herein include, but are not limited to, Gly, Ser, Ala, Leu, Ile, d-Ile, nLeu, Val, nVal, Aib, Abu, Met and 6-aminohexanoyl Do not. In another embodiment, the P2 ′ residue is Ser, hSer, Abu, nLeu, nVal, CHA, hCHA, (allyl) Gly or (hexyl) Gly. In another embodiment, the P2 ′ residue is Gly or Ala. In another embodiment, the P2 ′ residue is Leu, Abu, nLeu, nVal, CHA, hCHA, (hexyl) Gly, (allyl) Gly, (propargyl) Gly or (cyclopropyl) Ala. In further embodiments, the P2 ′ residue is Ala, Gly, Ile or d-Ile.

h. P3 'residue

In other aspects herein, the peptidic substrate used in the conjugates provided herein comprises a P3 'residue. P3 ′ residues for use herein include, but are not limited to, Gly, Ser, Ala, Leu, Ile, nLeu, Val, nVal, Aib, Abu, Met, 6-aminohexanoyl, CHA, and allylGly. In another embodiment, the P2 ′ residue is Ser, hSer, Abu, nLeu, nVal, CHA, hCHA, (allyl) Gly or (hexyl) Gly. In another embodiment, P3 ′ residues for use herein include, but are not limited to, Gly, Ser, Ala, Leu, Ile, nLeu, Val, nVal, Aib, Abu, Met and 6-aminohexanoyl. In another embodiment, the P3 ′ residue is Gly or Ala. In another embodiment, the P3 ′ residue is Leu, Abu, nLeu, nVal, CHA, hCHA, (hexyl) Gly, (allyl) Gly, (propargyl) Gly or (cyclopropyl) Ala.

i. P4 'residue

In other aspects herein, the peptidic substrate used in the conjugates provided herein comprises a P4 'moiety. P4 ′ residues for use herein include, but are not limited to, Gly, Ser, Ala, Leu, Ile, nLeu, Val, nVal, Aib, Abu, Met, 6-aminohexanoyl, CHA and allylGly. In other embodiments, P4 ′ residues for use herein include, but are not limited to, Gly, Ser, Ala, Leu, Ile, nLeu, Val, nVal, Aib, Abu, Met and 6-aminohexanoyl. In another embodiment, the P4 ′ residue is Gly or Ala. In another embodiment, the P4 ′ residue is Leu, Abu, nLeu, nVal, CHA, hCHA, (hexyl) Gly, (allyl) Gly, (propargyl) Gly or (cyclopropyl) Ala. In another embodiment, the P4 ′ residue is Leu.

j. cap

1) X ⁿ (N-terminal cap)

In embodiments in which the therapeutic agent is conjugated to the C-terminus of the peptidic substrate (ie the conjugate is a compound of Formula I), the N-terminus of the peptidic substrate is optionally capped with an acyl, sulfonyl or carbamoyl derivative. The cap selects, in certain embodiments, to increase the hydrophilicity of the conjugate. In embodiments in which the peptidic substrate-therapeutic conjugate is sufficiently hydrophilic not requiring additional hydrophilicity, a non-hydrophilic N-terminal cap such as an acyl group can be used. In embodiments where increased hydrophilicity is preferred, the N-terminal amino acids are modified using hydrophilic blocking groups. Such blocking groups are selected based on the presence of hydrophilic functional groups. Blocking such terminal amino groups may reduce or eliminate the enzymatic degradation of such peptidyl therapeutics by the action of exogenous amino peptidase present in the plasma of warm blooded animals.

N-terminal blocking groups that increase the water solubility of the conjugate by increasing the hydrophilicity of the conjugate include, but are not limited to, hydroxylated alkanoyls, polyhydroxylated alkanoyls, polyethylene glycols, glycosylates, sugars, and crown ethers It doesn't work.

In certain embodiments herein, the N-terminal blocking group is one of the groups of formula a or b.

In the above formula,

R ¹ and R ² are selected from (i) or (ii):

(i) R ¹ and R ² are each independently a) hydrogen; b) unsubstituted or substituted aryl, unsubstituted or substituted heterocyclyl, C ₃ -C ₁₀ cycloalkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, halogen, C ₁ -C ₆ purple Luoroalkyl, R ⁴ O-, R ³ C (O) NR ^3- , (R ³ ) ₂ NC (O)-, (R ³ ) ₂ NC (NR ³ )-, R ⁴ S (O) _e NH -, -CN, -NO ₂ , R ³ C (O)-, -N ₃ , -N (R ³ ) ₂ , or R ⁴ OC (O) NR _3- ; c) unsubstituted C ₁ -C ₆ alkyl; d) substituted C ₁ -C ₆ alkyl wherein the substituent on the substituted C ₁ -C ₆ alkyl is unsubstituted or substituted aryl, unsubstituted or substituted heterocyclyl, C ₃ -C ₁₀ cycloalkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, R ³ O-, R ⁴ S (O) _e NH-, R ₃ C (O) NR ^3- , (R ³ ) ₂ NC (O)-, (R ³ ) ₂ NC (NR ³ )-, -CN, R ³ C (O)-, -N ₃ , -N (R ³ ) ₂ and R ⁴ OC (O) -NR ^3- , or

(ii) R ¹ and R ² together represent — (CH ₂ ) _f —, wherein one carbon atom is —O—, —S (O) _e —, —NC (O) —, —NH— and —N ( Optionally substituted by a moiety selected from COR ⁴ )-, and

R ³ is selected from hydrogen, unsubstituted or substituted aryl, unsubstituted or substituted heterocyclyl, C ₁ -C ₆ alkyl, and C ₃ -C ₁₀ cycloalkyl,

R ⁴ is selected from unsubstituted or substituted aryl, unsubstituted or substituted heterocyclyl, C ₁ -C ₆ alkyl, and C ₃ -C ₁₀ cycloalkyl,

e is 0, 1 or 2,

a is 1, 2, 3 or 4,

b is 0 or an integer from I to 100,

c is 0 to 10, provided that when b is 0, c is 1 to 10,

f is 3, 4 or 5.

In certain embodiments, R ¹ and R ² are each independently hydrogen, OH, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₁ -C ₆ aralkyl or aryl. In this embodiment, a is 1, 2, 3 or 4, b is 0 or an integer from 1 to 100, c is 0 to 10, provided that b is 0, c is 1 to 10.

In another embodiment, the N-terminal cap (X ⁿ ) is hydrogen or a compound of formula i, ii, iii or iv.

In the above formula,

R ¹ and R ² are each independently hydrogen, C ₁ -C ₆ alkyl, and aryl,

a is 1, 2, 3 or 4,

a 'is 0, 1, 2 or 3,

b is 0 or an integer from 1 to 14,

c is 0 or 1, provided that when b is 0, c is 1.

In another embodiment, X ⁿ is R ³⁰ OC (O) —, R ³¹ R ³² NC (O) —, R ³³ (CH ₂ ) _k C (O) — or HC (O) —, wherein k is from 1 to An integer of 4 or 1 or 2, R ³⁰ is alkyl, aryl, heteroaryl, aralkyl or heteroaralkyl, R ³¹ and R ³² are each independently hydrogen, alkyl, aryl, heteroaryl, aralkyl or heteroar Alkyl, R ³³ is hydrogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, aralkyl, aralkoxy, heteroaralkyl or heteroaralkoxy.

In certain embodiments herein, X ⁿ is hydrogen, acetyl, hydroxyacetyl, 2,3-dihydroxypropionyl, 2,3,4-trihydroxybutanoyl, PEG (1), PEG (2), PEG (4), PEG (6), PEG (14), PEG (15), PEG (16), PEG (17), PEG (18) or PEG (19). In other embodiments herein, X ⁿ is hydrogen, acetyl, hydroxyacetyl, succinyl, quinyl, galyl, 4-imidazolylacetyl, cotininyl, 3-phosphonylpropionyl, gluronyl, 4-phosphonyl Butyryl, glutaryl, ethoxyscooaryl or PEG (2). In further embodiments, X ⁿ is hydrogen, acetyl, —C (O) NH ₂ , HOCH ₂ CH ₂ C (O) —, diaminopropanoyl or NH ₂ — (CH ₂ ) ₅ —C (O) — to be. In another embodiment, X ⁿ is hydrogen, acetyl, succinyl, glutaryl, PEG (2) or malonyl. In another embodiment, X ⁿ is hydrogen, acetyl, succinyl, glutaryl, PEG (2), malonyl, methoxycarbonyl, phenylsulfonyl, 3-methoxypropanoyl, ethoxycarbonyl, isobu Oxycarbonyl, benzyloxycarbonyl, tert-butoxycarbonyl, 4-oxopentanoyl, 2- (2-methoxyethoxy) ethoxy) acetyl, 3,4-methylenedioxyphenylacetyl, 2- Pyridylacetyl, phenoxyacetyl, phenylacetyl, methoxyacetyl, 2-methoxyethoxycarbonyl, 2-methoxyethoxyacetyl, 3-phenyl-2-hydroxypropanoyl, pent-4-inoyl , 1-naphthylacetyl, hydroxyacetyl, 3-methoxycarbonylpropanoyl or formyl.

In certain embodiments herein, the N-terminal cap (X ⁿ ) is acetyl, glutaryl or related acyl, sulfonyl or carbamoyl derivatives. Capping groups include, but are not limited to, simple N-acetyl residues through larger residues that affect the overall physicochemical properties of the conjugate of the conjugate. The capping group can be appropriately selected to allow any relatively hydrophilic or hydrophobic molecule to be delivered to the target site. In one embodiment, X ⁿ is acetyl.

2) X ^c (C-terminal cap)

In embodiments in which the therapeutic agent is conjugated to the N-terminus of the peptidic substrate (ie, the conjugate is a compound of Formula II), the C-terminus of the peptidic substrate is a carboxylic acid or carboxamide derivative. The capping group can be appropriately selected to allow any relatively hydrophilic or hydrophobic molecule to be delivered to the target site.

In one embodiment, X ^c is a formula -C (O) NR ^d R ^e wherein the carbonyl group is bonded to wherein R ^d and R ^e are selected from (i) or (ii): (i) R ^d and R ^e are each independently hydrogen, C ₁ -C ₆ -alkyl, -C ₁ -C ₆ -alkyl-OH, -C ₁ -C ₆ -alkyl-di-OH, -C ₁ -C _6- Alkyl-tri-OH and (Where b is 0 or an integer from 1 to 100, c is 0 or 1, provided that when b is 0, c is 1) or at least one of R ^d and R ^e is hydrogen or C ₁ -C _{Not 6} -alkyl, or (ii) R ^d and R ^e together form a —CH ₂ CH ₂ OCH ₂ CH ₂ -diradal].

2. Linker

The conjugate optionally contains a linker that covalently binds the peptidic substrate to the therapeutic agent (ie, L, L ¹ or L ² of Formulas I, II and III). The linker causes the peptidic moiety in the conjugate to be a conjugate that is a substrate for cell surface proteases and is substantially inert and active in active form when the therapeutic agent is in such conjugate or substantially by the environment of the cell, tissue or targeted tissue. It can be anything that allows it to be released in an activated form.

For example, the linker may comprise a linker of carbohydrate, peptide, diamine, arylamine, and / or hydrocarbon core structure. The linker is preferably obtainable by synthesis and provides a self-stable product and does not have any inherent biological activity that interferes with conjugate activity. They may add desirable properties such as increased solubility or the action of assisting in transporting the cleaved therapeutic agent intracellularly. In certain embodiments, some linkers may be enzymatically cleaved in vitro and in vivo and may be fragments that release an active therapeutic agent or activatable therapeutic agent. In embodiments where the therapeutic agent is doxorubicin, the linker is a sugar and / or peptide, such as, for example, the amino sugar daunosamine.

In one embodiment, a linker for use herein comprises a amine moiety on a therapeutic agent linked to a linker unit to form an amide bond and the amino terminus of the peptidic substrate to be linked to the other end of the linker unit to form an amide bond as well. Including but not limited to biscarbonyl alkyl diradicals. In contrast, diaminoalkyl diradical linker units are also provided such that the carbonyl moiety on the therapeutic agent is covalently linked to one of the amines of the linker unit and the remaining amine of the linker unit is covalently attached to the C-terminus of the peptidic substrate. Can be useful. Such other linkers that are stable in the physiological environment when not in the presence of cell surface protease, but cleavable when cleaved at the cell surface protease proteolytic cleavage site are intended for use herein. In addition, linker units may be used that remain bound to the therapeutic agent upon cleavage of the cell surface protease proteolytic cleavage site but do not significantly reduce the therapeutic activity of the therapeutic agent after such cleavage compared to the unmodified therapeutic agent.

In other embodiments, the linker is a diamine containing a cyclic alkyl moiety, and in certain embodiments the diamine contains a bicycloalkylene moiety. Examples of such diamine linkers include 1,4-bis (aminomethyl) cyclohexane, 1,4-bis (aminomethyl) -cycloheptane, 1,3-bis (aminomethyl) cyclopentane, 1-amino-4- ( Amino-methyl) cyclohexane, 1,4-diaminocyclohexane and 1,4-bis (aminomethyl) -biscyclo [2.2.2] octane.

Other linkers include, but are not limited to, 1, ω-diaminoalkanes, including but not limited to 1,3-diaminopropane, and oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, and pivalic acid 1, ω-dicarbonylalkanes which are not included.

Additional linkers for use in the conjugates provided herein include , , And [Wherein A is NH or O, D is N (H or alkyl) or O and R ²⁵ is at least one selected from H, alkyl, cycloalkyl, cycloalkylalkyl, aryl, or halo, halo alkyl and alkyl For example, heteroaryl, aralkyl, heteroaralkyl, alkenyl containing 1 to 2 double bonds optionally substituted by 1 to 3 substituents, alkys containing 1 to 2 triple bonds Nil, alk (en) (yn) yl groups, halo, pseudohalo, cyano, hydroxy, haloalkyl and polyhaloalkyl (e.g. halo lower alkyl, in particular trifluoromethyl), formyl, alkylcarbo Arylcarbonyl, heteroarylcarbonyl, carboxy, alkoxycarbonyl, aryloxy optionally substituted by one or more, for example one to three substituents selected from, for example, halo, halo alkyl and alkyl Carbonyl, Aminoimino, Alkoxycarbonylamino, Aryloxyca Nylamino, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, arylaminocarbonyl, diarylaminocarbonyl, aralkylaminocarbonyl, alkoxy, aryloxy, perfluoroalkoxy, alkenyloxy, alky Neyloxy, arylalkoxy, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, arylaminoalkyl, amino, alkylamino, dialkylamino, arylamino, alkylarylamino, alkylcarbonylamino, arylcarbonylamino, azido , Nitro, mercapto, alkylthio, arylthio, perfluoroalkylthio, thiocyano, isothiocyano, alkylsulfinyl, alkylsulfonyl, arylsulfinyl, arylsulfonyl, aminosulfonyl, alkylaminosul Self-removing linkers, such as the linker of phonyl, dialkylaminosulfonyl, or arylaminosulfonyl, and y is an integer from 1 to 3.

3. Therapeutics

Conjugates aim to modify various biological responses. Thus, a therapeutic agent is any agent that includes proteins and polypeptides, small molecules, and other molecules that possess or enhance the desired biological activity. Such molecules include, but are not limited to, toxins such as abrin, lysine A, Pseudomonas exotoxin, shiga toxin, diphtheria toxin and other toxins and toxic moieties and / or subunits or chains thereof; Proteins including, but not limited to, tumor necrosis factor, α-interferon, γ-interferon, nerve growth factor, growth factor derived from platelets, tissue plasminogen activator; Or for example lymphokines, interleukin-I (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte macrophage colony stimulating factor (GMCSF), granulocyte colony stimulating factor (G-CSF), erythropoietin (EPO), procoagulant (e.g. tissue factor and tissue factor variant), apoptosis promoter (e.g. FAS-ligand), fibroblast growth factor (FGF), nerve growth factor And biological response regulators such as other growth factors. Each must be in a form that can enter the cell or exert a therapeutic effect when in the vicinity of the cell.

Thus, therapeutic agents include, but are not limited to, anticancer agents, antiangiogenic agents, apoptosis promoters, anticancer agents, and antimitotic agents. They are optionally conjugated via a linker to a substrate, such as a peptidic substrate, which is a substrate for proteases.

Among the therapeutic agents there are generally cytotoxic agents including but not limited to acylation agents, toxins, antiproliferative agents and tubulin binding agents. Cytotoxic agent classes for use herein include, for example, anthracycline-based drugs, vinca drugs, mitomycin, bleomycin, cytotoxic nucleosides, pteridine-based drugs, dyinenes, maytansinoids , Epothilones, taxanes and grapephytotoxins.

Exemplary members of this class include, for example, doxorubicin, carminomycin, daunorubicin, aminopterin, methotrexate, methotterin, dichloro- methotrexate, mitomycin C, porphyromycin, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, grapephytotoxin or grapephytotoxin derivatives (eg etoposide or etoposide phosphate), melphalan, vinblastine, vincristine, leusodine, vindesine, leuuro Cin, maytansinol, epothilone A or B, taxotere and taxol and the like. Other such therapeutic agents include estradiol, cisplatin, combretastatin and analogs and cyclophosphamide. One skilled in the art can chemically modify the desired therapeutic agent to make the reaction of these compounds convenient for the purpose of preparing the conjugates.

Specific therapeutic agents include the following drugs. Those skilled in the art will understand that such structural formulas are merely exemplary and that such compounds or derivatives or analogs thereof have common or common names in the art.

a. Methotrexate group of Formula 1:

In the above formula,

R ¹² is amino or hydroxy,

R ⁷ is hydrogen or methyl,

R ⁸ is hydrogen, fluoro, chloro, bromo or iodo,

R ⁹ is a moiety that completes a salt of hydroxy or carboxylic acid.

b. Mitomycin group of formula 2:

In the above formula,

R ¹⁰ is hydrogen or methyl.

c. Bleomycin group of formula 3:

In the above formula,

R ¹¹ is hydroxy, amino, C ₁ -C ₃ alkylamino, di (C ₁ -C ₃ alkyl) amino, C ₄ -C ₆ polymethylene amino, -NHCH ₂ CH ₂ CH ₂ CH ₂ NH-C (NH ) NH ₂ or -NHCH ₂ CH ₂ CH ₂ S ⁺ (CH ₃ ) ₂ .

d. Melphalan of formula 4:

e. Mercaptopurine of Formula 5:

f. Cytosine arabinoside of Formula 6:

g. Grape filotoxin of formula 7:

In the above formula,

R ¹³ is hydrogen or methyl,

R ¹⁴ is methyl or thyethyl or a phosphate salt thereof.

h. Vinca alkaloid drug group of formula 8:

In the above formula,

When R ¹⁷ and R ¹⁸ are independently present, R ¹⁵ is H, CH ₃ or CHO, R ¹⁸ is H, one of R ¹⁶ and R ¹⁷ is ethyl, the other is H or OH,

When R ¹⁷ and R ¹⁸ are present with the carbon to which they are attached, they form an oxirane ring, in which case R ¹⁶ is ethyl,

R ¹⁹ is hydrogen, (C ₁ -C ₃ alkyl) -CO or chlorosubstituted (C ₁ -C ₃ alkyl) -CO.

The conjugate provided herein, wherein the therapeutic agent is vinca alkaloid vinblastine, comprises a compound of the formula: or a pharmaceutically acceptable derivative thereof.

In the above formula,

Peptide substrates are as described for Formulas I and II,

L is a linker such as -NH- (CH ₂ ) uT- (CH ₂ ) u-NH-,

X ⁿ is a) hydrogen, b)-(C = O) R ^1a , c) , d) , e) f) ethoxysquarate; And g) cotininyl,

R ¹ and R ² are independently hydrogen, OH, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₁ -C ₆ aralkyl and aryl,

R ^1a is C ₁ -C ₆ -alkyl, hydroxylated C ₃ -C ₈ -cycloalkyl, polyhydroxylated C ₃ -C ₈ -cycloalkyl, hydroxylated aryl, polyhydroxylated aryl Or aryl,

R ¹⁹ is hydrogen, (C ₁ -C ₃ alkyl) -CO or chlorosubstituted (C ₁ -C ₃ alkyl) -CO,

T is selected from cyclopentyl, cyclohexyl, cycloheptyl or bicyclo [2.2.2] octanyl,

a is 1, 2, 3 or 4,

b is 0 or an integer from 1 to 100,

c is 0 or 1, except that c is 1 when b is 0,

g is 1, 2 or 3,

u is 0, 1, 2 or 3.

i. Difluoronucleosides of Formula 9

In the above formula,

R ²¹ is a chemical formula or Is one of the bases,

R ²² is hydrogen, methyl, bromo, fluoro, chloro or iodo,

R ²³ is -OH or -NH ₂ ,

R ²⁴ is hydrogen, bromo, chloro or iodo.

j. Estramustine of Formula 10:

k. Cyclophosphamide of Formula 11:

l. Anthracycline antibiotic of Formula 12:

In the above formula,

R ^a is —CH ₃ , —CH ₂ 0H, —CH ₂ OCO (CH ₂ ) ₃ CH ₃ or —CH ₂ OCOCH (OC ₂ H ₅ ) ₂ ,

R ^b is —OCH ₃ , —OH or —H,

R ^c is -NH ₂ , -NHCOCF ₃ , 4-morpholinyl, 3-cyano-4-morpholinyl, 1-piperidinyl, 4-methoxy-1-piperidinyl, benzylamine, dibenzyl Amine, cyanomethylamine or 1-cyano-2-methoxyethyl amine,

R ⁵ is -OH -OTHP or -H,

R ⁶ is —OH or —H, provided that R ⁶ is not —OH when R ⁵ is —OH or —OTHP.

Table 2 below provides a number of anthracycline drugs and their generic or generic names.

compound R ^a R ^b R ^c R ⁵ R ⁶ Doxorubicin ^a CH ₃ OCH ₃ NH ₂ OH H Doxorubicin ^b CH ₂ OH OCH ₃ NH ₂ OH H Detorrubicin CH ₂ OCOCH (OC ₂ H ₅ ) ₂ OCH ₃ NH ₂ OH H Carminomycin CH ₃ OH NH ₂ OH H Idarubicin CH ₃ H NH ₂ OH H Epirubicin CH ₂ OH OCH ₃ NH ₂ OH OH Esorubicin CH ₂ OH OCH ₃ NH ₂ H H THP CH ₂ OH OCH ₃ NH ₂ OTHP H AD-32 CH ₂ OCO (CH ₂ ) ₃ CH ₃ OCH ₃ NHCOCF ₃ OH H ^a daunorubicin is an alternative name for daunomycin. ^b Doxorubicin is an alternative name for adriamycin.

In one embodiment when the therapeutic agent is doxorubicin it is conjugated to the peptidic group via the amino group of the aminoglycoside residue of doxorubicin.

m. Maytansinol

n. Epothilone A or B

A

B

o. Taxol

In the above formula,

R is PhC (O) or t-BuOC (O).

In one embodiment, when the therapeutic agent is Taxol (R = C (O) Ph), the peptidic substrate is conjugated to the secondary hydroxyl group of the cyclohexane residue of Taxol.

p. Ribosome-Inactivated Protein

Ribosome-inactivated proteins (RIPs), including lysine, abrine and saporin, are plant proteins that catalytically inactivate eukaryotic ribosomes. RIP inactivates ribosomes by interfering with the protein stretching step of protein synthesis. For example, RIP saporin (hereinafter also referred to as SAP) has been shown to enzymatically inactivate 60S ribosomes by cleaving the n-glycosidic bonds of adenine at position 4324 in rat 28S ribosomal RNA (rRNA). Some RIPs, such as the toxins abrin and lysine, contain two component chains: cell-binding chains that mediate binding to cell surface receptors and internalization of molecules and enzymatically active chains that are associated with protein synthesis inhibitory activity. . This RIP is a type II RIP. Other RIPs, such as saporin, are Japanese chains and are termed type I RIPs. This RIP is less toxic to the whole cell than the RIP with two chains because the cell-binding chain is deleted. Unless one chain is further conjugated to an agent such as a growth factor that mediates binding and internalization, a double stranded RIP is generally used for the conjugates herein.

Several structurally related RIPs have been isolated from the seeds and leaves of the plant Saponaria officinalis (Soapgrass). Of these, SAP-6 is the most active and abundant and corresponds to 7% of the total seed protein. Saporin is very stable, has a high isoelectric point, does not contain carbohydrates, and is resistant to denaturing agents such as sodium dodecyl sulfate (SDS) and various proteases. The amino acid sequences of several saporin-6 isotypes from seeds are known and appear to be of the saporin RIP family, which differ from one another in several amino acid residues. Saporin does not have a cell-binding chain because it is a type I RIP. Thus, its toxicity to whole cells is very low compared to other toxins such as lysine and abrin. However, when internalized by eukaryotic cells, the saponin's toxicity is 100 to 1000 times stronger than the lysine A chain.

4. Exemplary Conjugates

The conjugates provided herein are prepared by identifying suitable peptide substrates for targeted cell surface proteases or soluble, secreted or released forms thereof, and forming conjugates of the peptide substrate (s) with therapeutic agents. For example, exemplary conjugates containing peptide substrates designed for cleavage by MTSP1, endotheliase 1 and urokinase are described. Cell-attached and cell-stations present in or attached to cells that are involved in or in the vicinity of cells or tissues involved in or associated with a disease or other state of interest. When identifying cell surface proteases, including purified protease or soluble, secreted or released forms of cell surface proteases, experimentally designed suitable peptide substrates for such proteases and then conjugating to the therapeutic agents exemplified herein. I will understand.

In certain embodiments, conjugates for use in the compositions and methods provided herein include:

Ac-Leu-Arg-Ala-Quat-Gly-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 46);

Ac-Leu-Arg-Ala-Quat-Ala-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 47);

Ac-Leu-Arg-Ser-Quat-Gly-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 48);

Ac-Leu-Arg-Ser-Quat-Ala-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 49);

Ac-Leu-Arg-Pro-Arg-Phe-Lys-Ile-Ile- (therapeutic) (SEQ ID NO: 50);

Ac-Arg-Pro-Arg-Phe-Lys-Ile-Ile- (therapeutic) (SEQ ID NO: 51);

Ac-Pro-Arg-Phe-Lys-Ile-Ile- (therapeutic) (SEQ ID NO: 52);

Ac-Leu-Arg-Ser-Lys-Ser-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 53);

Ac-Arg-Ser-Lys-Ser-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 54);

Ac-Ser-Lys-Ser-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 55);

Ac-Leu-Arg-Pro-Arg-Phe-Arg-Ile-Ile- (therapeutic) (SEQ ID NO: 56);

Ac-Arg-Pro-Arg-Phe-Arg-Ile-Ile- (therapeutic) (SEQ ID NO: 57);

Ac-Pro-Arg-Phe-Arg-Ile-Ile- (therapeutic) (SEQ ID NO: 58);

Ac-Leu-Arg-Ser-Arg-Ser-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 59);

Ac-Arg-Ser-Arg-Ser-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 60) and

Ac-Ser-Arg-Ser-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 61).

In further embodiments herein, the conjugate is as follows:

Ac-Leu-Arg-Ala-Quat-Gly-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 62);

Ac-Leu-Arg-Ala-Quat-Ala-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 63);

Ac-Leu-Arg-Ser-Quat-Gly-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 64) and

Ac-Leu-Arg-Ser-Quat-Ala-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 65).

In other embodiments herein, the conjugates are as follows:

Ac-Leu-Arg-Pro-Arg-Phe-Lys-Ile-Ile- (therapeutic) (SEQ ID NO: 66);

Ac-Arg-Pro-Arg-Phe-Lys-Ile-Ile- (therapeutic) (SEQ ID NO: 67);

Ac-Pro-Arg-Phe-Lys-Ile-Ile- (therapeutic) (SEQ ID NO: 68);

Ac-Leu-Arg-Ser-Lys-Ser-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 69);

Ac-Arg-Ser-Lys-Ser-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 70);

Ac-Ser-Lys-Ser-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 71);

Ac-Leu-Arg-Pro-Arg-Phe-Arg-Ile-Ile- (therapeutic) (SEQ ID NO: 72);

Ac-Arg-Pro-Arg-Phe-Arg-Ile-Ile- (therapeutic) (SEQ ID NO: 73);

Ac-Pro-Arg-Phe-Arg-Ile-Ile- (therapeutic) (SEQ ID NO: 74);

Ac-Leu-Arg-Ser-Arg-Ser-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 75);

Ac-Arg-Ser-Arg-Ser-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 76) and

Ac-Ser-Arg-Ser-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 77).

In another embodiment, conjugates for use herein include:

Fatigue Glu-Pro-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 78);

CH ₃ SO ₂ -D-HHT-Gly-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 79);

N-p-tosyl-Gly-Pro-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 80);

Benzoyl-Val-Gly-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 81);

CH ₃ SO ₂ -D-HHT-Gly-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 82);

N-α-Z-D-Arg-Gly-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 83) (Z = benzyloxycarbonyl);

Fatigue Glu-Gly-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 84);

H-D-Ile-Pro-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 85);

Cbo-L- (γ) Glu (α-t-BuO) -Gly-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 86) (Cbo = carbobenzox);

H-D-Pro-Phe-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 87);

H-D-Val-Leu-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 88);

Bz-Ile-Glu (γ-OH) -Gly-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 89) (Bz = benzoyl);

Bz-Ile-Glu (γ-OMe) -Gly-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 90);

Benzoyl-Pro-Phe-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 91);

H-D-Phe-Pip-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 92);

H-D-Val-Leu-Lys-Ala-Ala- (therapeutic) (SEQ ID NO: 93);

H-D-Nle-HHT-Lys-Ala-Ala- (therapeutic) (SEQ ID NO: 94);

Pyr-Arg-Thr-Lys-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 95);

H-Arg-Gln-Arg-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 96);

Boc-Gln-Gly-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 97);

Z-Arg-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 98);

H-D-HHT-Ala-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 99);

H-D-CHT-Gly-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 100);

MeSO ₂ -d Phe-Pro-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 101);

δ-Z-D-Lys-Pro-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 102) and

CH ₃ SO ₂ -D-CHA-But-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 103).

In another embodiment, conjugates for use in the compositions and methods provided herein include:

Ac-Arg-Gln-Ser-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 104);

Ac-Arg-Arg-Gln-Ser-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 105);

Ac-Leu-Arg-Arg-Gln-Ser-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 106);

Ac-Arg-Gln-Ser-Arg-Ala- (therapeutic) (SEQ ID NO: 107);

Ac-Arg-Arg-Gln-Ser-Arg-Ala- (therapeutic) (SEQ ID NO: 108);

Ac-Leu-Arg-Arg-Gln-Ser-Arg-Gly-Gly- (therapeutic) (SEQ ID NO: 109);

Ac-Leu-Arg-Arg-Gln-Ser-Arg-Ala- (therapeutic) (SEQ ID NO: 110);

Ac-Arg-Arg-Gln-Ser-Arg-Ile- (therapeutic) (SEQ ID NO: 111) and

Ac-Leu-Arg-Arg-Gln-Ser-Arg-Ala-Ile- (therapeutic) (SEQ ID NO: 112).

In certain embodiments, conjugates for use in the compositions and methods provided herein include:

Ac-Leu-Arg-Ala-Quat-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 113);

Ac-Leu-Arg-Ala-Quat-Ala-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 114);

Ac-Leu-Arg-Ser-Quat-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 115);

Ac-Leu-Arg-Ser-Quat-Ala-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 116);

Ac-Leu-Arg-Pro-Arg-Phe-Lys-Ser-Leu- (therapeutic) (SEQ ID NO: 117);

Ac-Arg-Pro-Arg-Phe-Lys-Ser-Leu- (therapeutic) (SEQ ID NO: 118);

Ac-Pro-Arg-Phe-Lys-Ser-Leu- (therapeutic) (SEQ ID NO: 119);

Ac-Leu-Arg-Ser-Lys-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 120);

Ac-Arg-Ser-Lys-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 121);

Ac-Ser-Lys-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 122);

Ac-Leu-Arg-Pro-Arg-Phe-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 123);

Ac-Arg-Pro-Arg-Phe-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 124);

Ac-Pro-Arg-Phe-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 125);

Ac-Leu-Arg-Ser-Arg-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 126);

Ac-Arg-Ser-Arg-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 127) and

Ac-Ser-Arg-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 128).

In a further embodiment, the conjugate is as follows:

Ac-Leu-Arg-Ala-Quat-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 129);

Ac-Leu-Arg-Ala-Quat-Ala-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 130);

Ac-Leu-Arg-Ser-Quat-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 131) and

Ac-Leu-Arg-Ser-Quat-Ala-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 132).

In another embodiment, the conjugate is as follows:

Ac-Leu-Arg-Pro-Arg-Phe-Lys-Ser-Leu- (therapeutic) (SEQ ID NO: 133);

Ac-Arg-Pro-Arg-Phe-Lys-Ser-Leu- (therapeutic) (SEQ ID NO: 134);

Ac-Pro-Arg-Phe-Lys-Ser-Leu- (therapeutic) (SEQ ID NO: 135);

Ac-Leu-Arg-Ser-Lys-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 136);

Ac-Arg-Ser-Lys-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 137);

Ac-Ser-Lys-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 138);

Ac-Leu-Arg-Pro-Arg-Phe-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 139);

Ac-Arg-Pro-Arg-Phe-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 140);

Ac-Pro-Arg-Phe-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 141);

Ac-Leu-Arg-Ser-Arg-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 142);

Ac-Arg-Ser-Arg-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 143) and

Ac-Ser-Arg-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 144).

In other embodiments, conjugates for use herein include the following.

Fatigue Glu-Pro-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 145);

CH ₃ S0 ₂ -D-HHT-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 146);

N-p-tosyl-Gly-Pro-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 147);

Benzoyl-Val-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 148);

CH ₃ S0 ₂ -D-HHT-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 149);

N-α-Z-D-Arg-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 150) (Z = benzyloxycarbonyl);

Fatigue Glu-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 151);

H-D-Ile-Pro-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 152);

Cbo-L- (γ) Glu (α-t-BuO) -Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 153) (Cbo = carbobenzox);

H-D-Pro-Phe-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 154);

H-D-Val-Leu-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 155);

Bz-Ile-Glu (γ-OH) -Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 156) (Bz = benzoyl);

Bz-Ile-Glu (γ-OMe) -Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 157);

Benzoyl-Pro-Phe-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 158);

H-D-Phe-Pip-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 159);

H-D-Val-Leu-Lys-Ser-Leu- (therapeutic) (SEQ ID NO: 160);

H-D-Nle-HHT-Lys-Ser-Leu- (therapeutic) (SEQ ID NO: 161);

Pyr-Arg-Thr-Lys-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 162);

H-Arg-Gln-Arg-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 163);

Boc-Gln-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 164);

Z-Arg-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 165);

H-D-HHT-Ala-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 166);

H-D-CHT-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 167);

MeSO ₂ -d Phe-Pro-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 168);

δ-Z-D-Lys-Pro-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 169) and

CH ₃ SO ₂ -D-CHA-But-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 170).

In another embodiment, conjugates for use in the compositions and methods provided herein include:

Ac-Arg-Gln-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 171);

Ac-Arg-Arg-Gln-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 172);

Ac-Leu-Arg-Arg-Gln-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 173);

Ac-Arg-Gln-Ser-Arg-Leu- (therapeutic) (SEQ ID NO: 174);

Ac-Arg-Arg-Gln-Ser-Arg-Leu- (therapeutic) (SEQ ID NO: 175);

Ac-Leu-Arg-Arg-Gln-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 176);

Ac-Leu-Arg-Arg-Gln-Ser-Arg-Leu- (therapeutic) (SEQ ID NO: 177);

Ac-Arg-Arg-Gln-Ser-Arg-Leu- (therapeutic) (SEQ ID NO: 178) and

Ac-Leu-Arg-Arg-Gln-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 179).

In other embodiments, conjugates provided herein include the following:

Ac-Arg-Gln-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 180);

Ac-Arg-Gln-Ala-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 181);

Ac-Arg-Gln-Phe-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 182);

Ac-Arg-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 183);

Ac-Arg-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 184);

Ac-Arg-Ala-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 185);

Ac-Arg-Phe-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 186);

Ac-Gln-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 187);

Ac-Gln-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 188);

Ac-Gln-Ala-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 189) and

Ac-Gln-Phe-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 190).

