EP1069906A1 - Conjugues convenant au traitement du cancer de la prostate - Google Patents

Conjugues convenant au traitement du cancer de la prostate

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Publication number
EP1069906A1
EP1069906A1 EP99911146A EP99911146A EP1069906A1 EP 1069906 A1 EP1069906 A1 EP 1069906A1 EP 99911146 A EP99911146 A EP 99911146A EP 99911146 A EP99911146 A EP 99911146A EP 1069906 A1 EP1069906 A1 EP 1069906A1
Authority
EP
European Patent Office
Prior art keywords
ser
variant
xaa
amino acid
prt
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99911146A
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German (de)
English (en)
Inventor
Dong-Mei Feng
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Merck and Co Inc
Original Assignee
Merck and Co Inc
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Filing date
Publication date
Priority claimed from GBGB9815855.3A external-priority patent/GB9815855D0/en
Application filed by Merck and Co Inc filed Critical Merck and Co Inc
Publication of EP1069906A1 publication Critical patent/EP1069906A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/08—Drugs for disorders of the urinary system of the prostate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/1013—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing O or S as heteroatoms, e.g. Cys, Ser
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1016—Tetrapeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • prostate cancer In 1996 cancer of the prostate gland was expected to be diagnosed in 317,000 men in the U.S. and 42,000 American males die from this disease (Garnick, M.B. (1994). The Dilemmas of Prostate Cancer. Scientific American, April:72-81). Thus, prostate cancer is the most frequently diagnosed malignancy (other than that of the skin) in U.S. men and the second leading cause of cancer-related deaths (behind lung cancer) in that group.
  • Prostate specific Antigen is a single chain 33 kDa glycoprotein that is produced almost exclusively by the human prostate epithelium and occurs at levels of 0.5 to 2.0 mg/ml in human seminal fluid (Nadji, M, Taber, S.Z., Castro, A., et al. (1981) Cancer 48: 1229; Papsidero, L., Kuriyama, M., Wang, M., et al. (1981). JNCI 66:37; Qui, S.D., Young, C.Y.F., Bihartz, D.L., et al. (1990), J. Urol.
  • PSA protease with chymotrypsin-like specificity (Christensson, A., Laurell, C.B., Lilja, H. (1990). Eur. J. Biochem. 194:755-763).
  • PSA is mainly responsible for dissolution of the gel structure formed at ejaculation by proteolysis of the major proteins in the sperm entrapping gel, Semenogelin I and Semenogelin II, and fibronectin (Lilja, H. (1985). J. Clin. Invest. 76: 1899; Lilja, H., Oldbring, J., Rannevik, G., et al. (1987). J. Clin. Invest. 80:281; McGee, R.S., Herr, J.C. (1988). Biol. Reprod. 39:499).
  • PSA proteolytically degrade IGFBP-3 (insulin-like growth factor binding protein 3) allowing IGF to stimulate specifically the growth of PSA secreting cells (Cohen et al., (1992) J. Clin. Endo.
  • PSA complexed to alpha 1 - antichymotrypsin is the predominant molecular form of serum PSA and may account for up to 95% of the detected serum PSA (Christensson, A., Bjork, T., Nilsson, O., et al. (1993). J. Urol. 150: 100-105; Lilja, H., Christensson, A., Dahlen, U. (1991). Clin. Chem. 37: 1618-1625; Stenman, U.H., Leinoven, J., Alfthan, H., et al. (1991). Cancer Res. 51:222-226).
  • the prostatic tissue normal, benign hyperplastic, or malignant tissue
  • the prostatic tissue is implicated to predominantly release the mature, enzymatically active form of PSA, as this form is required for complex formation with alpha 1 - antichymotrypsin (Mast, A.E., Enghild, J.J., Pizzo, S.V., et al. (1991). Biochemistry 30: 1723-1730; Perlmutter, D.H., Glover, G.I., Rivetna, M., et al. (1990). Proc. Natl. Acad. Sci. USA 87:3753-3757).
  • PSA in the microenvironment of prostatic PSA secreting cells the PSA is believed to be processed and secreted in its mature enzymatically active form not complexed to any inhibitory molecule.
  • PSA also forms stable complexes with alpha 2 - macroglobulin, but as this results in encapsulation of PSA and complete loss of the PSA epitopes, the in vivo significance of this complex formation is unclear.
  • a free, noncomplexed form of PSA constitutes a minor fraction of the serum PSA (Christensson, A., Bjork, T., Nilsson, O., et al. (1993). J. Urol. 150: 100-105; Lilja, H., Christensson, A., Dahlen, U. (1991).
  • Serum measurements of PSA are useful for monitoring the treatment of adenocarcinoma of the prostate (Duffy, M.S. (1989). Ann. Clin. Biochem. 26:379-387; Brawer, M.K. and Lange, P.H. (1989). Urol. Suppl. 5: 11-16; Hara, M. and Kimura, H. (1989). J. Lab. Clin. Med. 113:541-548), although above normal serum concentrations of PSA have also been reported in benign prostatic hyperplasia and subsequent to surgical trauma of the prostate (Lilja, H., Christensson, A., Dahlen, U. (1991). Clin. Chem. 37: 1618-1625).
  • Prostate metastases are also known to secrete immunologically reactive PSA since serum PSA is detectable at high levels in prostatectomized patients showing widespread metatstatic prostate cancer (Ford, T.F., Butcher, D.N., Masters, R.W., et al. (1985). Brit. J. Urology 57:50- 55). Therefore, a cytotoxic compound that could be activated by the proteolytic activity of PSA should be prostate cell specific as well as specific for PSA secreting prostate metastases.
  • PSA prostate specific antigen
  • Another object of this invention is to provide a method of treating prostate cancer which comprises administration of the novel anti-cancer composition.
  • a further object of the invention is to provide novel cytotoxic derivatives of vinca alkaloid cytotoxic agents.
  • Chemical conjugates which comprise oligopeptides, having amino acid sequences that are selectively proteolytically cleaved by free prostate specific antigen (PSA), and a cytotoxic agent are disclosed.
