WO1997014416A1 - Conjugues utiles pour traiter l'adenome prostatique benin - Google Patents

Conjugues utiles pour traiter l'adenome prostatique benin Download PDF

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Publication number
WO1997014416A1
WO1997014416A1 PCT/US1996/016490 US9616490W WO9714416A1 WO 1997014416 A1 WO1997014416 A1 WO 1997014416A1 US 9616490 W US9616490 W US 9616490W WO 9714416 A1 WO9714416 A1 WO 9714416A1
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WIPO (PCT)
Prior art keywords
seq
dox
ser
peptide
type
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PCT/US1996/016490
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English (en)
Inventor
Deborah Defeo-Jones
Raymond E. Jones
Allen I. Oliff
Edward M. Scolnick
Victor M. Garsky
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Merck & Co., Inc.
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Priority claimed from GBGB9602903.8A external-priority patent/GB9602903D0/en
Application filed by Merck & Co., Inc. filed Critical Merck & Co., Inc.
Priority to CA 2234763 priority Critical patent/CA2234763A1/fr
Priority to US09/051,759 priority patent/US6177404B1/en
Priority to EP96936504A priority patent/EP0855910A4/fr
Priority to AU74321/96A priority patent/AU708475B2/en
Priority to JP9515930A priority patent/JP2000506494A/ja
Publication of WO1997014416A1 publication Critical patent/WO1997014416A1/fr

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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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • 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
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Benign prostate hyperplasia (or "prostatism") can be seen in almost 100 percent of all men over the age of 80, and changes in the prostate can be discovered in about 50 percent of men by the time they reach the age of 60.
  • Many men with benign prostate hyperplasia (BPH) remain without symptoms, others show slow progression, while others remain stable.
  • BPH benign prostate hyperplasia
  • some 400,000 men a year have symptoms severe enough to require surgery.
  • transurethral resection is effective in relieving the symptoms of BPH, although side-effects, including morbidity from the operation itself, mild to severe urinary incontinence and some degree of erectile or ejaculatory dysfunction, have been reported in a limited number of patients.
  • the prostate Normally the prostate remains stable until after the age of 45, when the tissue begins to change, growing and causing the size of the prostate to increase.
  • the enlarging prostate squeezes the urethra, producing the symptoms that characterize BPH. These include difficulty in starting urination (hesitancy), a weak urinary stream, dribbling after urination, and increased frequency or urgency to urinate during the sleep period. Sometimes urination may be painful. The symptoms of obstruction of the urethra can often become more severe if a urinary infection develops, one of the common complications of BPH.
  • 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- (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. & Meta. 75: 1046-1053).
  • 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., Bj ⁇ rk, 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., Bj ⁇ rk, T., Nilsson, O., et al. (1993). J. Urol.
  • 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). 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).
  • 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. Such a specific agent may be effective against BPH without causing the side-effects associated with other therapies.
  • a novel pharmaceutical composition useful for the treatment of benign prostatic hyperplasia which comprises novel oligopeptides, which are selectively cleaved by enzymatically active PSA, in conjugation with a cytotoxic agent.
  • Another object of this invention is to provide a method of treating benign prostatic hyperplasia which comprises administration of the novel pharmaceutical composition.
  • Novel pharmaceutical compositions useful for the treatment of adverse conditions of the prostate, in particular benign prostatic hyperplasia, which comprise novel oligopeptides, which are selectively cleaved by enzymatically active PSA, in conjugation with a pharmaceutical agent are described. Methods of treating such conditions of the prostate are also disclosed.
  • FIGURES 1 and IA Primary Amino Acid Sequence of Semenogelin I: The primary amino acid sequence of Semenogelin I is shown. (SEQ.ID.NO.: 1) The PSA proteolytic cleavage sites ("CS") are shown (numbered in order of the relative affinity of a site towards PSA hydrolysis) and the protein fragments are numbered sequentially starting at the amino terminus.
  • SEQ.ID.NO. 1
  • CS PSA proteolytic cleavage sites
  • FIGURE 2 Cleavage Affinity of Synthetic Oligopeptides: A nested set of synthetic oligopeptides was prepared and the oligopeptides were digested with enzymatically active free PSA for various times. The results are shown in Table 2. All of the oligopeptides were tested as trifluoroacetate salts.
  • FIGURES 3, 3 A and 3B Cleavage Affinity of Synthetic Oligopeptides: Synthetic oligopeptides were prepared and the oligopeptides were digested with enzymatically active free PSA for four (4) hours. The percentage of the oligopeptide that is cleaved in this period of time is listed. The results are shown in Table 4. Table 4a shows the amount of time (in minutes) required for 50% cleavage of the noted oligopeptides with enzymatically active free PSA. If no salt is indicated for an oligopeptide, the free base was tested.
  • FTGURE 4 Cytotoxicity Data of Non-cleavable Oligopeptide- Doxorubicin Conjugates:
  • the data of the figure shows comparative cytotoxicity of doxorubicin and a conjugate of doxorubicin covalently bound to an oligopeptide
  • Compound 12d that does not contain the free PSA proteolytic cleavage site.
  • the EC50 for doxorubicin is 0.3 ⁇ M, while the acetylated oligopeptide modified doxorubicin has an EC50 that has been reduced by greater than 300 fold.