In further embodiments, conjugates for use in the compositions and methods provided herein include:

Ac-Leu-Arg-Ala-Quat-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 191);

Ac-Leu-Arg-Ala-Quat-Ala-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 192);

Ac-Leu-Arg-Ser-Quat-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 193);

Ac-Leu-Arg-Ser-Quat-Ala-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 194);

Ac-Leu-Arg-Pro-Arg-Phe-Lys-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 195);

Ac-Arg-Pro-Arg-Phe-Lys-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 196);

Ac-Pro-Arg-Phe-Lys-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 197);

Ac-Leu-Arg-Ser-Lys-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 198);

Ac-Arg-Ser-Lys-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 199);

Ac-Ser-Lys-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 200);

Ac-Leu-Arg-Pro-Arg-Phe-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 201);

Ac-Arg-Pro-Arg-Phe-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 202);

Ac-Pro-Arg-Phe-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 203);

Ac-Leu-Arg-Ser-Arg-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 204);

Ac-Arg-Ser-Arg-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 205) and

Ac-Ser-Arg-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 206).

In a further embodiment, the conjugate is as follows:

Ac-Leu-Arg-Ala-Quat-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 207);

Ac-Leu-Arg-Ala-Quat-Ala-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 208);

Ac-Leu-Arg-Ser-Quat-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 209) and

Ac-Leu-Arg-Ser-Quat-Ala-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 210).

In another embodiment, the conjugate is as follows:

Ac-Leu-Arg-Pro-Arg-Phe-Lys-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 211);

Ac-Arg-Pro-Arg-Phe-Lys-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 212);

Ac-Pro-Arg-Phe-Lys-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 213);

Ac-Leu-Arg-Ser-Lys-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 214);

Ac-Arg-Ser-Lys-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 215);

Ac-Ser-Lys-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 216);

Ac-Leu-Arg-Pro-Arg-Phe-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 217);

Ac-Arg-Pro-Arg-Phe-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 218);

Ac-Pro-Arg-Phe-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 219);

Ac-Leu-Arg-Ser-Arg-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 220);

Ac-Arg-Ser-Arg-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 221) and

Ac-Ser-Arg-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 222).

In other embodiments, conjugates for use herein include:

Fatigue Glu-Pro-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 223);

CH ₃ SO ₂ -D-HHT-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 224);

N-p-tosyl-Gly-Pro-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 225);

Benzoyl-Val-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 226);

CH ₃ S0 ₂ -D-HHT-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 227);

N-α-Z-D-Arg-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 228) (Z = benzyloxycarbonyl);

Fatigue Glu-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 229);

H-D-Ile-Pro-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 230);

Cbo-L- (γ) Glu (α-t-BuO) -Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 231) (Cbo = carbobenzox);

H-D-Pro-Phe-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 232);

H-D-Val-Leu-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 233);

Bz-Ile-Glu (γ-OH) -Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 234) (Bz = benzoyl);

Bz-Ile-Glu (γ-OMe) -Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 235);

Benzoyl-Pro-Phe-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 236);

H-D-Phe-Pip-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 237);

H-D-Val-Leu-Lys-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 238);

H-D-Nle-HHT-Lys-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 239);

Pyr-Arg-Thr-Lys-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 240);

H-Arg-Gln-Arg-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 241);

Boc-Gln-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 242);

Z-Arg-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 243);

H-D-HHT-Ala-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 244);

H-D-CHT-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 245);

MeSO 2 -d Phe-Pro-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 246);

δ-Z-D-Lys-Pro-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 247) and

CH ₃ SO ₂ -D-CHA-But-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 248).

In another embodiment, conjugates for use in the compositions and methods provided herein include:

Ac-Arg-Gln-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 249);

Ac-Arg-Arg-Gln-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 250);

Ac-Leu-Arg-Arg-Gln-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 251);

Ac-Arg-Gln-Ser-Arg-Leu- (therapeutic) (SEQ ID NO: 252);

Ac-Arg-Arg-Gln-Ser-Arg-Leu- (therapeutic) (SEQ ID NO: 253);

Ac-Leu-Arg-Arg-Gln-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 254);

Ac-Leu-Arg-Arg-Gln-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 255);

Ac-Arg-Arg-Gln-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 256) and

Ac-Leu-Arg-Arg-Gln-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 257).

In other embodiments, conjugates provided herein include the following:

Ac-Arg-Gln-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 258);

Ac-Arg-Gln-Ala-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 259);

Ac-Arg-Gln-Phe-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 260);

Ac-Arg-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 261);

Ac-Arg-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 262);

Ac-Arg-Ala-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 263);

Ac-Arg-Phe-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 264);

Ac-Gln-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 265);

Ac-Gln-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 266);

Ac-Gln-Ala-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 267) and

Ac-Gln-Phe-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 268).

In another embodiment, the conjugates provided herein include:

Ac-Gly-d Ser-Ala-Arg-Ser-Ala- (therapeutic) (SEQ ID NO: 569);

Ac-Arg-Gly-dSer-Ala-Arg-Ser-Ala- (therapeutic) (SEQ ID NO: 570);

Ac-Gly-Ser-Gly-Arg-Ser-Ala- (therapeutic) (SEQ ID NO: 571);

Ac-Arg-Gly-Ser-Gly-Arg-Ser-Ala- (therapeutic) (SEQ ID NO: 572);

Ac-Leu-Arg-Gly-Ser-Gly-Arg-Ser-Ala- (therapeutic) (SEQ ID NO: 573);

Ac-Leu-Arg-Gly-dSer-Ala-Arg-Ser-Ala- (therapeutic) (SEQ ID NO: 574);

Ac-Cys (Me) -Pro-Gly-Arg-Val-Val- (therapeutic) (SEQ ID NO: 575);

Ac-Arg-Cys (Me) -Pro-Gly-Arg-Val-Val- (therapeutic) (SEQ ID NO: 577);

Ac-Arg-Arg-Cys (Me) -Pro-Gly-Arg-Val-Val- (therapeutic) (SEQ ID NO: 578);

Ac-Val-Ser-Ala-Arg-Met-Ala- (therapeutic) (SEQ ID NO: 579);

Ac-Ile-Val-Ser-Ala-Arg-Met-Ala- (therapeutic) (SEQ ID NO: 580);

Ac-Val-Ile-Val-Ser-Ala-Arg-Met-Ala- (therapeutic) (SEQ ID NO: 581);

Ac-Val-Ile-Val-Ser-Ala-Arg-nLeu-Ala- (therapeutic) (SEQ ID NO: 582);

Ac-Val-Ser-Ala-Arg-nLeu-Ala- (therapeutic) (SEQ ID NO: 583);

Ac-Ile-Val-Ser-Ala-Arg-nLeu-Ala- (therapeutic) (SEQ ID NO: 584);

Ac-Gly-Ser-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 585);

Ac-Gly-Ser-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 586);

Ac-Gly-Ser-Ala-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 587);

Ac-Ser-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 588);

Ac-Ser-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 589);

Ac-Ser-Ala-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 590);

Ac-Arg-Gly-Ser-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 591);

Ac-Arg-Gly-Ser-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 592);

Ac-Arg-Gly-Ser-Ala-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 593);

Ac-Leu-Arg-Gly-Ser-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 594);

Ac-Leu-Arg-Gly-Ser-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 595) and

Ac-Leu-Arg-Gly-Ser-Ala-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 596).

In another embodiment, the conjugate provided herein is selected from:

Ac-R-Q-G-R-S-L- (therapeutic) (SEQ ID NO: 491);

Ac-R-Q-G-R-S-S-L- (therapeutic) (SEQ ID NO: 492);

Ac-R-Q-G-R-S-nL- (therapeutic) (SEQ ID NO: 493);

Ac-R-Q-G-R-S-nV- (therapeutic) (SEQ ID NO: 494);

Ac-R-Q-G-R-S-F- (therapeutic) (SEQ ID NO: 495);

Ac-R-Q-G-R-A-L- (therapeutic) (SEQ ID NO: 496);

Ac-R-Q-G-R-A-L- (therapeutic) (SEQ ID NO: 497);

Ac-R-Q-G-R-A-nL- (therapeutic) (SEQ ID NO: 498);

Ac-R-Q-G-R-A-nL- (therapeutic) (SEQ ID NO: 499);

Ac-R-Q-G-R-A-nV- (therapeutic) (SEQ ID NO: 500);

Ac-R-Q-G-R-A-Cha- (therapeutic) (SEQ ID NO: 501);

Ac-R-Q-G-R-A-F- (therapeutic) (SEQ ID NO: 502);

Ac-R-N-G-R-S-L- (therapeutic) (SEQ ID NO: 503);

Ac-R-N-G-R-A-nL- (therapeutic) (SEQ ID NO: 504);

Ac-R-Q-A-R-S-L- (therapeutic) (SEQ ID NO: 505);

Ac-R-Q-A-R-S-nL- (therapeutic) (SEQ ID NO: 506);

Ac-R-Q-A-R-S-nV- (therapeutic) (SEQ ID NO: 507);

Ac-R-Q-A-A-S-Cha- (therapeutic) (SEQ ID NO: 508);

Ac-R-Q-A-R-S-S-Cha- (therapeutic) (SEQ ID NO: 509);

Ac-R-Q-A-R-T-nL- (therapeutic) (SEQ ID NO: 510);

Ac-R-Q-A-R-A-L- (therapeutic) (SEQ ID NO: 511);

Ac-R-Q-A-R-A-nL- (therapeutic) (SEQ ID NO: 512);

Ac-R-Q-A-R-A-nV- (therapeutic) (SEQ ID NO: 513);

Ac-R-Q-A-R-A-Cha- (therapeutic) (SEQ ID NO: 514);

Ac-R-Q-S-R-A-A- (therapeutic) (SEQ ID NO: 515);

Ac-R-Q-S-R-A- (therapeutic) (SEQ ID NO: 516);

Ac-R-Q-S-R-A-nL- (therapeutic) (SEQ ID NO: 517);

Ac-R-Q-S-R-A-L- (therapeutic) (SEQ ID NO: 518);

Ac-R-Q-S-R-A-nV- (therapeutic) (SEQ ID NO: 519);

Ac-R-Q-S-R-A-Cha- (therapeutic) (SEQ ID NO: 520);

Ac-R-Q-S-R-S-S-L- (therapeutic) (SEQ ID NO: 521);

Ac-R-Q-S-R-S-L- (therapeutic) (SEQ ID NO: 522);

Ac-R-Q-S-R-S-nL- (therapeutic) (SEQ ID NO: 523);

Ac-R-Q-S-R-S-nL- (therapeutic) (SEQ ID NO: 524);

Ac-R-Q-S-R-S-nV- (therapeutic) (SEQ ID NO: 525);

Ac-R-Q-S-R-S-allylG- (therapeutic) (SEQ ID NO: 526);

Ac-R-Q-S-R-S-Cha- (therapeutic) (SEQ ID NO: 527);

Ac-R-Q-S-R-T-nL- (therapeutic) (SEQ ID NO: 528);

Ac-R-Q-T-R-S-S-L- (therapeutic) (SEQ ID NO: 529);

Ac-R-Q-T-R-S-L- (therapeutic) (SEQ ID NO: 530);

Ac-R-N-S-R-S-nL- (therapeutic) (SEQ ID NO: 531);

Ac-R-Q-F-R-S-L- (therapeutic) (SEQ ID NO: 532);

Ac-R-Q-F-R-S-nL- (therapeutic) (SEQ ID NO: 534);

Ac-R-Q-F-R-S-nV- (therapeutic) (SEQ ID NO: 535);

Ac-R-Q-F-R-S-nL- (therapeutic) (SEQ ID NO: 536);

Ac-R-Q-F-R-S-Cha- (therapeutic) (SEQ ID NO: 537);

Ac-R-Q-F-R-A-L- (therapeutic) (SEQ ID NO: 538);

Ac-R-Q-F-R-A-nL- (therapeutic) (SEQ ID NO: 539);

Ac-R-Q-F-R-A-nV- (therapeutic) (SEQ ID NO: 540);

Ac-R-Q-F-R-A-Cha- (therapeutic) (SEQ ID NO: 541);

Ac-Q-S-R-S-S-nL- (therapeutic) (SEQ ID NO: 542);

MeOCO-Quat2-G-R-S-L- (therapeutic) (SEQ ID NO: 483);

MeOCO-Quat3-G-R-S-L- (therapeutic) (SEQ ID NO: 484);

MeOCO-Quat-G-R-S-L- (therapeutic) (SEQ ID NO: 485);

MeOCO-Quat4-G-R-S-L- (therapeutic) (SEQ ID NO: 486);

MeOCO-Quat5-G-R-S-L- (therapeutic) (SEQ ID NO: 487);

MeOCO-Quat2-G-R-S-S-L- (therapeutic) (SEQ ID NO: 488);

MeOCO-Quat4-G-R-S-L- (therapeutic) (SEQ ID NO: 489);

MeOCO-Quat2-G-R-S-L- (therapeutic) (SEQ ID NO: 490);

Ac-Q-G-R-S-L- (therapeutic) (SEQ ID NO: 445);

Ac-Q-G-R-S-S-L- (therapeutic) (SEQ ID NO: 446);

Ac-Q-G-R-A-S-L- (therapeutic) (SEQ ID NO: 447);

Ac-N-G-R-S-S-L- (therapeutic) (SEQ ID NO: 448);

Ac-Q-G-R-S-S-nL- (therapeutic) (SEQ ID NO: 449);

Ac-Q-G-R-S-S-nV- (therapeutic) (SEQ ID NO: 450);

Ac-Q-G-R-S-S-Cha- (therapeutic) (SEQ ID NO: 451);

Ac-Q-G-R-S-S-allylG- (therapeutic) (SEQ ID NO: 452);

Ac-Q-G-R-S-S-allylG- (therapeutic) (SEQ ID NO: 453);

Ac-Q-A-R-S-L- (therapeutic) (SEQ ID NO: 454);

Ac-Q-A-R-S-S-L- (therapeutic) (SEQ ID NO: 455);

Ac-Q-S-R-S-L- (therapeutic) (SEQ ID NO: 456);

Ac-Q-S-R-S-S-nV- (therapeutic) (SEQ ID NO: 457);

Ac-Q-S-R-S-S-Cha- (therapeutic) (SEQ ID NO: 458);

Ac-Q-S-R-S-S-L- (therapeutic) (SEQ ID NO: 459);

Ac-Q-T-R-S-S-L- (therapeutic) (SEQ ID NO: 460);

Ac-Q-Aib-R-S-S-Cha- (therapeutic) (SEQ ID NO: 461);

Ac-Q-Aib -R-S-S-L- (therapeutic) (SEQ ID NO: 462);

Ac-Q-Abu-R-S-S-Cha- (therapeutic) (SEQ ID NO: 463);

Ac-Q-Abu-R-S-S-L- (therapeutic) (SEQ ID NO: 464);

Ac-Q-Cha-R-S-S-Cha- (therapeutic) (SEQ ID NO: 465);

Ac-Q-F-R-S-L- (therapeutic) (SEQ ID NO: 466);

Ac-Q-F-R-S-S-L- (therapeutic) (SEQ ID NO: 467);

Ac-Q-Y-R-S-S-L- (therapeutic) (SEQ ID NO: 468);

Ac-R-G-R-S-L- (therapeutic) (SEQ ID NO: 469);

Ac-R-G-R-S-S-L- (therapeutic) (SEQ ID NO: 470);

Ac-R-G-R-S-S-Cha- (therapeutic) (SEQ ID NO: 471);

Ac-R-G-R-S-Cha- (therapeutic) (SEQ ID NO: 472);

Ac-R-A-R-S-L- (therapeutic) (SEQ ID NO: 473);

Ac-R-A-R-S-S-L- (therapeutic) (SEQ ID NO: 474);

Ac-R-S-R-S-L- (therapeutic) (SEQ ID NO: 475);

Ac-R-S-R-S-S-L- (therapeutic) (SEQ ID NO: 476);

Ac-R-S-R-S-Cha- (therapeutic) (SEQ ID NO: 477);

Ac-R-S-R-S-S-Cha- (therapeutic) (SEQ ID NO: 478);

Ac-R-F-R-S-L- (therapeutic) (SEQ ID NO: 479);

Ac-R-F-R-S-Cha- (therapeutic) (SEQ ID NO: 480);

Ac-Y-G-R-S-S-L- (therapeutic) (SEQ ID NO: 481);

Ac-M (O 2) -S-R-S-L- (therapeutic) (SEQ ID NO: 482);

Ac-R-R-Q-S-R-A-A- (therapeutic) (SEQ ID NO: 105);

Ac-R-R-Q-S-R-I- (therapeutic) (SEQ ID NO: 610);

Ac-R-R-Q-S-R-S-S-L- (therapeutic) (SEQ ID NO: 543);

Ac-R-R-Q-S-R-S-L- (therapeutic) (SEQ ID NO: 544);

Ac-R-G-S-G-R-S-L- (therapeutic) (SEQ ID NO: 545);

Ac-R-G-S-G-R--S-nL- (therapeutic) (SEQ ID NO: 546);

Ac-R-G-S-G-R-A-nL- (therapeutic) (SEQ ID NO: 547);

Ac-R-G-S-G-R-S-S-L- (therapeutic) (SEQ ID NO: 548);

Ac-I-V-S-G-R-A-S-L- (therapeutic) (SEQ ID NO: 549);

Ac-R-R-Q-S-R-A- (therapeutic) (SEQ ID NO: 108);

Ac-R-R-Q-S-R-I- (therapeutic) (SEQ ID NO: 111);

Ac-L-R-R-Q-S-R-A-A- (therapeutic) (SEQ ID NO: 106);

Ac-L-R-R-Q-S-R-G-G- (therapeutic) (SEQ ID NO: 109);

Ac-L-R-R-Q-S-R-A- (therapeutic) (SEQ ID NO: 110);

Ac-L-R-R-Q-S-R-A-I- (therapeutic) (SEQ ID NO: 112);

Ac-L-R-R-Q-S-R-A-I- (therapeutic) (SEQ ID NO: 611);

Ac-L-R-R-Q-S-R-S-S-L- (therapeutic) (SEQ ID NO: 550) and

Ac-L-R-R-Q-S-R-S-L- (therapeutic) (SEQ ID NO: 551).

In another embodiment, the conjugate provided herein is selected from:

Ac-S-G-R-S-L- (therapeutic) (SEQ ID NO: 362);

Ac-S-G-R-S-S-L- (therapeutic) (SEQ ID NO: 363);

Ac-S-G-R-S-S-S-L- (therapeutic) (SEQ ID NO: 364);

Ac-S-G-R-S-nL- (therapeutic) (SEQ ID NO: 365);

Ac-S-G-R-S-nV- (therapeutic) (SEQ ID NO: 366); Isomer 1

Ac-S-G-R-S-nV- (therapeutic) (SEQ ID NO: 367); Isomer 2

Ac-S-G-R-S-G (hex)-(therapeutic) (SEQ ID NO: 368);

Ac-S-G-R-S-Cha- (therapeutic) (SEQ ID NO: 369);

Ac-S-G-R-S-hCha- (therapeutic) (SEQ ID NO: 370);

Ac-S-A-R-S-L- (therapeutic) (SEQ ID NO: 371);

Ac-S-A-R-S-S-L- (therapeutic) (SEQ ID NO: 372);

Ac-S-S-R-S-nL- (therapeutic) (SEQ ID NO: 373);

Ac-T-G-R-S-Abu- (therapeutic) (SEQ ID NO: 374);

Ac-T-G-R-S-L- (therapeutic) (SEQ ID NO: 375);

Ac-T-G-R-S-nV- (therapeutic) (SEQ ID NO: 376);

Ac-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 377);

Ac-T-G-R-S-G (hex)-(therapeutic) (SEQ ID NO: 378);

Ac-T-G-R-S-Cha- (therapeutic) (SEQ ID NO: 379);

Ac-T-G-R-S-hCha- (therapeutic) (SEQ ID NO: 380);

Ac-T-G-R-T-Abu- (therapeutic) (SEQ ID NO: 381);

Ac-T-G-R-hS-nL- (therapeutic) (SEQ ID NO: 382);

Ac-T-G-R-Abu-nL- (therapeutic) (SEQ ID NO: 383);

Ac-T-G-R-Abu-nV- (therapeutic) (SEQ ID NO: 384);

Ac-T-G-F (Gn) -S-nL- (therapeutic) (SEQ ID NO: 385);

Ac-T-G-F (Gn) -S-Cha- (therapeutic) (SEQ ID NO: 386);

Ac-T-G-F (Gn) -Abu-nV- (therapeutic) (SEQ ID NO: 387);

Ac-T-G-K (alloc) -S-nL- (therapeutic) (SEQ ID NO: 388);

Ac-T-G-K-S-nL- (therapeutic) (SEQ ID NO: 389);

Ac-T-G-hR-S-nL- (therapeutic) (SEQ ID NO: 390);

Ac- (hS) G-G-R-S-nL- (therapeutic) (SEQ ID NO: 391);

MeOCO-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 392);

PhSO2-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 393);

MeOEtCO-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 394);

MeO (EtO) 2 Ac-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 395);

4-oxo-pentanoyl-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 396);

3,4-methyldioxyPhAc-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 397);

2-pyridylAc-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 398);

PhOAc-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 399);

L-3-Phlactyl-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 400);

MeOAc-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 401);

PhAc-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 402);

MeOEtOCO-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 403);

MeOEtOAc-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 404);

HOOCButa-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 405);

Z-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 406);

EtOCO-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 407);

βA-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 408);

Pent-4-inoyl-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 409);

NapAc-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 410);

iBoc-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 411);

HOAc-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 412);

MeSucc-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 413);

N, N-diMeGly-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 414);

Succ-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 415);

HCO-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 416);

Ac-T-A-R-S-nL- (therapeutic) (SEQ ID NO: 417);

Ac-T-A-F (Gn) -S-nL- (therapeutic) (SEQ ID NO: 418);

Ac-T-A-R-Abu-nV- (therapeutic) (SEQ ID NO: 419);

Ac-T-A-R-S-Abu- (therapeutic) (SEQ ID NO: 420);

Ac-T-A-R-T-Abu- (therapeutic) (SEQ ID NO: 421);

Ac-T-S (O-Me) -R-S-nL- (therapeutic) (SEQ ID NO: 422);

Ac-T-hS-R-S-nL- (therapeutic) (SEQ ID NO: 423);

Ac-T- (1-Me) H-R-S-nL- (therapeutic) (SEQ ID NO: 424);

Ac-T- (3-Me) H-R-S-nL- (therapeutic) (SEQ ID NO: 425);

Ac-T-H-R-S-nL- (therapeutic) (SEQ ID NO: 426);

Ac-T-Sar-R-S-nL- (therapeutic) (SEQ ID NO: 427);

Ac-T-nV-R-S-nL- (therapeutic) (SEQ ID NO: 428);

Ac-T-nL-R-S-nL- (therapeutic) (SEQ ID NO: 429);

Ac-T-A-R-S-Cha- (therapeutic) (SEQ ID NO: 430);

Ac-T-Abu-R-S-nL- (therapeutic) (SEQ ID NO: 431);

Ac-4,4-diMeThr-G-R-S-nL- (therapeutic) (SEQ ID NO: 432);

Ac-hS-G-R-S-nL- (therapeutic) (SEQ ID NO: 433);

Ac-hS-G-R-hS-Cha- (therapeutic) (SEQ ID NO: 434);

Ac-hS-G-R-S-Cha- (therapeutic) (SEQ ID NO: 435);

Ac-hS-G-R-T-Cha- (therapeutic) (SEQ ID NO: 436);

Ac-hS-A-R-S-Cha- (therapeutic) (SEQ ID NO: 437);

Ac-N-G-R-S-nL- (therapeutic) (SEQ ID NO: 438);

Ac-Y-G-R-S-S-L- (therapeutic) (SEQ ID NO: 439);

Ac-Y-G-R-S-Cha- (therapeutic) (SEQ ID NO: 440);

Ac-Q-G-R-S-S-nL- (therapeutic) (SEQ ID NO: 441);

Ac-Q-G-R-S-S-nV- (therapeutic) (SEQ ID NO: 442);

Ac-L-R-G-S-G-R-S-A- (therapeutic) (SEQ ID NO: 573);

Ac-L-R-G-S-G-R-S-L- (therapeutic) (SEQ ID NO: 342);

Ac-L-R-G-S-G-R-S-L- (therapeutic) (SEQ ID NO: 343);

Ac-L-R-G-S-G-R-S-S-nL- (therapeutic) (SEQ ID NO: 344);

Ac-L-R-G-S-G-R-S-S-Cha- (therapeutic) (SEQ ID NO: 345);

Ac-L-R-G-dS-A-R-S-A- (therapeutic) (SEQ ID NO: 574);

Ac-L-R-G-S-A-R-S-S-L- (therapeutic) (SEQ ID NO: 346);

Ac-L-R-G-S-A-R-S-L- (therapeutic) (SEQ ID NO: 347);

Ac-L-R-G-S-A-R-S-S-Cha- (therapeutic) (SEQ ID NO: 348);

Ac-L-R-G-S-A-R-S-S-nV- (therapeutic) (SEQ ID NO: 349);

Ac-L-R-G-S-A-R-S-S-nL- (therapeutic) (SEQ ID NO: 350);

Ac-V-I-V-S-G-R-A-L- (therapeutic) (SEQ ID NO: 351);

Ac-V-I-V-S-A-R-S-L- (therapeutic) (SEQ ID NO: 352);

Ac-V-I-V-S-G-R-S-S-L- (therapeutic) (SEQ ID NO: 353);

Ac-V-I-V-S-A-R-M-A- (therapeutic) (SEQ ID NO: 354);

Ac-V-I-V-S-A-R-nL-A- (therapeutic) (SEQ ID NO: 355);

Ac-V-I-V-S-A-R-S-nL- (therapeutic) (SEQ ID NO: 356);

Ac-V-I-V-S-A-R-S-Cha- (therapeutic) (SEQ ID NO: 357);

Ac-V-I-V-S-A-R-S-Cha- (therapeutic) (SEQ ID NO: 358);

Ac-V-I-V-S-A-R-S-S-Cha- (therapeutic) (SEQ ID NO: 359);

Ac-R-R- (Me) C-P-G-R-V-V- (therapeutic) (SEQ ID NO: 360);

Ac-R-R-nV-P-A-R-S-L- (therapeutic) (SEQ ID NO: 361);

Ac-R-G-dS-A-R-S-A- (therapeutic) (SEQ ID NO: 309);

Ac-R-G-S-G-R-S-A- (therapeutic) (SEQ ID NO: 310);

Ac-R-G-S-G-R-A-L- (therapeutic) (SEQ ID NO: 311);

Ac-R-G-S-G-R-S-L- (therapeutic) (SEQ ID NO: 312);

Ac-R-G-S-G-R--S-nL- (therapeutic) (SEQ ID NO: 313);

Ac-R-G-S-G-R-A-nL- (therapeutic) (SEQ ID NO: 314);

Ac-R-G-S-G-R-S-S-L- (therapeutic) (SEQ ID NO: 315);

Ac-R-G-S-G-R-S-Cha- (therapeutic) (SEQ ID NO: 316);

Ac-R-G-S-G-R-S-S-Cha- (therapeutic) (SEQ ID NO: 317);

Ac-R-G-S-A-R-S-Cha- (therapeutic) (SEQ ID NO: 318);

Ac-R-G-S-A-R-S-S- (therapeutic) (SEQ ID NO: 319);

Ac-R-G-S-A-R-S-nV- (therapeutic) (SEQ ID NO: 320);

Ac-R-G-S-A-R-S-S-nV-(therapeutic agent) (SEQ ID NO: 321);

Ac-R-G-S-A-R-S-L- (therapeutic) (SEQ ID NO: 322);

Ac-R- (Me) C-P-G-R-V-V- (therapeutic) (SEQ ID NO: 323);

Ac-R- (Me) C-P-G-R-V-V- (therapeutic) (SEQ ID NO: 324);

Ac-R-C (Me) -P-G-R-S-L- (therapeutic) (SEQ ID NO: 325);

Ac-R-L-P-G-R-S-L- (therapeutic) (SEQ ID NO: 326);

Ac-R-V-P-G-R-S-L- (therapeutic) (SEQ ID NO: 327);

Ac-R-V-P-G-R-S-L- (therapeutic) (SEQ ID NO: 328);

Ac-R-nL-P-G-R-S-L- (therapeutic) (SEQ ID NO: 329);

Ac-R-G (tBu) -P-A-R-S-L- (therapeutic) (SEQ ID NO: 330);

Ac-R-L-P-A-R-S-L- (therapeutic) (SEQ ID NO: 331);

Ac-R-V-P-A-R-S-L- (therapeutic) (SEQ ID NO: 332);

Ac-R-nL-P-A-R-S-L- (therapeutic) (SEQ ID NO: 333);

Ac-I-V-S-G-R-A-L- (therapeutic) (SEQ ID NO: 334);

Ac-I-V-S-G-R-S-S-L- (therapeutic) (SEQ ID NO: 335);

Ac-I-V-S-G-R-A-S-L- (therapeutic) (SEQ ID NO: 336);

Ac-I-V-S-A-R-M-A- (therapeutic) (SEQ ID NO: 337);

Ac-I-V-S-A-R-nL-A- (therapeutic) (SEQ ID NO: 338);

Ac-I-V-S-A-R-S-L- (therapeutic) (SEQ ID NO: 339);

Ac-I-V-S-A-R-S-nL- (therapeutic) (SEQ ID NO: 340);

Ac-I-V-S-A-R-S-S-L- (therapeutic) (SEQ ID NO: 341);

Ac-G-S-G-R-S-A- (therapeutic) (SEQ ID NO: 585);

Ac-G-S-G-R-S-L- (therapeutic) (SEQ ID NO: 277);

Ac-G-S-G-R-A-L- (therapeutic) (SEQ ID NO: 278);

Ac-G-S-G-R-S-S-L- (therapeutic) (SEQ ID NO: 279);

Ac-G-S-G-R-L- (therapeutic) (SEQ ID NO: 280);

Ac-G-S-G- (4-guan) Phg-S-L- (therapeutic) (SEQ ID NO: 281);

Ac-G-S-G-R-S-S-Cha- (therapeutic) (SEQ ID NO: 282);

Ac-G-S-G-R-A-S-L- (therapeutic) (SEQ ID NO: 283);

Ac-G-S-G-R-S-nL- (therapeutic) (SEQ ID NO: 284);

Ac-G-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 285);

Succ-bA-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 286);

Ac-G-T-G-R-S-hCha- (therapeutic) (SEQ ID NO: 287);

Ac-G-hS-G-R-S-nL- (therapeutic) (SEQ ID NO: 288);

Ac-G-dS-A-R-S-A- (therapeutic) (SEQ ID NO: 289);

Ac-G-S-A-R-S-L- (therapeutic) (SEQ ID NO: 290);

Ac-G-S-A-R-S-S-Cha- (therapeutic) (SEQ ID NO: 291);

Ac-G-S-A-R-S-S-L- (therapeutic) (SEQ ID NO: 292);

Ac-G-S-A-R-A-S-L- (therapeutic) (SEQ ID NO: 293);

Ac-V-S-G-R-S-L- (therapeutic) (SEQ ID NO: 294);

Ac-V-S-G-R-A-L- (therapeutic) (SEQ ID NO: 295);

Ac-V-S-G-R-A-S-L- (therapeutic) (SEQ ID NO: 296);

Ac-V-S-G-R-S-S-L- (therapeutic) (SEQ ID NO: 297);

Ac-V-S-A-R-M-A- (therapeutic) (SEQ ID NO: 298);

Ac-V-S-A-R-nL-A- (therapeutic) (SEQ ID NO: 299);

Ac-V-S-A-R-S-nL- (therapeutic) (SEQ ID NO: 300);

Ac-V-S-A-R-S-L- (therapeutic) (SEQ ID NO: 301);

Ac- (Me) C-P-G-R-V-V- (therapeutic) (SEQ ID NO: 302);

Ac- (Me) C-P-G-R-V-V- (therapeutic) (SEQ ID NO: 303);

Ac-C (Me) -P-G-R-A-L- (therapeutic) (SEQ ID NO: 304);

Ac-C (Me) -P-G-R-S-L- (therapeutic) (SEQ ID NO: 305);

Ac-C (Me) -P-A-R-S-L- (therapeutic) (SEQ ID NO: 306);

Ac-C (Me) -P-A-R-A-S-L- (therapeutic) (SEQ ID NO: 307) and

Ac-G (tBu) -P-G-R-S-L- (therapeutic) (SEQ ID NO: 308).

In another embodiment, the conjugate provided herein is selected from the following.

Ac-Q-S-R-A-A- (therapeutic) (SEQ ID NO: 552);

Ac-Q-S-R-S-A- (therapeutic) (SEQ ID NO: 553);

Ac-Q-S-R-S-G- (therapeutic) (SEQ ID NO: 554);

Ac-R-S-R-A-A- (therapeutic) (SEQ ID NO: 555);

Ac-R-Q-S-R-A-A- (therapeutic) (SEQ ID NO: 556);

Ac-R-Q-S-R-S-A- (therapeutics) (SEQ ID NO: 557) and

Ac-R-Q-S-R-S-A-A- (therapeutic) (SEQ ID NO: 558).

In another embodiment, the conjugate provided herein is selected from:

Ac-R-G-S-G-R-S-A- (therapeutic) (SEQ ID NO: 559);

Ac-S-G-R-A-A- (therapeutic) (SEQ ID NO: 560);

Ac-S-G-R-S-A- (therapeutic) (SEQ ID NO: 561);

Ac-S-G-R-S-S-A- (therapeutic) (SEQ ID NO: 562);

Ac-S-G-R-A-S-A- (therapeutic) (SEQ ID NO: 563);

Ac-S-G-R-S-G- (therapeutic) (SEQ ID NO: 564);

Ac-S-G-R-S-S-G- (therapeutic) (SEQ ID NO: 565);

Ac-S-G-R-S-G-A- (therapeutic) (SEQ ID NO: 566);

Ac-S-G-R-S-G-G- (therapeutic) (SEQ ID NO: 567) and

Ac-G-T-G-R-S-G-G- (therapeutic) (SEQ ID NO: 568).

In another embodiment, the conjugate provided herein is selected from:

Ac-L-R-R-Q-S-R-A-A- (therapeutic) (SEQ ID NO: 597);

MeSO ₂ -dA (Chx) -Abu-RSL- (therapeutic) (SEQ ID NO: 598);

Ac-R-A-R-S-L- (therapeutic) (SEQ ID NO: 599);

Ac-dA (Chx) -Abu-R-S-L- (therapeutic) (SEQ ID NO: 600);

Ac-dA (Chx) -Abu-R-S-S-L- (therapeutic) (SEQ ID NO: 601);

Ac-Q-G-R-S-S-L- (therapeutic) (SEQ ID NO: 602);

MeOCO-dhF-P (OH) -R-S-S-L- (therapeutic) (SEQ ID NO: 603);

MeOCO-Quat4-G-R-S-L- (therapeutic) (SEQ ID NO: 604);

Ac-dCha-P (OH) -R-S-S-L- (therapeutic) (SEQ ID NO: 605);

Ac-dCha-Abu-R-S-S-A- (therapeutic) (SEQ ID NO: 606);

MeOCO-Quat2-G-R-S-L- (therapeutic) (SEQ ID NO: 607);

MeOCO-Quat3-G-R-S-L- (therapeutic) (SEQ ID NO: 608) and

MeOCO-Quat-G-R-S-L- (therapeutic) (SEQ ID NO: 609).

It will also be appreciated that the conjugates containing the peptidic moiety can be prepared using other capping groups instead of Ac (see description for capping group X ⁿ ). Therapeutic agents for use in the conjugates include, for example, cytotoxic agents, including but not limited to toxins such as abrin, lysine A, Pseudomonas exotoxin, cigar toxin, diphtheria toxin and other toxins and toxic moieties thereof; Proteins such as tumor necrosis factor, interferon (e.g. α-interferon and gamma-interferon), precoagulant factors (e.g. tissue and tissue factor variants), nerve growth factor, platelet-derived growth factor, tissue plasminogen activator ; For example, lymphokines, interleukin-I (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte macrophage colony stimulating factor (GMCSF), granulocyte colony stimulating factor ( G-CSF), biological response modifiers such as fibroblast growth factor (FGF) and other growth factors, methotrexate drug groups, anthracycline-based drugs, vinca alkaloid drugs, mitomycin, bleomycin, cytosine arabinosides and difluoro Cytotoxic nucleosides including nucleosides, pteridine-based drugs, dienenes, taxanes and grapephytotoxins. All such conjugates are within the scope of the present invention and can be prepared and used as described herein.