  • the conjugates of the invention are characterized by a hydroxyalkyl- amine linker between the oligopeptide and a vinca alkaloid drug.
  • Such conjugates are useful in the treatment of prostatic cancer and benign prostatic hyperplasia (BPH).
  • novel cytotoxic derivatives of vinca alkaloid drugs wherein the C-23 ester of the vinca alkaloid is replaced with an unsubstituted or suitably substituted hydroxyalkylamide.
  • the instant invention relates to novel anti-cancer compositions useful for the treatment of prostate cancer.
  • Such compositions comprise the oligopeptides covalently bonded through a chemical linker to cytotoxic agent, preferably a vinca drug.
  • the oligopeptides are chosen from oligomers that are selectively recognized by the free prostate specific antigen (PSA) and are capable of being proteolytically cleaved by the enzymatic activity of the free prostate specific antigen.
  • PSA prostate specific antigen
  • Such a combination of an oligopeptide and cytotoxic agent may be termed a conjugate.
  • the conjugates of the instant invention are characterized by a linker between the C-terminus of the oligopeptide and the vinca drug.
  • the linker is a hydroxyalkylamine moiety, which is optionally substituted, and most preferably, the linker comprises a sterically hindered hydroxyalkylamine moiety.
  • the attachment of the oligopeptide to the linker is through an ester bond with the hydroxyl moiety of the linker.
  • the cytotoxic activity of the vinca drug is greatly reduced or absent when the oligopeptide containing the PSA proteolytic cleavage site is bonded through the chemical linker to the cytotoxic agent and is intact. Also ideally, the cytotoxic activity of the cytotoxic agent increases significantly or returns to the activity of the unmodified cytotoxic agent upon proteolytic cleavage of the attached oligopeptide at the cleavage site.
  • the vinca drug with the chemical linker intact exhibits cytotoxic activity that is at least 75% of the cytotoxicity of the unmodified vinca drug against the target cancer cells.
  • cytotoxic activity that is at least 75% of the cytotoxicity of the unmodified vinca drug against the target cancer cells.
  • Such a derivative of the vinca drug wherein the chemical linker is still covalently bound to the vinca drug may itself be considered a cytotoxic agent.
  • the oligopeptide is selected from oligopeptides that are not cleaved or are cleaved at a much slower rate in the presence of non-PSA proteolytic enzymes, such as those enzymes endogenous to human serum, when compared to the cleavage of the oligopeptides in the presence of free enzymatically active PSA.
  • the oligopeptide comprises a short peptide sequence, preferably less than ten amino acids. Most preferably the oligopeptide comprises seven or six amino acids.
  • the solubility of the conjugate may be influenced to a greater extent by the generally hydrophobic character of the cytotoxic agent component. Therefore, amino acids with hydrophilic substituents may be incorporated in the oligopeptide sequence or N-terminus blocking groups may be selected to offset or diminish such a hydrophobic contribution by the cytotoxic agent. Combinations of amino acids with hydrophilic substituents and N-terminus blocking groups that enhance solubility may also be employed in a single conjugate.
  • an embodiment of this invention is a conjugate wherein the oligopeptide and the chemical linker are detached from the cytotoxic agent by the proteolytic activity of the free PSA and any other native proteolytic enzymes present in the tissue proximity, thereby presenting the cytotoxic agent, or a cytotoxic agent that retains part of the oligopeptide/linker unit but remains cytotoxic, into the physiological environment at the place of proteolytic cleavage.
  • Pharmaceutically acceptable salts of the conjugates are also included.
  • the oligopeptide, that is conjugated to the cytotoxic agent through a chemical linker does not need to be the oligopeptide that has the greatest recognition by free PSA and is most readily proteolytically cleaved by free PSA.
  • the oligopeptide that is selected for incorporation in such an anti-cancer composition will be chosen both for its selective, proteolytic cleavage by free PSA and for the cytotoxic activity of the cytotoxic agent-proteolytic residue conjugate (or, in what is felt to be an ideal situation, the unmodified cytotoxic agent) which results from such a cleavage.
  • proteolytic PSA cleavage means a greater rate of cleavage of an oligopeptide component of the instant invention by free PSA relative to cleavage of an oligopeptide which comprises a random sequence of amino acids. Therefore, the oligopeptide component of the instant invention is a prefered substrate of free PSA.
  • selective also indicates that the oligopeptide is proteolytically cleaved by free PSA between two specific amino acids in the oligopeptide.
  • oligopeptide components of the instant invention are selectively recognized by the free prostate specific antigen (PSA) and are capable of being proteolytically cleaved by the enzymatic activity of the free prostate specific antigen.
  • PSA prostate specific antigen
  • Such oligopeptides comprise an oligomer selected from:
  • ChgGlnlSerLeu (SEQ.ID.NO.: 12);
  • Haa is a cyclic amino acid substituted with a hydrophilic moiety
  • hArg is homoarginine
  • Xaa is any amino acid
  • Cha is cyclohexylalanine
  • Abu is 2-aminobutyric acid
  • Chg is cyclohexylglycine.
  • the oligopeptide comprises an oligomer that is selected from:
  • the oligopeptide comprises an oligomer selected from:
  • SerSerChgGlnlSerPro (SEQ.ID.NO.: 45);
  • SerSerChgGlnlSerSer (SEQ.ID.NO.: 46);
  • SerSerSerChgGlnlSerLeu SEQ.ID.NO.: 47;
  • SerSerSerChgGlnlSerVal SEQ.ID.NO.: 48
  • SerSerSerChgGlnlSerPro SEQ.ID.NO.: 49
  • SerSerSerChgGlnlSerPro SEQ.ID.NO.: 49
  • SerSerSerChgGlnlSerSer (SEQ.ID.NO.: 50);
  • SerAlaSerChgGlnlSerLeu (SEQ.ID.NO.: 51);
  • oligomers that comprise an amino acid sequence describes oligomers of from about 3 to about 100 amino acids residues which include in their amino acid sequence the specific amino acid sequence decribed and which are therefore proteolytically cleaved within the amino acid sequence described by free PSA.
  • the oligomer is from 5 to 10 amino acid residues.