  • This conjugate had no HPLC detectable contamination with unmodified doxorubicin.
  • the oligopeptide alone had no detectable cell killing activity.
  • FIGURES 5 and 5 A Cleavage Affinity of Oligopeptides in Conjugation with Doxorubicin by Free PSA
  • Qligopeptides-doxorubicin conjugates were prepared and the conjugates were digested with enzymatically active free PSA for four (4) hours. The percentage conjugate that is enzymatically cleaved in the oligopeptide in this period of time is listed. The results are shown in Table 5.
  • Table 5a shows the amount of time (in minutes) required for 50% cleavage of the noted oligopeptide-cytotoxic agent conjugates with enzymatically active free PSA. If no salt is indicated for the conjugate, the free conjugate was tested.
  • FIGURE 6 Cleavage Affinity of Oligopeptides in Conjugation with Doxorubicin in Cell Conditioned Media:
  • Oligopeptides-doxorubicin conjugates were reacted for four (4) hours with cell culture media that had been conditioned by exposure to LNCaP cells (which are known to secrete free PSA) or DuPRO cell (which do not secrete free PSA). The percentage conjugate that is enzymatically cleaved in the oligopeptide in this period of time is listed. The results are shown in Table 6.
  • FIGURE 7 Cytotoxicity Data of Cleavable Oligopeptide -Doxorubicin Conjugates: The data in Table 7 shows cytotoxicity (as EC50) of conjugates of doxorubicin covalently bound to an oligopeptide that contain a free PSA proteolytic cleavage site against a cancer cell line that is known to secrete free PSA. Also shown for selected conjugates is the cytotoxicity of the conjugate against a cell line (DuPRO) which does not secrete free PSA. If no salt is indicated for the conjugate, the free conjugate was tested.
  • cytotoxicity as EC50
  • the present invention relates to pharmaceutical compositions that comprise conjugates that contain oligopeptides, which are specifically 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, and pharmaceutical agents covalently linked to such oligopeptides directly or through a linker unit, or pharmaceutically acceptable salts thereof.
  • PSA free prostate specific antigen
  • this invention is directed to such conjugates wherein the pharmaceutical agent is a cytotoxic agent.
  • the present invention also relates to a novel method of treating adverse conditions of the prostate, in particular benign prostatic hyperplasia, which utilizes these compositions.
  • Such oligopeptides include oligomers that comprise an amino acid sequence selected from:
  • hArg is homoarginine
  • Cha is cyclohexylalanine
  • Xaa is any natural amino acid
  • the oligopeptides include oligomers that comprise an amino acid sequence that is selected from:
  • the oligopeptides include oligomers that comprise an amino acid sequence that is selected from:
  • the oligopeptides include oligomers that comprise an amino acid sequence that is selected from:
  • oligomers that comprise an amino acid sequence as used hereinabove, and elsewhere in the Detailed Description of the Invention, describes oligomers of from about 6 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 following oligomer GlnLeuAspAsnLysIleSerTyrGlnlSerSerSerThrHisGlnSerSer (SEQ.ID.NO.: 20) comprises the amino acid sequence: AsnLysIleSerTyrGlnlSerSerThr (SEQ.ID.NO.: 10) and would therefore come within the instant invention. It is understood that such oligomers do not include semenogelin I and semenogelin II.
  • the instant invention includes oligomers wherein the N-terminus amino acid or the C-terminus amino acid, or both terminus amino acids are modified. Such modifications include, but are not limited to, acylation of the amine group at the N- terminus and formation of an amide to replace the carboxylic acid at the C-terminus. Addition of such moieties may be performed during solid- phase synthesis of the oligomer; thus, attachment of the C-terminus amino acid to a solid phase resin may be through an amine which results in an amide moiety upon acidic cleavage of the oligomer from the resin.
  • oligomers that comprise an amino acid sequence as used hereinabove and are meant to be illustrative and are not limiting:
  • 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.
  • 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:
  • 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: GlyGluGlnGlyValGlnLysAspValSerGlnSerSerlleTyrlSerGlnThrGlu (SEQ.ID.NO.: 45) ,
  • hR or hArg homoarginine
  • hY or hTyr homotyrosine
  • O-Me-Y O-methyltyrosine
  • compositions whose pharmaceutical activity is specific for cells that secrete enzymatically active PSA.
  • compositions comprise the oligopeptides described herein above covalently bonded directly, or through a linker unit, to a pharmaceutical agent.
  • a pharmaceutical agent such a combination of an oligopeptide and pharmaceutical agent may be termed a conjugate.
  • the pharmaceutical agent component of the conjugate may be selected from known compounds useful for treating conditions of the prostate, whose site of biological activity or the desired target of the biological activity is within the prostate or in close proximity to the prostate.
  • pharmaceutical agents include, but are not limited to cytotoxic agents.
  • the method of treatment of the instant invention utilizes cytotoxic compositions whose cytotoxicity is specific for cells that secrete enzymatically active PSA.
  • cytotoxic compositions whose cytotoxicity is specific for cells that secrete enzymatically active PSA.
  • Such compositions comprise the oligopeptides, described herein above, covalently bonded directly, or through a linker unit, to a cytotoxic agent.
  • the cytotoxic activity of the cytotoxic agent is greatly reduced or absent when the oligopeptide containing the PSA proteolytic cleavage site is bonded directly, or through a chemical linker, to the cytotoxic agent and is intact.