Thus, as the conjugate provided herein, the therapeutic agent may be, for example, doxorubicin, carminomycin, daunorubicin, detorrubicin, idarubicin, epirubicin, esorubicin, THP, AD-32, amino acids. Terrins, methotrexate, methotterin, dichloromethoprexate, mitomycin C, porphyromycin, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, grapephytotoxin or grapephytotoxin derivatives (e.g., etoposide Or etoposide phosphate), melphalan, vinblastine, vincristine, leurosine, vindesine, leucine, taxol, esturamustine, cisplatin, combretastatin and analogs, and cyclophosphamide Described conjugates include, but are not limited to. In one embodiment, the therapeutic agent is doxorubicin. In another embodiment, the therapeutic agent is Taxol.

Any conjugate corresponding to the above conjugates or any conjugate whose P1 'and / or P2' residues are Ile instead of Ala is within the scope of the present invention and can be prepared and used as described herein.

All peptide substrates formed by exchange and selection of amino acids from those set forth in the definitions of P groups are contemplated.

D. Preparation of Conjugates

Peptide substrates of the conjugates provided herein are synthesized from their component amino acids using conventional peptide synthesis techniques such as solid phase techniques. The peptidic substrate is then purified by reverse phase high performance liquid chromatography (HPLC).

Peptide acids can be prepared from its component Fmoc-amino acids. Standard methods for synthesizing peptides are described, for example, in Synthesis Notes Section, Nova Biochem Catalog 2002/3, Schroeder et al., “The Peptides”, Vol. 1, Academic Press 1965; Bodansky et al., “Peptide Synthesis”, Interscience Publishers, 1966; McOmie (ed.) "Protective Group in Organic Chemistry", Plenum Press, 1973, Barany et al., "The Peptides: Analysis, Synthesis, Biology" 2, Chapter 1, Academic Press, 1990, and Stewart et al., " Solid Phase Peptide Synthesis ", Second Edition, Pierce Chemical Company, 1994. The contents of this reference are incorporated herein by reference.

Pharmaceutically acceptable salts of the conjugates provided herein include, for example, conventional non-toxic salts of the conjugates, formed from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include salts derived from inorganic acids (e.g. hydrochloric acid, bromohydroic acid, sulfuric acid, sulfamic acid, phosphoric acid and nitric acid, etc.) and organic acids (e.g. acetic acid, propionic acid, succinic acid, glycolic acid, Stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenyl-acetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, 2-acetoxy-benzoic acid, fumaric acid, toluenesulfonic acid Salts derived from methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethionic acid and trifluoroacetic acid, and the like.

Conjugates provided herein containing a peptidic moiety containing a cell surface protease cleavage site and a therapeutic agent can be similarly synthesized using techniques known to those skilled in the art. For example, the free amine residue on the therapeutic agent may be covalently linked to the peptidic substrate at the carboxyl terminus so that an amide bond is formed. Similarly, amide bonds can be formed by covalently coupling the amine moiety of the peptidic substrate with the carboxyl moiety of the therapeutic agent. For this purpose, 2- (1H-benzotriazol-1-yl) -1,3,3-tetramethyl-uronium hexafluorophosphate (known as HBTU) and 1-hydric in combination or used alone Roxybenzotriazole hydrate (known as HOBT), dicyclohexyl-carbodiimide (DCC), N-ethyl-N- (3-dimethylaminopropyl) -carbodiimide (EDC), diphenyl-phosphoryl Reagents such as zide (DPPA) and benzotriazol-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphate (BOP) and the like can be used.

In addition, such conjugates may be formed by non-peptidyl bonds between cell surface protease cleavage sites and the therapeutic agent. For example, a therapeutic agent may be covalently linked to the carboxyl terminus of the peptidic substrate via hydroxyl residues on such therapeutic agent to form ester linkages. For this purpose, blends of HBTU and HOBT, blends of BOP and imidazole, blends of DCC and DMAP and the like can be used. It is also possible to form nitro-phenyl esters and the like to activate the carboxylic acid and react in the presence of DBU (1,8-diazabicyclo [5,4, O] undec-7-ene).

Such conjugates may also be formed by binding the peptidic substrate to the therapeutic agent via a linker unit. Such linker units include, for example, biscarbonyls such that the amine residue on the therapeutic agent is linked to the linker unit to form an amide bond and the amino terminus of the peptidic substrate is linked to the other end of the linker unit to form an amide bond as well. Alkyl diradicals are included. In contrast, diaminoalkyl diradical linker units are also provided such that the carbonyl moiety on the therapeutic agent is covalently linked to one of the amines of the linker unit and the remaining amine of the linker unit is covalently attached to the C-terminus of the peptidic substrate. Can be useful. Such other linkers are intended that are stable to the physiological environment when not in the presence of cell surface proteases or soluble, secreted or released forms thereof, but cleavable upon cleavage of cell surface protease proteolytic cleavage sites. In addition, linker units may be used that remain bound to the therapeutic agent upon cleavage of the cell surface protease proteolytic cleavage site but do not significantly reduce the therapeutic activity of the therapeutic agent after such cleavage compared to the unmodified therapeutic agent. .

One skilled in the art understands that while synthesizing the conjugates provided herein, there may be a need to protect various reactive functional groups on the starting compounds and intermediates, while the desired reactions occur for other parts of the molecule. After the desired reaction is completed or at any desired time point, typically such protecting groups can be removed, for example, by hydrolysis or hydrogenolysis means. Such protection and deprotection steps are common in organic chemistry. One of ordinary skill in the art would like to teach protective groups that may be useful when preparing the conjugates provided herein, see Protective Groups in Organic Chemistry, McOmie, ed., Plenum Press, NY, NY (1973); and, Protective Groups in Organic Synthesis, Greene, ed., John Wiley & Sons, NY, NY (1991).

By way of example only, useful amino-protecting groups include, for example, C ₁ -C ₁₀ alkanoyl groups such as formyl, acetyl, dichloroacetyl, propionyl, hexanoyl, 3,3-diethylhexanoyl and γ -Chlorobutyryl and the like); C ₁ -C ₁₀ alkoxycarbonyl and C ₅ -C ₁₅ aryloxycarbonyl groups (eg tert-butoxycarbonyl, benzyloxycarbonyl, allyloxycarbonyl, 4-nitrobenzyloxycarbonyl, fluore) Nylmethyloxycarbonyl and cinnamoyloxycarbonyl); Halo (C ₁ -C ₁₀ ) -alkoxycarbonyl (eg 2,2,2-trichloroethoxy-carbonyl); And C ₁ -C ₁₅ arylalkyl and alkenyl groups such as benzyl, phenethyl, allyl and trityl and the like. Other amino-protecting groups used syntactically include those in the form of enamines prepared using β-keto-esters such as methyl or ethyl acetoacetate.

Useful carboxy-protective groups include, for example, C ₁ -C ₁₀ alkyl groups (eg methyl, tert-butyl, decyl); Halo C ₁ -C ₁₀ alkyl such as 2,2,2-trichloroethyl and 2-iodoethyl; C ₅ -C ₁₅ arylalkyl (eg benzyl, 4-methoxybenzyl, 4-nitrobenzyl, triphenylmethyl, diphenyl-methyl); C ₁ -C ₁₀ alkanoyloxymethyl (eg, acetoxy-methyl and propionoxymethyl, etc.); And phenacyl, 4-halofenacyl, allyl, dimethylallyl, tri- (C ₁ -C ₃ alkyl) silyl (eg trimethylsilyl), β-p-toluenesulfonylethyl, β-p-nitrophenyl-thio Groups such as ethyl, 2,4,6-trimethylbenzyl, β-methylthioethyl, phthalimidomethyl, 2,4-dinitro-phenylsulphenyl, 2-nitrobenzhydryl and related groups may be included.

Similarly, useful hydroxy protecting groups include, for example, formyl group, chloroacetyl group, benzyl group, benzhydryl group, trityl group, 4-nitrobenzyl group, trimethylsilyl group, phenacyl group, tertiary -Butyl group, methoxymethyl group, tetrahydropyranyl group, tert-butyldimethylsilyl group and the like.

Regarding the embodiment of the peptidic substrate in combination with the anthracycline antibiotic doxorubicin, the following schemes describe the synthesis of the conjugates provided herein.

Scheme VI describes the preparation of a conjugate of a peptide substrate and a vinca alkaloid cytotoxic agent vinblastine, wherein the binding of vinblastine is located at the C-terminus of the peptide substrate. The use of 1,3-diaminopropane linkers is merely illustrative and other linker units between the carbonyl of vinblastine and the C-terminus of the peptidic substrate are also contemplated (eg, (CH ₂ ) _u -T- (CH ₂ ) _u ). Acyl azide starting materials are prepared by reacting vinblastine with hydrazine (60-65 ° C., MeOH) and then with HCl / DMF / isoamyl nitrite. Scheme VI also describes the synthesis of the conjugates, wherein the C4-hydroxy moiety is reacetylated after addition of the linker unit. It is known that the desacetyl vinblastine conjugate is also effective and can be prepared by protecting the primary amine of the linker, reacting the intermediate with acetic anhydride and then deprotecting the amine, as shown in Scheme VI. [Reference: International Patent Publication No. WO 98/10651].

Conjugation of a functional group of vinblastine with a peptide substrate at another position can be readily accomplished by those skilled in the art and provides a conjugate that is a substrate for cell surface proteases, or soluble, secreted or released forms thereof. It is also expected to.

Scheme VII illustrates the preparation of specific conjugates for use in the compositions and methods provided herein, wherein the peptide substrate is combined with the vinca alkaloid cytotoxic agent vinblastine. The N-terminus binding of the peptidic substrate to vinblastine is described. S.P. Kandukuri et al. (1985) J. Med. Chem. 28: 1079-1088].

The N-terminus of the peptidic substrate used in the compositions and methods provided herein is combined with one therapeutic agent, such as a cytotoxic agent (e.g. vinblastine), while at the same time another cytotoxicity with the same or different C-terminus. It is also understood that it is possible to prepare conjugates bound to doxorubicin, for example. Scheme VIII illustrates the synthesis of such multicytotoxic agent conjugates. Such multicytotoxic agent conjugates can provide many advantages over conjugates containing only one cytotoxic agent.

Regarding the embodiment of the peptidic substrate in combination with desacetylvinblastine, the following schemes IX and X illustrate the synthesis of the conjugates provided herein.

Scheme IX is a conjugate provided herein containing a peptidic substrate provided herein and a vinca alkaloid cytotoxic agent vinblastine, wherein the binding of the oxygen of 4-desacetylvinblastine to the C-terminus of the peptidic substrate Location). Other reaction sequences may be useful for forming such conjugates, but it is known that it is typical to first bind a single amino acid to 4-oxygen and subsequently to the remaining peptidic substrate sequence to said amino acid. Patent Publication WO 99/28345]. It is also known that 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (ODHBT) can be used in place of HOA in the final coupling step.

Scheme X illustrates the preparation of a conjugate of a peptidic substrate, wherein hydroxy alkanoyl acid is used as a linker between the peptidic substrate and vinca drug provided herein.

Taxol conjugates provided herein can be prepared by the general methods provided below. The preparation of 7-Ala-taxol and 7-Gly-taxol is described in Mathew et al. (1992) J. Med. Chem. 35: 145-151.

E. Formulation and Administration of Pharmaceutical Compositions

The conjugates and compositions provided herein are treated by targeting cells or tissues that express at a higher level the soluble, secreted or released form of cell surface proteases, particularly serine proteases, or cell surface proteases thereof, as compared to other cells. It is used to treat, prevent or alleviate one or more symptoms of any disease or disorder that may be. Such diseases or disorders include appropriate cell surface proteases including hyperproliferative diseases (eg cancer), all diseases associated with abnormal or excessive angiogenesis, autoimmune diseases, inflammatory diseases and cell-attached and cell-localized proteases. All other diseases that can be identified include, but are not limited to.

The pharmaceutical compositions provided herein provide a therapeutically effective amount of one or more conjugates provided herein useful for preventing, treating or alleviating one or more symptoms of a disease or disorder associated with undesirable and / or uncontrolled angiogenesis or angiogenesis. It contains. Such diseases or disorders include solid neoplasms, including lung cancer, colon cancer, esophageal cancer, breast cancer, ovarian cancer and prostate cancer; Hemangiofibromas, hemangiolipomas, atherosclerosis, restenosis / reperfusion injury, arteriovenous malformations, angiomatosis and vascular narrowing, cartilage dysplasia with hemangioma, hereditary hemorrhagic capillary dilatation and Von Hippel-Lindau Vascular malformations and cardiovascular disorders, including but not limited to syndromes; Diabetes mellitus, hemophilia joints, inflammatory bowel disease, incurable fractures, rapidly progressing periodontitis, psoriasis, juvenile periodontitis, rheumatoid arthritis, venous congestive ulcers, granules-burns, hypertrophic scars, sclerosis, radiation osteonecrosis, postoperative adhesions, purulent Chronic inflammatory diseases and abnormal wound healing, including but not limited to granulomas and systemic sclerosis; Circulatory disorders, including but not limited to Raynaud's phenomenon; Crest syndrome, including but not limited to calcification, esophageal dyskinesia, dendritic sclerosis and capillary telangiectasia; Systemic vasculitis, scleroderma, necrotic pyoderma, angiopathy, venous artery ulcer, Sturge-Weber syndrome, staphylococcal lesion, blue rubber foam nevi syndrome, Klippel-Trenaunay-Weber syndrome and Skin diseases including but not limited to Osler-Weber-Rendu syndrome; And blindness caused by ocular neovascular disease, corneal graft neovascularization, intraocular macular degeneration, neovascular glaucoma, trachoma, diabetic retinopathy, myopia, prematurity retinopathy, posterior capsular fibrosis and corneal neovascularization. Eye disorders, including but not limited to, but not limited to.

The composition contains one or more conjugates provided herein. Such conjugates may be, for example, oral administration solutions, suspensions, tablets, dispersible tablets, pills, capsules, powders, sustained release formulations or elixirs or sterile solutions or suspensions for parenteral administration, as well as transdermal patch preparations. And suitable pharmaceutical preparations such as dry powder inhalants. Typically, the conjugates described above are formulated into pharmaceutical compositions using techniques and procedures well known in the art. Ansel (1985) Introduction to Pharmaceutical Dosage Forms, Fourth Edition, p. 126)]. Effective concentrations can be determined experimentally using animal models, in vitro models or test subjects.

In the composition, one or more conjugates or pharmaceutically acceptable salts thereof in an effective concentration are mixed with a suitable pharmaceutical carrier or vehicle. The conjugate may be derivatized as a corresponding salt, ester, enol ether or ester, acid, base, solvate or hydrate prior to formulating as described above. The concentration of the conjugate in the composition is effective to deliver an amount that, when administered, treats, prevents or alleviates one or more symptoms of a disease or disorder associated with undesirable and / or uncontrolled angiogenesis or angiogenesis. Such diseases or disorders include solid neoplasms; Hemangiofibroma, hemangiolipoma, atherosclerosis, restenosis / reperfusion injury, arteriovenous malformation, angiomatosis and vascular narrowing, cartilage dysplasia with hemangioma, hereditary hemorrhagic capillary dilatation and von Hippel-Lindau syndrome Vascular malformations and cardiovascular disorders; Diabetes mellitus, hemophilia joints, inflammatory bowel disease, injured skeleton, rapidly progressing periodontitis, juvenile periodontitis, psoriasis, rheumatoid arthritis, venous congestion ulcers, granule-burn, hypertrophic scars, liver cirrhosis, radiation osteonecrosis, postoperative stenosis, pyogenic granulomas And chronic inflammatory diseases and abnormal wound healing, including but not limited to systemic sclerosis; Circulatory disorders, including but not limited to Raynaud's phenomenon; Crest syndrome, including but not limited to calcification, esophageal dyskinesia, scleroderma and capillary dilatation; Including but not limited to systemic vasculitis, scleroderma, necrotic pyoderma, angiopathy, venous arterial ulcers, Sturgi-Weber syndrome, staphylococcal lesions, blue rubber foam nevus syndrome, Klipel Trenonni Weber syndrome and Osler-Weber-rendu syndrome Skin diseases that are not; And blindness caused by ocular neovascular disease, corneal graft neovascularization, intraocular macular degeneration, neovascular glaucoma, trachoma, diabetic retinopathy, myopia degeneration, prematurity retinopathy, posterior capsular fibrosis and corneal neovascularization. Eye disorders, including but not limited to, but not limited to.

The conjugates herein can be formulated into pharmaceutical compositions suitable for topical, topical, intravenous and systemic applications. One or more conjugates of an effective concentration are mixed with a suitable pharmaceutical carrier or vehicle. An effective concentration or amount of conjugate, when administered, requires delivery of an amount that alleviates the symptoms, alleviates the disease, or treats the disease. Typically, the composition is formulated for single dose administration. The therapeutically effective concentrations and amounts can be determined experimentally by testing the conjugates in known in vitro and in vivo systems as described above, and then dosages for humans or other animals can be estimated therefrom.

When mixing the conjugate (s) with the vehicle or adding the vehicle to the conjugate, the resulting mixture can be a solution, suspension, emulsion or other composition. The type of mixture obtained depends on a number of factors including the intended mode of administration and the solubility of the conjugate in the carrier or vehicle of choice. Effective concentrations are sufficient to alleviate the symptoms of the disease, disorder or condition to be treated and can be determined experimentally based on in vitro and / or in vivo data such as data from a mouse xenograft model for rabbits or a rabbit eye model. have. If desired, pharmaceutically acceptable salts or other derivatives of the conjugates can be prepared.

Pharmaceutical carriers or vehicles suitable for administering the conjugates provided herein include any carrier known to those skilled in the art, suitable for a particular mode of administration. In addition, the conjugates may be formulated as a sole pharmaceutically active ingredient in the preparation or combined with other active ingredients.

The conjugates may be administered, for example, in liquid, semi-liquid or solid form by routes suitable for oral, parenteral, intravenous, intradermal, subcutaneous or topical administration, and formulated in a manner suitable for the respective route of administration. . Exemplary mode of administration depends on the indication to be treated. Skin and ocular signs can typically be treated locally, while tumor and vascular proliferative disorders can be conventionally treated by systemic, intradermal and intramuscular modes of administration.

The conjugate is included in a pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the patient to be treated. It will be appreciated that the number and extent of side effects will depend on the conditions under which the conjugate is administered. For example, certain toxic and undesirable side effects that are acceptable in the treatment of life-threatening diseases such as tumors may not be acceptable in the treatment of less severe diseases.

The concentration of the conjugate in the composition depends on the rate of absorption, inactivation rate, release rate, dosing schedule, amount administered, as well as other factors known to those skilled in the art.

Typically, a therapeutically effective amount should produce from about 0.1 ng / ml to about 50 to 100 μg / ml of serum concentration of the active ingredient. The pharmaceutical composition should typically provide a dosage of from about 0.01 mg to about 100 to 2000 mg of the conjugate depending on the selected conjugate as adjusted according to body surface area and / or body weight. Typically, for intravenous or systemic treatment, a daily dose of about 0.05 to 0.5 mg / kg will be sufficient. Topical application to ocular disorders should provide about 1 ng to 100 μg, generally about 1 μg to about 10 μg, per single dose administration. It will be appreciated that the amount administered is a function of the conjugate selected, the symptoms to be treated and the possible side effects that can be tolerated. Dosages can be determined experimentally using models recognized for each disorder.

Typically, the composition is formulated for single dose administration. To formulate the composition, the weight fraction of the conjugate is dissolved, suspended, dispersed or mixed in a selected vehicle at an effective concentration such that the condition to be treated is alleviated or alleviated. Pharmaceutical carriers or vehicles suitable for administration of the conjugates provided herein include any carrier known to those of skill in the art that is suitable for the particular mode of administration.

In addition, the conjugates may be formulated as the only active ingredient in the composition or combined with other active ingredients. Tissue-targeted liposomes, in particular liposome suspensions comprising tumor-targeted liposomes, may also be suitable as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art. For example, liposome formulations can be prepared as described in US Pat. No. 4,522,811. Briefly stated, liposomes such as multiple thin film vesicles (MLV) can be formed by drying egg phosphatidyl choline and brain phosphatidyl serine (7: 3 molar ratio) inside the flask. Provided herein is a solution of the conjugate in phosphate buffered saline (PBS) lacking divalent cations and shaking the flask until the lipid membrane is dispersed. The resulting vesicles are washed to remove unencapsulated conjugates, pelleted by centrifugation, and then resuspended in PBS.

The conjugate is included in a pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically effective effect in the absence of undesirable side effects on the patient to be treated. The therapeutically effective concentration can be determined experimentally by testing the conjugates in the in vitro and in vivo systems described herein (Examples 3 and 4), and then the dosage for humans or other animals can be estimated therefrom. .

The concentration of the conjugate in the pharmaceutical composition depends on the rate of absorption, inactivation and excretion of the conjugate, the physicochemical characteristics of the conjugate, the schedule of administration and the amount administered, as well as other factors known to those skilled in the art. For example, the amount delivered is sufficient to alleviate one or more symptoms of a disease or disorder associated with undesirable and / or uncontrolled angiogenesis or neovascularization as described herein.

Typically, a therapeutically effective amount should produce from about 0.1 ng / ml to about 50 to 100 μg / ml of serum concentration of the active ingredient. The pharmaceutical composition typically should provide a dosage of about 0.01 mg / kg body weight / day to about 2000 mg / kg body weight / day of the conjugate. Pharmaceutical dosage unit forms are prepared to provide from about 1 mg to about 1000 mg and generally from about 10 mg to about 500 mg of the essential active ingredient or combination of essential ingredients per dosage unit form.

The conjugate may be administered at one time or may be divided into a number of smaller doses administered at intervals of time. It will be appreciated that the exact dosage and duration of administration are a function of the disease to be treated and can be determined experimentally using known test protocols or by estimation from in vivo or in vitro test data. It will be appreciated that concentrations and dosage values may vary depending on the severity of the condition to be alleviated. For any particular subject, a particular dosage regimen should be adjusted over time depending on individual needs and the professional judgment of the person administering the composition or administering the composition, and the concentration ranges presented herein are illustrative only. It will be further understood that the scope or use of the claimed compositions and combinations containing them is not limited.

Exemplary pharmaceutically acceptable derivatives include acid, base, enol ethers and esters, salts, esters, hydrates, solvates and conjugate forms. The derivative is chosen such that its pharmacokinetic properties are superior to the corresponding natural conjugates.

Thus, to form a pharmaceutical composition, an effective concentration or amount of the conjugates or pharmaceutically acceptable derivatives thereof described herein is mixed with a pharmaceutical carrier or vehicle suitable for systemic, topical or topical administration. The conjugate is included in an amount effective to alleviate or treat or prevent one or more symptoms of a disease or disorder associated with undesirable and / or uncontrolled angiogenesis or angiogenesis as described herein. . The concentration of the conjugate in the composition depends on the rate of absorption, inactivation rate, release rate, dosing schedule, amount administered, the particular formulation, as well as other factors known to those skilled in the art.

The composition will be administered by any suitable route including oral, parenteral, rectal, topical and topical administration. For oral administration, capsules and tablets are generally used. The compositions are in liquid, semi-liquid or solid form and are formulated in a manner suitable for the respective route of administration. Exemplary modes of administration are parenteral and oral.

Liquids or suspensions for parenteral, intradermal, subcutaneous or topical application may comprise any of the following ingredients: sterile diluents, for example water for injection, saline solution, fixed oils, polyethylene glycols, glycerin , Propylene glycol or other synthetic solvents; Antimicrobial agents such as benzyl alcohol and methyl parabens; Antioxidants such as ascorbic acid and sodium bisulfite; Chelating agents such as ethylenediaminetetraacetic acid (EDTA); Buffers such as acetate, citrate and phosphate; And osmotic pressure regulators such as sodium chloride or dextrose. Parenteral preparations may be enclosed in ampoules, disposable syringes, or vials of single or several doses made of glass, plastic or other suitable material.

If the conjugate exhibits insufficient solubility, a method of solubilizing the conjugate can be used. Such methods are known to those skilled in the art and include methods using cosolvents (e.g. dimethylsulfoxide (DMSO)), methods using surfactants (e.g. Tween ^® ) or dissolving in aqueous sodium bicarbonate. Including but not limited to. Derivatives of the conjugates can also be used to formulate effective pharmaceutical compositions.

Upon mixing or addition of the conjugate (s), the resulting mixture may be a solution, suspension or emulsion, or the like. The form of the resulting mixture will depend on a number of factors including the intended mode of administration and the solubility of the compound in the carrier or vehicle of choice. Effective concentrations are sufficient to alleviate the symptoms of the disease, disorder or condition to be treated and can be determined experimentally.

The pharmaceutical composition may be in unit dosage form containing a suitable amount of a suitable or pharmaceutically acceptable derivative thereof, such as tablets, capsules, pills, powders, granules, sterile parenteral solutions or suspensions, and oral It is provided for administration to humans and animals in the form of solutions or suspensions, and lactating emulsions. Conjugates and derivatives thereof are typically formulated and administered in unit dosage form or multi-dose form. Unit-dosage form as used herein means physically discrete units suitable for human and animal patients and individually packaged as is known in the art. Each unit dose contains a predetermined amount of therapeutically active compound sufficient to provide the desired therapeutic effect, mixed with the required pharmaceutical carrier, vehicle or diluent. Examples of unit dosage forms include ampoules and syringes and individually packaged tablets or capsules. Unit-dosage forms can be administered as fractions or in multiples thereof. Multi-dosage forms are a number of identical unit-dosage forms packaged in a single container for administration in separate unit-dosage forms. Examples of multi-dose forms include vials, bottles or bottles of pints or gallons of tablets or capsules. Thus, multiple dosage forms are multiple unit-dosage forms that are not isolated at the time of packaging.

The composition may contain the following together with a conjugate: diluents such as lactose, sucrose, dicalcium phosphate or carboxymethylcellulose; Lubricants such as magnesium stearate, calcium stearate and talc; Binders such as starch, natural rubber (rubber acacia gelatin), glucose, molasses, polyvinylpyrrolidone, cellulose and derivatives thereof, povidone, crospovidone and other binders known to those skilled in the art. Pharmaceutically administrable liquid compositions can be prepared by, for example, dissolving, dispersing or mixing the conjugate as defined above and optionally pharmaceutical adjuvant in a carrier such as water, saline, aqueous dextrose, glycerol, glycol, ethanol and the like. It can be prepared by forming a solution or suspension. If desired, the pharmaceutical compositions to be administered are wetting agents, emulsifiers or solubilizers and pH buffers, such as, for example, acetates, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, And small amounts of non-toxic auxiliary substances such as other agents. Practical methods for preparing such dosage forms are known and will be apparent to those skilled in the art (eg, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 15th Edition, 1975). The composition or formulation to be administered will in any case contain a conjugate in an amount sufficient to alleviate the symptoms of the patient being treated.

Dosage forms or compositions may be prepared containing the remainder consisting of the active ingredient in the range of 0.005 to 100% and the non-toxic carrier. For oral administration, the pharmaceutically acceptable non-toxic composition may be any of the excipients commonly used, for example pharmaceutical mannitol, lactose, starch, magnesium stearate, talc, cellulose derivatives, sodium croscarmellose, glucose, It is formed by incorporating sucrose, magnesium carbonate or sodium saccharin. Such compositions include, but are not limited to, sustained release formulations and biodegradable biocompatible polymers (eg, collagen, ethylene vinyl acetate), including, but not limited to, solutions, suspensions, tablets, capsules, powders, and implants and microencapsulated delivery systems. , Polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid and the like). Methods of making these compositions are known to those skilled in the art. The intended composition may contain from 0.001% to 100% active ingredient, such as from 0.1% to 85%, for example from 75% to 95%.

Conjugates or pharmaceutically acceptable derivatives may be prepared as sustained release formulations or coatings using a carrier which prevents the conjugate from being rapidly removed from the body. The composition may include other conjugates to obtain the desired combination of properties. The conjugates provided herein or pharmaceutically acceptable derivatives thereof as described herein refer to the above diseases or disorders associated with angiogenesis or neovascularization which are undesirable and / or uncontrolled for therapeutic or prophylactic purposes. It may also be advantageously administered with another pharmacological agent known in the art, which is useful for treating one or more of the diseases or medical conditions. It will be appreciated that such combination therapies constitute additional aspects of the compositions and methods of treatment provided herein.

1. Compositions for Oral Administration

Oral pharmaceutical dosage forms are solids, gels or liquids. Solid dosage forms are tablets, capsules, granules, and mixed powders. Types of oral tablets include compressed chewable lozenges and tablets that can be enteric coated, sugar coated or encapsulated. Capsules may be hard or soft gelatin capsules, and granules and powders may be provided in non-foamable or effervescent form with other ingredients known to those skilled in the art.

In certain embodiments, the dosage form is a solid dosage form, eg, a capsule or tablet. Tablets, pills, capsules, troches, and other dosage forms may contain, for example, compounds of any of the following ingredients or similar properties. May contain: a binder; diluent; Disintegrants; slush; Glidants; Sweeteners and flavoring agents.

Examples of binders include microcrystalline cellulose, tragacanth rubber, glucose solution, acacia rubber, gelatin solution, sucrose and starch paste. Lubricants include talc, starch, magnesium or calcium stearate, lycopodium and stearic acid. Diluents include, for example, lactose, sucrose, starch, kaolin, salts, mannitol and dicalcium phosphate. Glidants include, but are not limited to, colloidal silicon dioxide. Disintegrants include croscarmellose sodium, sodium starch glycolate, alginic acid, corn starch, potato starch, bentonite, methylcellulose, agar and carboxymethylcellulose. Colorants include, for example, any approved water soluble FD & C dyes, mixtures thereof; And water insoluble FD & C dyes suspended on alumina hydrate. Sweetening agents include sucrose, lactose, mannitol and artificial sweeteners such as saccharin and any number of partially dried flavors. Flavoring agents include methyl salicylates and synthetic blends of natural flavors extracted from plants such as fruits and conjugates (including but not limited to peppermint) that impart a refreshing sensation. Wetting agents include propylene glycol mosttearate, sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylene lauryl ether. Enteric coatings include fatty acids, fats, waxes, shellac, ammonia shellac and cellulose acetate phthalate. Coating agents include hydroxyethyl cellulose, sodium carboxymethyl cellulose, polyethylene glycol 4000 and cellulose acetate phthalate.

If oral administration is desired, the conjugate may be provided as a composition that protects it from the acidic environment of the stomach. For example, the composition can be formulated as an enteric coating that retains its native form in the stomach and releases the conjugate in the intestine. The compositions may also be formulated in combination with antacids or other ingredients.

When the dosage unit form is a capsule, it may contain a liquid carrier such as fatty oil in addition to the above types of substances. In addition, dosage unit forms may contain various other materials that modify the physical form of the dosage unit, such as sugar coatings and other enteric coatings. The conjugate may also be administered as a component such as elixirs, suspensions, syrups, wafers, sprinkles or chewing gums. Syrups may contain, in addition to the conjugate, sucrose as a sweetening agent and certain preservatives, dyes and colorants and flavors.

The conjugate may also be mixed with other active ingredients that do not impair the desired action or with substances that complement the desired action, such as antacids, H2 blockers and diuretics. Higher concentrations, up to about 98% by weight of the conjugate may be included.

Pharmaceutically acceptable carriers included in tablets are binders, lubricants, diluents, disintegrants, colorants, flavors and wetting agents. Enteric coated tablets are resistant to the action of gastric acid due to the enteric coating and dissolve or disintegrate in the neutral or alkaline intestine. Sugar coated tablets are compressed tablets to which different layers of pharmaceutically acceptable materials have been applied. Encapsulated tablets are compressed tablets coated with a polymer or other suitable coating. Multiple compressed tablets are compressed tablets made by one or more compression cycles using the pharmaceutically acceptable materials already mentioned. Coloring agents can also be used in the dosage forms described above. Flavoring and sweetening agents are used in compressed tablets, sugar coated tablets, multiple compressed and chewable tablets. Flavoring and sweetening agents are particularly useful when forming chewable tablets and lozenges.

Liquid oral dosage forms include suspensions reconstituted from aqueous liquids, emulsions, suspensions, solutions and / or non-foamable granules and effervescent preparations reconstituted from effervescent granules. Aqueous liquids include, for example, elixirs and syrups. Emulsions are oil-in-water or water-in-oil.

Elixir is a sweet, transparent, hydroalcoholic agent. Pharmaceutically acceptable carriers used in elixirs include solvents. Syrups are concentrated aqueous solutions of sugars such as sucrose and may contain a preservative. Emulsions are two-phase systems in which one liquid is dispersed in the form of small spheres throughout another liquid. Pharmaceutically acceptable carriers used in emulsions are non-aqueous liquids, emulsifiers and preservatives. Suspensions use pharmaceutically acceptable suspending agents and preservatives. Pharmaceutically acceptable materials used in non-foamable granules to be reconstituted in liquid or oral dosage forms include diluents, sweeteners and wetting agents. Pharmaceutically acceptable materials used in effervescent granules to be reconstituted in liquid or oral dosage forms include organic acids and carbon dioxide sources. Colorants and flavoring agents are used in all of the above dosage forms.

Solvents include glycerin, sorbitol, ethyl alcohol and syrup. Examples of preservatives include glycerin, methyl and propylparabens, benzoic acid, sodium benzoate and alcohols. Examples of non-aqueous liquids used in emulsions include mineral oil and cottonseed oil. Examples of emulsifiers include gelatin, acacia, tragacanth, bentonite and surfactants such as polyoxyethylene sorbitan monooleate. Examples of suspending agents include sodium carboxymethylcellulose, pectin, tragacanth, Veegum and acacia. Diluents include lactose and sucrose. Sweetening agents include sucrose, syrup, glycerin and artificial sweeteners (saccharin). Wetting agents include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylene lauryl ether. Organic acids include citric acid and tartaric acid. Sources of carbon dioxide include sodium bicarbonate and sodium carbonate. Colorants include any approved water soluble FD & C dyes and mixtures thereof. Flavoring agents include synthetic blends of natural flavors extracted from plants such as fruits and conjugates that give a refreshing aesthetic.

For solid dosage forms, for example, solutions or suspensions in propylene carbonate, vegetable oils or triglycerides, for example formulations, may be enclosed in gelatin capsules. Such solutions and their preparation and encapsulation are described in US Pat. No. 4,328,245; 4,409,239; 4,409,239; And 4,410,545. For liquid dosage forms, for example, liquid solutions in polyethylene glycol can be diluted with a sufficient amount of a pharmaceutically acceptable liquid carrier, for example water, which is readily measured for administration.

In addition, liquid or semisolid oral formulations dissolve or disperse conjugates or derivatives thereof in vegetable oils, glycols, triglycerides, propylene glycol esters (such as propylene carbonate) and other carriers, and disperse these solutions or suspensions in hard or soft gelatin capsule shells. It can be produced by encapsulation. Other useful formulations include formulations set forth in US Pat. Nos. 28,819 and 4,358,603. Briefly stated, such formulations include conjugates provided herein, 1,2-dimethoxymethane, diglyme, triglyme, tetraglyme, polyethylene glycol-350-dimethyl ether, polyethylene glycol-550-dimethyl ether, polyethylene glycol Dialkylated mono- or poly-alkylene glycols and one or more antioxidants, including, but not limited to, -750-dimethyl ether, where 350, 550, and 750 mean the approximate average molecular weight of polyethylene glycol Butylated Hydroxytolene (BHT), Butylated Hydroxyanisole (BHA), Propyl Gallate, Vitamin E, Hydroquinone, Hydroxycoumarin, Ethanolamine, Lecithin, Cephalin, Ascorbic Acid, Malic Acid, Sorbitol , Phosphoric acid, thiodipropionic acid and esters thereof and dithiocarbamate).

Other formulations include, but are not limited to, aqueous alcoholic solutions comprising pharmaceutically acceptable acetals. The alcohol used in such formulations is any pharmaceutically acceptable water miscible solvent having one or more hydroxyl groups, including but not limited to propylene glycol and ethanol. Acetals include but are not limited to di (lower alkyl) acetals of lower alkyl aldehydes such as acetaldehyde diethyl acetal.

In all embodiments, tablets and capsules of the formulation may be coated as known to those skilled in the art to modify or maintain dissolution of the conjugate. Thus, for example, tablets and capsules may be coated with conventional coatings that are digestible in the gut, such as phenylsalicylate, wax and cellulose acetate phthalate.