  • the following oligomer hArgSerAlaChgGlnlSerLeu (SEQ.ID.NO.: 67); comprises the amino acid sequence: ChgGlnlSerLeu (SEQ.ID.NO.: 12); and would therefore come within the instant invention.
  • oligomer hArgSer4- HypChgGlnlSerLeu (SEQ.ID.NO.: 68); comprises the amino acid sequence: 4-HypChgGlnlSerLeu (SEQ.ID.NO.: 69); and would therefore come within the instant invention. It is understood that such oligomers do not include semenogelin I and semenogelin II.
  • tyrosine may be replaced by 3-iodotyrosine, 2-methyltyrosine, 3-fluorotyrosine, 3-methyltyrosine and the like.
  • lysine may be replaced with N'- (2-imidazolyl)lysine and the like.
  • amino acid replacements is meant to be illustrative and is not limiting: Original Amino Acid Replacement Amino Acid(s)
  • oligopeptides may be synthesized by techniques well known to persons of ordinary skill in the art and would be expected to be proteolytically cleaved by free PSA:
  • the compounds of the present invention may have asymmetric centers and occur as racemates, racemic mixtures, and as individual diastereomers, with all possible isomers, including optical isomers, being included in the present invention.
  • named amino acids are understood to have the natural "L" stereoconfiguration
  • amino acids which are disclosed are identified both by conventional 3 letter and single letter abbreviations as indicated below:
  • hR or hArg homoarginine
  • hY or hTyr homotyrosine
  • DPL 2-(4,6-dimethylpyrimidinyl)lysine
  • Me2P03-Y O-dimethylphosphotyrosine
  • O-Me-Y O-methyltyrosine
  • TFA trifluoroacetic acid
  • AA acetic acid
  • Trt trityl
  • peptidyl therapeutic agents such as the instant oligopeptide-cytotoxic agent conjugates preferably have the terminal amino moiety of any oligopeptide substituent protected with a suitable protecting group, such as acetyl, benzoyl, pivaloyl and the like.
  • a suitable protecting group such as acetyl, benzoyl, pivaloyl and the like.
  • Such protection of the terminal amino group reduces or eliminates the enzymatic degradation of such peptidyl therapeutic agents by the action of exogenous amino peptidases which are present in the blood plasma of warm blooded animals.
  • protecting groups also include hydrophilic blocking groups, which are chosen based upon the presence of hydrophilic functionality.
  • Blocking groups that increase the hydrophilicity of the conjugates and therefore increase the aqueous solubility of the conjugates include but are not limited to hydroylated alkanoyl, polyhydroxylated alkanoyl, polyethylene glycol, glycosylates, sugars and crown ethers. N-Terminus unnatural amino acid moieties may also ameleorate such enzymatic degradation by exogenous amino peptidases.
  • N-terminus protecting group is selected from a) acetyl
  • Rl and R ⁇ are independently selected from: a) hydrogen, b) unsubstituted or substituted aryl, unsubstituted or substituted heterocycle, C3-C10 cycloalkyl, C2-C6 alkenyl,
  • Rl and R2 are combined to form - (CH2)s - wherein one of the carbon atoms is optionally replaced by a moiety selected from: O, S(0)m, -NC(O)-, NH and -N(COR7)- ;
  • R6 is selected from: hydrogen, aryl, substituted aryl, heterocycle, substituted heterocycle, C1-C6 alkyl and C3-C10 cycloalkyl
  • R7 is selected from: aryl, substituted aryl, heterocycle, substituted heterocycle, Cl-C6 alkyl and C3-C10 cycloalkyl
  • the cytotoxic agent that is utilized in the conjugates of the instant invention may be selected from alkylating agents, antiprolifer- ative agents, tubulin binding agents and the like.
  • Preferred classes of cytotoxic agents which may be linked to cleavable oligomers via the hydroxyalkylamine linker include, for example, the methotrexates, the vinca drugs (also known as vinca alkaloid cytotoxic agents), the mitomycins and the bleomycins.
  • Particularly useful members of those classes include, for example, aminopterin, methotrexate, methopterin, dichloro-methotrexate, mitomycin C, porfiromycin, melphalan, vinblastine, vincristine, leurosidine, vindesine, leurosine and the like.
  • Other useful cytotoxic agents include cisplatin and cyclophosphamide.
  • One skilled in the art may make chemical modifications to the desired cytotoxic agent in order to make reactions of that compound more convenient for purposes of preparing conjugates of the invention.
  • the preferred cytotoxic agents include, in general, the vinca alkaloid cytotoxic agents.
  • Particularly useful members of this class include, for example, vinblastine, desacetylvinblastine, vincristine, leurosidine, vindesine, vinorelbine, navelbine, leurosine and the like.
  • One skilled in the art may make chemical modifications to the desired cytotoxic agent in order to make reactions of that compound more convenient for purposes of preparing conjugates of the invention.
  • cytotoxic agents for the present invention include drugs of the following formulae: THE VINCA ALKALOID GROUP OF DRUGS OF FORMULA (1):
  • Rl5 is H, CH3 or CHO; when Rl7 and Rl8 are taken singly, Rl8 is H, and one of Rl6 and
  • Rl7 is ethyl and the other is H or OH; when Rl7 and Rl8 are taken together with the carbons to which they are attached, they form an oxirane ring in which case Rl6 is ethyl; R9 is hydrogen, (C1-C3 alkyl)-CO, or chlorosubstituted (C1-C3 alkyl)-CO.