  • 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.
  • a preferred embodiment of this aspect of the invention is a conjugate wherein the oligopeptide, and the linker unit if present, 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 releasing unmodified cytotoxic agent into the physiological environment at the place of proteolytic cleavage.
  • Pharmaceutically acceptable salts of the conjugates are also included.
  • the oligopeptide of the instant invention that is conjugated to the cytotoxic agent 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-BPH 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.
  • cytotoxic agent is not to be construed as limited to classical chemical therapeutic agents.
  • the cytotoxic agent may be a protein or polypeptide possessing a desired biological activity.
  • Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, ⁇ -interferon, ⁇ -interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example, lymphokines, interleukin-l ("IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM- CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.
  • IL-1 interleukin-l
  • IL-2 interleukin-2
  • IL-6 interleukin-6
  • GM- CSF granulocyte macrophage colony stimulating factor
  • G-CSF granulocyte colony stimulating factor
  • the preferred cytotoxic agents include, in general, alkylating agents, antiproliferative agents, tubulin binding agents and the like.
  • Preferred classes of cytotoxic agents include, for example, the anthracycline family of drugs, the vinca drugs, the mitomycins. the bleomycins, the cytotoxic nucleosides, the pteridine family of drugs, diynenes, the taxanes and the podophyllotoxins.
  • Particularly useful members of those classes include, for example, doxorubicin, carminomycin, daunorubicin, aminopterin, methotrexate, methopterin, dichloro-methotrexate, mitomycin C, porfiromycin, 5-fluorouracil, 6- mercaptopurine, cytosine arabinoside, podophyllotoxin, or podo- phyllotoxin derivatives such as etoposide or etoposide phosphate, melphalan, vinblastine, vincristine, leurosidine, vindesine, leurosine, taxol and the like.
  • Other useful cytotoxic agents include estramustine, 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.
  • cytotoxic agents for the present invention include drugs of the following formulae:
  • Rl2 is amino or hydroxy
  • R7 is hydrogen or methyl
  • R8 is hydrogen, fluoro, chloro, bromo or iodo
  • R9 is hydroxy or a moiety which completes a salt of the carboxylic acid
  • R 10 is hydrogen or methyl
  • Rl 1 is hydroxy, amino, C1-C3 alkylamino, di(Cl-C3 alkyl)amino, C4-C6 polymethylene amino, NH
  • Rl3 is hydrogen or methyl
  • Rl4 is methyl or thienyl; or a phosphate salt thereof;
  • Rl5 is H, CH3 or CHO; when R 1 7 and R 1 8 are taken singly; RlS is H, and one of Rl6 and R 17 is ethyl and the other is H or OH; when Rl7 and R 18 are taken together with the carbons to which they are attached, they form an oxirane ring in which case Rl 6 is ethyl;
  • R19 IS hydrogen, (C1-C3 alkyl )-CO, or chlorosubstituted (Cl-C3 alkyl)-CO;
  • R22 is hydrogen, methyl, bromo, fluoro, chloro or iodo;
  • R23 is -OH or -NH2;
  • R i hydrogen, bromo, chloro or iodo
  • Rl is -CH3, -CH20H, -CH2 ⁇ CO(CH2)3CH3, or
  • R3 is -OCH3, -OH or -H;
  • R4 is -NH2, -NHCOCF3, 4-mo ⁇ holinyl, 3-cyano-4- mo ⁇ holinyl, 1-piperidinyl, 4-methoxy-l-piperidinyl, benzylamine, dibenzylamine, cyanomethylamine, or 1- cyano-2-methoxyethyl amine;
  • R5 is -OH -OTHP or -H; and
  • R 6 is -OH or -H provided that
  • R6 is not -OH when R5 is -OH or -OTHP.
  • the most highly preferred drugs are the anthracycline antiobiotic agents of Formula (10), described previously.
  • this structural formula includes compounds which are drugs, or are derivatives of drugs, which have acquired in the art different generic or trivial names.
  • Table 1, which follows, represents a number of anthracycline drugs and their generic or trivial names and which are especially preferred for use in the present invention.
  • daunomycin is an alternative name for daunorubicin
  • adriamycin is an alternative name for doxorubicin
  • cytotoxic agents are doxorubicin, vinblastine and desacetylvinblastine.
  • Doxorubicin (also referred to herein as "DOX") is that anthracycline of Formula (10) in which Rl is -CH2OH, R3 is -OCH3, R4 is -NH2, R5 is -OH, and R6 is -H.
  • oligopeptides, peptide subunits and peptide derivatives (also termed "peptides") incorporated in the conjugates utilized in the method of treatment of the present invention can be synthesized from their constituent amino acids by conventional peptide synthesis techniques, preferably by solid-phase technolog ⁇ .
  • the peptides are then purified by reverse-phase high performance liquid chromatography (HPLC).
  • the pharmaceutically acceptable salts of the compounds incorporated in the conjugates utilized in the method of treatment 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, phenyl-acetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxy-benzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, trifluoroacetic and the
  • conjugates utilized in the method of treatment 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- 1 -yl)- 1 ,3,3-tetramethyluronium hexafluorophosphate (known as HBTU) and 1-hyroxybenzotriazole hydrate (known as HOBT), dicyclohexyl- carbodiimide (DCC), N-ethyl-N-(3- dimethylaminopropyl)- carbodiimide (EDC), diphenylphosphorylazide (DPP A), benzotriazol- 1 -yl-oxy-tris-(dimethylamino)phosphonium hexafluorophosphate (BOP) and the like, used in combination or singularly, may be utilized.