2. Injections, solutions and emulsions

Parenteral administration, generally characterized by subcutaneous, intramuscular or intravenous injection, is also contemplated herein. Injectables can be prepared in conventional forms such as liquid solutions or suspensions, solid forms or emulsions suitable for dissolving or suspending in liquids prior to injection. Suitable excipients are, for example, water, saline, dextrose, glycerol or ethanol. In addition, in some cases, the pharmaceutical composition to be administered may contain a small amount, such as wetting or emulsifying agents, pH buffers, stabilizers, solubility enhancers and other agents, such as sodium acetate, sodium monolaurate, triethanolamine oleate and cyclodextrins. It may also contain non-toxic auxiliary substances. Implantation of sustained release or sustained release systems such that constant dosage levels are maintained (see US Pat. No. 3,710,795) is also contemplated herein. Briefly stated, the conjugates provided herein are insoluble in body fluids and contain external polymeric membranes such as polyethylene, polypropylene, ethylene / propylene copolymers, ethylene / ethyl acetate copolymers, ethylene / vinylacetate copolymers, silicone rubbers, Polydimethyl siloxane, neoprene rubber, chlorinated polyethylene, polyvinylchloride, vinyl acetate, vinylidene chloride, vinyl chloride copolymers with ethylene and propylene, ionomer polyethylene terephthalate, butyl rubber epichlorohydrin rubber, ethylene / vinyl alcohol air Solid internal matrix surrounded by copolymers, ethylene / vinyl acetate / vinyl alcohol terpolymers and ethylene / vinyloxyethanol copolymers, such as polymethylmethacrylate, polybutylmethacrylate, plasticized or unplasticized Polyvinylchloride, plasticization Nylon, plasticized polyethylene terephthalate, natural rubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene, ethylene-vinylacetate copolymer, silicone rubber, polydimethylsiloxane, silicone carbonate copolymer, hydrophilic polymer (e.g. Hydrogels of esters of acrylic acid and methacrylic acid) dispersed in collagen, crosslinked polyvinyl alcohol and crosslinked partially hydrolyzed polyvinyl acetate. The conjugate is dispersed throughout the outer polymeric membrane in the release rate control step. The proportion of polymer contained in such parenteral compositions depends largely on its specific properties as well as the activity of the conjugate and the needs of the patient.

Parenteral administration of the composition includes intravenous, subcutaneous and intramuscular administration. Formulations for parenteral administration may be immediately formulated with an immediate injectable sterile solution, including a subcutaneous tablet, an immediate injectable sterile suspension, a sterile insoluble product that can be immediately combined with the vehicle immediately before use, and a sterile emulsion, and a solvent immediately before use. Sterile anhydrous water soluble products, such as lyophilized powders.

When administered intravenously, suitable carriers include physiological saline or phosphate buffered saline (PBS) and solutions containing thickeners and solubilizers such as glucose, polyethylene glycol and polypropylene glycol and mixtures thereof.

Pharmaceutically acceptable carriers used in parenteral preparations include aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, metal ion sequestrants or chelating agents, and the like. Pharmaceutically acceptable substances.

Examples of aqueous vehicles include naclium chloride injections, Ringer's injections, isotonic dextrose injections, sterile water injections, dextrose and lactate Ringer's injections. Non-aqueous parenteral vehicles include fixed oils, cottonseed oil, corn oil, sesame oil and peanut oil of vegetable origin. Antimicrobial agents of bacteriostatic or fungal concentrations should be added to parenteral preparations packaged in multi-dose containers, antimicrobial agents being phenol or cresol, mercury, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, Thimerosal, benzalkonium chloride and benzetonium chloride. Buffers include phosphate and citrate. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending agents and dispersing agents include sodium carboxymethylcellulose, hydroxypropyl methylcellulose and polyvinylpyrrolidone. Emulsifiers include Polysorbate 80 (Tween ^® 80). Metal ion sequestrants or chelating agents include EDTA. Pharmaceutically acceptable carriers also include ethyl alcohol, polyethylene glycol and propylene glycol for water-miscible vehicles and include sodium hydroxide, hydrochloric acid, citric acid or lactic acid for pH adjustment.

The concentration of the conjugate is adjusted to such an extent that the injection provides an effective amount that produces the desired pharmacological effect. The exact dosage depends on the patient or animal age, weight and condition as is known in the art.

Unit dosage parenteral formulations are packaged in ampoules, vials or syringes with needles. All formulations for parenteral administration should be sterile as known and practiced in the art.

As an example, intravenous or intraarterial infusion of sterile aqueous solutions containing conjugates is an effective mode of administration. Another embodiment is a sterile aqueous solution or oily solution or suspension containing the conjugate to be injected as needed to produce the desired pharmacological effect.

Injectables are designed for topical and systemic administration. Typically, a therapeutically effective amount is formulated to contain a concentration of about 0.1% w / w to about 90% w / w or more, generally 1% w / w or more of the conjugate to the tissue (s) to be treated. The conjugate may be administered at one time or may be divided into a number of smaller doses administered at intervals of time. It will be appreciated that the exact dosage and duration of treatment are a function of the tissue to be treated and can be determined experimentally using known test protocols or by estimation from in vivo or in vitro test data. It will be appreciated that concentrations and dosage values may vary depending on the severity of the condition to be alleviated. For any particular subject, a particular dosage regimen should be adjusted over time depending on the needs of the individual and the professional judgment of the person administering or administering the dosage form and the concentration ranges presented herein are illustrative only. It will be further understood that it is not intended to limit the scope or practice of the claimed formulations.

The conjugates can be suspended in fine or other suitable form or derivatized to produce more stable products. The form of the mixture obtained depends on a number of factors including the intended mode of administration and the solubility of the conjugate in the selected carrier or vehicle. Effective concentrations are sufficient to alleviate the symptoms of the condition and can be determined experimentally.

3. Lyophilized Powder

Of interest herein are lyophilized powders that can be reconstituted for administration as solutions, emulsions and other mixtures. Lyophilized powder can be reconstituted and formulated as a solid or gel.

Sterile lyophilized powders are prepared by dissolving the conjugates provided herein or pharmaceutically acceptable derivatives thereof in a suitable solvent. The solvent may contain excipients that improve the stability or other pharmacological component of the powder or reconstituted solution prepared from such powder. Excipients that can be used include, but are not limited to, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other stable agents. The solvent may also contain buffers such as citrate, sodium phosphate or potassium phosphate or other buffers, typically of about neutral pH, known to those skilled in the art. The solution is subsequently sterile filtered and then lyophilized under standard conditions known to those skilled in the art to prepare the desired formulation. In general, the resulting solution can be dispensed into lyophilized vials. Each vial may contain a single dose (eg, 10-1000 mg, eg, 100-500 mg) or multiple doses of the conjugate. The lyophilized powder can be stored under appropriate conditions such as about 4 ° C. to room temperature.

This lyophilized powder is reconstituted with water for injection to obtain a formulation for parenteral administration. For reconstitution, generally about 1-50 mg, for example 5-35 mg or about 9-30 mg, of lyophilized powder per ml of sterile water or other suitable carrier is added. The exact amount depends on the conjugate selected, the intended subject, and other experimentally measurable parameters. Thus, the amount can be determined experimentally.

4. Topical Administration

Topical mixtures are prepared as described for topical and systemic administration. The resulting mixture may be a solution, suspension or emulsion, and the like, creams, gels, ointments, emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes, foams, aerosols, irrigations, sprays, It may be formulated in suppositories, bandages, transdermal patches or any other formulation suitable for topical administration.

Conjugates or pharmaceutically acceptable derivatives thereof can be formulated as aerosols for topical application by inhalation. See US Pat. No. 4,044,126, which describes aerosols for the delivery of steroids useful for treating inflammatory diseases, especially asthma. 4,414,209, and 4,364,923. These formulations for administration to the respiratory tract may be in the form of aerosols or solutions for nebulizers or fine powders for blowers, either alone or in combination with an inert carrier such as lactose. In such cases, the particles of the formulation may typically be less than 50 microns, typically less than 10 microns in diameter.

The conjugates may be formulated in the form of gels, creams and lotions for topical or topical application, for example for application to the skin and mucous membranes (such as the eye), and applied to the eye or intratracheal or spinal cord. It may be formulated for application. Topical administration is contemplated for transdermal delivery and administration to the ocular or mucosa or inhalation therapy. Nasal solutions of the conjugates, alone or in combination with other pharmaceutically acceptable excipients, can also be used.

Such solutions, in particular those intended for ocular use, can be formulated as 0.01% to 10% isotonic solutions with a pH of about 5 to 7 with suitable salts.

5. Compositions for Other Routes of Administration

Other routes of administration are also contemplated herein, such as topical application, transdermal patches and rectal administration.

For example, pharmaceutical dosage forms for rectal administration are rectal suppositories, capsules and tablets for systemic effects. Rectal suppositories as used herein are solids for insertion into the rectum that melt or soften at body temperature to release one or more conjugates. Pharmaceutically acceptable substances used in rectal suppositories are agents that raise the substrate or vehicle and melting point. Examples of substrates include cocoa butter (theobroma oil), glycerin-gelatin, carbowax (polyoxyethylene glycol) and suitable mixtures of mono-, di- and triglycerides of fatty acids. Formulations that raise the melting point of suppositories include spermatozoa and waxes. Rectal suppositories can be prepared by compression methods or by molding. Typical weights of rectal suppositories are about 2 to 3 gm.

Tablets and capsules for rectal administration are prepared in the same manner as formulations for oral administration using the same pharmaceutically acceptable materials.

6. Products

Conjugates or pharmaceutically acceptable derivatives are conjugates or pharmaceutically acceptable derivatives thereof provided herein for use in the treatment, prevention or alleviation of one or more symptoms associated with packaging materials, proliferative diseases or disorders and the derivatives or pharmaceutically It may be packaged as a product containing a label indicating that an acceptable derivative thereof is used for the treatment, prevention or alleviation of one or more symptoms associated with a proliferative disease or disorder.

The products provided herein contain packaging materials. Packaging materials used to package pharmaceutical products are well known to those skilled in the art (US Pat. Nos. 5,323,907, 5,052,558 and 5,033,252). Examples of pharmaceutical packaging materials include, but are not limited to, transparent packaging, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes, bottles, and selected formulations and packaging materials suitable for the intended mode of administration and treatment. Extensive formulation of the conjugates and compositions provided herein is contemplated as well as various therapies for any disorder involving cell surface proteases, their soluble, secreted or released forms.

F. Evaluation of Conjugate Activity

Standard physiological, pharmacological and biochemical procedures can be used to test conjugates to identify conjugates that have therapeutic activity upon action in cell surface proteases or soluble, secreted or released forms thereof. In vitro and in vivo assays that can be used to assess the therapeutic activity of the conjugate, eg, cytotoxicity, will depend on the therapeutic agent to be tested.

Exemplary assays are briefly discussed below in connection with cytotoxic conjugates (see Examples). The specific activity assayed will depend on the therapeutic agent conjugated.

1. In vitro assay

Therapeutic activity, eg, cytotoxicity, of the conjugates provided herein can be assessed by any assay commonly used to assess therapeutic activity, eg, cytotoxicity, of nonconjugated therapeutic agents. Numerous such assays are known, for example, assays can utilize cells expressing targeted cell surface proteases to evaluate the therapeutic activity of a therapeutic agent. For example, cytotoxicity can be assessed by measuring cell viability or measuring cell proliferation by incorporating labeled nucleotides or other labels. In general, activity is compared to cells that do not express the targeted protease.

For example, the cell can be any cell that expresses a targeted MTSP or endoteliase. Such cells may be selected for cells known to express cell surface proteases by measuring tissue expression profiles as discussed above, or labeled for proteases in the presence and absence of known substrates of targeted proteases, e.g. , By screening with an antibody against targeted protease or protease activity in the presence of a chromogenic substrate.

In addition, a cell line expressing a protease of interest can be prepared by introducing a nucleic acid encoding a protease into a cell line that does not express a protease and expressing it in the cell line. The recombinant cells obtained can be used in cytotoxicity assays.

2. In vivo assay

Numerous animal models are known for assessing therapeutic activity. Any suitable in vivo model can be used. Examples include mouse xenograft models and chick embryo models.

Chick embryo model

CAM model [Chick Embryo choriocapillary model; Ossowski (1998), J. Cell Biol., 107: 2437-2445 provide another method for assessing the inhibitory activity of test compounds. In the CAM model, tumor cells invade through the chorionic villus containing CAM, while tumor cells in the presence of some serine protease inhibitors have had low or no invasion through this membrane. Thus, CAM assays are performed using CAM and tumor cells in the presence and absence of various concentrations of test compound. Invasiveness of tumor cells is measured under conditions that provide an indication of the inhibitory activity of the compounds. Compounds with inhibitory activity correlate with lower tumor invasion.

Thus, CAM assays are performed using CAM and tumor cells in the presence and absence of various concentrations of test compound. Compounds having activity correlate with changes in tumor invasion and / or tumor growth.

For example, the activity of a cell surface protease or a conjugate that releases a therapeutic agent such as a cytotoxic agent can be assessed using this model. If the therapeutic agent is released from the compound and the therapeutic agent is an inhibitor, tumor invasion will be lower and tumor size will be reduced. If the therapeutic agent is inactive in the conjugate, it will not affect tumor invasion.

The CAM model is also used in standard assays for angiogenesis (ie, effects on the formation of neovascularization) (Brooks et al. (1999) Methods in Molecular Biology, 129: 257-269). According to this model, a filter disc containing angiogenic inducers such as basic fibroblast growth factor (bFGF) is placed on the CAM. The diffusion of the cytokine into the CAM induces local angiogenesis, which can be measured in several ways, such as by counting the number of blood vessel branching points in the CAM just below the filter disc. The ability of the identified compounds to inhibit cytokine-induced angiogenesis can be tested using this model. Test compounds may be added to filter discs containing angiogenic inducers and placed directly on the membrane or administered systemically. The degree of neovascularization in the presence and / or absence of the test compound can be compared using this model. Less neovascularization in the presence of test compounds will be indicative of anti-angiogenic activity.

It may be adapted for use with the conjugates herein to evaluate (1) the activity of the therapeutic agent in the conjugate, and (2) the ability of a particular cell surface protease to release the therapeutic agent from the conjugate.

Mouse xenograft model

In vivo activity can be assessed using recognized animal models, such as well known anti-tumor activity mouse xenograft models. Beitz et al. (1992) Cancer Research 52: 227-230; Houghton et al. (1982) Cancer Res. 42: 535-539; Bogden et al. (1981) Cancer (Philadelphia) 48: 10-20; Hoogenhout et al. (1983) Int. J. Radiat. Oncol., Biol. Phys. 9: 871-879; Stastny et al. (1993) Cancer Res. 53: 5740-5744. In vivo mouse solid tumor xenograft models are used in assays to test the ability of agents to inhibit tumor cell growth and / or spontaneous metastasis. For example, the conjugate is evaluated in a mouse tumor xenograft model for antitumor activity against any tumor subtype that expresses a targeted cell surface protease, such as an ovarian tumor. Nude mice are injected intravenously with one or more conjugates, for example four times. Dosage substances are prepared by mixing the test substance with an appropriate volume, eg, PBS / 0.1% / BSA, to achieve the desired dosage. During the experimental period, day 1 is designated as the day in which tumor cells are injected into the mouse, for example, IV injection (250-300 μl) into the tail vein in mice on days 5, 12, 19 and 26. Dosage is fixed or normalized according to differences in body weight. Tumor volume is measured twice a week for the selected period.

Thymus-free female Balb / c nu / nu mice from 8 to 12 weeks old (Roger Williams Hospital Animal Facility, Providence, RI) are suitable mice. These mice are maintained in a sterile environment and selected to have a body weight in the range of about 25-30 g on the day before dosing. Animals are kept in quarantine and domesticated under sterile conditions. Food and water are provided at random. Suitable tumor cells can be obtained, for example, from the American Type Culture Collection (Rockville, MD) and grown in modified Eagle's medium supplemented with 10% fetal calf serum. Mice are injected subcutaneously with tumor cells into the right posterior flank 5 days prior to the selected day, eg, 5 days prior to injection of the test substance.

Calipers are used to measure the area of each tumor. Measurements of the maximum and minimum width in mm are performed prior to injecting the test substance at selected intervals during the experiment, eg, twice a week. Tumor volume (mm ³ ) can be calculated, for example, using the following formula:

Volume = [(Width) ² (Length)] / 2

G. How to Identify Proteases for Targets

Also provided is a method of identifying a protease that targets a conjugate for treating a disease. The method involves identifying cell surface protease related diseases by identifying cells involved in the disease process or cells in the vicinity of cells involved in the disease process. For example, when a disease is associated with a particular tumor, a tumor presented on a cell located at or near the particular tumor is identified. Subsequently, cell surface proteases on the cells for targeting and substrates thereof are identified. Subsequently, a conjugate can be prepared that targets the protease as provided herein.

The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

Example 1

General Procedure for the Preparation of Peptide-Doxorubicin Conjugates

Step A: Preparation of Peptides on Wang Resin

Peptides were automatically prepared using an ABI 431A Peptide Synthesizer (Perseptive Biosystems) on pre-added Wang resin (0.25 mmol). ABI 431A uses HOBT, HBTU, DIEA activation. Synthesis of N-acetyl (or other amide) capped peptides involves the use of AcOH (or other representative carboxylic acid) during the final coupling step in ABI 431A. Other N-terminal caps manually bound using the following reagents: For carbamate and sulfonamides, the peptides were capped with ROCOCl or RSO ₂ Cl and DIEA (4 equivalents, 1 hour each) in DMF (3 mL).

Step B: Cleavage of Peptides from Wang Resin

Cleavage of the peptide from Wang resin involves shaking the resin with 2 mL TFA / H 2 O (95: 5) for 45 minutes. The resin is filtered off and the filtrate is left for additional time. The solution is concentrated to a residue. The crude peptide is analyzed by analytical HPLC (System A). Typical purity of the crude peptide is in the range of 80% to 95%. Peptides are purified by preparative HPLC (System B) using an appropriate gradient (typically 10-30%). The pure fractions are then lyophilized to give the desired peptide as a white solid. Typical yields are 20-50% and purity is 96-99%.

Analytical HPLC Conditions (System A)

Column: Chromolith RP-18e 4.6 mm x 100 mm from EM science

Gradient: 5-50% B in A over 6 minutes

Flow rate: 4 mL / min

Solvent A: 0.1% TFA in water

Solvent B: 0.1% TFA in acetonitrile

Wavelength: 210 nm, 280 nm

Preparative HPLC Conditions (System B)

Column: Ultro 120 5 C18Q 150 x 20 manufactured by Peeke Scientific

Gradient: 0-20% B, or 10-30% B or 20-40% B in A over 40 minutes

Flow rate: 18mL / min

Solvent A: 0.1% TFA in water

Solvent B: Acetonitrile

Wavelength: 214 nm

Step C: Coupling of Peptide Acids to Doxorubicin

To a mixture of peptide acid (0.052 mmol, 1.2 equiv), doxorubicin hydrochloride (0.043 mmol, 25 mg) and HATU (0.0604 mmol, 22.9 mg, 1.4 equiv), DMF (1 mL) followed by 2,6-lutidine (0.17 mmol, 20 μl, 4 equiv) is added. The mixture is mixed until a homogeneous solution is obtained. After 4 to 24 hours (monitored by HPLC System A), the reaction is diluted with water (9 mL) and purified directly by preparative HPLC (System D). The pure fractions are then lyophilized to give the desired peptide doxorubicin conjugates as fluffy red solids. The amount of final conjugate is confirmed by analytical HPLC (System C) and mass spectrometry. Typical yields are 10-30% and purity 95-99%. (Note: DIEA is substituted as base if peptide acid contains histidine residues and reaction time is shortened to 1 when peptide acid is replaced with base)

Deprotection of Fluorenylmethylester of Peptide Doxorubicin Conjugate: When free carboxylic acid is present in the conjugate, fluorenyl methyl ester is used to protect the carboxylic acid while the C-terminus of the peptide acid is coupled to doxorubicin. The fluorenylmethyl group is removed using 10% morpholine in DMF for 1 hour.

Analytical HPLC Conditions (System C)

Column: Chromolith RP-18e 4.6 mm x 100 mm from EM science

Gradient: 5-50% B in A over 6 minutes

Flow rate: 4 mL / min

Solvent A: 0.1% TFA in water

Solvent B: 0.1% TFA in acetonitrile

Wavelength: 210 nm, 280 nm

Example of residence time (minutes)

Doxorubicin 4.05

Ac-Gly-Ser-Gly-Arg-Ser-nLeu-Dox 4.34

MeOCO-Thr-Gly-Arg-Ser-nLeu-Dox 4.39

PhSO2-Thr-Gly-Arg-Ser-nLeu-Dox 4.83

N, N-dimethylglycine-Thr-Gly-Arg-Ser-nLeu-Dox 4.27

Ac-Thr-Gly-Arg-Ser-nLeu-Dox 4.32

Preparative HPLC Conditions (System D)

Column: Ultro 120 5 C18Q 150 x 20 mm from Peeke Scientific

Gradient: 10-30% B in A over 40 minutes

Flow rate: 18mL / min

Solvent A: 0.1% acetic acid in water

Solvent B: Acetonitrile

Wavelength: 214 nm

Example 2

Preparation of Ac-Gly-Ser-Gly-Arg-Ser-nLeu-Dox

Step A: Passive Synthesis of Ac-Gly-Ser (tBu) -Gly-Arg (Pbf) -Ser (tBu) -nLeu-Wang Resin

In a 25 mL sintered peptide synthesis vessel equipped with a nitrogen stirrer and vacuum assisted drain, Fmoc-nL-Wang resin (nova-biochem, 3.3 g, 0.9 mmol / g, 3 mmol) was preswollen for 30 minutes using DMF. . Subsequently, the peptide is extended with the following four steps of Fmoc amino acids in a total of five repetitions: Fmoc-Ser (tBu) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Gly-OH, Fmoc-Ser (tBu) -OH, Fmoc-Gly-OH.

Iterative Coupling Process

1. Mix the resin with 20% piperidine in DMF (100 mL) for 5 minutes and then drain (repeat 3 times).

2. Stir the resin with DMF (100 mL) for 30 seconds and then drain (repeat 3 times).

3. In a mixture of Fmoc-amino acid (12 mmol), HOBT (12 mmol, 4 equiv, 1.622 g), TBTU (11.7 mmol, 3.9 equiv, 3.757 g), DMF (10 mL) and NMP (90 mL), 12 mmol, 4 equiv, 2.10 mL) is added. After stirring for 5 minutes to preactivate, the solution is added to the synthesis vessel. Check if the reaction is complete by the ninhydrin test and then discharge (if the ninhydrin test shows blue, double coupling (step 3 repeat)) is performed.

4. Stir the resin with DMF (100 mL) for 30 seconds, then drain (repeat 3 times)

The stretched resin (Fmoc-Gly-Ser (tBu) -Gly-Arg (Pbf) -Ser (tBu) -nLeu-Wang resin) is introduced in steps 1 and 2 to remove the Fmoc group. A solution of acetic anhydride (15 mmol, 5 equiv, 1.42 mL), DIEA (15 mmol, 5 equiv, 2.62 mL), DMF (10 mL) and NMP (90 mL) is added to the reaction vessel. After 1 hour, the resin is washed with DMF (100 mL, 3 times), CH ₂ Cl ₂ (100 mL, 3 times) and MeOH (100 mL, 3 times). The resin is dried under vacuum for 15 hours.

Step B: Preparation of Ac-Gly-Ser-Gly-Arg-Ser-nLeu-OH

TFA / H ₂ O (95: 5, 50 mL) is added to the synthesis vessel containing Ac-Gly-Ser (tBu) -Gly-Arg (Pbf) -Ser (tBu) -nLeu-Wang resin (3 mmol). . After gentle stirring for 45 minutes, the cleavage solution is collected and the filtrate is left for an additional 90 minutes. The solution is concentrated to a residue. The crude peptide is analyzed by analytical HPLC (System A, RT = 1.73, purity = 90%). The residue is dissolved in water (50 mL) and hexane (10 mL) and mixed. The hexane layer is removed and the aqueous layer is bubbled with nitrogen to evaporate all remaining hexane. The crude peptide is purified by preparative HPLC (System E). The pure fractions are then lyophilized to give Ac-Gly-Ser-Gly-Arg-Ser-n Leu-OH (1.04 g, 1.68 mmol, 56%) as a white solid. Purity is assessed by analytical HPLC (System A, RT = 1.73 min, 97% purity) and composition is assessed by mass spectrometry (ions observed in 617.9).

Preparative HPLC Conditions (System E)

Column: Waters Delta-Pak Radial Compression Column, 15um, 100A

Gradient: 5-15% B in A over 40 minutes

Flow rate: 80mL / min

Solvent A: 0.1% acetic acid in water

Solvent B: Acetonitrile

Wavelength: 214 nm

Step C: Preparation of Ac-Gly-Ser-Gly-Arg-Ser-nLeu-Dox

A mixture of Ac-Gly-Ser-Gly-Arg-Ser-nLeu-OH (1.68 mmol, 1.04 g, 1.1 equiv), doxorubicin hydrochloride (1.53 mmol, 887.8 mg) and HATU (1.76 mmol, 669.6 mg, 1.15 equiv) To DMF (40 mL) is added followed by 2,6-lutidine (6.12 mmol, 709 μl, 4 equiv). The solution is stirred for 18 hours. The reaction is diluted with water (100 mL) and acidified with acetic acid (400 μl) and then immediately purified into three batches by preparative HPLC (System E). Each red fraction is analyzed by analytical HPLC (System F). Subsequently, the fractions having a purity of 95% or more are combined. Acetonitrile is removed in vacuo and the residual solution is lyophilized to give Ac-Gly-Ser-Gly-Arg-Ser-nLeu-Dox (0.682 mmol, 780 mg, 45%) as a Maulip red solid. Purity is assessed by analytical HPLC (System F, RT = 3.51 min, 95% purity) and composition is assessed by mass spectrometry (ions observed at 1143.5).

Analytical HPLC Conditions (System F)

Column: Chromolith RP-18e 4.6 mm x 100 mm from EM science

Gradient: 20-40% B in A over 6 minutes

Flow rate: 4 mL / min

Solvent A: 0.1% TFA in water

Solvent B: 0.1% TFA in acetonitrile

Wavelength: 210 nm, 280 nm

Preparative HPLC Conditions (System E)

Column: Waters Delta-Pak Radial Compression Column, 15um, 100A

Gradient: 15-25% B in A over 40 minutes

Flow rate: 80mL / min

Solvent A: 0.1% acetonitrile in water

Solvent B: Acetonitrile

Wavelength: 214nm

Example 3

General Procedure for Preparing Peptide-Taxol Conjugates

Step A: Preparation of Peptides on Wang Resin

See step A of example 1.

Step B: Cleavage of Peptides from Wang Resin

See step B of Example 1.

Step C: Coupling of Peptide Acids to 7-Gly-Taxol or 7-Ala-Taxol

To a mixture of peptide acid (0.0121 mmol, 1.1 equiv), 7-Gly-taxol or 7-Ala-taxol (0.011 mmol) and HATU (0.0154 mmol, 5.9 mg, 1.4 equiv) then DMF (0.3 mL) followed by 2,6 -Lutidine (0.044 mmol, 5.1 μl, 4 equiv) is added. The mixture is mixed until a homogeneous solution is obtained. After 4 to 24 hours (monitored by HPLC system H), the reaction is diluted with water (9 mL) and purified directly by preparative HPLC (System I). The pure fractions are then lyophilized to afford the desired peptide taxol conjugates as parent whit white solids. The amount of final conjugate is confirmed by analytical HPLC (System H) and mass spectrometry. Typical yields are 30-50% and purity 96-99%.

Analytical HPLC Conditions (System H)

Column: Chromolith RP-18e 4.6 mm x 100 mm from EM science

Gradient: 5 to 90% B in A over 6 minutes

Flow rate: 4 mL / min

Solvent A: 0.1% TFA in water

Solvent B: 0.1% TFA in acetonitrile

Wavelength: 210nm, 280nm

Example of residence time (minutes)

Ac-Gln-Ser-Arg-Ala-Ala-Taxol 2.86

Ac-Gln-Ser-Arg-Ser-Ala-Ala-Taxol 2.79

Ac-Ser-Gly-Arg-Ala-Ser-Ala-Taxol 2.87

Ac-Arg-Ser-Arg-Ala-Ala-Taxol 2.80

Ac-Ser-Gly-Arg-Ser-Ser-Ala-Taxol 2.81

Preparative HPLC Conditions (System I)

Column: Ultro 120 5 C18Q 150 x 20 mm from Peeke Scientific

Gradient: 20-45% B in A over 40 minutes

Flow rate: 18mL / min

Solvent A: 0.1% TFA in water

Solvent B: Acetonitrile

Wavelength: 214 nm

Example 4

Preparation of N-Ac-Arg-Gln-Ser-Arg-Ala-Ala-DOX

Step A: N-Ac-Arg-Gln-Ser-Arg-Ala-Ala-OH

Using the following general procedure, the N-acetyl peptidic substrate N-Ac-Arg-Gln-Ser-Arg-Ala-Ala-OH is synthesized in a peptide synthesis flask. Commercially available Fmoc-Ala-Wang resin (0.35 g, 0.84 mmol, Nova) is started with standard Fmoc-deprotection with 20% piperidine, followed by a sequential iterative coupling-Fmoc deprotection strategy. Each coupling uses threefold excess (2.52 mmol) of Fmoc-Ala, Fmoc-Arg (Boc) ₂ , Fmoc-Ser (tBu), Fmoc-Gln (Trt) and Fmoc-Arg (Boc) ₂ , respectively. . Coupling is achieved using PyBOP (2.52 mmol) and DIEA (2.52 mmol) in DMF solvent. During each coupling cycle, the Fmoc protecting group is removed using 20% piperidine in DMF. After removal of the N-terminal Fmoc group, capping with acetic anhydride (1.43 mmol, 1.7 equiv), DMAP (0.25 mmol, 0.3 equiv) and DIEA (1.26 mmole, 1.5 equiv) affords the resin-bound N-acetyl intermediate . The protected peptide resin was treated with 50% TFA in methylene chloride for 30 minutes to cleave the Wang resin and then removed with 70% TFA in Boc, Trt and t-Bu protecting group methylene chloride. The solvent and other volatile byproducts are evaporated under reduced pressure and the crude product is dissolved in water and lyophilized to give the title compound as an almost colorless amorphous solid. Mass spectral analysis confirms the desired molecular weight. HPLC analysis shows that the purity of the product is about 95%. Peptide carboxylic acid intermediates can optionally be milled or further purified using preparative HPLC.

Step B: N-Ac-Arg-Gln-Ser-Arg-Ala-Ala-DOX

The intermediate from Step A (20 mg, 0.027 mmol) is dissolved in anhydrous DMF (0.8 mL) and stirred at room temperature under a nitrogen atmosphere. To this solution is added doxorubicin hydrochloride (15.6 mg, 0.027 mmol), EDC (6.8 mg, 0.035 mmol), HOAt (4.8 mg, 0.035 mmol) and 2,6-lutidine (7.3 μl, 0.06 mmol). Agitation is continued until coupling is complete, as monitored by analytical HPLC (System J, see below). The solution is filtered and the crude product is purified by C18 RP-HPLC (A = 0.1% AcOH / H ₂ O; B = CH ₃ CN, gradient eluent 100% to 60% A over 60 minutes). The homogeneous product fraction (evaluated by HPLC, system J) is collected and lyophilized to give the title product as a pale red solid.

HPLC conditions, system J:

Column: Phenomenex 15 cm # 00F-3033-E0, C18

Elution: gradient 95: 5 (A: B) to 25:75 (A: B) over 20 minutes.

A = 0.1% TFA / H2O, B = 0.1% TFA / acetonitrile

Flow rate: 1 mL / min.

Wavelength: 210 nm, 280 nm

Retention time: doxorubicin = 8.89 min.

N-Ac-Arg-Gln-Ser-Arg-Ala-Ala-Dox = 8.4 min.

Physical properties:

Molecular Formula: C ₅₅ H ₇₈ N ₁₄ O ₂₀

Molecular Weight: 1255.3

Low resolution mass spectrum: 628.2 (M + 2/2)

Table 2 lists data for additional peptidic substrate-doxorubicin conjugates. These conjugates were prepared from appropriate amino acid precursors prepared by the general procedure described in Example 4.

TABLE 2

Peptide Substrate-DOX Conjugates Mass spectrum HPLC-Retention Time (minutes) Acetyl-Arg-Arg-Gln-Ser-Arg-Ala-Ala-DOX 471.2 (M + 3/3) 8.23 Acetyl-Leu-Arg-Arg-Gln-Ser-Arg-Ala-Ala-DOX 509.2 (M + 3/3) 8.60

Example 5

Measurement of time to 50% cleavage of doxorubicin / peptide substrate conjugate by recombinant protease domain of MTSP1

1 mM stock solution for each peptidic substrate conjugate is prepared in double distilled water. The 100 μl conjugate was then purified from the recombinantly produced active single chain protease domain of MTSP1 in 29.2 mM Tris (pH 8.4), 29.2 mM imidazole, 217 mM NaCl (residues 615-855 of SEQ ID NO: 2, nucleotide of SEQ ID NO: 1). Cleavage reaction) (encoded by 1865 to 2582). Final volume is 200 μl. The reactions are incubated in a water bath at 37 ° C. At times ranging from 2 minutes to 128 minutes, 20 μl of the sample is aliquoted and trifluoroacetic acid is added to stop the enzymatic activity at 0.33%. The amount of hydrolysis in each sample is determined by reverse phase HPLC. The area under the product peak is then divided by the sum of the area under the product and product peaks to calculate% hydrolysis. The% non-hydrolyzed substrate is plotted against the log of reaction time and the plot is fitted to the sigmoid curve using Prism software (Graphpad Inc., San Diego, Calif.) Measure the time at which 50% is cut.

The results for the specific conjugates provided herein are shown in FIG. 1 (conditions: 1 mM MTSP and 100 at 37 ° C. in 12 mM tris (hydroxymethyl) aminomethane, pH 8.0), 25 mM NaCl, 0.5 mM CaCl ₂ . Μl conjugate; the reaction is cooled with 0.33% trifluoroacetic acid).

Example 6

In vitro assay of cytotoxicity of the conjugate

Cytotoxicity of the conjugate can also be tested to determine whether the conjugate acts as a prodrug. The conjugate is tested for cell lines known to be killed by unmodified cytotoxic agents using the Alamar Blue assay. LNCaP cells (Source: The American Type Culture Collection, Rockville, Maryland) expressing cell surface proteases such as MTSP1 or endotheliase were placed in 96-well plates at a density of 1 x 10 ⁴ cells / well (0.1 mL / well). Say it. Plates containing only medium are used as controls. Cells are incubated at 37 ° C. for 3 days and 20 μl of Alamar Blue is added to the assay well (s). After incubation for 7 hours, cell death is measured using an EL-310 plate reader at 570 and 600 nm. Cell lethality values are expressed as percent reduction of cell number relative to the media control.

Example 7

In vivo efficacy of the conjugate

Tumor cells are treated with trypsin, resuspended in growth medium and centrifuged at 200 × g for 6 minutes. Cells are resuspended in serum free α-MEM and counted. The appropriate volume of the solution containing the desired number of cells is then transferred to a conical centrifuge tube, centrifuged as described above and resuspended in a cold 1: 1 mixture of the appropriate volume of α-MEM-Matrigel. Store suspensions on ice until animals are inoculated.

10-week-old male nude mice are used. Mice are weighed individually and classified into groups (n = 10 per group) with a weight difference of 2 g or less between individual mice in each group. On day 1, mice are inoculated subcutaneously with tumor cell lines. Each mouse is inoculated with, for example, 0.5 mL of 0.5 x 10 ⁶ to 10 ⁸ tumor cells / mL in a 60% solution of ice cold Matrigel and α-MEM. Then, after 24 hours, the conjugate is started to be injected. Vehicle-treated mice are injected with 5% dextrose in water. At the end of the scheduled time, such as days 18 to 2 months or more, mice are sacrificed and tumor size and mass or other parameters are measured. Tumor size and mass or other parameters for conjugate-treated mice are compared to vehicle-treated mice to determine the efficacy of the conjugates.

Mice are inoculated with tumor cells and treated under one of the following three protocols.