  • oligopeptide-cytotoxic agent conjugate of the instant invention wherein the cytotoxic agent is the preferred cytotoxic agent vinblastine may be described by the general formula I below:
  • oligopeptide is an oligopeptide which is specifically recognized by the free prostate specific antigen (PSA) and is capable of being proteolytically cleaved by the enzymatic activity of the free prostate specific antigen,
  • PSA prostate specific antigen
  • X L is selected from - NH - (CR 3 2 ) U (CR 4 2 ) V - O - and
  • d R2 are independently selected from: a) hydrogen, b) unsubstituted or substituted aryl, unsubstituted or substituted heterocycle, C3-C10 cycloalkyl, C2-C6 alkenyl,
  • R* a is Cl-C6-alkyl, hydroxylated C3-C8-cycloalkyl, polyhydroxylated C3-C8-cycloalkyl, hydroxylated aryl, polyhydroxylated aryl or aryl,
  • R3 and R4 are independently selected from: hydrogen, Cl-C6-alkyl, hydroxylated C3-C8-cycloalkyl, polyhydroxylated C3-C8- cycloalkyl, hydroxylated aryl, polyhydroxylated aryl and aryl, or one R3 and one R4 are combined to form a -(CH2)w- > which is unsubstituted or substituted with one or two substituents selected from OH and C1-C6 alkyl; or an R3 is combined with another R3 on the same carbon to form a
  • R5 is selected from OH and C1-C6 alkyl
  • R6 is selected from: hydrogen, aryl, substituted aryl, heterocycle, substituted heterocycle, C1-C6 alkyl and C3-C10 cycloalkyl;
  • R7 is selected from: aryl, substituted aryl, heterocycle, substituted heterocycle, C1-C6 alkyl and C3-C10 cycloalkyl;
  • R9 is hydrogen, (C1-C3 alkyl)-CO, or chlorosubstituted (C1-C3 alkyl)-CO;
  • u is 1 and v is 1.
  • At least one R is selected from phenyl, cyclohexyl and cyclopentyl.
  • At least one R is selected from phenyl, cyclohexyl, cyclopentyl and Ci -C alkyl.
  • R and R are independently selected from: hydrogen, OH, C j -Cg alkyl, C ⁇ -Cg alkoxy, C j -Cg aralkyl and aryl.
  • attachment of the group X ⁇ to the C-23 carbonyl of the vinca alkaloid cytotoxic agent is through the nitrogen of the XL group.
  • X ⁇ is selected from the following group:
  • X ⁇ is selected from the following group:
  • oligopeptides of the instant conjugates comprise a cyclic amino acid substituted with a hydrophilic moiety, previously represented by the term "Haa”, which may also be represented by the formula:
  • R5 is selected from HO- and C ⁇ -Cg alkoxy
  • R is selected from hydrogen, halogen, C j -C 6 alkyl, HO- and C j -Cg alkoxy;
  • t 3 or 4.
  • cyclic amine moiety having 5 or 6 members in the ring, such a cyclic amine which may be optionally fused to a phenyl or cyclohexyl ring.
  • a cyclic amine moiety include, but are not limited to, the following specific structures:
  • cycloalkyl moieties When one R and one R are combined to form a -(CH2) W -, a cycloalkyl moiety having 5-7 members in the ring.
  • cycloalkyl moieties include, but are not limited to, the following specific structures:
  • the conjugates of the present invention may have asymmetric centers and occur as racemates, racemic mixtures, and as individual diastereomers, with all possible isomers, including optical isomers, being included in the present invention.
  • HO(CR R ) 2 - represents HOCH2CH2-
  • alkyl and the alkyl portion of aralkyl and similar terms, is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms; “alkoxy” represents an alkyl group of indicated number of carbon atoms attached through an oxygen bridge.
  • chlorosubstituted-alkyl is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms and being substituted with a chlorine atom. Examples include, but are not limited to chloromethyl, 1-chloroethyl, 2-chloroethyl, 1-chloropropyl, 2-chloropropyl and the like.
  • cycloalkyl is intended to include non- aromatic cyclic hydrocarbon groups having the specified number of carbon atoms.
  • examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like.
  • alkenyl groups include those groups having the specified number of carbon atoms and having one or several double bonds. Examples of alkenyl groups include vinyl, allyl, isopropenyl, pentenyl, hexenyl, heptenyl, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, 1-propenyl, 2-butenyl, 2-methyl-2-butenyl, isoprenyl, farnesyl, geranyl, geranylgeranyl and the like.
  • Alkynyl groups include those groups having the specified number of carbon atoms and having one triple bonds. Examples of alkynyl groups include acetylene, 2-butynyl, 2-pentynyl, 3-pentynyl and the like.
  • Halogen or “halo” as used herein means fluoro, chloro, bromo and iodo.
  • aryl and the aryl portion of aralkyl and aroyl, is intended to mean any stable monocyclic or bicyclic carbon ring of up to 7 members in each ring, wherein at least one ring is aromatic. Examples of such aryl elements include phenyl, naphthyl, tetrahydronaphthyl, indanyl, biphenyl, phenanthryl, anthryl or acenaphthyl.
  • heterocycle or heterocyclic represents a stable 5- to 7-membered monocyclic or stable 8- to 11-membered bicyclic heterocyclic ring which is either saturated or unsaturated, and which consists of carbon atoms and from one to four heteroatoms selected from the group consisting of N, O, and S, and including any bicyclic group in which any of the above-defined heterocyclic rings is fused to a benzene ring.
  • the heterocyclic ring may be attached at any heteroatom or carbon atom which results in the creation of a stable structure.
  • heterocyclic elements include, but are not limited to, azepinyl, benzimidazolyl, benzisoxazolyl, benzofurazanyl, benzopyranyl, benzothiopyranyl, benzofuryl, benzothiazolyl, benzothienyl, benzoxazolyl, chromanyl, cinnolinyl, dihydrobenzofuryl, dihydrobenzothienyl, dihydrobenzothiopyranyl, dihydrobenzothiopyranyl sulfone, furyl, imidazolidinyl, imidazolinyl, imidazolyl, indolinyl, indolyl, isochromanyl, isoindolinyl, isoquinolinyl, isothiazolidinyl, isothiazolyl, isothiazolidinyl, mo holinyl, naphthyridinyl, oxadiazolyl
  • substituted aryl and “substituted heterocycle” include moieties containing from 1 to 3 substituents in addition to the point of attachment to the rest of the compound. Such additional substituents are selected from F, Cl, Br, CF3, NH2, N(Ci-C6 alkyl)2, NO2, CN, (C1-C6 alkyl)0-, -OH, (C1-C6 alkyl)S(0) m -, (C1-C6 alkyl)C(0)NH-, H2N-C(NH)-, (C1-C6 alkyl)C(O)-, (C1-C6 alkyl)OC(O)-, N3, (C1-C6 alkyl)OC(0)NH- and C1-C20 alkyl.