  • HBTU 2-(lH- benzotriazol- 1 -yl)- 1 ,3,3-tetramethyluronium hexafluorophosphate
  • HOBT 1-hyroxybenzotriazole hydrate
  • 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 nitro- phenyl ester or the like and reacted in the presence of DBU (1,8- diazabicyclo[5,4,0]undec-7-ene.
  • the instant conjugate may also be formed by attachment of the oligopeptide to the cytotoxic agent via a linker unit.
  • linker units include, for example, a biscarbonyl alkyl diradical whereby an amine moiety on the cytotoxic agent is connected with the linker unit to form an amide bond and the amino terminus of the oligopeptide is connected with the other end of the linker unit also forming an amide bond.
  • a diaminoalkyl diradical linker unit whereby a carbonyl moiety on the cyctotoxic agent is covalently attacted to one of the amines of the linker unit while the other amine of the linker unit is covalently attached to the C terminus of the oligopeptide, may also be uselful.
  • Other such linker units which are stable to the physiological environment when not in the presence of free PSA, but are cleavable upon the cleavage of the PSA proteolytic cleavage site, are also envisioned.
  • linker units may be utilized that, upon cleavage of the PSA proteolytic cleavage site, remain attached to the cytotoxic agent but do not significantly decrease the cytotoxic activity of such a post-cleavage cytotoxic agent derivative when compared with an unmodified cytotoxic agent.
  • useful amino-protecting groups may include, for example, Cl-ClO alkanoyl groups such as formyl, acetyl, dichloroacetyl, propionyl, hexanoyl, 3,3-diethylhexanoyl, ⁇ - chlorobutryl, and the like; Cl-ClO alkoxycarbonyl and C5-C15 aryloxycarbonyl groups such as tert-butoxycarbonyl, benzyloxycarbonyl, aUyloxycarbonyl, 4-nitrobenzyloxycarbonyl, fluorenylmethyloxycarbonyl and cinnamoyloxycarbonyl; halo-(Cl-Cl ⁇ )-alkoxycarbonyl such as 2,2,2-trichloroethoxycarbonyl; and
  • Useful carboxy-protecting groups may include, for example, Cl-ClO alkyl groups such as methyl, tert-butyl, decyl; halo-Cl-ClO alkyl such as 2,2,2-trichloroethyl, and 2-iodoethyl; C5-C15 arylalkyl such as benzyl, 4-methoxybenzyl, 4-nitrobenzyl, triphenylmethyl, diphenyl- methyl; Cl-ClO alkanoyloxymethyl such as acetoxymethyl, propionoxymethyl and the like; and groups such as phenacyl, 4- halophenacyl, allyl, dimethylallyl, tri-(Cl-C3 alkyl)silyl, such as trimethylsilyl, ⁇ -p-toluenesulfonylethyl, ⁇ -p-nitrophenyl-thioethyl, 2,4,6- trimethylbenzyl, ⁇ -methyl
  • useful hydroxy protecting groups may include, for example, the formyl group, the chloroacetyl group, the benzyl group, the benzhydryl group, the trityl group, the 4-nitrobenzyl group, the trimethylsilyl group, the phenacyl group, the tert-butyl group, the methoxymethyl group, the tetrahydropyranyl group, and the like.
  • Reaction Scheme VI illustrates preparation of conjugates utilized in the instant method of treatment wherein the oligopeptides are combined with the vinca alkaloid cytotoxic agent vinblastine. Attachment of the N-terminus of the oligopeptide to vinblastine is illustrated (S.P. Kandukuri et al. J. Med. Chem. 28: 1079-1088 (1985)).
  • Reaction Scheme VII illustrates preparation of conjugates utilized in the instant method of treatment wherein the oligopeptides are combined with the vinca alkaloid cytotoxic agent vinblastine wherein the attachment of vinblastine is at the C-terminus of the oligopeptide.
  • 1,3-diaminopropane linker is illustrative only; other spacer units between the carbonyl of vinblastine and the C-terminus of the oligopeptide are also envisioned.
  • Scheme VII illustrates a synthesis of conjugates wherein the C-4-position hydroxy moiety is reacetylated following the addition of the linker unit.
  • the desacetyl vinblastine conjugate is also efficacious and may be prepared by eliminating the steps shown in Reaction Scheme VII of protecting the primary amine of the linker and reacting the intermediate with acetic anhydride, followed by deprotection of the amine.
  • Conjugation of the oligopeptide at other positions and functional groups of vinblastine may be readily accomplished by one of ordinary skill in the art and is also expected to provide compounds useful in the treatment of benign prostatic hyperplasia.
  • conjugates may be prepared wherein the N-terminus of the oligopeptide utilized in the instant method of treatment is combined with one cytotoxic agent, such as vinblastine, while the C-terminus is simultaneously attached to another cytotoxic agent, which is the same or different cytotoxic agent, such as doxorubicin.
  • Reaction Scheme VIII illustrates the synthesis of such a polycytotoxic agent conjugate.