Protocol A

One day after seeding the cells, the animals were treated with 1 to 100 μmol / kg or 3 to 50 μmol / kg or 5 to 25 μmol /, including 7.2 or 17.9 μmol / kg of the test conjugate, unmodified cytotoxic agent or vehicle control (sterile water). kg or 7-22 μmol / kg is administered. The dose of conjugate and cytotoxic agent may be titrated initially at maximum non-fatal dose but later at lower levels. The same dose is administered at 24 hour intervals for 5 days. At the end of 5.5 weeks or other suitable interval, the mice are killed and the weight of all tumors shown is measured. Animal weights are measured at the beginning and end of the assay.

Protocol B

From 14 to 15 days after cell inoculation, the animals are subjected to 1 to 100 μmol / kg or 3 to 50 μmol / kg or 5, including 7.2 or 17.9 μmol / kg of the test conjugate, unmodified cytotoxic agent or vehicle control (sterile water). To 25 μmol / kg or 7 to 22 μmol / kg. The dose of conjugate and cytotoxic agent may be titrated initially at maximum non-fatal dose but later at lower levels. The same dose is administered at 24 hour intervals for 5 days. At the end of 5.5 weeks or other suitable interval, the mice are killed and the weight of all tumors shown is measured. Animal weights are measured at the beginning and end of the assay.

Protocol C

One day after seeding the cells, the animals were treated with 1 to 100 μmol / kg or 3 to 50 μmol / kg or 5 to 25 μmol /, including 7.2 or 17.9 μmol / kg of the test conjugate, unmodified cytotoxic agent, or vehicle control (sterile water). kg or 7-22 μmol / kg is administered intraperitoneally. The dose of conjugate and cytotoxic agent can be titrated initially at maximum non-lethal dose but later at lower levels. The same dose is administered at 7 day intervals for 5 weeks. At the end of 5.5 weeks or other suitable interval, the mice are killed and the weight of all tumors shown is measured. Animal weights are measured at the beginning and end of the assay.

Example 8

Gene Expression Profiles and Domain Configuration of Exemplary MTSPs

Gene Expression Profile of MTSP1 in Normal Tissues, Cancer Cells, and Cancer Tissues

To obtain information on tissue distribution and gene expression levels of MTSP1, 76 different human tissues (catalog number) using DNA inserts from Pichia pastoris expression vector pPIC9K-MTSP1 containing coding nucleic acids Blots containing RNA from 7775-1; human multiple tissue expression (MTE) array; CLONTECH, Palo Alto, CA) were probed. Significant expression was observed in the colon (ascending colon, transverse colon and descending colon), rectum, trachea, esophagus and duodenum. Moderate levels of expression have been observed in the jejunum, ileum, synapse, stomach, prostate, pituitary, caecum, kidney, lung, placenta, pancreas, thyroid, salivary gland, mammary gland, fetal kidney and fetal lung. Low levels of expression were found in the spleen, thymus, peripheral blood leukocytes, lymph nodes, bone marrow, bladder, uterus, liver, adrenal gland, fetal heart, fetal liver, fetal spleen and fetal thymus. A significant amount of MTSP1 transcript was also detected in colorectal adenocarcinoma cell line (SW480), Burkitt's lymphoma cell line (Daudi) and leukemia cell line (HL-60). RT-PCR of MTSP1 transcripts in several human primary tumors xenografted to thymus-free nude mice were performed using gene-specific primers. High levels of MTSP1 transcript were detected in colon adenocarcinoma (CX-1) and pancreatic adenocarcinoma (GI-103). Moderate levels were observed in another colon adenocarcinoma (GI-112), ovarian carcinoma (GI-102), lung carcinoma (LX-1), and breast carcinoma (GI-101). Another lung carcinoma (GI-117) expressed lower levels of MTSP1 transcript. Similar RT-PCR was performed to detect the presence of MTSP1 transcripts in PC-3 and LNCaP cell lines. Both cell lines expressed significant amounts of MTSP1 transcripts. MTSP1 is also a marker of ovarian cancer.

Gene Expression Profile of Serine Protease MTSP3 in Normal and Tumor Tissues

To obtain information about the tissue distribution of MTSP3, 76 different human tissues (Cat. No. 7775-1; human multiple tissue expression (MTE) arrays; DNA clone encoding MTSP3 protease domain; manufactured by CLONTECH, Palo) RNA blot consisting of Alto, CA) was probed. The pattern of expression observed with reduced signal levels was as follows: organ = colon (descending colon) = esophagus> colon (ascending colon)> colon (transverse colon) = rectum> ileum> duodenum> jejunum> bladder> ligature> stomach > Kidneys> Caecum. It is also less abundantly expressed in fetal kidney and two tumor cell lines, HeLa S3 and leukemia K-562. Northern analysis using RNA blots (Catalog Nos. 7780-1, 7765-1 and 7782-1; human 12-lane, human muscle and human digestive system multi-tissue northern (MTN) blot; manufactured by CLONTECH) showed that the expression was most likely in the colon. It was confirmed that abundance was detected and moderately detected in the esophagus, small intestine, bladder and kidney and less abundantly in the stomach and rectum. A single transcript of about 2.2 kb was detected.

Amplification of the MTSP3 transcript in several human primary tumors transplanted into mice was performed using gene-specific primers. MTSP3 transcripts were detected in lung carcinoma (LX-1), colon adenocarcinoma (CX-1), colon adenocarcinoma (GI-112) and ovarian carcinoma (GI-102). No obvious signals were detected in other forms of lung carcinoma (GI-117), breast carcinoma (GI-101), pancreatic adenocarcinoma (GI-103) and prostate adenocarcinoma (PC3).

Gene Expression Profile of MTSP4 in Normal and Tumor Tissues

To obtain information about the gene expression profile of the MTSP4 transcript, 76 different human tissues (Catalog No. 7775-1; Human Multiple Tissue Expression (MTE)) were used using DNA fragments encoding portions of the LDL receptor domain and the protease domain. RNA blot consisting of an array; manufactured by Clontech) was probed. As in the northern gel blot analysis, a very strong signal was observed in the liver. Signals from other tissues were observed (at decreasing signal levels) as follows: liver> heart = kidney = adrenal = testes = fetal heart and kidney = skeletal muscle = bladder = placenta> brain = spinal cord = colon = stomach = spleen = Lymph node = bone marrow = organ = uterus = pancreas = salivary gland = mammary gland = lung. MTSP4 is also less abundantly expressed in several tumor cell lines, including: HeLa S3 = leukemia K-562 = Burkitt's lymphoma (Raji and Daudi) = colorectal adenocarcinoma (SW480)> lung carcinoma (A549) = leukemia MOLT-4 = leukemia HL-60. PCR of MTSP4 transcripts from a cDNA library (human tumor multi-tissue cDNA panel, catalog number K1522-1, manufactured by CLONTECH) made from several human primary tumors transplanted into nude mice was carried out using MTSP4-specific primers. Was performed. MTSP4 transcripts were detected in breast carcinoma (GI-101), lung carcinoma (LX-1), colon adenocarcinoma (GI-112) and pancreatic adenocarcinoma (GI-103). No obvious signals were detected in other forms of lung carcinoma (GI-117), colon adenocarcinoma (CX-1), ovarian carcinoma (GI-102) and prostate adenocarcinoma (PC3). MTSP4 transcript was also detected in the LNCaP and PC-3 prostate cancer cell lines as well as the HT-1080 human fibrosarcoma cell line.

Gene Expression Profile of MTSP6 in Normal and Tumor Tissues

To obtain information on the gene expression profile of the MTSP6 transcript, 76 different human tissues (Catalog No. 7775-1; 495 bp DNA fragments obtained from PCR reactions using primers Ch17-NSP-3 and NSP-4AS) were used; RNA blots consisting of human multiple tissue expression (MTE) array, CLONTECH were probed. The strongest signal was observed in the duodenum. Signals from other tissues were observed (at decreasing signal levels) as follows: stomach> organs = mammary gland = thyroid = salivary gland = pituitary gland = pancreas> kidneys> lung> jejunum = ileum = ligature = cecum = fetal kidney> fetus lungs. Very weak signals can also be observed in several other tissues. MTSP6 is also expressed in several tumor cell lines, including: HeLa S3> Colorectal Adenocarcinoma (SW480)> Leukemia MOLT-4> Leukemia K-562. PCR analysis of MTSP6 transcripts from cDNA libraries (human tumor multi-tissue cDNA panel, catalog number K1522-1, manufactured by CLONTECH) prepared from several human primary tumors transplanted into nude mice was performed using MTSP6-specific primers (Ch17). -NSP-3 and Ch17-NSP2AS). MTSP6 transcript was strongly detected in lung carcinoma (LX-1), moderately detected in pancreatic adenocarcinoma (GI-103), weakly detected in ovarian carcinoma (GI-102), and colon adenocarcinoma (GI-112 and CX-1). Weakly detected in breast carcinoma (GI-101), lung carcinoma (GI-117) and prostate adenocarcinoma (PC3). MTSP6 transcript was also detected in breast cancer cell line MDA-MB-231, prostate cancer cell line PC-3, but not in HT-1080 human fibrosarcoma cell line. MTSP6 is also expressed in mammary carcinoma cDNA (Clontech).

Gene Expression Profile of MTSP9 in Normal, Tumor Tissue, and Cell Lines

To obtain gene expression profiles of MTSP9 transcripts, using MTSP9 cDNA fragments obtained from the human pancreas, 76 different human tissues (human multiple tissue expression (MTE) arrays, manufactured by Clontech, Palo Alto, CA; catalog number Dot blot consisting of RNA extracted from 7775-1) was probed. The results of this analysis indicate that MTSP9 is highly expressed in the esophagus and at low levels in many other tissues. MTSP9 transcripts include kidneys (adults and fetuses), spleen (adults and fetuses), placenta, liver (adults and fetuses), thymus, peripheral blood leukocytes, lungs (adults and fetuses), pancreas, lymph nodes, bone marrow, organs, uterus, It is found in the prostate, esophagus, testes, ovaries and glandular organs (mammary gland, adrenal gland, thyroid gland, pituitary gland and salivary gland). MTSP9 is also expressed in esophageal tumor tissue and lung carcinoma (A549 cell line), and is characterized by colorectal carcinoma (SW480), lymphoma (Raji and Daudi), cervical carcinoma (HeLaS3) and leukemia (HL-60, K-562 and MOLT- 4) are expressed at low levels in cell lines.

Gene Expression Profile of MTSP10 in Normal and Tumor Tissues

To obtain information regarding the gene expression profile of the MTSP10 transcript, PCR analysis was performed using MTSP10-specific primers on cDNA prepared from several human adult tissue (Clontech, Cat. No. K1420-1) cDNA panels. MTSP10 transcript was detected in the pancreas, lung and kidney. MTSP10 transcript was also detected in small intestine marathon-ready cDNA (Clontech). PCR of MTSP10 transcripts from cDNA libraries (human tumor multi-tissue cDNA panel, Cat. No. K1522-1, CLONTECH) made from several human primary tumors implanted in nude mice is performed. MTSP10 transcripts were detected in breast carcinoma (GI-101), lung carcinoma (LX-1 and GI-117), ovarian carcinoma (GI-102) and pancreatic adenocarcinoma (GI-103). MTSP10 transcripts can be detected weakly in prostate adenocarcinoma (PC3). No obvious signal was detected in two types of colon adenocarcinoma (GI-112 and CX-1). MTSP10 transcript was also detected in CWR22R prostate tumors grown in nude mice.

Domain Composition and Gene Expression Profile of MTSP12 in Normal and Tumor Tissues

Domain composition of MTSP12PD1, -PD2 and -PD3 and homology to other serine proteases

Sequence and protein domain analysis of the transcribed MTSP12PD1, -PD2, and -PD3 nucleotide sequences indicate that these three serine proteases are in succession. The sequence sequence is as follows: MTSP12-PD1 is found at the N terminus following MTSP12-PD2, and MTSP12-PD3 is located at the C terminus. MTSP12-PD1 and -PD2 are protease activating cleavage sites (R236 ↓ I ₂₃₇ VGGMEAS ... and ... R ₅₃₇ ↓ V ₅₃₈ VGGFGAA ... for MTSP12-PD1 and -PD2, respectively, where | Catalytic triad residues in his three highly conserved regions of the catalytic domain (His ₂₇₇ , Asp ₃₂₆ and Ser _{421 in} MTSP12-PD1; His ₅₇₈ , Asp ₆₂₆ and Ser in MTSP12-PD2). ₇₂₁ ) and trypsin-type serine protease domains (aa 236 to aa 465 and aa 537 to aa 765 for MTSP12-PD1 and -PD2, respectively). MTSP12-PD3 contains a serine protease domain (aa 861 to aa 1087), which serine protease domain has a protease activating cleavage site (... R ₈₆₀ ↓ I ₈₆₁ VGGSAAG ...) and the catalytic His ₉₀₂ and Asp ₉₄₉ , but Ala ₁₀₄₃ instead of the conserved catalytic serine found in serine proteases. Several domains are found on top of the MTSP12-PD1 serine protease domain, which are transmembrane domains (aa 28 to aa 50), SEA (germ sperm protein-entokinase-agrin; s ea urchin sperm protein- e nterokinase- a grin) domains (aa 51-aa 170) and LDLa (low density lipoprotein receptor class a ) domains (aa 187-aa 225). There are five possible N-linked glycosylation sites (N ₁₁₆ SS, N ₅₈₁ HT, N ₆₇₂ AT, N ₆₉₇ FS and N ₈₂₀ ST). In the protease domain of MTSP12-PD1, there is an unpaired cysteine (C ₃₄₆ ) in the single-chain protease domain and the following following Cys pairs are noted: C ₂₆₂ -C ₂₇₈ ; C ₃₆₀ -C ₄₂₇ ; C ₄₁₇ -C ₄₄₆ ; C ₃₉₂ -C ₄₀₆ . In the protease domain of MTSP12-PD2, there is an unpaired cysteine (C ₆₄₆ ) in the single-chain protease domain and the following following Cys pairs are noted: C _563- C ₅₇₉ ; C ₆₆₀ -C ₇₂₇ ; C ₆₉₂ -C ₇₀₆ ; C ₇₁₇ -C ₇₄₆ . In the protease domain of MTSP12-PD3, there is an unpaired cysteine (C ₉₆₉ ) in the single-chain protease domain, followed by the following Cys pair: C ₈₈₇ -C ₉₀₃ ; C ₉₈₃ -C ₁₀₄₉ ; C ₁₀₁₄ -C ₁₀₂₈ ; C ₁₀₃₉ -C ₁₀₆₈ .

Alignment (blastp; http://www.ncbi.nlm.nih.gov/BLAST) of each MTSP12-PD1, MTSP12-PD2 and MTSP12-PD3 protein sequence for known serine proteases accumulated in published databases 45%, 45% and 48% identity with lipase, 44%, 43% and 41% identity with DESC1 / endotelias 44, 44%, 43% and 48% identity with prostamine (AB030036), spinesin ( 43%, 39% and 39% identity with TMPRSS5; NM_030770) and 40%, 38% and 38% identity with marafsin (NM_031948). The clones have about 93% homology with the clones and encoded proteins described in International PCT Application WO 02/00860 (see SEQ ID NOs: 38 and 97 of this document) at the nucleotide and encoded protein levels. However, the encoded protein described in the PCT application has the sequence set forth in SEQ ID NO: 271 between amino acids Leu373 and Val374 in SEQ ID NO: 20 as well as additionally extended amino acid sequences between amino acids Ala ₄₈ and Phe ₄₉ in SEQ ID NO: 20 and It lacks amino acids 91-124.

Gene and Tissue Expression Profiles of MTSP12

Three cDNA probes were prepared to obtain information regarding the tissue distribution profiles of the MTSP12PD1, -PD2 and -PD3 transcripts. The data show that MTSP12PD1, -PD2 and -PD3 transcripts are expressed at low levels in most of 76 tissues and cell lines, but at high levels in lymph nodes and testes.

To compare expression profiles of MTSP12PD1, -PD2, and -PD3 in the range of normal human and matched tumor tissue, a matched tumor / normal expression array consisting of 68 pairs of cDNA samples from each patient (Catalog No. 7840-1; http://www.clontech.com). The results indicate that MTSP12PD1, -PD2 and -PD3 transcripts are expressed at low levels in many tissues including breast, uterus, colon, ovary, lung, kidney and rectum, but not differently in all matching tissues. It also includes HeLa (cervical carcinoma), Daudi (bucket lymphoma), K562 (chronic myeloid leukemia), HL-60 (promyelocytic leukemia), G361 (melanoma), A549 (lung carcinoma), MOLT-4 (lymphoblast Low levels are expressed in several tumor cell lines, including leukemia), SW480 (colon adenocarcinoma), and Raji (bucket lymphoma).

Several SMART ^TM 5'-RACE cDNA libraries (Catalog No. K1811-1; http://www.clontech.com) prepared from normal breast, normal testis, normal prostate, prostate cancer cell lines, and breast cancer cell lines were used in two sets of genes- Specific primers were used to analyze the presence of MTSP12PD1, -PD2 and -PD3 transcripts by RT-PCR. MTSP12-PD2 and -PD3 transcripts were detected in normal prostate, PC-3, LNCaP, normal breast, MDA-MB-231, MDA-MB-361, MDA-MB-453 and DU4475, but higher levels were normal breast And in MDA-MB-231. MTSP12-PD1 transcript was detected in the same tissues and cell lines, but exceptionally higher levels were observed in normal breast, MDA-MB-231 and DU4475.

Gene Expression Profile of MTSP20 in Normal, Tumor Tissue, and Cell Lines

To obtain information about the gene expression profile of the MTSP20 transcript, 76 different human tissues (human multi-tissue expression (MTE) arrays; Clontech, Palo Alto, CA) were obtained using MTSP20 cDNA fragments obtained from human lung tissue; Dot blot consisting of RNA extracted from Cat. No. 7775-1) was probed. The results show that RNA encoding MTSP20 is expressed in various tissues. MTSP20 transcripts are found in the liver, lymph nodes, cerebellum, pancreas, prostate, uterus, testes, glands (adrenal glands, thyroid and salivary glands), thymus, kidneys and spleen. Low levels of transcripts can be found in the lungs, placenta, bladder, ovaries, digestive system, circulatory system and other parts of the brain. MTSP20 is also a specific tumor, including lung carcinoma (A519), colorectal carcinoma (SW480), lymphoma (Raji and Daudi), cervical carcinoma (HeLaS3) and leukemia (HL-60, K-562 and MOLT-4) cell lines. It is expressed in cell lines.

Gene Expression Profile of MTSP22 in Normal, Tumor Tissue, and Cell Lines

MTSP22 is expressed in uterine tissue, thymus, adipose tissue and lymph nodes. It can also be expressed in lung, stomach, uterus, breast, ovary, prostate tumors and other tumors. To obtain information regarding the gene expression profile of the MTSP22 transcript, 72 different human tissues (human multi-tissue expression (MTE) arrays; clontech, Palo Alto, using a cDNA fragment encoding the entire serine protease domain) were obtained. CA; normalization from 241 tumors from each patient and corresponding normal tissues (cancer profiling array, Clontech, catalog number 7841-1), as well as dot blots consisting of RNA extracted from catalog number 7776-1) Dot blots composed of cDNAs were probed. The results of the MTE analysis indicated that the MTSP22 transcript was mainly expressed in the esophagus. In cancer profiling array analysis, MTSP22 is highly expressed in 3 of 42 normal uterine tissue samples, but not in matching tumor samples. In one of the 42 uterine samples, MTSP22 is expressed in tumors and metastatic tissues thereof, but not in normal tissue counterparts. MTSP22 is also expressed in 2 of 17 gastric tissues and 2 of 21 lung tumors, but not in its normal tissue counterparts. MTSP22 is also expressed only in normal tissue matched cDNA pairs of the pancreas. In addition, cDNA panels from several human adult tissues (commercially available at Clontech, catalog numbers K1420-1 and K1420-2) and primary tumors (commercially available at Clontech, catalog number K1522-1), were also available for PCR analysis. It was performed using several marathon-ready cDNAs (Clontech).

MTSP22 cDNA was detected in the thymus, adipose tissue and lymph nodes. Homology for MTSP22 Serine Protease Domain and Other Proteases.

Sequence analysis of the transcribed MTSP22 protease domain sequence showed that the trypsin-type is characterized by the presence of catalytic triad residues (histidine, aspartate and serine) in the highly conserved region and protease activation cleavage site of the amino terminus of the MTSP22 domain. It has been shown to contain a serine protease domain. The alignment of the MTSP22 protein sequence and the protein sequence of endotheliase 1 (same as that of the serine protease DESC1 protein; Genbank Accession No. AF064819) indicates that the two proteins share 50% sequence identity in their protease domain.

Gene Expression Profile of MTSP25 in Normal, Tumor Tissue, and Cell Lines

MTSP25 is expressed in breast, colon, uterus, ovary, kidney, prostate and testicular cancer tissues. It can also be expressed in lung, stomach, prostate tumors and other tumors. To obtain information regarding the gene expression profile of the MTSP25 transcript, a 369 bp DNA fragment containing the MTSP25 protease domain sequence (obtained from the PCR reaction) was used to construct 72 different human tissues (human multiple tissue expression (MTE) arrays). ; Clontech, Palo Alto, CA; 241 tumors and corresponding normal tissues from each patient, as well as dot blots consisting of RNA extracted from catalog number 7776-1) (cancer profiling array, Clontech, catalog Dot blot consisting of normalized cDNA from No. 7841-1) was probed. The results of the MTE analysis indicate that MTSP25 transcript is weakly expressed in lymph nodes. In cancer profiling array analysis, MTSP25 is highly expressed in all four prostate samples (normal and cancer samples). In one of the 20 renal cDNA pairs, MTSP25 is highly expressed in tumor samples but not in its normal tissue counterparts. MTSP25 is also expressed in one of 50 breast cancer samples, but not in its normal tissue counterpart.

MTSP25 is also expressed in three of the 42 normal uterine samples, but not in its tumor counterparts. MTSP25 expression is also detected in 3 of 14 ovarian cancer samples. Of the three samples, expression of MTSP25 was also detected in one of the matched normal tissue counterparts. MTSP25 expression was also detected in five tumor samples within 34 colon cDNA pairs.

In addition, cDNA panels from several human adult tissues (commercially available at Clontech, catalog numbers K1420-1 and K1420-2) and primary tumors (commercially available at Clontech, catalog number K1522-1), were also available for PCR analysis. Several marathon-ready cDNAs (Clontech) were used. MTSP25 cDNA was strongly detected in testicular and mammary adenocarcinoma, weakly detected in brain, placenta, lung, spleen, prostate, small intestine, colon and leukocytes, and very weakly in heart, liver and pancreas.

Example 9

According to the procedures of Examples 1-4, conjugates prepared by conventional and only slightly modified procedures, such as using different Fmoc-amino acid building blocks, include:

Ac-R-Q-G-R-S-L- (Dox) (SEQ ID NO: 491);

Ac-R-Q-G-R-S-S-L- (Dox) (SEQ ID NO: 492);

Ac-R-Q-G-R-S-nL- (Dox) (SEQ ID NO: 493);

Ac-R-Q-G-R-S-nV- (Dox) (SEQ ID NO: 494);

Ac-R-Q-G-R-S-F- (Dox) (SEQ ID NO: 495);

Ac-R-Q-G-R-A-L- (Dox) (SEQ ID NO: 496);

Ac-R-Q-G-R-A-L- (Dox) (SEQ ID NO: 497);

Ac-R-Q-G-R-A-nL- (Dox) (SEQ ID NO: 498);

Ac-R-Q-G-R-A-nL- (Dox) (SEQ ID NO: 499);

Ac-R-Q-G-R-A-nV- (Dox) (SEQ ID NO: 500);

Ac-R-Q-G-R-A-Cha- (Dox) (SEQ ID NO: 501);

Ac-R-Q-G-R-A-F- (Dox) (SEQ ID NO: 502);

Ac-R-N-G-R-S-L- (Dox) (SEQ ID NO: 503);

Ac-R-N-G-R-A-nL- (Dox) (SEQ ID NO: 504);

Ac-R-Q-A-R-S-L- (Dox) (SEQ ID NO: 505);

Ac-R-Q-A-R-S-nL- (Dox) (SEQ ID NO: 506);

Ac-R-Q-A-R-S-nV- (Dox) (SEQ ID NO: 507);

Ac-R-Q-A-A-S-Cha- (Dox) (SEQ ID NO: 508);

Ac-R-Q-A-R-S-S-Cha- (Dox) (SEQ ID NO: 509);

Ac-R-Q-A-R-T-nL- (Dox) (SEQ ID NO: 510);

Ac-R-Q-A-R-A-L- (Dox) (SEQ ID NO: 511);

Ac-R-Q-A-R-A-nL- (Dox) (SEQ ID NO: 513);

Ac-R-Q-A-R-A-nV- (Dox) (SEQ ID NO: 514);

Ac-R-Q-A-R-A-Cha- (Dox) (SEQ ID NO: 515);

Ac-R-Q-S-R-A-A- (Dox) (SEQ ID NO: 516);

Ac-R-Q-S-R-A- (Dox) (SEQ ID NO: 517);

Ac-R-Q-S-R-A-nL- (Dox) (SEQ ID NO: 518);

Ac-R-Q-S-R-A-L- (Dox) (SEQ ID NO: 519);

Ac-R-Q-S-R-A-nV- (Dox) (SEQ ID NO: 520);

Ac-R-Q-S-R-A-Cha- (Dox) (SEQ ID NO: 521);

Ac-R-Q-S-R-S-S-L- (Dox) (SEQ ID NO: 522);

Ac-R-Q-S-R-S-L- (Dox) (SEQ ID NO: 523);

Ac-R-Q-S-R-S-dnL- (Dox) (SEQ ID NO: 524);

Ac-R-Q-S-R-S-nL- (Dox) (SEQ ID NO: 525);

Ac-R-Q-S-R-S-nV- (Dox) (SEQ ID NO: 526);

Ac-R-Q-S-R-S-allylG- (Dox) (SEQ ID NO: 527);

Ac-R-Q-S-R-S-Cha- (Dox) (SEQ ID NO: 528);

Ac-R-Q-S-R-T-nL- (Dox) (SEQ ID NO: 529);

Ac-R-Q-T-R-S-S-L- (Dox) (SEQ ID NO: 530);

Ac-R-Q-T-R-S-L- (Dox) (SEQ ID NO: 531);

Ac-R-N-S-R-S-nL- (Dox) (SEQ ID NO: 532);

Ac-R-Q-F-R-S-L- (Dox) (SEQ ID NO: 533);

Ac-R-Q-F-R-S-nL- (Dox) (SEQ ID NO: 534);

Ac-R-Q-F-R-S-nV- (Dox) (SEQ ID NO: 535);

Ac-R-Q-F-R-S-nL- (Dox) (SEQ ID NO: 536);

Ac-R-Q-F-R-S-Cha- (Dox) (SEQ ID NO: 537);

Ac-R-Q-F-R-A-L- (Dox) (SEQ ID NO: 538);

Ac-R-Q-F-R-A-nL- (Dox) (SEQ ID NO: 539);

Ac-R-Q-F-R-A-nV- (Dox) (SEQ ID NO: 540);

Ac-R-Q-F-R-A-Cha- (Dox) (SEQ ID NO: 541);

Ac-Q-S-R-S-S-nL- (Dox) (SEQ ID NO: 542);

MeOCO-Quat2-G-R-S-L-NH2 (SEQ ID NO: 483);

MeOCO-Quat3-G-R-S-L-NH 2 (SEQ ID NO: 484);

MeOCO-Quat-G-R-S-L-NH 2 (SEQ ID NO: 485);

MeOCO-Quat4-G-R-S-L-NH 2 (SEQ ID NO: 486);

MeOCO-Quat5-G-R-S-L-NH 2 (SEQ ID NO: 487);

MeOCO-Quat2-G-R-S-S-L-NH2 (SEQ ID NO: 488);

MeOCO-Quat4-G-R-S-L- (Dox) (SEQ ID NO: 489);

MeOCO-Quat2-G-R-S-L- (Dox) (SEQ ID NO: 490);

Ac-Q-G-R-S-L- (Dox) (SEQ ID NO: 445);

Ac-Q-G-R-S-S-L- (Dox) (SEQ ID NO: 446);

Ac-Q-G-R-A-S-L- (Dox) (SEQ ID NO: 447);

Ac-N-G-R-S-S-L- (Dox) (SEQ ID NO: 448);

Ac-Q-G-R-S-S-nL- (Dox) (SEQ ID NO: 449);

Ac-Q-G-R-S-S-nV- (Dox) (SEQ ID NO: 450);

Ac-Q-G-R-S-S-Cha- (Dox) (SEQ ID NO: 451);

Ac-Q-G-R-S-S-allyl G- (Dox) (SEQ ID NO: 452);

Ac-Q-G-R-S-S-allyl G- (Dox) (SEQ ID NO: 453);

Ac-Q-A-R-S-L- (Dox) (SEQ ID NO: 454);

Ac-Q-A-R-S-S-L- (Dox) (SEQ ID NO: 455);

Ac-Q-S-R-S-L- (Dox) (SEQ ID NO: 456);

Ac-Q-S-R-S-S-nV- (Dox) (SEQ ID NO: 457);

Ac-Q-S-R-S-S-Cha- (Dox) (SEQ ID NO: 458);

Ac-Q-S-R-S-S-L- (Dox) (SEQ ID NO: 459);

Ac-Q-T-R-S-S-L- (Dox) (SEQ ID NO: 460);

Ac-Q-Aib-R-S-S-Cha- (Dox) (SEQ ID NO: 461);

Ac-Q-Aib -R-S-S-L- (Dox) (SEQ ID NO: 462);

Ac-Q-Abu-R-S-S-Cha- (Dox) (SEQ ID NO: 463);

Ac-Q-Abu-R-S-S-L- (Dox) (SEQ ID NO: 464);

Ac-Q-Cha-R-S-S-Cha- (Dox) (SEQ ID NO: 465);

Ac-Q-F-R-S-L- (Dox) (SEQ ID NO: 466);

Ac-Q-F-R-S-S-L- (Dox) (SEQ ID NO: 467);

Ac-Q-Y-R-S-S-L- (Dox) (SEQ ID NO: 468);

Ac-R-G-R-S-L- (Dox) (SEQ ID NO: 469);

Ac-R-G-R-S-S-L- (Dox) (SEQ ID NO: 470);

Ac-R-G-R-S-S-Cha- (Dox) (SEQ ID NO: 471);

Ac-R-G-R-S-Cha- (Dox) (SEQ ID NO: 472);

Ac-R-A-R-S-L- (Dox) (SEQ ID NO: 473);

Ac-R-A-R-S-S-L- (Dox) (SEQ ID NO: 474);

Ac-R-S-R-S-L- (Dox) (SEQ ID NO: 475);

Ac-R-S-R-S-S-L- (Dox) (SEQ ID NO: 476);

Ac-R-S-R-S-Cha- (Dox) (SEQ ID NO: 477);

Ac-R-S-R-S-S-Cha- (Dox) (SEQ ID NO: 478);

Ac-R-F-R-S-L- (Dox) (SEQ ID NO: 479);

Ac-R-F-R-S-Cha- (Dox) (SEQ ID NO: 480);

Ac-Y-G-R-S-S-L- (Dox) (SEQ ID NO: 481);

Ac-M (O 2) -S-R-S-L- (Dox) (SEQ ID NO: 482);

Ac-R-R-Q-S-R-A-A- (Dox) (SEQ ID NO: 105);

Ac-R-R-Q-S-R-I- (Dox) (SEQ ID NO: 610);

Ac-R-R-Q-S-R-S-S-L- (Dox) (SEQ ID NO: 543);

Ac-R-R-Q-S-R-S-L- (Dox) (SEQ ID NO: 544);

Ac-R-G-S-G-R-S-L- (Dox) (SEQ ID NO: 545);

Ac-R-G-S-G-R--S-nL- (Dox) (SEQ ID NO: 546);

Ac-R-G-S-G-R-A-nL- (Dox) (SEQ ID NO: 547);

Ac-R-G-S-G-R-S-S-L- (Dox) (SEQ ID NO: 548);

Ac-I-V-S-G-R-A-S-L- (Dox) (SEQ ID NO: 549);

Ac-R-R-Q-S-R-A- (Dox) (SEQ ID NO: 108);

Ac-R-R-Q-S-R-I- (Dox) (SEQ ID NO: 111);

Ac-L-R-R-Q-S-R-A-A- (Dox) (SEQ ID NO: 106);

Ac-L-R-R-Q-S-R-G-G- (Dox) (SEQ ID NO: 109);

Ac-L-R-R-Q-S-R-A- (Dox) (SEQ ID NO: 110);

Ac-L-R-R-Q-S-R-A-I- (Dox) (SEQ ID NO: 112);

Ac-L-R-R-Q-S-R-A-I- (Dox) (SEQ ID NO: 611);

Ac-L-R-R-Q-S-R-S-S-L- (Dox) (SEQ ID NO: 550);

Ac-L-R-R-Q-S-R-S-L- (Dox) (SEQ ID NO: 551);

Ac-S-G-R-S-L- (Dox) (SEQ ID NO: 362);

Ac-S-G-R-S-S-L- (Dox) (SEQ ID NO: 363);

Ac-S-G-R-S-S-S-L- (Dox) (SEQ ID NO: 364);

Ac-S-G-R-S-nL- (Dox) (SEQ ID NO: 365);

Ac-S-G-R-S-nV- (Dox) (SEQ ID NO: 366); Isomer 1

Ac-S-G-R-S-nV- (Dox) (SEQ ID NO: 367); Isomer 2

Ac-S-G-R-S-G (hexyl)-(Dox) (SEQ ID NO: 368);

Ac-S-G-R-S-Cha- (Dox) (SEQ ID NO: 369);

Ac-S-G-R-S-hCha- (Dox) (SEQ ID NO: 370);

Ac-S-A-R-S-L- (Dox) (SEQ ID NO: 371);

Ac-S-A-R-S-S-L- (Dox) (SEQ ID NO: 372);

Ac-S-S-R-S-nL- (Dox) (SEQ ID NO: 373);

Ac-T-G-R-S-Abu- (Dox) (SEQ ID NO: 374);

Ac-T-G-R-S-L- (Dox) (SEQ ID NO: 375);

Ac-T-G-R-S-nV- (Dox) (SEQ ID NO: 376);

Ac-T-G-R-S-nL- (Dox) (SEQ ID NO: 377);

Ac-T-G-R-S-G (hexyl)-(Dox) (SEQ ID NO: 378);

Ac-T-G-R-S-Cha- (Dox) (SEQ ID NO: 379);

Ac-T-G-R-S-hCha- (Dox) (SEQ ID NO: 380);

Ac-T-G-R-T-Abu- (Dox) (SEQ ID NO: 381);

Ac-T-G-R-hS-nL- (Dox) (SEQ ID NO: 382);

Ac-T-G-R-Abu-nL- (Dox) (SEQ ID NO: 383);

Ac-T-G-R-Abu-nV- (Dox) (SEQ ID NO: 384);

Ac-T-G-F (Gn) -S-nL- (Dox) (SEQ ID NO: 385);

Ac-T-G-F (Gn) -S-Cha- (Dox) (SEQ ID NO: 386);

Ac-T-G-F (Gn) -Abu-nV- (Dox) (SEQ ID NO: 387);

Ac-T-G-K (alloc) -S-nL- (Dox) (SEQ ID NO: 388);

Ac-T-G-K-S-nL- (Dox) (SEQ ID NO: 389);

Ac-T-G-hR-S-nL- (Dox) (SEQ ID NO: 390);

Ac- (hS) G-G-R-S-nL- (Dox) (SEQ ID NO: 391);

MeOCO-T-G-R-S-nL- (Dox) (SEQ ID NO: 392);

PhSO2-T-G-R-S-nL- (Dox) (SEQ ID NO: 393);

MeOEtCO-T-G-R-S-nL- (Dox) (SEQ ID NO: 394);

MeO (EtO) 2Ac-T-G-R-S-nL- (Dox) (SEQ ID NO: 395);

4-oxo-pentanoyl-T-G-R-S-nL- (Dox) (SEQ ID NO: 396);

3,4-methyldioxyPhAc-T-G-R-S-nL- (Dox) (SEQ ID NO: 397);

2-pyridylAc-T-G-R-S-nL- (Dox) (SEQ ID NO: 398);

PhOAc-T-G-R-S-nL- (Dox) (SEQ ID NO: 399);