  • cyclic moieties so defined include, but are not limited to:
  • heteroatom-containing cyclic moieties so defined include, but are not limited to:
  • hydroxylated represents substitution on a substitutable carbon of the ring system being so described by a hydroxyl moiety.
  • polyhydroxylated represents substitution on two or more substitutable carbon of the ring system being so described by two, three or four hydroxyl moieties.
  • cotininyl represents the following structure: -
  • oligopeptides, peptide subunits and peptide derivatives can be synthesized from their constituent amino acids by conventional peptide synthesis techniques, preferably by solid-phase technology.
  • the peptides are then purified by reverse-phase high performance liquid chromatography (HPLC). Standard methods of peptide synthesis are disclosed, for example, in the following works: Schroeder et al, "The Peptides", Vol.
  • the pharmaceutically acceptable salts of the compounds of this invention include the conventional non-toxic salts of the compounds of this invention as formed, e.g., from non-toxic inorganic or organic acids.
  • such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like: and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, trifluoroacetic and the like.
  • conjugates of the instant invention which comprise the oligopeptide containing the PSA cleavage site and a cytotoxic agent may similarly be synthesized by techniques well known in the medicinal chemistry art.
  • a free amine moiety on the cytotoxic agent may be covalently attached to the oligopeptide at the carboxyl terminus such that an amide bond is formed.
  • an amide bond may be formed by covalently coupling an amine moiety of the oligopeptide and a carboxyl moiety of the cytotoxic agent.
  • a reagent such as 2-(lH-benzotriazol-l-yl)- 1,3,3-tetramethyluronium hexafluorophosphate (known as HBTU) and 1-hyroxybenzotriazole hydrate (known as HOBT), dicyclohexylcarbodiimide (DCC), N-ethyl-N-(3-dimethylamino- propyl)- carbodiimide (EDC), diphenylphosphorylazide (DPP A), benzotriazol-l-yl-oxy-tris-(dimethylamino)phosphonium hexafluorophosphate (BOP) and the like, used in combination or singularly, may be utilized.
  • HBTU 2-(lH-benzotriazol-l-yl)- 1,3,3-tetramethyluronium hexafluorophosphate
  • HOBT 1-hyroxybenzotriazole hydrate
  • DCC dicycl
  • the instant conjugate may be formed by a non-peptidyl bond between the PSA cleavage site and a cytotoxic agent.
  • the cytotoxic agent may be covalently attached to the carboxyl terminus of the oligopeptide via a hydroxyl moiety on the cytotoxic agent, thereby forming an ester linkage.
  • a reagent such as a combination of HBTU and HOBT, a combination of BOP and imidazole, a combination of DCC and DMAP, and the like may be utilized.
  • the carboxylic acid may also be activated by forming the nitrophenyl ester or the like and reacted in the presence of DBU (l,8-diazabicyclo[5,4,0]undec-7-ene.
  • useful amino-protecting groups may include, for example, Cl-Cl ⁇ alkanoyl groups such as formyl, acetyl, dichloroacetyl, propionyl, hexanoyl, 3,3- diethylhexanoyl, ⁇ -chlorobutryl, and the like; Cl-Cio alkoxycarbonyl and C5-C15 aryloxycarbonyl groups such as tert-butoxycarbonyl, benzyloxycarbonyl, allyloxycarbonyl, 4-nitrobenzyloxycarbonyl, fluorenylmethyloxycarbonyl and cinnamoyloxy carbonyl; halo-(Cl-Cl ⁇ )-alkoxycarbonyl such as 2,2,2-trichloroethoxycarbonyl; and C1-C15 arylalkyl and alkenyl group such as benzyl, phenethyl, allyl, trityl, and the
  • Useful carboxy-protecting groups may include, for example, Cl-ClO alkyl groups such as methyl, tert-butyl, decyl; halo-Cl-Cl ⁇ alkyl such as 2,2,2-trichloroethyl, and 2-iodoethyl; C5-C15 arylalkyl such as benzyl, 4-methoxybenzyl, 4-nitrobenzyl, triphenylmethyl, diphenylmethyl; Cl-ClO alkanoyloxymethyl such as acetoxy methyl, propionoxymethyl and the like; and groups such as phenacyl, 4-halophenacyl, allyl, dimethylallyl, tri-(Cl-C3 alkyl)silyl, such as trimethylsilyl, ⁇ -p-toluenesulfonylethyl, ⁇ -p-nitrophenylthioethyl, 2,4,6-trimethylbenzyl, ⁇ -methylthioe
  • useful hydroxy protecting groups may include, for example, the formyl group, the chloroacetyl group, the benzyl group, the benzhydryl group, the trityl group, the
  • Reaction Scheme I illustrates preparation of conjugates of the oligopeptides of the instant invention and the vinca alkaloid cytotoxic agent vinblastine derivative wherein the attachment of vinblastine is via the linker to the C-terminus of the oligopeptide. Furthermore, Scheme I illustrates a synthesis of conjugates wherein the C-4-position hydroxy moiety is reacetylated following the addition of the linker unit. Applicants have discovered that the desacetyl vinblastine conjugate is also efficacious and may be prepared by eliminating the steps of reacting the intermediate with acetic anhydride, followed by deprotection of the amine.
  • Addition of a single amino acid to the hydroxyalkylamine linker prior to the inco ⁇ oration of the remaining peptide portion of the oligopeptide may be advantageous if the functionality of the amino acids that comprise the oligopeptide would compete with the nucleophillic hydroxyl moiety. Alternatively, if no such competing functional groups are present on the oligopeptide, the oligopeptide may be attached to the linker in a single reaction step.