  • Such a polycytotoxic conjugate may offer advantages over a conjugate containing only one cytotoxic agent.
  • oligopeptide-cytotoxic agent conjugate utilized in the method of treatment of the instant invention wherein the cytotoxic agent is the preferred cytotoxic agent doxorubicin 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
  • XL is absent or is an amino acid selected from: a) phenylalanine, b) leucine, c) valine, d) isoleucine, e) (2-naphthyl)alanine, f) cyclohexylalanine, g) diphenylalanine, h) norvaline, i) norleucine, and j) 1,2,3,4-tetrahydroiso quinoline-3-carboxylic acid;
  • R 1 is C 1 -C6-alkyl or aryl
  • oligopeptide is an oligomer that comprises an amino acid sequence selected from:
  • Xaa is any natural amino acid
  • XL is absent or is an amino acid selected from: a) leucine, b) isoleucine, c) norleucine, and d) valine;
  • R is acetyl, pivaloyl or benzoyl
  • conjugates of an oligopeptide and doxorubicin wherein the N-terminus of the oligopeptide is acylated and the C-terminus of the oligopeptide is attached to the doxorubicin at the 3'- amine are as follows:
  • oligopeptide-cytotoxic agent conjugate utilized in the method of treatment of the instant invention wherein the cytotoxic agent is the preferred cytotoxic agent vinblastine or desacetylvinblastine 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
  • XL is absent or is an amino acid selected from: a) phenylalanine, b) leucine, c) valine, d) isoleucine, e) (2-naphthyl)alanine, f) cyclohexylalanine, g) diphenylalanine, h) norvaline, and i) norleucine, and j) l,2,3,4-tetrahydroisoquinoline-3-carboxylic acid; or
  • Rl is Cl-C6-alkyl or aryl
  • R 19 is hydrogen or acetyl
  • n 1, 2, 3, 4 or 5
  • peptidyl therapeutic agents such as the 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.
  • oligopeptide-cytotoxic agent conjugates utilized in the method of treatment of the instant invention are administered to the patient in the form of a pharmaceutical composition which comprises a conjugate of Formula (I) 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 warm-blooded 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.
  • compositions can be prepared using carriers, diluents or excipients familiar to one skilled in the art.
  • 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.
  • 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 .20 percent by weight of an antioxidant such as sodium metabisulfite or sodium bisulfite.
  • an antioxidant such as sodium metabisulfite or sodium bisulfite.
  • the composition preferably will be prepared so that the amount administered to the patient will be from about .01 to about 1 g of the conjugate. Preferably, the amount administered will be in the range of about .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.
  • Figure 1 we designed polymerase chain reaction primers to clone the semenogelin cDNA from a commercially available prostatic cDNA library (Clone- tech, Palo Alto, CA.).
  • the purified semenogelin cDNA was placed into the bacterial expression vector pTAC [Linemeyer, D.L., Kelly, L.J., Minke, J.G., Gimenez-Gallego, G., DeSalvo, J. and Thomas, K.A., (1987) Bio/Technology 5, 960-965].
  • the semenogelin cDNA was designed so that a tubulin epitope was placed at the carboxyl end of semenogelin protein..
  • the bacterially expressed semenogelin protein was purified on an anti-tubulin antibody column.
  • the purified semenogelin I protein was mixed with commercially prepared prostate-specific antigen (PSA) (York Biologicals International, Stony Brook, NY) in an 100 to 1 molar ratio (semenogelin I/PSA) in 12 mM Tris pH 8.0, 25 mM NaCl, 0.5 mM CaCl2, and incubated for various times.
  • PSA prostate-specific antigen
  • the digest was fractionated by polyacrylamide gel electrophoresis and transferred by electrophoresis to ProBlott filter paper (Applied Biosystems, Inc., Foster City, CA.) in CAPS buffer [Matsudaira, P., (1987) J. Biol. Chem.
  • the ProBlott filter paper was stained with coomassie blue to identify the novel PSA generated semenogelin I protein fragments.
  • the novel fragments were cut out of the filter with a scalpel and submitted for sequence determination. After the proteolytic fragments were identified by variable time digestion, a 10 minute digestion reaction was performed.
  • the relative affinities were derived from the comassie blue staining intensity of each PSA generated peptide fragment. These intensities had approximate - ratios of 3: 1:0.6:0.3.
  • Oligopeptides were prepared by solid-phase synthesis, using a double coupling protocol for the introduction of amino acids on the Applied Biosystems model 430A automated peptide synthesizer. Deprotection and removal of the oligopeptide from the resin support were achieved by treatment with liquid hydrofluoric acid. The oligopeptides were purified by preparative high pressure liquid chromatography on reverse phase C18 silica columns using an aqueous 0.1% trifluoroacetic acid/acetonitrile gradient. Identity and homogeneity of the oligopeptides were confirmed by amino acid composition analysis, high pressure liquid chromatography, and fast atom bombardment mass spectral analysis.
  • the oligopeptides prepared as described in Example 2 were individually dissolved in PSA digestion buffer (12 mM tris(hydroxymethyl)- aminomethane pH8.0, 25 mM NaCl, 0.5 mM CaCl2) and the solution added to PSA at a molar ration of 100 to 1.