L-3-Phlactyl-T-G-R-S-nL- (Dox) (SEQ ID NO: 400);

MeOAc-T-G-R-S-nL- (Dox) (SEQ ID NO: 401);

PhAc-T-G-R-S-nL- (Dox) (SEQ ID NO: 402);

MeOEtOCO-T-G-R-S-nL- (Dox) (SEQ ID NO: 403);

MeOEtOAc-T-G-R-S-nL- (Dox) (SEQ ID NO: 404);

HOOCButa-T-G-R-S-nL- (Dox) (SEQ ID NO: 405);

Z-T-G-R-S-nL- (Dox) (SEQ ID NO: 406);

EtOCO-T-G-R-S-nL- (Dox) (SEQ ID NO: 407);

βA-T-G-R-S-nL- (Dox) (SEQ ID NO: 408);

Pent-4-inoyl-T-G-R-S-nL- (Dox) (SEQ ID NO: 409);

NapAc-T-G-R-S-nL- (Dox) (SEQ ID NO: 410);

iBoc-T-G-R-S-nL- (Dox) (SEQ ID NO: 411);

HOAc-T-G-R-S-nL- (Dox) (SEQ ID NO: 412);

MeSucc-T-G-R-S-nL- (Dox) (SEQ ID NO: 413);

N, N-diMeGly-T-G-R-S-nL- (Dox) (SEQ ID NO: 414);

Succ-T-G-R-S-nL- (Dox) (SEQ ID NO: 415);

HCO-T-G-R-S-nL- (Dox) (SEQ ID NO: 416);

Ac-T-A-R-S-nL- (Dox) (SEQ ID NO: 417);

Ac-T-A-F (Gn) -S-nL- (Dox) (SEQ ID NO: 418);

Ac-T-A-R-Abu-nV- (Dox) (SEQ ID NO: 419);

Ac-T-A-R-S-Abu- (Dox) (SEQ ID NO: 420);

Ac-T-A-R-T-Abu- (Dox) (SEQ ID NO: 421);

Ac-T-S (O-Me) -R-S-nL- (Dox) (SEQ ID NO: 422);

Ac-T-hS-R-S-nL- (Dox) (SEQ ID NO: 423);

Ac-T- (1-Me) H-R-S-nL- (Dox) (SEQ ID NO: 424);

Ac-T- (3-Me) H-R-S-nL- (Dox) (SEQ ID NO: 425);

Ac-T-H-R-S-nL- (Dox) (SEQ ID NO: 426);

Ac-T-Sar-R-S-nL- (Dox) (SEQ ID NO: 427);

Ac-T-nV-R-S-nL- (Dox) (SEQ ID NO: 428);

Ac-T-nL-R-S-nL- (Dox) (SEQ ID NO: 429);

Ac-T-A-R-S-Cha- (Dox) (SEQ ID NO: 430);

Ac-T-Abu-R-S-nL- (Dox) (SEQ ID NO: 431);

Ac-4,4-diMeThr-G-R-S-nL- (Dox) (SEQ ID NO: 432);

Ac-hS-G-R-S-nL- (Dox) (SEQ ID NO: 433);

Ac-hS-G-R-hS-Cha- (Dox) (SEQ ID NO: 434);

Ac-hS-G-R-S-Cha- (Dox) (SEQ ID NO: 435);

Ac-hS-G-R-T-Cha- (Dox) (SEQ ID NO: 436);

Ac-hS-A-R-S-Cha- (Dox) (SEQ ID NO: 437);

Ac-N-G-R-S-nL- (Dox) (SEQ ID NO: 438);

Ac-Y-G-R-S-S-L- (Dox) (SEQ ID NO: 439);

Ac-Y-G-R-S-Cha- (Dox) (SEQ ID NO: 440);

Ac-Q-G-R-S-S-nL- (Dox) (SEQ ID NO: 441);

Ac-Q-G-R-S-S-nV- (Dox) (SEQ ID NO: 442);

Ac-L-R-G-S-G-R-S-A- (Dox) (SEQ ID NO: 573);

Ac-L-R-G-S-G-R-S-L- (Dox) (SEQ ID NO: 342);

Ac-L-R-G-S-G-R-S-L- (Dox) (SEQ ID NO: 343);

Ac-L-R-G-S-G-R-S-S-nL- (Dox) (SEQ ID NO: 344);

Ac-L-R-G-S-G-R-S-S-Cha- (Dox) (SEQ ID NO: 345);

Ac-L-R-G-dS-A-R-S-A- (Dox) (SEQ ID NO: 574);

Ac-L-R-G-S-A-R-S-S-L- (Dox) (SEQ ID NO: 346);

Ac-L-R-G-S-A-R-S-L- (Dox) (SEQ ID NO: 347);

Ac-L-R-G-S-A-R-S-S-Cha- (Dox) (SEQ ID NO: 348);

Ac-L-R-G-S-A-R-S-S-nV- (Dox) (SEQ ID NO: 349);

Ac-L-R-G-S-A-R-S-S-nL- (Dox) (SEQ ID NO: 350);

Ac-V-I-V-S-G-R-A-L- (Dox) (SEQ ID NO: 351);

Ac-V-I-V-S-A-R-S-L- (Dox) (SEQ ID NO: 352);

Ac-V-I-V-S-G-R-S-S-L- (Dox) (SEQ ID NO: 353);

Ac-V-I-V-S-A-R-M-A- (Dox) (SEQ ID NO: 354);

Ac-V-I-V-S-A-R-nL-A- (Dox) (SEQ ID NO: 355);

Ac-V-I-V-S-A-R-S-nL- (Dox) (SEQ ID NO: 356);

Ac-V-I-V-S-A-R-S-Cha- (Dox) (SEQ ID NO: 357);

Ac-V-I-V-S-A-R-S-Cha- (Dox) (SEQ ID NO: 358);

Ac-V-I-V-S-A-R-S-S-Cha- (Dox) (SEQ ID NO: 359);

Ac-R-R- (Me) C-P-G-R-V-V- (Dox) (SEQ ID NO: 360);

Ac-R-R-nV-P-A-R-S-L- (Dox) (SEQ ID NO: 361);

Ac-R-G-dS-A-R-S-A- (Dox) (SEQ ID NO: 309);

Ac-R-G-S-G-R-S-A- (Dox) (SEQ ID NO: 310);

Ac-R-G-S-G-R-A-L- (Dox) (SEQ ID NO: 311);

Ac-R-G-S-G-R-S-L- (Dox) (SEQ ID NO: 312);

Ac-R-G-S-G-R--S-nL- (Dox) (SEQ ID NO: 313);

Ac-R-G-S-G-R-A-nL- (Dox) (SEQ ID NO: 314);

Ac-R-G-S-G-R-S-S-L- (Dox) (SEQ ID NO: 315);

Ac-R-G-S-G-R-S-Cha- (Dox) (SEQ ID NO: 316);

Ac-R-G-S-G-R-S-S-Cha- (Dox) (SEQ ID NO: 317);

Ac-R-G-S-A-R-S-Cha- (Dox) (SEQ ID NO: 318);

Ac-R-G-S-A-R-S-S- (Dox) (SEQ ID NO: 319);

Ac-R-G-S-A-R-S-nV- (Dox) (SEQ ID NO: 320);

Ac-R-G-S-A-R-S-S-nV- (Dox) (SEQ ID NO: 321);

Ac-R-G-S-A-R-S-L- (Dox) (SEQ ID NO: 322);

Ac-R- (Me) C-P-G-R-V-V- (Dox) (SEQ ID NO: 323);

Ac-R- (Me) C-P-G-R-V-V- (Dox) (SEQ ID NO: 324);

Ac-R-C (Me) -P-G-R-S-L- (Dox) (SEQ ID NO: 325);

Ac-R-L-P-G-R-S-L- (Dox) (SEQ ID NO: 326);

Ac-R-V-P-G-R-S-L- (Dox) (SEQ ID NO: 327);

Ac-R-V-P-G-R-S-L- (Dox) (SEQ ID NO: 328);

Ac-R-nL-P-G-R-S-L- (Dox) (SEQ ID NO: 329);

Ac-R-G (tBu) -P-A-R-S-L- (Dox) (SEQ ID NO: 330);

Ac-R-L-P-A-R-S-L- (Dox) (SEQ ID NO: 331);

Ac-R-V-P-A-R-S-L- (Dox) (SEQ ID NO: 332);

Ac-R-nL-P-A-R-S-L- (Dox) (SEQ ID NO: 333);

Ac-I-V-S-G-R-A-L- (Dox) (SEQ ID NO: 334);

Ac-I-V-S-G-R-S-S-L- (Dox) (SEQ ID NO: 335);

Ac-I-V-S-G-R-A-S-L- (Dox) (SEQ ID NO: 336);

Ac-I-V-S-A-R-M-A- (Dox) (SEQ ID NO: 337);

Ac-I-V-S-A-R-nL-A- (Dox) (SEQ ID NO: 338);

Ac-I-V-S-A-R-S-L- (Dox) (SEQ ID NO: 339);

Ac-I-V-S-A-R-S-nL- (Dox) (SEQ ID NO: 340);

Ac-I-V-S-A-R-S-S-L- (Dox) (SEQ ID NO: 341);

Ac-G-S-G-R-S-A- (Dox) (SEQ ID NO: 585);

Ac-G-S-G-R-S-L- (Dox) (SEQ ID NO: 277);

Ac-G-S-G-R-A-L- (Dox) (SEQ ID NO: 278);

Ac-G-S-G-R-S-S-L- (Dox) (SEQ ID NO: 279);

Ac-G-S-G-R-L- (Dox) (SEQ ID NO: 280);

Ac-G-S-G- (4-guan) Phg-S-L-NH2 (SEQ ID NO: 281);

Ac-G-S-G-R-S-S-Cha- (Dox) (SEQ ID NO: 282);

Ac-G-S-G-R-A-S-L- (Dox) (SEQ ID NO: 283);

Ac-G-S-G-R-S-nL- (Dox) (SEQ ID NO: 284);

Ac-G-T-G-R-S-nL- (Dox) (SEQ ID NO: 285);

Succ-bA-T-G-R-S-nL- (Dox) (SEQ ID NO: 286);

Ac-G-T-G-R-S-hCha- (Dox) (SEQ ID NO: 287);

Ac-G-hS-G-R-S-nL- (Dox) (SEQ ID NO: 288);

Ac-G-dS-A-R-S-A- (Dox) (SEQ ID NO: 289);

Ac-G-S-A-R-S-L- (Dox) (SEQ ID NO: 290);

Ac-G-S-A-R-S-S-Cha- (Dox) (SEQ ID NO: 291);

Ac-G-S-A-R-S-S-L- (Dox) (SEQ ID NO: 292);

Ac-G-S-A-R-A-S-L- (Dox) (SEQ ID NO: 293);

Ac-V-S-G-R-S-L- (Dox) (SEQ ID NO: 294);

Ac-V-S-G-R-A-L- (Dox) (SEQ ID NO: 295);

Ac-V-S-G-R-A-S-L- (Dox) (SEQ ID NO: 296);

Ac-V-S-G-R-S-S-L- (Dox) (SEQ ID NO: 297);

Ac-V-S-A-R-M-A- (Dox) (SEQ ID NO: 298);

Ac-V-S-A-R-nL-A- (Dox) (SEQ ID NO: 299);

Ac-V-S-A-R-S-nL- (Dox) (SEQ ID NO: 300);

Ac-V-S-A-R-S-L- (Dox) (SEQ ID NO: 301);

Ac- (Me) C-P-G-R-V-V- (Dox) (SEQ ID NO: 302);

Ac- (Me) C-P-G-R-V-V- (Dox) (SEQ ID NO: 303);

Ac-C (Me) -P-G-R-A-L- (Dox) (SEQ ID NO: 304);

Ac-C (Me) -P-G-R-S-L- (Dox) (SEQ ID NO: 305);

Ac-C (Me) -P-A-R-S-L- (Dox) (SEQ ID NO: 306);

Ac-C (Me) -P-A-R-A-S-L- (Dox) (SEQ ID NO: 307);

Ac-G (tBu) -P-G-R-S-L- (Dox) (SEQ ID NO: 308);

Ac-Q-S-R-A-A- (taxol) (SEQ ID NO: 552);

Ac-Q-S-R-S-A- (taxol) (SEQ ID NO: 553);

Ac-Q-S-R-S-G- (taxol) (SEQ ID NO: 554);

Ac-R-S-R-A-A- (taxol) (SEQ ID NO: 555);

Ac-R-Q-S-R-A-A- (taxol) (SEQ ID NO: 556);

Ac-R-Q-S-R-S-A- (taxol) (SEQ ID NO: 557);

Ac-R-Q-S-R-S-A-A- (taxol) (SEQ ID NO: 558);

Ac-R-G-S-G-R-S-A- (taxol) (SEQ ID NO: 559);

Ac-S-G-R-A-A- (taxol) (SEQ ID NO: 560);

Ac-S-G-R-S-A- (taxol) (SEQ ID NO: 561);

Ac-S-G-R-S-S-A- (taxol) (SEQ ID NO: 562);

Ac-S-G-R-A-S-A- (taxol) (SEQ ID NO: 563);

Ac-S-G-R-S-G- (taxol) (SEQ ID NO: 564);

Ac-S-G-R-S-S-G- (taxol) (SEQ ID NO: 565);

Ac-S-G-R-S-G-A- (taxol) (SEQ ID NO: 566);

Ac-S-G-R-S-G-G- (taxol) (SEQ ID NO: 567);

Ac-G-T-G-R-S-G-G- (taxol) (SEQ ID NO: 568);

Ac-L-R-R-Q-S-R-A-A- (Dox) (SEQ ID NO: 597);

MeSO 2 -d A (Chx) -Abu-R-S-L- (Dox) (SEQ ID NO: 598);

Ac-R-A-R-S-L- (Dox) (SEQ ID NO: 599);

Ac-dA (Chx) -Abu-R-S-L- (Dox) (SEQ ID NO: 600);

Ac-dA (Chx) -Abu-R-S-S-L- (Dox) (SEQ ID NO: 601);

Ac-Q-G-R-S-S-L- (Dox) (SEQ ID NO: 602);

MeOCO-dhF-P (OH) -R-S-S-L- (Dox) (SEQ ID NO: 603);

MeOCO-Quat4-G-R-S-L- (Dox) (SEQ ID NO: 604);

As-dCha-P (OH) -R-S-S-L- (Dox) (SEQ ID NO: 605);

Ac-dCha-Abu-R-S-S-A- (taxol) (SEQ ID NO: 606);

MeOCO-Quat2-G-R-S-L-NH2 (SEQ ID NO: 607);

MeOCO-Quat3-G-R-S-L-NH2 (SEQ ID NO: 608) and

MeOCO-Quat-G-R-S-L-NH2 (SEQ ID NO: 609).

Example 10

Pharmacodynamic Study of Conjugates and Fractions of Dose Metabolized to Doxorubicin and Leucine-Doxorubicin in Non-Tumor and Tumor-bearing Mice

Tumor-free or tumor-bearing nude mice from 8 to 12 weeks of age were used for pharmacokinetic studies of test conjugates. Transplantation tumor cells were prepared using one of three protocols:

Protocol A Tumor Cells Collected from Tissue Culture

Tumor cells are treated with trypsin, resuspended in growth medium and centrifuged at 200 × g for 6 minutes. Cells are resuspended in serum free medium and counted. The appropriate volume of the solution containing the desired number of cells is then transferred to a conical centrifuge tube, centrifuged as described above and resuspended in a cold 1: 1 mixture of cells: Matrigel in the appropriate volume of phenol-free medium. Let's do it. Each mouse is inoculated subcutaneously or in situ with 0.2-0.5 mL containing 1 × 10 ⁶ to 1 × 10 ⁷ tumor cells.

Protocol B Tumor Cell Suspension

Established tumors (200-1000 mm ³ ) are dissected from the mice and weighed and washed in tumor cell growth medium. The tumor is passed through a still cell dissociation sieve. The cells are washed through a sieve with growth medium. The cells are centrifuged at 200 × g for 6 minutes and resuspended in a cold 1: 1 mixture of cells: Matrigel in the appropriate volume. Each mouse is subcutaneously or inoculated with 0.2-0.5 mL of tumor cells.

Protocol C Tumor Fragment

Alternatively, a tumor of about 800 mm ³ is excised from the mouse, washed in tumor cell growth medium and cut into 1-2 mm ³ fragments. Each fragment is subcutaneously or inoculated using a trocar.

Pharmacokinetic Studies

Tumor-free or tumor bearing mice are weighed individually and grouped into groups. Mice are administered from 1 to 100 μle / kg comprising test conjugate 30 μole / kg, 25 μole / kg or 21.5 μole / kg. Mice are sacrificed at some time between 5 minutes and 24 hours after administration of the compound. Blood is collected in a syringe containing inhibitors such as EDTA, AEBSF, Aprotinin, Leupeptin, Vestatin, Pepstanin A or E64 and transferred into heparin added blood collection in vitro. The plasma is prepared by centrifugation. Tumors are collected and ground in liquid nitrogen. The tumor powder obtained is stored at -80 ° C. The tumor powder and plasma are extracted and analyzed for parental test conjugates and their products, including leucine-doxorubicin (or norleucine-doxorubicin, etc.) and doxorubicin.

Consider the delivery of toxins to tumor cells and the levels of parent conjugates and toxins in the plasma (dox and nor-leu dox).

result

For example, test conjugates (21.5 μole / kg Ac-Gly-Ser-Gly-Arg-Ser-nLeu-Dox (see Example 2)) were intraperitoneally (IP) to non-tumor-bearing and tumor-bearing (TB) mice. Or intravenous (IV). One hour after administration, plasma and tumor tissues are collected from mice. The concentrations of the test conjugates and their products are compared. The results suggest that the conjugate did not enter the tumor and that the toxins (norleu and dox) were in μM concentrations in the tumor 1 hour after one (IP and IV) injection. The levels of dox and nor-leu dox were lower in plasma than in tumors.

Extraction, Chromatography LC / MS Conditions

Plasma: Plasma samples are prepared using acetonitrile protein precipitation. Standard curves are constructed by adding 5-20 μL of standard compound to 0.1 mL or 0.05 mL volume of iced plasma. Standard curves range from 10 ng / mL to 1 ug / mL or 100 ng / mL to 4 ug / mL of the standard compound. Immediately after the standard addition, acetonitrile is added to precipitate the protein. A plasma sample is prepared by thawing frozen plasma samples on ice. Aliquots are added directly to acetonitrile.

After sample precipitation, the samples are mixed using vortex mixing. The precipitate is pelleted using centrifugation. The supernatant is dried using vacuum centrifugation. Samples are reconstituted with 0.15 mL of 30% acetonitrile-70% (0.01 M ammonium acetate and 0.1% formic acid). 0.01 mL of sample is injected for LC-MS analysis. HPLC conditions range from 20% acetonitrile-80% (10 mM ammonium acetate and 0.1% formic acid) to 50% acetonitrile-50% (10 mM ammonium over 1 minute at 0.3 mL / min on a 30 x 2.1 mm Zorbax SB C18 HPLC column Acetate and 0.1% formic acid). Detection is provided by triple quad mass spectroscopy using electrospray ionization. Doxorubicin is monitored using m / z metastasis 544.1-396.8. Leucine-doxorubicin is monitored using 657.2-242.8. Exemplary parent conjugates are monitored using 1555.9 -1555.9. Injection LC-MS and fluorescence detection are used to identify cleavage products other than doxorubicin or leucine-doxorubicin (or norleucine-doxorubicin, etc.) in plasma.

Tumor: Immediately after dissection from the mouse, the tumor for analysis is weighed and placed into a mortar containing liquid nitrogen. Mortars located in a dry ice bed are used to crush the tumor into fine powder and to add thaw to further liquid nitrogen as needed to prevent thawing. Once a homogeneous tumor powder is achieved, the residual liquid nitrogen is boiled off. The tumor powder is transferred to a pre-cooled 15 mL centrifuge tube and placed on dry ice. Samples are stored at -70 ° C until analysis. The tumor powder is thawed on ice and vortex mixed with 0.01 M ammonium acetate at a concentration of mL of 1 g / ammonium acetate solution of the tumor to form a slurry. An aliquot of 0.1 mL of the tumor slurry is precipitated with 0.5 mL of acetonitrile. The supernatant is separated from the precipitated solids and then evaporated using vacuum centrifugation. Quantification of doxorubicin, leucine-doxorubicin (or norleucine-doxorubicin, etc.) is accomplished by reference to a standard curve constructed by mixing measured amounts of standard compounds (doxorubicin, leucine-doxorubicin, etc.) with a control tumor slurry. Typical standard curves range from 1 ng to 200 ng of compound per tumor Sliri aliquot. After the unknown and standard samples are processed and dried, the residue is reconstituted with 30% acetonitrile-70% (0.01 M ammonium acetate + 0.1% formic acid). 10 μl of solution is injected into a liquid chromatography-mass spectrometry system. HPLC conditions range from 20% acetonitrile-80% (10 mM ammonium acetate and 0.1% formic acid) to 50% acetonitrile-50% (10 mM ammonium over 1 minute at 0.3 mL / min on a 30 x 2.1 mm Zorbax SB C18 HPLC column Acetate and 0.1% formic acid). Detection is provided by a triple quadrupole mass spectrometer using electrospray ionization. Doxorubicin is monitored using m / z metastasis 544.1-396.8. Leucine-doxorubicin is monitored using 657.2-242.8.

Modifications will be apparent to those skilled in the art, meaning that the invention should be limited only by the scope of the appended claims.

Claims (148)