  • oligopeptide * is the cleavable oligopeptide without the C-terminus amino acid REACTION SCHEME I (continued)
  • novel cytotoxic agents of the instant invention which are derivatives of the vinca drug vinblastine may be described by the general formula II below:
  • X L is selected from - NH - (CR 3 2 ) U (CR 4 2 ) V - O - and
  • R3 and R4 are independently selected from: hydrogen, Cl-C6-alkyl, hydroxylated C3-C8-cycloalkyl, polyhydroxylated C3-C8- cycloalkyl, hydroxylated aryl, polyhydroxylated aryl and aryl, or one R3 and one R4 are combined to form a -(CH2)w- > which is unsubstituted or substituted with one or two substituents selected from OH and C1-C6 alkyl; or an R3 is combined with another R3 on the same carbon to form a -(CH 2 ) x -;or an R4 is combined with another R4 on the same carbon to form a
  • R5 is selected from OH and Cl-C6 alkyl
  • R9 is hydrogen, (C1-C3 alkyl)-CO, or chlorosubstituted (C1-C3 alkyl)-CO;
  • r is 1, 2 or 3; u and v are independently selected from: 0, 1, 2 or 3; w is 2, 3 or 4; x is 3, 4 or 5;
  • u is 1 and v is 1.
  • At least one R is selected from phenyl, cyclohexyl and cyclopentyl.
  • At least one R is selected from phenyl, cyclohexyl, cyclopentyl and C ⁇ -C ⁇ alkyl.
  • the pharmaceutically acceptable salts of the conjugates and novel cytotoxic agents of this invention include the conventional non- toxic salts of the compounds of this invention (also referred to as the compounds of the invention) as formed, e.g., from non-toxic inorganic or organic acids.
  • such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like: and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxy-benzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, trifluoroacetic and the like.
  • inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like
  • organic acids such as acetic, propionic, succinic, glycolic,
  • the pharmaceutically acceptable salts of the compounds of this invention can be synthesized from the compounds of this invention which contain a basic moiety by conventional chemical methods. Generally, the salts are prepared either by ion exchange chromatography or by reacting the free base with stoichiometric amounts or with an excess of the desired salt-forming inorganic or organic acid in a suitable solvent or various combinations of solvents.
  • the oligopeptide-cytotoxic agent conjugates of the invention are administered to the patient in the form of a pharmaceutical composition which comprises a conjugate of of the instant invention and a pharmaceutically acceptable carrier, excipient or diluent therefor.
  • pharmaceutically acceptable refers to those agents which are useful in the treatment or diagnosis of a warmblooded animal including, for example, a human, equine, procine, bovine, murine, canine, feline, or other mammal, as well as an avian or other warm-blooded animal.
  • the preferred mode of administration is parenterally, particularly by the intravenous, intramuscular, subcutaneous, intraperitoneal, or intralymphatic route.
  • Such formulations can be prepared using carriers, diluents or excipients familiar to one skilled in the art. In this regard, See, e ⁇ . Remington's Pharmaceutical Sciences.
  • compositions may include proteins, such as serum proteins, for example, human serum albumin, buffers or buffering substances such as phosphates, other salts, or electrolytes, and the like.
  • Suitable diluents may include, for example, sterile water, isotonic saline, dilute aqueous dextrose, a polyhydric alcohol or mixtures of such alcohols, for example, glycerin, propylene glycol, polyethylene glycol and the like.
  • the compositions may contain preservatives such as phenethyl alcohol, methyl and propyl parabens, thimerosal, and the like. If desired, the composition can include about 0.05 to about 0.20 percent by weight of an antioxidant such as sodium metabisulfite or sodium bisulfite.
  • composition is intended to encompass a product comprising the specified ingredients in the specific amounts, as well as any product which results, directly or indirectly, from combination of the specific ingredients in the specified amounts.
  • the composition preferably will be prepared so that the amount administered to the patient will be from about 0.01 to about 1 g of the conjugate.
  • the amount administered will be in the range of about 0.2 g to about 1 g of the conjugate.
  • the conjugates of the invention are effective over a wide dosage range depending on factors such as the disease state to be treated or the biological effect to be modified, the manner in which the conjugate is administered, the age, weight and condition of the patient as well as other factors to be determined by the treating physician. Thus, the amount administered to any given patient must be determined on an individual basis.
  • the clinical physician would administer them initially by the same route in the same vehicle and against the same types of tumors as for clinical use of leurocristine, vinblastine and vindesine. Differences in dosage levels would, of course, be based on the relative activity between the cytotoxic agents of formula II and the known vinca alkaloid drugs against the specific tumor type.
  • the specific cancers that the cytotoxic agents of formula II may be useful against include, but are not limited to, haemotological tumors (such as chronic myologenis leukemia (CML), and acute lympoblastic leukemia (ALL)), prostate cancer and ovarian cancer.
  • novel cytotoxic agents of formula II may be administered to mammals, preferably humans, either alone or, preferably, in combination with pharmaceutically acceptable carriers or diluents, optionally with known adjuvants, such as alum, in a pharmaceutical composition, according to standard pharmaceutical practice.
  • the compounds can be administered orally or parenterally, including the intravenous, intramuscular, intraperitoneal, subcutaneous, rectal and topical routes of administration.
  • the selected compound may be administered, for example, in the form of tablets or capsules, or as an aqueous solution or suspension.
  • carriers which are commonly used include lactose and corn starch, and lubricating agents, such as magnesium stearate, are commonly added.
  • useful diluents include lactose and dried corn starch.
  • aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring agents may be added.
  • sterile solutions of the active ingredient are usually prepared, and the pH of the solutions should be suitably adjusted and buffered.
  • the total concentration of solutes should be controlled in order to render the preparation isotonic.
  • the cytotoxic agents of formula II may be administered at the rate of from 0.01 to 1 mg./kg. and preferably from 0.1 to 1 mg./kg. of the mammalian body weight once or twice a week or every two weeks depending on both the activity and the toxicity of the drug.
  • An alternative method of arriving at a therapeutic dose is based on body surface area with a dose range of 0.1 to 10 mg./meter squared of mammalian body surface every 7 or 14 days.
  • cytotoxic agents of the instant invention may also be co-administered with other well known therapeutic agents that are selected for their particular usefulness against the condition that is being treated.
  • the instant compounds may be useful in combination with known anti-cancer and cytotoxic agents.