  • 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 %
  • LNCaP prostate tumor cells which are a human metastatic prostate adenocarcinoma isolated from a needle biopsy of a lymph node (LNCaP.FGC: American Type Culture Collection, ATCC CRL 1740) , or DuPRO cells in 96 well plates were 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 and then 20 ⁇ l of Alamar Blue was 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.
  • the crude reaction mixture was purified directly by preparative HPLC on a reversed-phase C-18 column using a 0.1% trifluoroacetic acid (TFA) in acetonitrile/0.1% TFA in water gradient.
  • TFA trifluoroacetic acid
  • a functionality was typically protected as the fluorenylmethyloxycarbonyl adduct, which was removed by treatment with a secondary amine, such as piperidine and the like, subsequent to conjugation with doxirubicin.
  • the instant conjugates have a structure of the general formula
  • Boc-Leu-PAM resin Starting with 0.5 mmol (0.67g) Boc-Leu-PAM resin, the protected peptide was synthesized on a 430 A ABI peptide synthesizer. The protocol used a 4 fold excess (2 mmol) of each of the following protected amino acids: Boc-Ser(OBzl), Boc-Gln, Boc-Tyr(BrZ), Boc- Lys(Fmoc). Coupling was achieved using DCC and HOBT activation in methyl-2-pyrrolidinone. Acetic acid was used for the introduction of the N terminal acetyl group. Removal of the Boc group was performed using 50% TFA in methylene chloride and the TFA salt neutralized with diisopropylethylamine. At the completion of the synthesis, the peptide resin was dried to yield 1.3g of (1).
  • Step B Ac-Lvs(FmocVTyr-Gln-Ser-Ser-Ser-Leu-OH (2)
  • the protected peptide resin (1) 1.3 g, was treated with HF
  • Step C Ac-Lvs(Fmoc)-Tyr-Gln-Ser-Ser-Ser-Leu-Dox (3)
  • Step D Ac-Lvs-Gln-Tyr-Ser-Ser-Ser-Leu-Dox (4).
  • Step A NH2 - Leu-Asn-Lys(Fmoc)-Ala-Ser-Tyr-Gln-Ser-Ser-Ser-
  • B 15% acetic acid-methanol.
  • the crude product was suspended in 400 ml of 10% B/90% A buffer, filtered and the filtrate loaded onto the column.
  • a step gradient of 10% B to 55% B was used at a flow rate of 75 ml/min.
  • Homogeneous product fractions were pooled, concentrated and freeze-dried from H2 ⁇ to yield (6).
  • Step B Deacetylvinblastin Monohvdrazide (7) lg of vinblastine sulfate was converted to the amine form by extraction in methylene chloride and saturated sodium bicarbonate. The methylene chloride layer was washed with H2O, dried over anhydrous MgS04 and concentrated to dryness. The vinblastine was then dissolved in anhydrous ethanol (20 ml) and anhydrous hydrazine added (20 ml). The solution was heated (60° C) under an N2 atmosphere for 17 hrs. The reaction was concentrated to an oil, dissolved in methylene chloride, extracted with H2O and dried over MgS04- After evaporation compound (7) was isolated. [Ref: K.S.P. Bhushana Rao et al., J. Med. Chem. (1985), 28: 1079.]
  • Step C Deacetylvinblastine Acid Azide (8).
  • Deacetylvinblastine monohvdrazide (7) (48 mg, 0.0624 mmol) was dissolved in DMF (3 ml), cooled (-15° C) and acidified to ⁇ 2.5 (pH paper) with HCl/dioxane. Isoamylnitrite (10 ⁇ l) was added followed by an additional 10 ⁇ l after 10 min. HPLC analysis indicated complete conversion of the hydrazide to azide after 5 min. The azide was maintained in solution at -15° C until ready for use.
  • Step D Deacetylvinblastinyl-Leu-Asn-Lys-Ala-Ser-Try-Gln-Ser- Ser-Ser-Leu-NH9* Acetate (5)
  • the oligopeptide product (6) from Step A 32 mg (0.0225 mmol), was dissolved in DMF (1 ml) and cooled (-15° C). To this solution was added a 1.5 ml DMF solution (0.031 mmol) of desacetylvinblastine acid azide (8). The pH was adjusted to - 7.5 (pH paper) with triethylamine and the reaction stirred at -5° C (2 hr), and 0° C for 18 hr. To the reaction was added H2 ⁇ (2 ml) and the solution evaporated to dryness. The intermediate was dissolved in DMF (4 ml), cooled (0° C) and 2 ml of piperidine added. The solution was concentrated to dryness and purified by preparative HPLC as described in Step A. The homogeneous fractions were pooled, concentrated and freeze-dried from H2O to yield (5).
  • Step A Deacetylvinblastinyl-Leu-Asn-Lys(Fmoc)-Ala- - Ser-Trv-Gln-Ser-Ser-Ser-Leu-Dox 'Acetate (9
  • the oligopeptide product (6) prepared as described in Example 9, Step A, (166 mg, 0.125 mmol), was dissolved in DMSO (3 ml) and cooled to -15° C. To this solution was added a DMF solution (0.125 mmol) of desacetylvinblastine acid azide (8) prepared as described in Example 9, Step C. The pH was adjusted to ⁇ 7.5 (pH paper) with triethylamine and the reaction stirred at -15° C for 90 mins.