A plasmin or prostate, comprising a therapeutic agent, and a peptidic substrate linked to the therapeutic agent, optionally via a linker, and proteolytically cleaved by cell surface protease or a soluble, released or secreted form thereof to liberate the therapeutic agent. A conjugate that is not substantially cleaved by a specific antigen (PSA). The conjugate of claim 1, wherein the released therapeutic agent is active. The conjugate of claim 1, wherein the cleavage releases the therapeutic agent in a form that requires further treatment for activity. The composition of claim 1, wherein the components (peptide substrate) _s , (linker) _q and (therapeutic) _t [where one or more peptide substrate substrate residues are linked to one or more therapeutic agents with or without the linker, and s is from 1 to 1 6, q is 0 to t, t is 1 to 6], wherein the cell surface protease cleaving the peptidic substrate (s) results in delivery of the therapeutic agent to the cell. The conjugate of claim 1, wherein the peptidic substrate comprises one or more amino acids and is in active form when the therapeutic agent obtained upon proteolytic cleavage of the conjugate is active or further processed. The conjugate of claim 1, wherein the cell surface protease is a serine protease. The conjugate of claim 1, wherein the cell surface protease is a type II transmembrane serine protease (MTSP) or an endotheliase. The cell surface protease of claim 1, wherein the cell surface protease is endothelial agent 1, endothelial agent 2, MTSP1, MTSP3, MTSP4, MTSP6, MTSP7, MTSP9, MTSP10, MTSP12, MTSP20, MTSP22, MTSP25, corin, enterokinase, human airway trypsin Conjugates selected from type protease (HAT), TMPRSS2, heptin, urokinase-type plasminogen activator (uPA) and TMPRSS4. The method of claim 1, wherein the cell surface protease is SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 29, 31, 33, 35, 37, 39, 41, 43, 45, 270, 272, 274 And a polypeptide comprising the amino acid sequence set forth in any one of 276, SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 28, 30, 32, 34, 36,38, 40, 42, 44, 269, 273 and 275 A polypeptide encoded by a nucleotide sequence that hybridizes under high stringency conditions to the nucleotide sequence set forth in any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 29, 31, 33, 35, 37, 39, 41, 43, 45, 270, 272, 274 And a polypeptide comprising an amino acid sequence having at least about 40% sequence identity with the amino acid sequence set forth in 276 and SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 29, 31, 33, 35, 37, 39, 41, 43, 45, 270, 272, 274 And a polypeptide selected from the group consisting of polypeptides encoded by splice variants of the nucleotide sequence set forth in any one of 276. The conjugate of claim 1, wherein the therapeutic agent is a toxin, a small organic molecule, a nucleic acid, a protein therapeutic agent, a cytokine or a growth factor. The conjugate of claim 1, wherein the therapeutic agent is an anticancer agent. The conjugate of claim 1, wherein the therapeutic agent is an antiangiogenic agent. The method of claim 1, wherein the therapeutic agent is abrin, lysine A, Pseudomonas exotoxin, shiga toxin, diphtheria toxin, tumor necrosis factor, α-interferon, γ-interferon, nerve growth factor, tissue factor and tissue factor variant, FAS -Ligand, growth factor derived from platelets, tissue plasminogen activator, interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte macrophage colony stimulating factor ( GMCSF), granulocyte colony stimulating factor (G-CSF), erythropoietin (EPO), nerve growth factor, fibroblast growth factor (FGF) and epidermal growth factor. The conjugate of claim 1, wherein the therapeutic agent is selected from alkylating agents, toxins, anti-proliferative agents, pro-apoptotic agents, procoagulants, cytotoxic nucleosides, and tubulin binders. The conjugate of claim 1, wherein the therapeutic agent is selected from the following classes of drugs or pharmaceutically acceptable derivatives thereof: a) anthracycline drugs, b) vinca alkaloid drugs, c) mitomycin, d) bleomycin, e) cytotoxic nucleosides, f) pterinic drugs, g) diinene, h) esturamustine, i) cyclophosphamide, j) taxane, k) grapephytotoxin, l) maytanzanoids, m) epothilones, and n) combretastatin and analogs. The conjugate of claim 1, wherein the therapeutic agent is selected from the following drugs or pharmaceutically acceptable derivatives thereof: a) doxorubicin, b) carminomycin, c) daunorubicin, d) aminopterin, e) methotrexate, f) metopeterin, g) dichloro methotrexate, h) mitomycin C, i) porphyromycin, j) 5-fluorouracil, k) 6-mercaptopurine, l) cytosine arabinoside, m) grapephytotoxin, n) etoposide, o) etoposide phosphate, p) melphalan, q) vinblastine, r) vincristine, s) leucinedin, t) bindesin, u) esturamustine, v) cisplatin, w) cyclophosphamide, x) taxol, y) leucine, z) 4-desacetylvinblastine, aa) epothilone B, bb) Taxotere, cc) maytanzanol, dd) epothilone A and ee) combretastatin and analogues. The conjugate of claim 1, further comprising a linker between the therapeutic agent and the peptidic substrate. 18. The conjugate of claim 17, wherein the linker comprises a carbohydrate, peptide and / or hydrocarbon core. 18. The biscarbonyl of claim 17, wherein the linker is such that the amine residue on the therapeutic agent is linked to the linker unit to form an amide bond and the amino terminus of the peptidic substrate is linked to the other end of the linker unit to form an amide bond as well. Diaminoalkyl diradical linkers such that the carbonyl moiety on the alkyl diradical or therapeutic agent is covalently bound to one of the amines of the linker unit and the remaining amines on the linker unit are covalently attached to the C-terminus of the peptidic substrate. Containing units, or , And [Wherein A is NH or O, D is N (H or alkyl) or O and R ²⁵ is at least one selected from H, alkyl, cycloalkyl, cycloalkylalkyl, aryl, or halo, halo alkyl and alkyl For example, heteroaryl, aralkyl, heteroaralkyl, alkenyl containing 1 to 2 double bonds optionally substituted by 1 to 3 substituents, alkys containing 1 to 2 triple bonds Nil, alk (en) (yn) yl group, halo, pseudohalo, cyano, hydroxy, haloalkyl and polyhaloalkyl (e.g. halo lower alkyl including trifluoromethyl), formyl, alkyl Carbonyl, or for example arylcarbonyl, heteroarylcarbonyl, carboxy, alkoxycarbonyl, optionally substituted by one or more substituents selected from, for example, halo, halo alkyl and alkyl, Aryloxycarbonyl, aminoimino, alkoxycarbonylamino, Aryloxycarbonylamino, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, arylaminocarbonyl, diarylaminocarbonyl, aralkylaminocarbonyl, alkoxy, aryloxy, perfluoroalkoxy, alkenyl Oxy, alkynyloxy, arylalkoxy, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, arylaminoalkyl, amino, alkylamino, dialkylamino, arylamino, alkylarylamino, alkylcarbonylamino, arylcarbonylamino , Azido, nitro, mercapto, alkylthio, arylthio, perfluoroalkylthio, thiocyano, isothiocyano, alkylsulfinyl, alkylsulfonyl, arylsulfinyl, arylsulfonyl, aminosulfonyl, Alkylaminosulfonyl, dialkylaminosulfonyl, or arylaminosulfonyl, and y is an integer from 1 to 3]. 18. The conjugate of claim 17, wherein the linker is a diamine comprising cyclic alkylene moieties. 18. The conjugate of claim 17, wherein the diamine contains a bicycloalkylene moiety. 18. The method of claim 17, wherein the linker is 1,4-bis (aminomethyl) cyclohexane, 1,4-bis (aminomethyl) cycloheptane, 1,3-bis (aminomethyl) cyclopentane, 1-amino-4- Conjugates selected from (aminomethyl) cyclohexane, 1,4-diaminocyclohexane and 1,4-bis (aminomethyl) bicyclo [2.2.2] octane. 18. The conjugate of claim 17, wherein the linker is 1, ω-diaminoalkane. 18. The conjugate of claim 17, wherein the linker is 1,3-diaminopropane. 18. The conjugate of claim 17, wherein the linker is 1, ω-dicarbonylalkane. The conjugate of claim 25, wherein the linker is selected from oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid and pivalic acid. The conjugate of claim 1, wherein the peptidic substrate comprises P1, which is any amino acid. The conjugate of claim 27, wherein P 1 is a natural amino acid. The conjugate of claim 27, wherein P 1 is an amino acid having an aromatic side chain, a branched side chain or a branched aromatic side chain. The conjugate of claim 1, wherein the peptidic substrate comprises P1 selected from Arg, Lys, Tyr, Phe, Trp, Ala, Val, Ile and Thr. The method of claim 1, wherein the peptidic substrate is a P1-P1 ′ bond that is a cleavage site by cell surface protease, wherein P1 is selected from Arg, Lys, Tyr, Phe, Trp, Ala, Val, Ile and Thr, and P1 Is Gly, Ser, Ala, Leu, Ile, d-Ile, nLeu, Val, nVal, Aib, Abu, Met or 6-aminohexanoyl. The conjugate of claim 1, wherein the peptidic substrate comprises P1, which is an Arg, Lys, or Arg surrogate. The conjugate of claim 1, further comprising a P2 residue selected from Phe, Ser, Gly, and Ala. The conjugate of claim 1, further comprising a P3 residue selected from Arg, Lys, Gln, Ser, Quat, and Arg surrogate. The conjugate of claim 1, further comprising a P4 residue selected from Pro, Arg, Ser, Ala, Lys, Gly, nLeu, Leu, Tyr, Glu, Phe, and Val. The conjugate of claim 1, further comprising a P5 residue selected from Arg and Arg surrogate. The conjugate of claim 1, further comprising a P6 residue selected from Leu, Ile and Val. The conjugate of claim 1, further comprising a P2 ′ residue selected from Gly, Ser, Ala, Leu, Ile, d-Ile, nLeu, Val, nVal, Aib, Abu, Met and 6-aminohexanoyl. The conjugate of claim 1, further comprising a P3 ′ residue selected from Gly, Ser, Ala, Leu, Ile, nLeu, Val, nVal, Aib, Abu, Met, and 6-aminohexanoyl. The method of claim 1, wherein the peptidic substrate is a pentamer of formula P4-P3-P2-P1-P1 'wherein P1 is selected from Arg, Lys, Tyr, Phe, Trp, Ala, Val, Ile and Thr, P2 is selected from Phe, Ser, Gly and Ala, P3 is selected from Arg, Lys, Gln, Quat and Arg surrogate, P4 is Pro, Arg, Ser, Ala, Lys, Gly, nLeu, Leu, Tyr, Glu, Phe and Val, and P1 'is Gly, Ser, Ala, Leu, Ile, d-Ile, nLeu, Val, nVal, Aib, Abu, Met or 6-aminohexanoyl. The method of claim 40, wherein the peptidic substrate is optionally one or more P5 or P2 ′ amino acid residues, wherein P5 is an Arg or Arg surrogate and P2 ′ is Gly, Ser, Ala, Leu, Ile, d-Ile, nLeu , Val, nVal, Aib, Abu, Met and 6-aminohexanoyl. 42. The method of claim 41 wherein the peptidic substrate further comprises a P6 amino acid residue optionally selected from Leu, Ile and Val when the peptidic substrate comprises a P5 amino acid residue and the peptidic substrate comprises a P2 'amino acid residue. Wherein the peptidic substrate optionally further comprises a P3 ′ amino acid residue selected from Gly, Ser, Ala, Leu, Ile, nLeu, Val, nVal, Aib, Abu, Met and 6-aminohexanoyl. The conjugate of claim 1, wherein the therapeutic agent is conjugated to the C-terminus of the peptidic substrate either directly or via a linker. The conjugate of claim 1, wherein the peptidic substrate comprises a cap at the N-terminus. The conjugate of claim 1, wherein the cap is a hydrophilic blocking group. The conjugate of claim 1, wherein the cap is an acyl, sulfonyl or carbonyl derivative. 46. The conjugate of claim 45, wherein the blocking group is selected from hydroxylated alkanoyls, polyhydroxylated alkanoyls, polyethylene glycols, glycosylates, sugars and crown ethers. 44. The conjugate of claim 43, which is a compound of formula (I) or a derivative thereof. Formula I X ^n- (P6) _m- (P5) _p- (P4) _i- (P3) _j- (P2) _l -P1- (P1 ') _u- (P2') _k- (P3 ') _r- (L ) _n -Z In the above formula, Z is a cure, L is a linker, l, j, i, p and m are chosen as follows: l is 0 or 1, when l is 0, j, i, p and m are 0, when l is 1 j is 0 or 1 and when j is 0 i, p and m are 0 I is 0 or 1 when j is 1, p and m are 0 when i is 0, p is 0 or 1 when i is 1, and m is 0 when p is 0 When p is 1, m is 0 or 1, u, k and r are selected as follows: u is 0 or 1, k and r are 0 when u is 0, k is 0 or 1 when u is 1, r is 0 when k is 0, and k is 1 r is 0 or 1, n is 0 or 1, X ⁿ is hydrogen or acyl, sulfonyl or carbamoyl cap, P1 is selected from Arg, Lys, Tyr, Phe, Trp, Ala, Val, Ile and Thr, P1 ′ is Gly, Ser, Ala, Leu, Ile, d-Ile, nLeu, Val, nVal, Aib, Abu, Met or 6-aminohexanoyl, P2 is selected from Phe, Ser, Gly and Ala, P3 is selected from Arg, Lys, Gln, Ser, Quat and Arg surrogate, P4 is selected from Pro, Arg, Ser, Ala, Lys, Gly, nLeu, Leu, Tyr, Glu, Phe and Val, P5 is selected from Arg and Arg surrogate, P6 is selected from Leu, Ile and Val, P2 'is selected from Gly, Ser, Ala, Leu, Ile, d-Ile, nLeu, Val, nVal, Aib, Abu, Met and 6-aminohexanoyl, P3 ′ is selected from Gly, Ser, Ala, Leu, Ile, nLeu, Val, nVal, Aib, Abu, Met and 6-aminohexanoyl. 49. The conjugate of claim 48, wherein Pl is an Arg, Lys or Arg surrogate. The conjugate of claim 1, wherein the therapeutic agent is conjugated to the N-terminus of the peptidic substrate either directly or via a linker. 51. The conjugate of claim 50, wherein the C-terminus of the peptidic substrate is a carboxylic acid or carboxamide derivative. 51. The conjugate of claim 50, which is a compound of Formula II or a derivative thereof. Formula II Z- (L) _n- (P6) _m- (P5) _p- (P4) _i- (P3) _j- (P2) _l -P1- (P1 ') _u- (P2') _k- (P3 ') _r -X ^c In the above formula, Z is a cure, L is a linker, l, j, i, p and m are chosen as follows: l is 0 or 1, when l is 0 j, i, p and m are 0, when l is 1 j is 0 or 1 and when j is 0 i, p and m are 0 I is 0 or 1 when j is 1, p and m are 0 when i is 0, p is 0 or 1 when i is 1, and m is 0 when p is 0 When p is 1, m is 0 or 1, u, k and r are selected as follows: u is 0 or 1, k and r are 0 when u is 0, k is 0 or 1 when u is 1, r is 0 when k is 0, and k is 1 r is 0 or 1, n is 0 or 1, X ^c together with the carbonyl group of the bound amino acid residue form a carboxylic acid or carboxamide group, P1 is selected from Arg, Lys, Tyr, Phe, Trp, Ala, Val, Ile and Thr, P1 ′ is Gly, Ser, Ala, Leu, Ile, d-Ile, nLeu, Val, nVal, Aib, Abu, Met or 6-aminohexanoyl, P2 is selected from Phe, Ser, Gly and Ala, P3 is selected from Arg, Lys, Gln, Ser, Quat and Arg surrogate, P4 is selected from Pro, Arg, Ser, Ala, Lys, Gly, nLeu, Leu, Tyr, Glu, Phe and Val, P5 is selected from Arg and Arg surrogate, P6 is selected from Leu, Ile and Val, P2 'is selected from Gly, Ser, Ala, Leu, Ile, d-Ile, nLeu, Val, nVal, Aib, Abu, Met and 6-aminohexanoyl, P3 ′ is selected from Gly, Ser, Ala, Leu, Ile, nLeu, Val, nVal, Aib, Abu, Met and 6-aminohexanoyl. 53. The conjugate of claim 52, wherein Pl is an Arg, Lys or Arg surrogate. The method of claim 1, wherein the first therapeutic agent is optionally linked to the N-terminus of the peptidic substrate via the first linker, and the second therapeutic agent, which is the same or different than the first therapeutic agent, optionally comprises a second linker that is the same or different from the first linker. Conjugates coupled to the C-terminus of the peptidic substrate through. 55. The conjugate of claim 54, wherein the conjugate is a compound of Formula III or a derivative thereof. Formula III Z ^1- (L ¹ ) _n- (P6) _m- (P5) _p- (P4) _i- (P3) _j- (P2) _l -P1- (P1 ') _u- (P2') _k- (P3 ') _r- (L ² ) _v -Z ² In the above formula, Z ¹ and Z ² are each a therapeutic agent and are the same or different, L ¹ and L ² are each a linker and are the same or different, l, j, i, p and m are chosen as follows: l is 0 or 1, when l is 0 j, i, p and m are 0, when l is 1 j is 0 or 1 and when j is 0 i, p and m are 0 I is 0 or 1 when j is 1, p and m are 0 when i is 0, p is 0 or 1 when i is 1, and m is 0 when p is 0 When p is 1, m is 0 or 1, u, k and r are selected as follows: u is 0 or 1, k and r are 0 when u is 0, k is 0 or 1 when u is 1, r is 0 when k is 0, and k is 1 r is 0 or 1, n and v are each independently 0 or 1, P1 is selected from Arg, Lys, Tyr, Phe, Trp, Ala, Val, Ile and Thr, P1 ′ is Gly, Ser, Ala, Leu, Ile, d-Ile, nLeu, Val, nVal, Aib, Abu, Met or 6-aminohexanoyl, P2 is selected from Phe, Ser, Gly and Ala, P3 is selected from Arg, Lys, Gln, Ser, Quat and Arg surrogate, P4 is selected from Pro, Arg, Ser, Ala, Lys, Gly, nLeu, Leu, Tyr, Glu, Phe and Val, P5 is selected from Arg and Arg surrogate, P6 is selected from Leu, Ile and Val, P2 'is selected from Gly, Ser, Ala, Leu, Ile, d-Ile, nLeu, Val, nVal, Aib, Abu, Met and 6-aminohexanoyl, P3 ′ is selected from Gly, Ser, Ala, Leu, Ile, nLeu, Val, nVal, Aib, Abu, Met and 6-aminohexanoyl. The conjugate of claim 55, wherein P 1 is an Arg, Lys, or Arg surrogate. The method according to any one of claims 1 to 57, Ac-Leu-Arg-Ala-Quat-Gly-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 46); Ac-Leu-Arg-Ala-Quat-Ala-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 47); Ac-Leu-Arg-Ser-Quat-Gly-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 48); Ac-Leu-Arg-Ser-Quat-Ala-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 49); Ac-Leu-Arg-Pro-Arg-Phe-Lys-Ile-Ile- (therapeutic) (SEQ ID NO: 50); Ac-Arg-Pro-Arg-Phe-Lys-Ile-Ile- (therapeutic) (SEQ ID NO: 51); Ac-Pro-Arg-Phe-Lys-Ile-Ile- (therapeutic) (SEQ ID NO: 52); Ac-Leu-Arg-Ser-Lys-Ser-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 53); Ac-Arg-Ser-Lys-Ser-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 54); Ac-Ser-Lys-Ser-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 55); Ac-Leu-Arg-Pro-Arg-Phe-Arg-Ile-Ile- (therapeutic) (SEQ ID NO: 56); Ac-Arg-Pro-Arg-Phe-Arg-Ile-Ile- (therapeutic) (SEQ ID NO: 57); Ac-Pro-Arg-Phe-Arg-Ile-Ile- (therapeutic) (SEQ ID NO: 58); Ac-Leu-Arg-Ser-Arg-Ser-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 59); Ac-Arg-Ser-Arg-Ser-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 60); Ac-Ser-Arg-Ser-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 61); Ac-Leu-Arg-Ala-Quat-Gly-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 62); Ac-Leu-Arg-Ala-Quat-Ala-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 63); Ac-Leu-Arg-Ser-Quat-Gly-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 64); Ac-Leu-Arg-Ser-Quat-Ala-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 65); Ac-Leu-Arg-Pro-Arg-Phe-Lys-Ile-Ile- (therapeutic) (SEQ ID NO: 66); Ac-Arg-Pro-Arg-Phe-Lys-Ile-Ile- (therapeutic) (SEQ ID NO: 67); Ac-Pro-Arg-Phe-Lys-Ile-Ile- (therapeutic) (SEQ ID NO: 68); Ac-Leu-Arg-Ser-Lys-Ser-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 69); Ac-Arg-Ser-Lys-Ser-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 70); Ac-Ser-Lys-Ser-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 71); Ac-Leu-Arg-Pro-Arg-Phe-Arg-Ile-Ile- (therapeutic) (SEQ ID NO: 72); Ac-Arg-Pro-Arg-Phe-Arg-Ile-Ile- (therapeutic) (SEQ ID NO: 73); Ac-Pro-Arg-Phe-Arg-Ile-Ile- (therapeutic) (SEQ ID NO: 74); Ac-Leu-Arg-Ser-Arg-Ser-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 75); Ac-Arg-Ser-Arg-Ser-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 76) and Ac-Ser-Arg-Ser-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 77) Fatigue Glu-Pro-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 78); CH ₃ SO ₂ -D-HHT-Gly-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 79); N-p-tosyl-Gly-Pro-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 80); Benzoyl-Val-Gly-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 81); CH ₃ SO ₂ -D-HHT-Gly-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 82); N-α-Z-D-Arg-Gly-Arg-Ala-Ala- (therapeutic), wherein Z is benzyloxycarbonyl (SEQ ID NO: 83); Fatigue Glu-Gly-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 84); H-D-Ile-Pro-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 85); Cbo-L- (γ) Glu (α-t-BuO) -Gly-Arg-Ala-Ala- (therapeutic), wherein Cbo is carbobenzoxyl (SEQ ID NO: 86); H-D-Pro-Phe-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 87); H-D-Val-Leu-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 88); Bz-Ile-Glu (γ-OH) -Gly-Arg-Ala-Ala- (therapeutic), wherein Bz is benzoyl (SEQ ID NO: 89); Bz-Ile-Glu (γ-OMe) -Gly-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 90); Bz-Pro-Phe-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 91); H-D-Phe-Pip-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 92); H-D-Val-Leu-Lys-Ala-Ala- (therapeutic) (SEQ ID NO: 93); H-D-Nle-HHT-Lys-Ala-Ala- (therapeutic) (SEQ ID NO: 94); Pyr-Arg-Thr-Lys-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 95); H-Arg-Gln-Arg-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 96); Boc-Gln-Gly-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 97); Z-Arg-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 98); H-D-HHT-Ala-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 99); H-D-CHT-Gly-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 100); MeSO ₂ -d Phe-Pro-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 101); δ-Z-D-Lys-Pro-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 102); CH ₃ SO ₂ -D-CHA-But-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 103); Ac-Arg-Gln-Ser-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 104); Ac-Arg-Arg-Gln-Ser-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 105); Ac-Leu-Arg-Arg-Gln-Ser-Arg-Ala-Ala- (therapeutic) (SEQ ID NO: 106); Ac-Arg-Gln-Ser-Arg-Ala- (therapeutic) (SEQ ID NO: 107); Ac-Arg-Arg-Gln-Ser-Arg-Ala- (therapeutic) (SEQ ID NO: 108); Ac-Leu-Arg-Arg-Gln-Ser-Arg-Gly-Gly- (therapeutic) (SEQ ID NO: 109); Ac-Leu-Arg-Arg-Gln-Ser-Arg-Ala- (therapeutic) (SEQ ID NO: 110); Ac-Arg-Arg-Gln-Ser-Arg-Ile- (therapeutic) (SEQ ID NO: 111); Ac-Leu-Arg-Arg-Gln-Ser-Arg-Ala-Ile- (therapeutic) (SEQ ID NO: 112); Ac-Leu-Arg-Ala-Quat-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 113); Ac-Leu-Arg-Ala-Quat-Ala-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 114); Ac-Leu-Arg-Ser-Quat-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 115); Ac-Leu-Arg-Ser-Quat-Ala-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 116); Ac-Leu-Arg-Pro-Arg-Phe-Lys-Ser-Leu- (therapeutic) (SEQ ID NO: 117); Ac-Arg-Pro-Arg-Phe-Lys-Ser-Leu- (therapeutic) (SEQ ID NO: 118); Ac-Pro-Arg-Phe-Lys-Ser-Leu- (therapeutic) (SEQ ID NO: 119); Ac-Leu-Arg-Ser-Lys-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 120); Ac-Arg-Ser-Lys-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 121); Ac-Ser-Lys-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 122); Ac-Leu-Arg-Pro-Arg-Phe-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 123); Ac-Arg-Pro-Arg-Phe-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 124); Ac-Pro-Arg-Phe-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 125); Ac-Leu-Arg-Ser-Arg-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 126); Ac-Arg-Ser-Arg-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 127); Ac-Ser-Arg-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 128); Ac-Leu-Arg-Ala-Quat-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 129); Ac-Leu-Arg-Ala-Quat-Ala-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 130); Ac-Leu-Arg-Ser-Quat-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 131); Ac-Leu-Arg-Ser-Quat-Ala-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 132); Ac-Leu-Arg-Pro-Arg-Phe-Lys-Ser-Leu- (therapeutic) (SEQ ID NO: 133); Ac-Arg-Pro-Arg-Phe-Lys-Ser-Leu- (therapeutic) (SEQ ID NO: 134); Ac-Pro-Arg-Phe-Lys-Ser-Leu- (therapeutic) (SEQ ID NO: 135); Ac-Leu-Arg-Ser-Lys-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 136); Ac-Arg-Ser-Lys-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 137); Ac-Ser-Lys-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 138); Ac-Leu-Arg-Pro-Arg-Phe-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 139); Ac-Arg-Pro-Arg-Phe-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 140); Ac-Pro-Arg-Phe-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 141); Ac-Leu-Arg-Ser-Arg-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 142); Ac-Arg-Ser-Arg-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 143); Ac-Ser-Arg-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 144); Fatigue Glu-Pro-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 145); CH ₃ S0 ₂ -D-HHT-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 146); N-p-tosyl-Gly-Pro-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 147); Benzoyl-Val-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 148); CH ₃ S0 ₂ -D-HHT-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 149); N-α-Z-D-Arg-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 150) (Z = benzyloxycarbonyl); Fatigue Glu-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 151); H-D-Ile-Pro-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 152); Cbo-L- (γ) Glu (α-t-BuO) -Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 153) (Cbo = carbobenzox); H-D-Pro-Phe-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 154); H-D-Val-Leu-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 155); Bz-Ile-Glu (γ-OH) -Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 156) (Bz = benzoyl); Bz-Ile-Glu (γ-OMe) -Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 157); Benzoyl-Pro-Phe-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 158); H-D-Phe-Pip-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 159); H-D-Val-Leu-Lys-Ser-Leu- (therapeutic) (SEQ ID NO: 160); H-D-Nle-HHT-Lys-Ser-Leu- (therapeutic) (SEQ ID NO: 161); Pyr-Arg-Thr-Lys-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 162); H-Arg-Gln-Arg-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 163); Boc-Gln-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 164); Z-Arg-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 165); H-D-HHT-Ala-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 166); H-D-CHT-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 167); MeSO ₂ -d Phe-Pro-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 168); δ-Z-D-Lys-Pro-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 169); CH ₃ SO ₂ -D-CHA-But-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 170); Ac-Arg-Gln-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 171); Ac-Arg-Arg-Gln-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 172); Ac-Leu-Arg-Arg-Gln-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 173); Ac-Arg-Gln-Ser-Arg-Leu- (therapeutic) (SEQ ID NO: 174); Ac-Arg-Arg-Gln-Ser-Arg-Leu- (therapeutic) (SEQ ID NO: 175); Ac-Leu-Arg-Arg-Gln-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 176); Ac-Leu-Arg-Arg-Gln-Ser-Arg-Leu- (therapeutic) (SEQ ID NO: 177); Ac-Arg-Arg-Gln-Ser-Arg-Leu- (therapeutic) (SEQ ID NO: 178); Ac-Leu-Arg-Arg-Gln-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 179); Ac-Arg-Gln-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 180); Ac-Arg-Gln-Ala-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 181); Ac-Arg-Gln-Phe-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 182); Ac-Arg-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 183); Ac-Arg-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 184); Ac-Arg-Ala-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 185); Ac-Arg-Phe-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 186); Ac-Gln-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 187); Ac-Gln-Gly-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 188); Ac-Gln-Ala-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 189); Ac-Gln-Phe-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 190). Ac-Leu-Arg-Ala-Quat-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 191); Ac-Leu-Arg-Ala-Quat-Ala-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 192); Ac-Leu-Arg-Ser-Quat-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 193); Ac-Leu-Arg-Ser-Quat-Ala-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 194); Ac-Leu-Arg-Pro-Arg-Phe-Lys-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 195); Ac-Arg-Pro-Arg-Phe-Lys-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 196); Ac-Pro-Arg-Phe-Lys-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 197); Ac-Leu-Arg-Ser-Lys-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 198); Ac-Arg-Ser-Lys-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 199); Ac-Ser-Lys-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 200); Ac-Leu-Arg-Pro-Arg-Phe-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 201); Ac-Arg-Pro-Arg-Phe-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 202); Ac-Pro-Arg-Phe-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 203); Ac-Leu-Arg-Ser-Arg-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 204); Ac-Arg-Ser-Arg-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 205); Ac-Ser-Arg-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 206); Ac-Leu-Arg-Ala-Quat-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 207); Ac-Leu-Arg-Ala-Quat-Ala-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 208); Ac-Leu-Arg-Ser-Quat-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 209); Ac-Leu-Arg-Ser-Quat-Ala-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 210); Ac-Leu-Arg-Pro-Arg-Phe-Lys-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 211); Ac-Arg-Pro-Arg-Phe-Lys-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 212); Ac-Pro-Arg-Phe-Lys-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 213); Ac-Leu-Arg-Ser-Lys-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 214); Ac-Arg-Ser-Lys-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 215); Ac-Ser-Lys-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 216); Ac-Leu-Arg-Pro-Arg-Phe-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 217); Ac-Arg-Pro-Arg-Phe-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 218); Ac-Pro-Arg-Phe-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 219); Ac-Leu-Arg-Ser-Arg-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 220); Ac-Arg-Ser-Arg-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 221); Ac-Ser-Arg-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 222); Fatigue Glu-Pro-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 223); CH ₃ SO ₂ -D-HHT-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 224); N-p-tosyl-Gly-Pro-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 225); Benzoyl-Val-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 226); CH ₃ S0 ₂ -D-HHT-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 227); N-α-Z-D-Arg-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 228) (Z = benzyloxycarbonyl); Fatigue Glu-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 229); H-D-Ile-Pro-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 230); Cbo-L- (γ) Glu (α-t-BuO) -Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 231) (Cbo = carbobenzox); H-D-Pro-Phe-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 232); H-D-Val-Leu-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 233); Bz-Ile-Glu (γ-OH) -Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 234) (Bz = benzoyl); Bz-Ile-Glu (γ-OMe) -Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 235); Benzoyl-Pro-Phe-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 236); H-D-Phe-Pip-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 237); H-D-Val-Leu-Lys-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 238); H-D-Nle-HHT-Lys-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 239); Pyr-Arg-Thr-Lys-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 240); H-Arg-Gln-Arg-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 241); Boc-Gln-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 242); Z-Arg-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 243); H-D-HHT-Ala-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 244); H-D-CHT-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 245); MeSO ₂ -d Phe-Pro-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 246); δ-Z-D-Lys-Pro-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 247); CH ₃ SO ₂ -D-CHA-But-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 248); Ac-Arg-Gln-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 249); Ac-Arg-Arg-Gln-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 250); Ac-Leu-Arg-Arg-Gln-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 251); Ac-Arg-Gln-Ser-Arg-Leu- (therapeutic) (SEQ ID NO: 252); Ac-Arg-Arg-Gln-Ser-Arg-Leu- (therapeutic) (SEQ ID NO: 253); Ac-Leu-Arg-Arg-Gln-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 254); Ac-Leu-Arg-Arg-Gln-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 255); Ac-Arg-Arg-Gln-Ser-Arg-Ser-Leu- (therapeutic) (SEQ ID NO: 256); Ac-Leu-Arg-Arg-Gln-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 257); Ac-Arg-Gln-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 258); Ac-Arg-Gln-Ala-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 259); Ac-Arg-Gln-Phe-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 260); Ac-Arg-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 261); Ac-Arg-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 262); Ac-Arg-Ala-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 263); Ac-Arg-Phe-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 264); Ac-Gln-Ser-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 265); Ac-Gln-Gly-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 266); Ac-Gln-Ala-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 267) and A conjugate selected from Ac-Gln-Phe-Arg-Ser-Ser-Leu- (therapeutic) (SEQ ID NO: 268). 36. The conjugate of claim 35, wherein P4 is selected from Pro, Arg, Ser, Ala, Lys, Gly, nLeu, Phe, and Val. The conjugate of claim 35, wherein P2, P3 and / or P4 are selected from Pro, Arg, Ser, Ala, Lys, Gly, nLeu, Leu, Tyr, GLu, Phe and Val. 36. The conjugate of claim 35, wherein P2, P3 and / or P4 is selected from Pro, Arg, Ser, Ala, Lys, Gly, nLeu, Tyr, Glu, Leu, Phe and Val, and P1 is any amino acid. 61. The conjugate of claim 60, wherein Pl is a natural amino acid. 61. The conjugate of claim 60, wherein Pl is an amino acid having an aromatic side chain, a branched side chain or a branched aromatic side chain. 61. The conjugate of claim 60, wherein Pl is selected from Arg, Lys, Tyr, Phe, Trp, Ala, Val, Ile and Thr. 61. The conjugate of claim 60, wherein Pl is an Arg, Lys or Arg surrogate. The conjugate of claim 1, wherein the protease is located at the cell surface due to specific binding interactions with the receptor thereto. 66. The conjugate of claim 65, wherein the cell surface protease is a urokinase plasminogen activator (u-PA) bound to the urokinase plasminogen activator receptor (u-PAR). The method of claim 1, wherein the peptide substrate of the formula P6-P5-P4-P3-P2-P1-P1'-P2'-P3 ', wherein P1, P2, P3, P4, P5, P6, P1' and P2 Is selected from the residues shown in FIGS. 1 and 2, respectively, and P6, P5, P4, P2 'and P3' are optional. The method of claim 67, P6 is optionally selected from L, V or R, P5 is optionally selected from R, I or L, P4 is optionally selected from G, C or V, P3 is selected from S, dS, P, A or G, P2 is selected from A or G, P1 is R, P1 'is S, V, M or nL, P2 'is optionally selected from S, L, A or V, Wherein the P3 'is optionally L. A conjugate selected from the conjugates shown in FIGS. 1-5, wherein the therapeutic agent Doxorubicin (Dox) or Taxol is optionally replaced with any therapeutic agent. 66. The conjugate of claim 65, wherein the therapeutic agent is a toxin, small organic molecule, nucleic acid, protein therapeutic, cytokine, or growth factor. 66. The conjugate of claim 65, wherein the therapeutic agent is an anticancer agent. 66. The conjugate of claim 65, wherein the therapeutic agent is an antiangiogenic agent. 67. The method of claim 65, wherein the therapeutic agent is abrin, lysine A, Pseudomonas exotoxin, cigar toxin, diphtheria toxin, tumor necrosis factor, α-interferon, γ-interferon, nerve growth factor, tissue factor and tissue factor variant, FAS-ligand, Platelet derived growth factors, tissue plasminogen activator, interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte macrophage colony stimulating factor (GMCSF), Conjugates selected from granulocyte colony stimulating factor (G-CSF), erythropoietin (EPO), nerve growth factor, fibroblast growth factor (FGF) and epidermal growth factor. 66. The conjugate of claim 65, wherein the therapeutic agent is selected from alkylating agents, toxins, antiproliferative agents, apoptosis promoters, procoagulants, cytotoxic nucleosides, and tubulin binders. 66. The conjugate of claim 65, wherein the therapeutic agent is selected from the following classes of drugs or pharmaceutically acceptable derivatives thereof: a) anthracycline drugs, b) vinca alkaloid drugs, c) mitomycin, d) bleomycin, e) cytotoxic nucleosides, f) pterinic drugs. g) diinene, h) esturamustine, i) cyclophosphamide, j) taxane, k) grapephytotoxin, l) maytanzanoids, m) epothilones and n) combretastatin and analogs. 