  • Step A 4-des- Acetylvinblastine-23-hydrazide (1-1) A sample of 6.0 g (6.6 mmol) of vinblastine sulfate
  • Step B (lS,2R)-(+)-2-Hydroxy-3-Cyclohexylisopropylamine
  • Step C Preparation of 4-des- Acetylvinblastine-23-(lS,2R)-(+)-2- Hydroxy-3 -Cyclohexy lisopropylamide (HC AP- (dAc)vinblastine (1-3)
  • Table 3 shows the compound described in Example 1 and other vinca drug derivatives that were prepared by the procedure described in Example 1, but utilizing the appropriate amine in Step C. Unless otherwise indicated, the trifluoroacetate salt of the conjugate was prepared and tested.
  • Step B N-Boc-(lS.2R -(+VNorephedrine (2-2 ⁇ A solution of 1.51 g (10 mmol) of (lS,2R)-(+)-
  • Norephedrine in a mixture of 1,4 dioxane (20 ml), water (10 ml) and IN NaOH (10 ml) was stirred and cooled in an ice- water bath.
  • Di-(t-butyl) dicarbonate (2.4 g, 11 mmol) was added in portions over approx. 20 min.
  • the reaction was stirred in the cold for 2hrs., then at room temp, for an additional lh.
  • the solution was concentrated to remove most of the dioxane, cooled in an ice bath and covered with a layer of ethyl acetate (30 ml) and acidified to pH 2 with IN KHSO4.
  • Step C N-Boc-HCAP (2-3) A solution of 2.38 g of N-Boc-(lS,2R)-(+)-Norephedrine
  • Step D N-Benzyloxycarbonyl-Ser-N-t-Boc-HCAP ester (2-4) A solution of 1.95 g (6.6 mmol) of N-Z-Ser(tBu)-OH, 1.54g (6.0 mmol) of N-Boc-HCAP (2-3), 1.26 g (6.6 mmol) of EDC, and 146 mg (1.2 mmol) of DMAP in 30 ml of anh. CH2C12 was treated and the resulting solution stirred at room temp, in an N2 atmosphere for
  • Step F N-Acetyl-4-trans-L-Hyp-Ser-Ser-Chg-Gln-Ser-HCAP amine (2-6) (SEQ.ID.NO. 82)
  • N-Hydroxyacetyl-Abu-Ser-Ser-Chg-Gln-Ser- Pro-HCAP amine (3-4) (SEQ.ID.NO. 89) by coupling of N-Hydroxyacetyl-Abu-Ser-Ser-Chg-Gln-Ser-OH (3-1) with H-Pro-N-t-Boc-HCAP ester (3-3) followed by deprotection.
  • Step G 4-des- Acetylvinblastine-23-(N-Ac-4-trans-L-Hyp-Ser-Ser-
  • Ser-Ser-Chg-Gln-Ser-Pro-HCAP) amide (3-5) by coupling 4- es- Acetylvinblastine-23-hydrazide (1-1) with OH- Acetyl-Abu-Ser-Ser-Chg-Gln-Ser-Pro-HCAP amine (3-4) 4-des- Acetylvinblastine-23-(N-hvdroxyl-Ac-Abu-Ser-Ser-Chg-Gln-Ser- HCAP) amide acetate salt (3-5)
  • Step A N-Acetyl-Ser-Chg-Gln-Ser-Ser-OH (2A-1)
  • Step B N-Boc-(lS.2R)-(+)-Norephedrine (2A-2)
  • Step D N-Benzyloxycarbonyl-Pro-N-t-Boc-HCAP ester (2A-4)
  • Step F N-Acetyl -Ser-Chg-Gln-Ser-Ser-Pro-HCAP amine (2A-6)
  • Step G 4-des- Acetylvinblastine-23-(N-Ac -Ser-Chg-Gln-Ser-Ser- Pro-HCAP) amide acetate salt (2A-7)
  • Table 4 shows the compounds described in Examples 2 and 2A and other peptide-vinca drug conjugates that were prepared by the procedures described in Examples 2 and 2A, but utilizing the appropriate amino acid residues and blocking group acylation. Unless otherwise indicated, the acetate salt of the conjugate was prepared and tested.
  • Pheol is phenylalaninol
  • PIP is pipecolinic acid
  • Abu 2-aminobutyric acid
  • gammaAbu is 4-aminobutyric acid
  • PSA digestion buffer 50 mM tris(hydroxymethyl)-aminomethane pH7.4, 140 mM NaCl
  • the PSA digestion buffer utilized is 50 mM tris(hydroxymethyl)- aminomethane pH7.4, 140 mM NaCl.
  • the reaction is quenched after various reaction times by the addition of trifluoroacetic acid (TFA) to a final 1% (volume/volume).
  • TFA trifluoroacetic acid
  • lOmM ZnC12 The quenched reaction was analyzed by HPLC on a reversed-phase C18 column using an aqueous 0.1 %TFA acetonitrile gradient.
  • Table 4 shows the amount of time (in minutes) required for 50% cleavage of the noted oligopeptide-cytotoxic agent conjugates with enzymatically active free PSA. Unless otherwise indicated, the acetate salt of the conjugate was tested.
  • the cytotoxicities of the vinca alkaloid derivatives, prepared as described in Example 1, and the cleaveable oligopeptide- vinca drug conjugates, prepared as described in Examples 2 and 2 A, against a line of cells which is known to be killed by unmodified vinca drug was assessed with an Alamar Blue assay.
  • cell culmres of LNCap prostate tumor cells, Colo320DM cells (also designated C320), T24 bladder carcinoma cells or T47D breast carcinoma cells in 96 well plates was diluted with medium containing various concentrations of a given conjugate (final plate well volume of 200 ⁇ l). The cells were incubated for 3 days at 37°C, 20 ⁇ l of Alamar Blue is added to the assay well. The cells were further incubated and the assay plates were read on a EL-310 ELISA reader at the dual wavelengths of 570 and 600 nm at 4 and 7 hours after addition of Alamar Blue.