  • Step B Deacetylvinblastinyl-Leu-Asn-Lys-Ala-Ser-Try- Gln-Ser-Ser-Ser-Leu-Dox 'Acetate (10).
  • Step A Deacetylvinblastine-3-aminopropyl amide (11)
  • Step B Deacetylvinblastine-3-aminopropylamide- norleucine amide (12)
  • Step C Ac-Lvs(Fmoc)-Tyr-Gln-Ser-Ser-Ser-Nle-OH (13)
  • Step D Ac-Lys-Tyr-Gln-Ser-Ser-Ser-Nle-NH-(CH2)3 NH- deacetyl vinblastine amide (14)
  • the conjugates prepared as described in Examples 7-9 were individually dissolved in PSA digestion buffer (12 mM tris(hydroxymethyl)- aminomethane pH8.0, 25 mM NaCl, 0.5 mM CaCl2) and the solution added to PSA at a molar ration of 100 to 1.
  • 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%
  • LNCaP.FGC or DuPRO- 1 cells are trypsinized, resuspended in the growth medium and centifuged for 6 mins. at 200xg. The cells are resuspended in serum-free ⁇ -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 ⁇ -MEM- Matrigel. The suspension is kept on ice until the animals are inoculated.
  • mice 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 5x10$ DuPRO cells or 1.5x10 7 LNCaP.FGC cells.
  • Protocol A One day after cell inoculation the animals are dosed with a 0.1-0.5 mL volume of test conjugate, doxorubicin or vehicle control (sterile water). Dosages of the conjugate and doxorubicin 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.
  • Protocol A One day after cell inoculation the animals are dosed with a 0.1-0.5 mL volume of test conjugate, doxorubicin or vehicle control (sterile water). Dosages of the conjugate and doxorubicin are initially the maximum non- lethal amount, but may be subsequently
  • 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, doxorubicin or vehicle control (sterile water). Dosages of the conjugate and doxorubicin 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.
  • ADDRESSEE DAVID A. MUTHARD
  • Gin Glu His Ser Gin Lys Ala Asn Lys lie Ser Tyr Gin Ser Ser Ser 340 345 350
  • Val Ser Gin Arg Ser lie Tyr Ser Gin Thr Glu Lys Leu Val Ala Gly 370 375 380
  • Lys Ser Gin lie Gin Ala Pro Asn Pro Lys Gin Glu Pro Trp His Gly 385 390 395 400
  • Gly Lys Gly lie Ser Ser Gin Tyr Ser Asn Thr Glu Glu Arg Leu 1 5 10 15
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FRAGMENT TYPE internal
  • Gly Arg Lys Ala Asn Lys lie Ser Tyr Gin Ser Ser Ser Thr Glu Glu 1 5 10 15
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FRAGMENT TYPE internal
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FRAGMENT TYPE internal
  • Lys lie Ser Tyr Gin Ser Ser Ser Thr Glu
  • Ala Asn Lys lie Ser Tyr Gin Ser Ser Ser Thr Glu
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FRAGMENT TYPE internal
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FRAGMENT TYPE internal
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FRAGMENT TYPE internal
  • Ala Asn Lys lie Ser Tyr Gin Ser Ser Ser Ser
  • Lys lie Ser Tyr Gin Ser Ser Ser Thr Glu 1 5 10
  • Gin Leu Asp Asn Lys lie Ser Tyr Gin Ser Ser Ser Thr His Gin Ser 1 5 10 15
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FRAGMENT TYPE internal
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FRAGMENT TYPE internal
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FRAGMENT TYPE internal -83-
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FRAGMENT TYPE internal
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FRAGMENT TYPE internal
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FRAGMENT TYPE internal
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FRAGMENT TYPE internal
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FRAGMENT TYPE internal
  • MOLECULE TYPE peptide *
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FRAGMENT TYPE internal
  • Gin Lys lie Ser Tyr Gin Thr Ser Ser Thr 1 5 10
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FRAGMENT TYPE internal
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FRAGMENT TYPE internal
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FRAGMENT TYPE internal
  • MOLECULE TYPE peptide iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FRAGMENT TYPE internal
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FRAGMENT TYPE internal
  • Gly Lys Gly lie Ser Ser Gin Tyr Ser Asn Thr Asp Glu Arg Leu 1 5 10 15
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FRAGMENT TYPE internal
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FRAGMENT TYPE internal
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FRAGMENT TYPE internal

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Abstract

Nouvelles compositions pharmaceutiques utiles pour traiter l'adénome prostatique bénin, comprenant de nouveaux oligopeptides coupés sélectivement par une activité enzymatique de l'antigène spécifique de la prostate (PSA) et conjugués à un agent cytotoxique. Procédés de traitement de l'adénome prostatique bénin.