66. The conjugate of claim 65, wherein the therapeutic agent is selected from the following drugs or pharmaceutically acceptable derivatives thereof. a) doxorubicin, b) carminomycin, c) daunorubicin, d) aminopterin, e) methotrexate, f) metopeterin, g) dichloro methotrexate, h) mitomycin C, i) porphyromycin, j) 5-fluorouracil, k) 6-mercaptopurine, l) cytosine arabinoside, m) grapephytotoxin, n) etoposide, o) etoposide phosphate, p) melphalan, q) vinblastine, r) vincristine, s) leucinedin, t) bindesin, u) esturamustine, v) cisplatin, w) cyclophosphamide, x) taxol, y) leucine, z) 4-desacetylvinblastine, aa) epothilone B, bb) Taxotere, cc) maytanzanol, dd) epothilone A and ee) combretastatin and analogues. 66. The conjugate of claim 65, further comprising a linker between the therapeutic agent and the peptidic substrate. 66. The conjugate of claim 65, wherein the linker comprises a carbohydrate, peptide and / or hydrocarbon core. 78. The biscarbonyl of claim 77, wherein the linker is such that the amine residue on the therapeutic agent is linked to the linker unit to form an amide bond and the amino terminus of the peptidic substrate is linked to the other end of the linker unit to form an amide bond as well. Diaminoalkyl diradical linkers such that the carbonyl moiety on the alkyl diradical or therapeutic agent is covalently bound to one of the amines of the linker unit and the remaining amine of the linker unit is covalently attached to the C-terminus of the peptidic substrate. Containing units, or , And [Wherein A is NH or O, D is N (H or alkyl) or O and R ²⁵ is at least one selected from H, alkyl, cycloalkyl, cycloalkylalkyl, aryl, or halo, halo alkyl and alkyl For example, heteroaryl, aralkyl, heteroaralkyl, alkenyl containing 1 to 2 double bonds optionally substituted by 1 to 3 substituents, alkys containing 1 to 2 triple bonds Nil, alk (en) (yn) yl group, halo, pseudohalo, cyano, hydroxy, haloalkyl and polyhaloalkyl (e.g. halo lower alkyl including trifluoromethyl), formyl, alkyl Carbonyl, or for example arylcarbonyl, heteroarylcarbonyl, carboxy, alkoxycarbonyl, optionally substituted by one or more substituents selected from, for example, halo, halo alkyl and alkyl, Aryloxycarbonyl, aminoimino, alkoxycarbonylamino, Aryloxycarbonylamino, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, arylaminocarbonyl, diarylaminocarbonyl, aralkylaminocarbonyl, alkoxy, aryloxy, perfluoroalkoxy, alkenyl Oxy, alkynyloxy, arylalkoxy, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, arylaminoalkyl, amino, alkylamino, dialkylamino, arylamino, alkylarylamino, alkylcarbonylamino, arylcarbonylamino , Azido, nitro, mercapto, alkylthio, arylthio, perfluoroalkylthio, thiocyano, isothiocyano, alkylsulfinyl, alkylsulfonyl, arylsulfinyl, arylsulfonyl, aminosulfonyl, Alkylaminosulfonyl, dialkylaminosulfonyl, or arylaminosulfonyl, and y is an integer from 1 to 3]. 78. The conjugate of claim 77, wherein the linker is a diamine comprising a cyclic alkylene moiety. 78. The conjugate of claim 77, wherein the diamine contains a bicycloalkylene moiety. 78. The method of claim 77, wherein the linker is 1,4-bis (aminomethyl) cyclohexane, 1,4-bis (aminomethyl) cycloheptane, 1,3-bis (aminomethyl) cyclopentane, 1-amino-4- Conjugates selected from (aminomethyl) cyclohexane, 1,4-diaminocyclohexane and 1,4-bis (aminomethyl) bicyclo [2.2.2] octane. 78. The conjugate of claim 77, wherein the linker is 1, ω-diaminoalkane. 78. The conjugate of claim 77, wherein the linker is 1,3-diaminopropane. 78. The conjugate of claim 77, wherein the linker is 1, ω-dicarbonylalkane. 78. The conjugate of claim 77, wherein the linker is selected from oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, and pivalic acid. 31. The P1-P1 'binding of any one of claims 1 to 30, wherein the peptidic substrate is a cleavage site by cell surface protease, wherein P1 is Arg, Lys, Tyr, Phe, Trp, Ala, Val, Is selected from Ile and Thr, and P1 ′ is Gly, Ser, hSer, Thr, Ala, Leu, Ile, d-Ile, nLeu, Val, nVal, Aib, Abu, Met or 6-aminohexanoyl] Conjugate. 33. The method of any one of claims 1-32, wherein Phe, Ser, Gly, Ala, Ser (OMe), hSer, 1-methylHis, 3-methylHis, His, nVal, nLeu, Abu, (hS) A conjugate further comprising a P2 residue selected from Gly, Thr, Aib, CHA and Tyr. 34. The compound according to any one of claims 1 to 33, wherein Arg, Lys, Gln, Quat, Arg surrogate, Ser, Thr, hSer, dSer, Pro, (hS) Gly, Tyr, 4,4-dimethyl Thr, A conjugate further comprising a P3 residue selected from Asn, Met (O ₂ ), Quat ² , Quat ³ , Quat ⁴ and Quat ⁵ . The method of claim 1, wherein Pro, Arg, Ser, Ala, Lys, Gly, nLeu, Leu, Tyr, Glu, Phe, Val, N, N-dimethylGly, β-Ala, Cys A conjugate further comprising a P4 residue selected from (Me), Gln, t-butyl Gly and nVal. 36. The conjugate of any one of claims 1 to 35, further comprising a P5 residue selected from Ile, Arg and Arg surrogate. The conjugate of any one of claims 1 to 36, further comprising a P6 residue selected from Val, Leu, Ile and Val. 38. The compound according to any one of claims 1 to 37, wherein Gly, Ser, Ala, Leu, Ile, d-Ile, nLeu, Val, nVal, Aib, Abu, Met, 6-aminohexanoyl, hCHA, CHA, A conjugate further comprising a P2 ′ residue selected from hexyl Gly, allyl Gly, and Phe. 39. P3 according to any one of the preceding claims, selected from Gly, Ser, Ala, Leu, Ile, nLeu, Val, nVal, Aib, Abu, Met, 6-aminohexanoyl, CHA and allylGly. 'Conjugates further comprising residues. 40. The P4 according to any one of claims 1 to 39, selected from Gly, Ser, Ala, Leu, Ile, nLeu, Val, nVal, Aib, Abu, Met, 6-aminohexanoyl, CHA and allylGly. 'Conjugates further comprising residues. 40. The conjugate of any one of the preceding claims, wherein P4 'is Gly, Ser, Ala, Leu, Ile, nLeu, Val, nVal, Aib, Abu, Met and 6-aminohexanoyl. 40. The conjugate of any one of the preceding claims, wherein P4 'is Leu. The method of claim 1, wherein the peptidic substrate is a pentamer of formula P4-P3-P2-P1-P1 'wherein P1 is Arg, Lys, Tyr, Phe, Trp, Ala, Val. , Ile and Thr, P2 is selected from Phe, Ser, Gly, Ala, Ser (OMe), hSer, 1-methylHis, 3-methylHis, His, nVal, nLeu, Abu, (hS) Gly, Thr, Aib, CHA and Tyr, P3 is Arg, Lys, Gln, Quat, Arg surrogate, Ser, Thr, hSer, dSer, Pro, (hS) Gly, Tyr, 4,4-dimethylThr, Asn, Met (O ₂ ), Quat ² , Quat ³ , Quat ⁴ and Quat ⁵ , P4 is selected from Pro, Arg, Ser, Ala, Lys, Gly, nLeu, Leu, Tyr, Glu, Phe, Val, N, N- DimethylGly, β-Ala, Cys (Me), Gln, t-butylGly and nVal, P1 'is Gly, Ser, hSer, Thr, Ala, Leu, Ile, d-Ile, nLeu, Val, nVal , Aib, Abu, Met or 6-aminohexanoyl. 41. The method of claim 40, wherein the peptidic substrate is optionally one or more P5 or P2 'amino acid residues, wherein P5 is an Ile, Arg or Arg surrogate, and P2' is Gly, Ser, Ala, Leu, Ile, d-Ile. , nLeu, Val, nVal, Aib, Abu, Met, and 6-aminohexanoyl. 42. The method of claim 41, wherein when the peptidic substrate comprises a P5 amino acid residue, the peptidic substrate further comprises a P6 amino acid residue, optionally selected from Arg, Leu, Ile, and Val, wherein the peptidic substrate comprises a P2 'amino acid residue. When included, the peptidic substrate optionally further comprises a P3 ′ amino acid residue selected from Gly, Ser, Ala, Leu, Ile, nLeu, Val, nVal, Aib, Abu, Met, 6-aminohexanoyl, CHA and allylGly. Wherein the peptidic substrate optionally comprises Gly, Ser, Ala, Leu, Ile, nLeu, Val, nVal, Aib, Abu, Met, 6-aminohexanoyl, CHA and A conjugate further comprising a P4 'amino acid residue selected from allyl Gly. 48. The conjugate of any one of claims 43-47, which is a compound of Formula IV or a derivative thereof. Formula IV X ^n- (P6) _m- (P5) _p- (P4) _i- (P3) _j- (P2) _l -P1- (P1 ') _u- (P2') _k- (P3 ') _r- (P4 ') _s- (L) _n -Z In the above formula, Z is a cure, L is a linker, l, j, i, p and m are chosen as follows: l is 0 or 1, when l is 0 j, i, p and m are 0, when l is 1 j is 0 or 1 and when j is 0 i, p and m are 0 I is 0 or 1 when j is 1, p and m are 0 when i is 0, p is 0 or 1 when i is 1, and m is 0 when p is 0 When p is 1, m is 0 or 1, u, k, r and s are selected as follows: u is 0 or 1, when u is 0, k, r and s are 0, when u is 1, k is 0 or 1, when k is 0, r and s are 0 and k is R is 0 or 1 when 1, s is 0 when r is 0, s is 0 or 1 when r is 1, n is 0 or 1, X ⁿ is hydrogen or acyl, sulfonyl or carbamoyl cap, P1 is selected from Arg, Lys, Tyr, Phe, Trp, Ala, Val, Ile and Thr, P1 ′ is Gly, Ser, Ala, Leu, Ile, d-Ile, nLeu, Val, nVal, Aib, Abu, Met or 6-aminohexanoyl, P2 is selected from Phe, Ser, Gly and Ala, P3 is selected from Arg, Lys, Gln, Ser, Quat and Arg surrogate, P4 is selected from Pro, Arg, Ser, Ala, Lys, Gly, nLeu, Leu, Tyr, Glu, Phe and Val, P5 is selected from Arg and Arg surrogate, P6 is selected from Leu, Ile and Val, P2 'is selected from Gly, Ser, Ala, Leu, Ile, d-Ile, nLeu, Val, nVal, Aib, Abu, Met and 6-aminohexanoyl, P3 'is selected from Gly, Ser, Ala, Leu, Ile, nLeu, Val, nVal, Aib, Abu, Met and 6-aminohexanoyl, P4 ′ is selected from Gly, Ser, Ala, Leu, Ile, nLeu, Val, nVal, Aib, Abu, Met and 6-aminohexanoyl. The conjugate of claim 50 or 51, which is a compound of Formula V or a derivative thereof. Formula V Z- (L) _n- (P6) _m- (P5) _p- (P4) _i- (P3) _j- (P2) _l -P1- (P1 ') _u- (P2') _k- (P3 ') _r- (P4 ') _s -X ^c In the above formula, Z is a cure, L is a linker, l, j, i, p and m are chosen as follows: l is 0 or 1, when l is 0 j, i, p and m are 0, when l is 1 j is 0 or 1 and when j is 0 i, p and m are 0 I is 0 or 1 when j is 1, p and m are 0 when i is 0, p is 0 or 1 when i is 1, and m is 0 when p is 0 When p is 1, m is 0 or 1, u, k, r and s are selected as follows: u is 0 or 1, when u is 0, k, r and s are 0, when u is 1, k is 0 or 1, when k is 0, r and s are 0 and k is R is 0 or 1 when 1, s is 1 when r is 0, s is 0 or 1 when r is 1, n is 0 or 1, X ^c together with the carbonyl group of the bound amino acid residue form a carboxylic acid or carboxamide group, P1 is selected from Arg, Lys, Tyr, Phe, Trp, Ala, Val, Ile and Thr, P1 ′ is Gly, Ser, Ala, Leu, Ile, d-Ile, nLeu, Val, nVal, Aib, Abu, Met or 6-aminohexanoyl, P2 is selected from Phe, Ser, Gly and Ala, P3 is selected from Arg, Lys, Gln, Ser, Quat and Arg surrogate, P4 is selected from Pro, Arg, Ser, Ala, Lys, Gly, nLeu, Leu, Tyr, Glu, Phe and Val, P5 is selected from Arg and Arg surrogate, P6 is selected from Leu, Ile and Val, P2 'is selected from Gly, Ser, Ala, Leu, Ile, d-Ile, nLeu, Val, nVal, Aib, Abu, Met and 6-aminohexanoyl, P3 'is selected from Gly, Ser, Ala, Leu, Ile, nLeu, Val, nVal, Aib, Abu, Met and 6-aminohexanoyl, P4 ′ is selected from Gly, Ser, Ala, Leu, Ile, nLeu, Val, nVal, Aib, Abu, Met and 6-aminohexanoyl. 55. The conjugate of claim 54, wherein the conjugate is a compound of Formula VI or a derivative thereof. Formula VI Z ^1- (L ¹ ) _n- (P6) _m- (P5) _p- (P4) _i- (P3) _j- (P2) _l -P1- (P1 ') _u- (P2') _k- (P3 ') _r- (P4') _s- (L ² ) _v -Z ² In the above formula, Z ¹ and Z ² are each a therapeutic agent and are the same or different, L ¹ and L ² are each a linker and are the same or different, l, j, i, p and m are chosen as follows: l is 0 or 1, when l is 0 j, i, p and m are 0, when l is 1 j is 0 or 1 and when j is 0 i, p and m are 0 I is 0 or 1 when j is 1, p and m are 0 when i is 0, p is 0 or 1 when i is 1, and m is 0 when p is 0 When p is 1, m is 0 or 1, u, k, r and s are selected as follows: u is 0 or 1, when u is 0, k, r and s are 0, when u is 1, k is 0 or 1, when k is 0, r and s are 0 and k is R is 0 or 1 when 1, s is 0 when r is 0, s is 0 or 1 when r is 1, n and v are each independently 0 or 1, P1 is selected from Arg, Lys, Tyr, Phe, Trp, Ala, Val, Ile and Thr, P1 ′ is Gly, Ser, Ala, Leu, Ile, d-Ile, nLeu, Val, nVal, Aib, Abu, Met or 6-aminohexanoyl, P2 is selected from Phe, Ser, Gly and Ala, P3 is selected from Arg, Lys, Gln, Ser, Quat and Arg surrogate, P4 is selected from Pro, Arg, Ser, Ala, Lys, Gly, nLeu, Leu, Tyr, Glu, Phe and Val, P5 is selected from Arg and Arg surrogate, P6 is selected from Leu, Ile and Val, P2 'is selected from Gly, Ser, Ala, Leu, Ile, d-Ile, nLeu, Val, nVal, Aib, Abu, Met and 6-aminohexanoyl, P3 'is selected from Gly, Ser, Ala, Leu, Ile, nLeu, Val, nVal, Aib, Abu, Met and 6-aminohexanoyl, P4 ′ is selected from Gly, Ser, Ala, Leu, Ile, nLeu, Val, nVal, Aib, Abu, Met and 6-aminohexanoyl. The method according to any one of claims 1 to 49, Ac-R-Q-G-R-S-L- (therapeutic) (SEQ ID NO: 491); Ac-R-Q-G-R-S-S-L- (therapeutic) (SEQ ID NO: 492); Ac-R-Q-G-R-S-nL- (therapeutic) (SEQ ID NO: 493); Ac-R-Q-G-R-S-nV- (therapeutic) (SEQ ID NO: 494); Ac-R-Q-G-R-S-F- (therapeutic) (SEQ ID NO: 495); Ac-R-Q-G-R-A-L- (therapeutic) (SEQ ID NO: 496); Ac-R-Q-G-R-A-L- (therapeutic) (SEQ ID NO: 497); Ac-R-Q-G-R-A-nL- (therapeutic) (SEQ ID NO: 498); Ac-R-Q-G-R-A-nL- (therapeutic) (SEQ ID NO: 499); Ac-R-Q-G-R-A-nV- (therapeutic) (SEQ ID NO: 500); Ac-R-Q-G-R-A-Cha- (therapeutic) (SEQ ID NO: 501); Ac-R-Q-G-R-A-F- (therapeutic) (SEQ ID NO: 502); Ac-R-N-G-R-S-L- (therapeutic) (SEQ ID NO: 503); Ac-R-N-G-R-A-nL- (therapeutic) (SEQ ID NO: 504); Ac-R-Q-A-R-S-L- (therapeutic) (SEQ ID NO: 505); Ac-R-Q-A-R-S-nL- (therapeutic) (SEQ ID NO: 506); Ac-R-Q-A-R-S-nV- (therapeutic) (SEQ ID NO: 507); Ac-R-Q-A-A-S-Cha- (therapeutic) (SEQ ID NO: 508); Ac-R-Q-A-R-S-S-Cha- (therapeutic) (SEQ ID NO: 509); Ac-R-Q-A-R-T-nL- (therapeutic) (SEQ ID NO: 510); Ac-R-Q-A-R-A-L- (therapeutic) (SEQ ID NO: 511); Ac-R-Q-A-R-A-nL- (therapeutic) (SEQ ID NO: 512); Ac-R-Q-A-R-A-nV- (therapeutic) (SEQ ID NO: 513); Ac-R-Q-A-R-A-Cha- (therapeutic) (SEQ ID NO: 514); Ac-R-Q-S-R-A-A- (therapeutic) (SEQ ID NO: 515); Ac-R-Q-S-R-A- (therapeutic) (SEQ ID NO: 516); Ac-R-Q-S-R-A-nL- (therapeutic) (SEQ ID NO: 517); Ac-R-Q-S-R-A-L- (therapeutic) (SEQ ID NO: 518); Ac-R-Q-S-R-A-nV- (therapeutic) (SEQ ID NO: 519); Ac-R-Q-S-R-A-Cha- (therapeutic) (SEQ ID NO: 520); Ac-R-Q-S-R-S-S-L- (therapeutic) (SEQ ID NO: 521); Ac-R-Q-S-R-S-L- (therapeutic) (SEQ ID NO: 522); Ac-R-Q-S-R-S-nL- (therapeutic) (SEQ ID NO: 523); Ac-R-Q-S-R-S-nL- (therapeutic) (SEQ ID NO: 524); Ac-R-Q-S-R-S-nV- (therapeutic) (SEQ ID NO: 525); Ac-R-Q-S-R-S-allylG- (therapeutic) (SEQ ID NO: 526); Ac-R-Q-S-R-S-Cha- (therapeutic) (SEQ ID NO: 527); Ac-R-Q-S-R-T-nL- (therapeutic) (SEQ ID NO: 528); Ac-R-Q-T-R-S-S-L- (therapeutic) (SEQ ID NO: 529); Ac-R-Q-T-R-S-L- (therapeutic) (SEQ ID NO: 530); Ac-R-N-S-R-S-nL- (therapeutic) (SEQ ID NO: 531); Ac-R-Q-F-R-S-L- (therapeutic) (SEQ ID NO: 532); Ac-R-Q-F-R-S-nL- (therapeutic) (SEQ ID NO: 534); Ac-R-Q-F-R-S-nV- (therapeutic) (SEQ ID NO: 535); Ac-R-Q-F-R-S-nL- (therapeutic) (SEQ ID NO: 536); Ac-R-Q-F-R-S-Cha- (therapeutic) (SEQ ID NO: 537); Ac-R-Q-F-R-A-L- (therapeutic) (SEQ ID NO: 538); Ac-R-Q-F-R-A-nL- (therapeutic) (SEQ ID NO: 539); Ac-R-Q-F-R-A-nV- (therapeutic) (SEQ ID NO: 540); Ac-R-Q-F-R-A-Cha- (therapeutic) (SEQ ID NO: 541); Ac-Q-S-R-S-S-nL- (therapeutic) (SEQ ID NO: 542); MeOCO-Quat2-G-R-S-L- (therapeutic) (SEQ ID NO: 483); MeOCO-Quat3-G-R-S-L- (therapeutic) (SEQ ID NO: 484); MeOCO-Quat-G-R-S-L- (therapeutic) (SEQ ID NO: 485); MeOCO-Quat4-G-R-S-L- (therapeutic) (SEQ ID NO: 486); MeOCO-Quat5-G-R-S-L- (therapeutic) (SEQ ID NO: 487); MeOCO-Quat2-G-R-S-S-L- (therapeutic) (SEQ ID NO: 488); MeOCO-Quat4-G-R-S-L- (therapeutic) (SEQ ID NO: 489); MeOCO-Quat2-G-R-S-L- (therapeutic) (SEQ ID NO: 490); Ac-Q-G-R-S-L- (therapeutic) (SEQ ID NO: 445); Ac-Q-G-R-S-S-L- (therapeutic) (SEQ ID NO: 446); Ac-Q-G-R-A-S-L- (therapeutic) (SEQ ID NO: 447); Ac-N-G-R-S-S-L- (therapeutic) (SEQ ID NO: 448); Ac-Q-G-R-S-S-nL- (therapeutic) (SEQ ID NO: 449); Ac-Q-G-R-S-S-nV- (therapeutic) (SEQ ID NO: 450); Ac-Q-G-R-S-S-Cha- (therapeutic) (SEQ ID NO: 451); Ac-Q-G-R-S-S-allylG- (therapeutic) (SEQ ID NO: 452); Ac-Q-G-R-S-S-allylG- (therapeutic) (SEQ ID NO: 453); Ac-Q-A-R-S-L- (therapeutic) (SEQ ID NO: 454); Ac-Q-A-R-S-S-L- (therapeutic) (SEQ ID NO: 455); Ac-Q-S-R-S-L- (therapeutic) (SEQ ID NO: 456); Ac-Q-S-R-S-S-nV- (therapeutic) (SEQ ID NO: 457); Ac-Q-S-R-S-S-Cha- (therapeutic) (SEQ ID NO: 458); Ac-Q-S-R-S-S-L- (therapeutic) (SEQ ID NO: 459); Ac-Q-T-R-S-S-L- (therapeutic) (SEQ ID NO: 460); Ac-Q-Aib-R-S-S-Cha- (therapeutic) (SEQ ID NO: 461); Ac-Q-Aib -R-S-S-L- (therapeutic) (SEQ ID NO: 462); Ac-Q-Abu-R-S-S-Cha- (therapeutic) (SEQ ID NO: 463); Ac-Q-Abu-R-S-S-L- (therapeutic) (SEQ ID NO: 464); Ac-Q-Cha-R-S-S-Cha- (therapeutic) (SEQ ID NO: 465); Ac-Q-F-R-S-L- (therapeutic) (SEQ ID NO: 466); Ac-Q-F-R-S-S-L- (therapeutic) (SEQ ID NO: 467); Ac-Q-Y-R-S-S-L- (therapeutic) (SEQ ID NO: 468); Ac-R-G-R-S-L- (therapeutic) (SEQ ID NO: 469); Ac-R-G-R-S-S-L- (therapeutic) (SEQ ID NO: 470); Ac-R-G-R-S-S-Cha- (therapeutic) (SEQ ID NO: 471); Ac-R-G-R-S-Cha- (therapeutic) (SEQ ID NO: 472); Ac-R-A-R-S-L- (therapeutic) (SEQ ID NO: 473); Ac-R-A-R-S-S-L- (therapeutic) (SEQ ID NO: 474); Ac-R-S-R-S-L- (therapeutic) (SEQ ID NO: 475); Ac-R-S-R-S-S-L- (therapeutic) (SEQ ID NO: 476); Ac-R-S-R-S-Cha- (therapeutic) (SEQ ID NO: 477); Ac-R-S-R-S-S-Cha- (therapeutic) (SEQ ID NO: 478); Ac-R-F-R-S-L- (therapeutic) (SEQ ID NO: 479); Ac-R-F-R-S-Cha- (therapeutic) (SEQ ID NO: 480); Ac-Y-G-R-S-S-L- (therapeutic) (SEQ ID NO: 481); Ac-M (O 2) -S-R-S-L- (therapeutic) (SEQ ID NO: 482); Ac-R-R-Q-S-R-A-A- (therapeutic) (SEQ ID NO: 105); Ac-R-R-Q-S-R-I- (therapeutic) (SEQ ID NO: 610); Ac-R-R-Q-S-R-S-S-L- (therapeutic) (SEQ ID NO: 543); Ac-R-R-Q-S-R-S-L- (therapeutic) (SEQ ID NO: 544); Ac-R-G-S-G-R-S-L- (therapeutic) (SEQ ID NO: 545); Ac-R-G-S-G-R--S-nL- (therapeutic) (SEQ ID NO: 546); Ac-R-G-S-G-R-A-nL- (therapeutic) (SEQ ID NO: 547); Ac-R-G-S-G-R-S-S-L- (therapeutic) (SEQ ID NO: 548); Ac-I-V-S-G-R-A-S-L- (therapeutic) (SEQ ID NO: 549); Ac-R-R-Q-S-R-A- (therapeutic) (SEQ ID NO: 108); Ac-R-R-Q-S-R-I- (therapeutic) (SEQ ID NO: 111); Ac-L-R-R-Q-S-R-A-A- (therapeutic) (SEQ ID NO: 106); Ac-L-R-R-Q-S-R-G-G- (therapeutic) (SEQ ID NO: 109); Ac-L-R-R-Q-S-R-A- (therapeutic) (SEQ ID NO: 110); Ac-L-R-R-Q-S-R-A-I- (therapeutic) (SEQ ID NO: 112); Ac-L-R-R-Q-S-R-A-I- (therapeutic) (SEQ ID NO: 611); Ac-L-R-R-Q-S-R-S-S-L- (therapeutic) (SEQ ID NO: 550); Ac-L-R-R-Q-S-R-S-L- (therapeutic) (SEQ ID NO: 551); Ac-S-G-R-S-L- (therapeutic) (SEQ ID NO: 362); Ac-S-G-R-S-S-L- (therapeutic) (SEQ ID NO: 363); Ac-S-G-R-S-S-S-L- (therapeutic) (SEQ ID NO: 364); Ac-S-G-R-S-nL- (therapeutic) (SEQ ID NO: 365); Ac-S-G-R-S-nV- (therapeutic) (SEQ ID NO: 366); Isomer 1 Ac-S-G-R-S-nV- (therapeutic) (SEQ ID NO: 367); Isomer 2 Ac-S-G-R-S-G (hex)-(therapeutic) (SEQ ID NO: 368); Ac-S-G-R-S-Cha- (therapeutic) (SEQ ID NO: 369); Ac-S-G-R-S-hCha- (therapeutic) (SEQ ID NO: 370); Ac-S-A-R-S-L- (therapeutic) (SEQ ID NO: 371); Ac-S-A-R-S-S-L- (therapeutic) (SEQ ID NO: 372); Ac-S-S-R-S-nL- (therapeutic) (SEQ ID NO: 373); Ac-T-G-R-S-Abu- (therapeutic) (SEQ ID NO: 374); Ac-T-G-R-S-L- (therapeutic) (SEQ ID NO: 375); Ac-T-G-R-S-nV- (therapeutic) (SEQ ID NO: 376); Ac-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 377); Ac-T-G-R-S-G (hex)-(therapeutic) (SEQ ID NO: 378); Ac-T-G-R-S-Cha- (therapeutic) (SEQ ID NO: 379); Ac-T-G-R-S-hCha- (therapeutic) (SEQ ID NO: 380); Ac-T-G-R-T-Abu- (therapeutic) (SEQ ID NO: 381); Ac-T-G-R-hS-nL- (therapeutic) (SEQ ID NO: 382); Ac-T-G-R-Abu-nL- (therapeutic) (SEQ ID NO: 383); Ac-T-G-R-Abu-nV- (therapeutic) (SEQ ID NO: 384); Ac-T-G-F (Gn) -S-nL- (therapeutic) (SEQ ID NO: 385); Ac-T-G-F (Gn) -S-Cha- (therapeutic) (SEQ ID NO: 386); Ac-T-G-F (Gn) -Abu-nV- (therapeutic) (SEQ ID NO: 387); Ac-T-G-K (alloc) -S-nL- (therapeutic) (SEQ ID NO: 388); Ac-T-G-K-S-nL- (therapeutic) (SEQ ID NO: 389); Ac-T-G-hR-S-nL- (therapeutic) (SEQ ID NO: 390); Ac- (hS) G-G-R-S-nL- (therapeutic) (SEQ ID NO: 391); MeOCO-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 392); PhSO2-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 393); MeOEtCO-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 394); MeO (EtO) 2 Ac-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 395); 4-oxo-pentanoyl-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 396); 3,4-methyldioxyPhAc-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 397); 2-pyridylAc-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 398); PhOAc-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 399); L-3-Phlactyl-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 400); MeOAc-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 401); PhAc-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 402); MeOEtOCO-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 403); MeOEtOAc-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 404); HOOCButa-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 405); Z-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 406); EtOCO-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 407); βA-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 408); Pent-4-inoyl-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 409); NapAc-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 410); iBoc-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 411); HOAc-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 412); MeSucc-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 413); N, N-diMeGly-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 414); Succ-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 415); HCO-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 416); Ac-T-A-R-S-nL- (therapeutic) (SEQ ID NO: 417); Ac-T-A-F (Gn) -S-nL- (therapeutic) (SEQ ID NO: 418); Ac-T-A-R-Abu-nV- (therapeutic) (SEQ ID NO: 419); Ac-T-A-R-S-Abu- (therapeutic) (SEQ ID NO: 420); Ac-T-A-R-T-Abu- (therapeutic) (SEQ ID NO: 421); Ac-T-S (O-Me) -R-S-nL- (therapeutic) (SEQ ID NO: 422); Ac-T-hS-R-S-nL- (therapeutic) (SEQ ID NO: 423); Ac-T- (1-Me) H-R-S-nL- (therapeutic) (SEQ ID NO: 424); Ac-T- (3-Me) H-R-S-nL- (therapeutic) (SEQ ID NO: 425); Ac-T-H-R-S-nL- (therapeutic) (SEQ ID NO: 426); Ac-T-Sar-R-S-nL- (therapeutic) (SEQ ID NO: 427); Ac-T-nV-R-S-nL- (therapeutic) (SEQ ID NO: 428); Ac-T-nL-R-S-nL- (therapeutic) (SEQ ID NO: 429); Ac-T-A-R-S-Cha- (therapeutic) (SEQ ID NO: 430); Ac-T-Abu-R-S-nL- (therapeutic) (SEQ ID NO: 431); Ac-4,4-diMeThr-G-R-S-nL- (therapeutic) (SEQ ID NO: 432); Ac-hS-G-R-S-nL- (therapeutic) (SEQ ID NO: 433); Ac-hS-G-R-hS-Cha- (therapeutic) (SEQ ID NO: 434); Ac-hS-G-R-S-Cha- (therapeutic) (SEQ ID NO: 435); Ac-hS-G-R-T-Cha- (therapeutic) (SEQ ID NO: 436); Ac-hS-A-R-S-Cha- (therapeutic) (SEQ ID NO: 437); Ac-N-G-R-S-nL- (therapeutic) (SEQ ID NO: 438); Ac-Y-G-R-S-S-L- (therapeutic) (SEQ ID NO: 439); Ac-Y-G-R-S-Cha- (therapeutic) (SEQ ID NO: 440); Ac-Q-G-R-S-S-nL- (therapeutic) (SEQ ID NO: 441); Ac-Q-G-R-S-S-nV- (therapeutic) (SEQ ID NO: 442); Ac-L-R-G-S-G-R-S-A- (therapeutic) (SEQ ID NO: 573); Ac-L-R-G-S-G-R-S-L- (therapeutic) (SEQ ID NO: 342); Ac-L-R-G-S-G-R-S-L- (therapeutic) (SEQ ID NO: 343); Ac-L-R-G-S-G-R-S-S-nL- (therapeutic) (SEQ ID NO: 344); Ac-L-R-G-S-G-R-S-S-Cha- (therapeutic) (SEQ ID NO: 345); Ac-L-R-G-dS-A-R-S-A- (therapeutic) (SEQ ID NO: 574); Ac-L-R-G-S-A-R-S-S-L- (therapeutic) (SEQ ID NO: 346); Ac-L-R-G-S-A-R-S-L- (therapeutic) (SEQ ID NO: 347); Ac-L-R-G-S-A-R-S-S-Cha- (therapeutic) (SEQ ID NO: 348); Ac-L-R-G-S-A-R-S-S-nV- (therapeutic) (SEQ ID NO: 349); Ac-L-R-G-S-A-R-S-S-nL- (therapeutic) (SEQ ID NO: 350); Ac-V-I-V-S-G-R-A-L- (therapeutic) (SEQ ID NO: 351); Ac-V-I-V-S-A-R-S-L- (therapeutic) (SEQ ID NO: 352); Ac-V-I-V-S-G-R-S-S-L- (therapeutic) (SEQ ID NO: 353); Ac-V-I-V-S-A-R-M-A- (therapeutic) (SEQ ID NO: 354); Ac-V-I-V-S-A-R-nL-A- (therapeutic) (SEQ ID NO: 355); Ac-V-I-V-S-A-R-S-nL- (therapeutic) (SEQ ID NO: 356); Ac-V-I-V-S-A-R-S-Cha- (therapeutic) (SEQ ID NO: 357); Ac-V-I-V-S-A-R-S-Cha- (therapeutic) (SEQ ID NO: 358); Ac-V-I-V-S-A-R-S-S-Cha- (therapeutic) (SEQ ID NO: 359); Ac-R-R- (Me) C-P-G-R-V-V- (therapeutic) (SEQ ID NO: 360); Ac-R-R-nV-P-A-R-S-L- (therapeutic) (SEQ ID NO: 361); Ac-R-G-dS-A-R-S-A- (therapeutic) (SEQ ID NO: 309); Ac-R-G-S-G-R-S-A- (therapeutic) (SEQ ID NO: 310); Ac-R-G-S-G-R-A-L- (therapeutic) (SEQ ID NO: 311); Ac-R-G-S-G-R-S-L- (therapeutic) (SEQ ID NO: 312); Ac-R-G-S-G-R--S-nL- (therapeutic) (SEQ ID NO: 313); Ac-R-G-S-G-R-A-nL- (therapeutic) (SEQ ID NO: 314); Ac-R-G-S-G-R-S-S-L- (therapeutic) (SEQ ID NO: 315); Ac-R-G-S-G-R-S-Cha- (therapeutic) (SEQ ID NO: 316); Ac-R-G-S-G-R-S-S-Cha- (therapeutic) (SEQ ID NO: 317); Ac-R-G-S-A-R-S-Cha- (therapeutic) (SEQ ID NO: 318); Ac-R-G-S-A-R-S-S- (therapeutic) (SEQ ID NO: 319); Ac-R-G-S-A-R-S-nV- (therapeutic) (SEQ ID NO: 320); Ac-R-G-S-A-R-S-S-nV-(therapeutic agent) (SEQ ID NO: 321); Ac-R-G-S-A-R-S-L- (therapeutic) (SEQ ID NO: 322); Ac-R- (Me) C-P-G-R-V-V- (therapeutic) (SEQ ID NO: 323); Ac-R- (Me) C-P-G-R-V-V- (therapeutic) (SEQ ID NO: 324); Ac-R-C (Me) -P-G-R-S-L- (therapeutic) (SEQ ID NO: 325); Ac-R-L-P-G-R-S-L- (therapeutic) (SEQ ID NO: 326); Ac-R-V-P-G-R-S-L- (therapeutic) (SEQ ID NO: 327); Ac-R-V-P-G-R-S-L- (therapeutic) (SEQ ID NO: 328); Ac-R-nL-P-G-R-S-L- (therapeutic) (SEQ ID NO: 329); Ac-R-G (tBu) -P-A-R-S-L- (therapeutic) (SEQ ID NO: 330); Ac-R-L-P-A-R-S-L- (therapeutic) (SEQ ID NO: 331); Ac-R-V-P-A-R-S-L- (therapeutic) (SEQ ID NO: 332); Ac-R-nL-P-A-R-S-L- (therapeutic) (SEQ ID NO: 333); Ac-I-V-S-G-R-A-L- (therapeutic) (SEQ ID NO: 334); Ac-I-V-S-G-R-S-S-L- (therapeutic) (SEQ ID NO: 335); Ac-I-V-S-G-R-A-S-L- (therapeutic) (SEQ ID NO: 336); Ac-I-V-S-A-R-M-A- (therapeutic) (SEQ ID NO: 337); Ac-I-V-S-A-R-nL-A- (therapeutic) (SEQ ID NO: 338); Ac-I-V-S-A-R-S-L- (therapeutic) (SEQ ID NO: 339); Ac-I-V-S-A-R-S-nL- (therapeutic) (SEQ ID NO: 340); Ac-I-V-S-A-R-S-S-L- (therapeutic) (SEQ ID NO: 341); Ac-G-S-G-R-S-A- (therapeutic) (SEQ ID NO: 585); Ac-G-S-G-R-S-L- (therapeutic) (SEQ ID NO: 277); Ac-G-S-G-R-A-L- (therapeutic) (SEQ ID NO: 278); Ac-G-S-G-R-S-S-L- (therapeutic) (SEQ ID NO: 279); Ac-G-S-G-R-L- (therapeutic) (SEQ ID NO: 280); Ac-G-S-G- (4-guan) Phg-S-L- (therapeutic) (SEQ ID NO: 281); Ac-G-S-G-R-S-S-Cha- (therapeutic) (SEQ ID NO: 282); Ac-G-S-G-R-A-S-L- (therapeutic) (SEQ ID NO: 283); Ac-G-S-G-R-S-nL- (therapeutic) (SEQ ID NO: 284); Ac-G-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 285); Succ-bA-T-G-R-S-nL- (therapeutic) (SEQ ID NO: 286); Ac-G-T-G-R-S-hCha- (therapeutic) (SEQ ID NO: 287); Ac-G-hS-G-R-S-nL- (therapeutic) (SEQ ID NO: 288); Ac-G-dS-A-R-S-A- (therapeutic) (SEQ ID NO: 289); Ac-G-S-A-R-S-L- (therapeutic) (SEQ ID NO: 290); Ac-G-S-A-R-S-S-Cha- (therapeutic) (SEQ ID NO: 291); Ac-G-S-A-R-S-S-L- (therapeutic) (SEQ ID NO: 292); Ac-G-S-A-R-A-S-L- (therapeutic) (SEQ ID NO: 293); Ac-V-S-G-R-S-L- (therapeutic) (SEQ ID NO: 294); Ac-V-S-G-R-A-L- (therapeutic) (SEQ ID NO: 295); Ac-V-S-G-R-A-S-L- (therapeutic) (SEQ ID NO: 296); Ac-V-S-G-R-S-S-L- (therapeutic) (SEQ ID NO: 297); Ac-V-S-A-R-M-A- (therapeutic) (SEQ ID NO: 298); Ac-V-S-A-R-nL-A- (therapeutic) (SEQ ID NO: 299); Ac-V-S-A-R-S-nL- (therapeutic) (SEQ ID NO: 300); Ac-V-S-A-R-S-L- (therapeutic) (SEQ ID NO: 301); Ac- (Me) C-P-G-R-V-V- (therapeutic) (SEQ ID NO: 302); Ac- (Me) C-P-G-R-V-V- (therapeutic) (SEQ ID NO: 303); Ac-C (Me) -P-G-R-A-L- (therapeutic) (SEQ ID NO: 304); Ac-C (Me) -P-G-R-S-L- (therapeutic) (SEQ ID NO: 305); Ac-C (Me) -P-A-R-S-L- (therapeutic) (SEQ ID NO: 306); Ac-C (Me) -P-A-R-A-S-L- (therapeutic) (SEQ ID NO: 307); Ac-G (tBu) -P-G-R-S-L- (therapeutic) (SEQ ID NO: 308); Ac-Q-S-R-A-A- (therapeutic) (SEQ ID NO: 552); Ac-Q-S-R-S-A- (therapeutic) (SEQ ID NO: 553); Ac-Q-S-R-S-G- (therapeutic) (SEQ ID NO: 554); Ac-R-S-R-A-A- (therapeutic) (SEQ ID NO: 555); Ac-R-Q-S-R-A-A- (therapeutic) (SEQ ID NO: 556); Ac-R-Q-S-R-S-A- (therapeutic) (SEQ ID NO: 557); Ac-R-Q-S-R-S-A-A- (therapeutic) (SEQ ID NO: 558); Ac-R-G-S-G-R-S-A- (therapeutic) (SEQ ID NO: 559); Ac-S-G-R-A-A- (therapeutic) (SEQ ID NO: 560); Ac-S-G-R-S-A- (therapeutic) (SEQ ID NO: 561); Ac-S-G-R-S-S-A- (therapeutic) (SEQ ID NO: 562); Ac-S-G-R-A-S-A- (therapeutic) (SEQ ID NO: 563); Ac-S-G-R-S-G- (therapeutic) (SEQ ID NO: 564); Ac-S-G-R-S-S-G- (therapeutic) (SEQ ID NO: 565); Ac-S-G-R-S-G-A- (therapeutic) (SEQ ID NO: 566); Ac-S-G-R-S-G-G- (therapeutic) (SEQ ID NO: 567); Ac-G-T-G-R-S-G-G- (therapeutic) (SEQ ID NO: 568); Ac-G-S-G-R-S-G-G- (therapeutic) (SEQ ID NO: 243) Ac-L-R-R-Q-S-R-A-A- (therapeutic) (SEQ ID NO: 597); MeSO2-dA (Chx) -Abu-R-S-L- (therapeutic) (SEQ ID NO: 598); Ac-R-A-R-S-L- (therapeutic) (SEQ ID NO: 599); Ac-dA (Chx) -Abu-R-S-L- (therapeutic) (SEQ ID NO: 600); Ac-dA (Chx) -Abu-R-S-S-L- (therapeutic) (SEQ ID NO: 601); Ac-Q-G-R-S-S-L- (therapeutic) (SEQ ID NO: 602); MeOCO-dhF-P (OH) -R-S-S-L- (therapeutic) (SEQ ID NO: 603); MeOCO-Quat4-G-R-S-L- (therapeutic) (SEQ ID NO: 604); Ac-dCha-P (OH) -R-S-S-L- (therapeutic) (SEQ ID NO: 605); Ac-dCha-Abu-R-S-S-A- (therapeutic) (SEQ ID NO: 606); MeOCO-Quat2-G-R-S-L- (therapeutic) (SEQ ID NO: 607); MeOCO-Quat3-G-R-S-L- (therapeutic) (SEQ ID NO: 608) and The conjugate selected from MeOCO-Quat-G-R-S-L- (therapeutic) (SEQ ID NO: 609). 57. The compound of any of claims 35-56, wherein P4 is Pro, Arg, Ser, Ala, Lys, Gly, nLeu, Leu, Tyr, Glu, Phe, Val, N, N-dimethylGly, β-Ala , Cys (Me), Gln, t-butylGly and nVal. 67. The peptidic substrate of claim 1 or 66, wherein the peptidic substrate of formula P6-P5-P4-P3-P2-P1-P1'-P2'-P3'-P4 ', wherein P1, P2, P3, P4, P5 , P6, P1 'and P2' are selected from the residues shown in Figures 1 and 2, respectively, and P6, P5, P4, P2 ', P3' and P4 'are optional. The method of claim 67, P6 is optionally selected from L, V or R, P5 is optionally selected from R, I or L, P4 is optionally selected from G, C or V, P3 is selected from S, dS, P, A or G, P2 is selected from A or G, P1 is R, P1 'is S, V, M or nL, P2 'is optionally selected from S, L, A or V, P3 'is optionally L, Conjugates where P4 'is optionally L. A plasmin or prostate specific comprising a therapeutic agent and a nucleic acid substrate linked to the therapeutic agent via a peptidic linker that is proteolytically cleaved by cell surface protease or a soluble, released or secreted form thereof to release the therapeutic agent. A conjugate that is not substantially cleaved by an antigen (PSA). 109. The conjugate of claim 108, wherein the nucleic acid is DNA. 109. The conjugate of claim 108, wherein the nucleic acid is RNA. 109. The conjugate of claim 108, wherein the nucleic acid is double stranded RNA. The method of claim 67, P6 is optionally selected from L, V or R, P5 is selected from R, I or L, P4 is optionally selected from G, C or V, P3 is selected from S, dS, P, A or G, P2 is selected from A or G, P1 is R, P1 'is T, Abu, hS, nV or A, P2 'is optionally selected from S, L, A or V, P 3 ′ is optionally L, nL, nV, G (hexyl), G (allyl), CHA, hCHA or Abu, A conjugate wherein P4 'is optionally L, nL, nV, G (hexyl), G (allyl), CHA, hCHA or Abu. The method of claim 67, P6 is optionally selected from L, V and R, P5 is optionally selected from R, I and L, P4 is optionally selected from G, C and V, P3 is selected from S, dS, P, A or G, P2 is selected from A or G, P1 is R, P1 'is S, G or A, P2 'is optionally selected from G or A, P 3 ′ is optionally L, nL, nV, G (hexyl), G (allyl), CHA, hCHA or Abu, A conjugate wherein P4 'is optionally L, nL, nV, G (hexyl), G (allyl), CHA, hCHA or Abu. 115. The conjugate of any one of claims 1-113, wherein the therapeutic agent is taxol. 115. The conjugate of any one of claims 1-113, wherein the therapeutic agent is doxorubicin. 117. A method of treating cell surface protease related diseases, comprising administering the conjugate of any one of claims 1-113 to a patient suffering from cell surface protease related diseases. 116. The method of claim 116, wherein the disease is selected from the group consisting of autoimmune diseases, inflammatory diseases, infectious diseases and endocrine diseases. 116. The method of claim 116, wherein the disease is a proliferative disease. A cell surface protease comprises a conjugate comprising a therapeutic agent and a peptide substrate that is linked to the therapeutic agent, optionally via a linker, and that is proteolytically cleaved by cell surface protease or a soluble, released or secreted form thereof to release the therapeutic agent. A method of treating cell surface related diseases, including administering to a patient exhibiting symptoms of a related disorder. 119. The method of claim 119, wherein the disease is selected from the group consisting of autoimmune diseases, inflammatory diseases, infectious diseases and endocrine diseases. 119. The method of claim 119, wherein the disease is a proliferative disease. 119. The method of any one of claims 114-119, wherein the patient is a mammal. 121. The method of claim 120, wherein the mammal is a human. 121. The method of claim 118 or 121, wherein the disease is cancer. 121. The method of claim 118 or 121, wherein the disease is selected from eye disorders, cardiovascular disorders, chronic inflammatory diseases, wounds, circulatory disorders, skin diseases and cancer. 121. The disease according to claim 118 or 121, wherein the disease is rheumatoid arthritis, psoriasis, diabetic retinopathy, pterygium recurrence, scars due to excimer laser surgery, scars due to glaucoma removal surgery, macular degeneration of the anterior eye, crest syndrome, solid kidney Method selected from biological and vascular tumors. 121. The method of claim 118 or 121, wherein the disease is selected from lung cancer, colon cancer, pancreatic cancer, esophageal cancer, breast cancer, ovarian cancer, prostate cancer, melanoma and Kaposi's sarcoma. 129. The method of any one of claims 116-127, wherein the therapeutic agent is Taxol. 127. The method of any one of claims 116-127, wherein the therapeutic agent is doxorubicin. 117. A pharmaceutical composition comprising the conjugate according to any one of claims 1 to 113 or a pharmaceutically acceptable derivative thereof in a pharmaceutically acceptable carrier. 131. The pharmaceutical composition of claim 130, formulated for single dose administration. 113. A conjugate or pharmaceutically acceptable according to any one of claims 1 to 113 contained in a packaging material for use in the treatment, prevention or alleviation of one or more symptoms associated with the packaging material, cell surface protease related disease or disorder. A product comprising a label indicating that the derivative thereof and the conjugate or pharmaceutically acceptable derivative thereof is used for the treatment, prevention or alleviation of one or more symptoms associated with a cell surface protease related disease or disorder. 119. The conjugate of any one of claims 1-113, which is used for the treatment of cell surface protease related diseases. 133. The conjugate of claim 133, wherein the disease is a proliferative disease. 136. The conjugate of claim 134, wherein the proliferative disease is cancer. 136. The conjugate of claim 134, wherein the proliferative disease is selected from eye disease, cardiovascular disease, chronic inflammatory disease, wound, circulatory disease, skin disease, and cancer. 135. The method of claim 134, wherein the proliferative disease is rheumatoid arthritis, psoriasis, diabetic retinopathy, pterygium recurrence, scars due to excimer laser surgery, scars due to glaucoma removal surgery, macular degeneration of the anterior eye, crest syndrome, solid neoplasms and Conjugates selected from vascular tumors. 136. The conjugate of claim 134, wherein the proliferative disease is selected from lung cancer, colon cancer, pancreatic cancer, esophageal cancer, breast cancer, ovarian cancer, prostate cancer, melanoma and Kaposi's sarcoma. Use of the conjugate according to any one of claims 1 to 113 for the manufacture of a medicament for the treatment of cell surface protease related diseases. 139. The use of claim 139, wherein the disease is a proliferative disease. 141. The use of claim 140, wherein the proliferative disease is cancer. 141. The use according to claim 140, wherein the proliferative disease is selected from eye disease, cardiovascular disease, chronic inflammatory disease, wound, circulatory disease, skin disease and cancer. 140. The method of claim 140, wherein the proliferative disease is rheumatoid arthritis, psoriasis, diabetic retinopathy, pterygium recurrence, scars due to excimer laser surgery, scars due to glaucoma removal surgery, macular degeneration of the anterior eye, crest syndrome, solid neoplasms and Use selected from vascular tumors. 141. The use according to claim 140, wherein the proliferative disease is selected from lung cancer, colon cancer, pancreatic cancer, esophageal cancer, breast cancer, ovarian cancer, prostate cancer, melanoma and Kaposi's sarcoma. (a) synthesizing a peptidic substrate, (b) optionally capping the peptide substrate at the N-terminus or C-terminus, (c) optionally linking the uncapped end of the peptidic substrate to the linker, (d) coupling the peptidic substrate to the therapeutic agent, optionally via a linker, to form a conjugate, and (e) Optionally a method of making a conjugate according to any one of claims 1 to 113, comprising deprotecting the conjugate if it is protected. 145. The method of claim 145, comprising identifying the peptidic substrate for the protease before step (a). (a) selecting a disease, (b) identifying cells involved in the disease process or cells in the vicinity of the cells involved in the disease process, and (c) identifying the protease to target the conjugate for disease treatment, comprising identifying a cell surface protease on the cell. 147. The method of claim 147, further comprising preparing a conjugate that targets the protease.