  • LNCaP.FGC or C320 cells are trypsinized, resuspended in the growth medium and centifuged for 6 mins. at 200xg. The cells are resuspended in serum-free a-MEM and counted. The appropriate volume of this solution containing the desired number of cells is then transferred to a conical centrifuge tube, centrifuged as before and resuspended in the appropriate volume of a cold 1 : 1 mixture of a-MEM-Matrigel. The suspension is kept on ice until the animals are inoculated.
  • Harlan Sprague Dawley male nude mice (10-12 weeks old) are restrained without anesthesia and are inoculated with 0.5 mL of cell suspension on the left flank by subcutaneous injection using a 22G needle. Mice are either given approximately 5x105 DuPRO cells or 1.5x107 LNCaP.FGC cells.
  • mice Following inoculation with the tumor cells the mice are treated under one of two protocols:
  • test conjugate 0.1-0.5 mL volume of test conjugate, vinca drug or vehicle control (sterile water).
  • doses of the conjugate and vinca drug are initially the maximum non-lethal amount, but may be subsequently titrated lower. Identical doses are administered at 24 hour intervals for 5 days. After 10 days, blood samples are removed from the mice and the serum level of PSA is determined. Similar serum PSA levels are determined at 5-10 day intervals. At the end of 5.5 weeks the mice are sacrificed and weights of any tumors present are measured and serum PSA again determined.The animals' weights are determined at the beginning and end of the assay.
  • mice Ten days after cell inoculation,blood samples are removed from the animals and serum levels of PSA are determined. Animals are then grouped according to their PSA serum levels. At 14-15 days after cell inoculation, the animals are dosed with a 0.1-0.5 mL volume of test conjugate, vinca drug or vehicle control (sterile water). Dosages of the conjugate and vinca drug are initially the maximum non-lethal amount, but may be subsequently titrated lower. Identical doses are administered at 24 hour intervals for 5 days. Serum PSA levels are determined at 5-10 day intervals. At the end of 5.5 weeks the mice are sacrificed, weights of any tumors present are measured and serum PSA again determined. The animals' weights are determined at the beginning and end of the assay.
  • Step A Preparation of proteolytic tissue extracts All procedures are carried out at 4 C. Appropriate animals are sacrificed and the relevant tissues are isolated and stored in liquid nitrogen. The frozen tissue is pulverized using a mortar and pestle and the pulverized tissue is transfered to a Potter-El vej eh homogenizer and 2 volumes of Buffer A (50 mM Tris containing 1.15% KCl, pH 7.5) are added. The tissue is then disrupted with 20 strokes using first a lose fitting and then a tight fitting pestle.
  • Buffer A 50 mM Tris containing 1.15% KCl, pH 7.5
  • the homogenate is centrifuged at 10,000 x g in a swinging bucket rotor (HB4-5), the pellet is discarded and the re-supernatant centrifuged at 100,000 x g (Ti 70). The supernatant (cytosol) is saved.
  • the pellet is resuspended in Buffer B (10 mM EDTA containing 1.15% KCl, pH 7.5) using the same volume used in step as used above with Buffer A.
  • Buffer B (10 mM EDTA containing 1.15% KCl, pH 7.5)
  • the suspension is homogenized in a dounce homogenizer and the solution centrifuged at 100,000x g.
  • the supernatant is discarded and the pellet resuspended in Buffer C (10 mM potassium phosphate buffer containing ⁇ .25 M sucrose, pH 7.4), using 1/2 the volume used above, and homogenized with a dounce homogenizer.
  • Protein content of the two solutions is determine using the Bradford assay. Assay aliquots are then removed and frozen in liquid N2. The aliquots are stored at -70°C.
  • Step B Proteolytic cleavage assay For each time point, 20 microgram of peptide- vinca drug conjugate and 150 micrograms of tissue protein, prepared as described in Step A and as determined by Bradford in reaction buffer are placed in solution of final volume of 200 microliters in buffer (50 mM TRIS, 140 mM NaCl, pH 7.2). Assay reactions are run for 0, 30, 60, 120, and 180 minutes and are then quenched with 9 microliters of 0.1 M ZnCl2 and immediately placed in boiling water for 90 seconds. Reaction products are analyzed by HPLC using a VYDAC C18 15 cm column in water / acetonitrile (5% to 50% acetonitrile over 30 minutes).

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Abstract

L'invention concerne des conjugués qui comprennent des oligopeptides portant des séquences d'acides aminés subissant sélectivement un clivage protéolytique sous l'effet d'un antigène libre spécifique de la prostate et d'agents cytotoxiques connus. Les conjugués de l'invention se caractérisent par une liaison hydroxylalkylamino entre l'oligopeptide et la vinblastine. De tels conjugués contiennent au traitement du cancer de la prostate et de l'hypertrophie prostatique bénigne. L'invention concerne également des agents cytotoxiques dérivés des alcaloïdes de la pervenche.
EP99911146A 1998-03-05 1999-03-04 Conjugues convenant au traitement du cancer de la prostate Withdrawn EP1069906A1 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US7686098P 1998-03-05 1998-03-05
US76860P 1998-03-05
GBGB9815855.3A GB9815855D0 (en) 1998-07-21 1998-07-21 Conjugates useful in the treatment of prostate cancer
GB9815855 1998-07-21
PCT/US1999/004882 WO1999044628A1 (fr) 1998-03-05 1999-03-04 Conjugues convenant au traitement du cancer de la prostate

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GB9924759D0 (en) * 1999-10-19 1999-12-22 Merck Sharp & Dohme Process for preparing peptide intermediates

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US4203898A (en) * 1977-08-29 1980-05-20 Eli Lilly And Company Amide derivatives of VLB, leurosidine, leurocristine and related dimeric alkaloids
US4639456A (en) * 1980-06-10 1987-01-27 Omnichem S.A. Vinblastin-23-oyl amino acid derivatives
US5599686A (en) * 1994-06-28 1997-02-04 Merck & Co., Inc. Peptides
US6143864A (en) * 1994-06-28 2000-11-07 Merck & Co., Inc. Peptides

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WO1999044628A8 (fr) 1999-10-14
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