PCT/US1996/016490 1995-10-18 1996-10-15 Conjugues utiles pour traiter l'adenome prostatique benin WO1997014416A1 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
CA 2234763 CA2234763A1 (fr) 1995-10-18 1996-10-15 Conjugues utiles pour traiter l'adenome prostatique benin
US09/051,759 US6177404B1 (en) 1996-10-15 1996-10-15 Conjugates useful in the treatment of benign prostatic hyperplasia
EP96936504A EP0855910A4 (fr) 1995-10-18 1996-10-15 Conjugues utiles pour traiter l'adenome prostatique benin
AU74321/96A AU708475B2 (en) 1995-10-18 1996-10-15 Conjugates useful in the treatment of benign prostatic hyperplasia
JP9515930A JP2000506494A (ja) 1995-10-18 1996-10-15 良性前立腺過形成の治療に有用な複合体

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US566495P 1995-10-18 1995-10-18
US60/005,664 1995-10-18
GBGB9602903.8A GB9602903D0 (en) 1996-02-13 1996-02-13 Conjugates useful in the treatment of benign prostatic hyperplasia
GB9602903.8 1996-02-13

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WO1997014416A1 true WO1997014416A1 (fr) 1997-04-24

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JP (1) JP2000506494A (fr)
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WO1998010651A1 (fr) * 1996-09-12 1998-03-19 Merck & Co., Inc. Conjugues utiles dans le traitement du cancer de la prostate
WO1998018493A2 (fr) * 1996-10-30 1998-05-07 Merck & Co., Inc. Conjugues utilises dans le traitement du cancer de la prostate
WO1999028345A1 (fr) * 1997-12-02 1999-06-10 Merck & Co., Inc. Conjugues utiles dans le traitement du cancer de la prostate
US5948750A (en) * 1996-10-30 1999-09-07 Merck & Co., Inc. Conjugates useful in the treatment of prostate cancer
WO2000071571A2 (fr) * 1999-05-14 2000-11-30 Boehringer Ingelheim Pharma Kg Composes anti-tumoraux actives par la proteine fap
US6174858B1 (en) 1998-11-17 2001-01-16 Merck & Co., Inc. Conjugates useful in the treatment of prostate cancer
WO2001030804A2 (fr) * 1999-10-27 2001-05-03 Merck & Co., Inc. Sel d'un conjugue utile pour traiter le cancer de la prostate
WO2002038590A1 (fr) * 2000-11-10 2002-05-16 Boehringer Ingelheim Pharma Gmbh & Co. Kg Composes antitumoraux actives par la proteine fap-alpha
WO2002038591A1 (fr) * 2000-11-10 2002-05-16 Boehringer Ingelheim Pharma Gmbh & Co. Kg Composes anticancereux actives par la fap
WO2003094972A2 (fr) * 2002-05-10 2003-11-20 Boehringer Ingelheim Pharma Gmbh & Co Kg Promedicaments antitumoraux actives par la fap
US6844318B2 (en) 2000-03-15 2005-01-18 Bristol Myers Squibb Pharma Company Peptidase-cleavable, targeted antineoplastic drugs and their therapeutic use
US6855689B2 (en) 1999-05-14 2005-02-15 Boehringer Ingelheim Pharmaceuticals, Inc. Enzyme-activated anti-tumor prodrug compounds
WO2022136586A1 (fr) 2020-12-22 2022-06-30 Cobiores Nv Composés comprenant une fraction tétrapeptidique
WO2022167664A1 (fr) 2021-02-07 2022-08-11 Cobiores Nv Composés comprenant une fraction tétrapeptidique

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Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998010651A1 (fr) * 1996-09-12 1998-03-19 Merck & Co., Inc. Conjugues utiles dans le traitement du cancer de la prostate
WO1998018493A2 (fr) * 1996-10-30 1998-05-07 Merck & Co., Inc. Conjugues utilises dans le traitement du cancer de la prostate
WO1998018493A3 (fr) * 1996-10-30 1998-07-23 Merck & Co Inc Conjugues utilises dans le traitement du cancer de la prostate
US5948750A (en) * 1996-10-30 1999-09-07 Merck & Co., Inc. Conjugates useful in the treatment of prostate cancer
WO1999028345A1 (fr) * 1997-12-02 1999-06-10 Merck & Co., Inc. Conjugues utiles dans le traitement du cancer de la prostate
US6174858B1 (en) 1998-11-17 2001-01-16 Merck & Co., Inc. Conjugates useful in the treatment of prostate cancer
WO2000071571A3 (fr) * 1999-05-14 2001-09-07 Boehringer Sohn Ingelheim Composes anti-tumoraux actives par la proteine fap
WO2000071571A2 (fr) * 1999-05-14 2000-11-30 Boehringer Ingelheim Pharma Kg Composes anti-tumoraux actives par la proteine fap
US6613879B1 (en) 1999-05-14 2003-09-02 Boehringer Ingelheim Pharma Kg FAP-activated anti-tumour compounds
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WO2003094972A2 (fr) * 2002-05-10 2003-11-20 Boehringer Ingelheim Pharma Gmbh & Co Kg Promedicaments antitumoraux actives par la fap
WO2003094972A3 (fr) * 2002-05-10 2004-03-18 Boehringer Ingelheim Pharma Promedicaments antitumoraux actives par la fap
WO2022136586A1 (fr) 2020-12-22 2022-06-30 Cobiores Nv Composés comprenant une fraction tétrapeptidique
WO2022167664A1 (fr) 2021-02-07 2022-08-11 Cobiores Nv Composés comprenant une fraction tétrapeptidique

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AU708475B2 (en) 1999-08-05
AU7432196A (en) 1997-05-07
EP0855910A4 (fr) 2000-07-05
JP2000506494A (ja) 2000-05-30
EP0855910A1 (fr) 1998-08-05

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