Title of Invention

"SOMATOSTATIN PEPTIDE ANALOG"

Abstract The present invention provides a somatostatin analog peptide of the following general formula: X-D-Phe-Cys-Tyr-D-Trp-Al-A2-A3-Thr-NH2 wherein X is Acetyl or straight, branched, or cyclic alkanoyl group from 3 to 18 carbon atoms, or is deleted; A1 = Orn or Lys; A2 = Aib or Deg or Dpg or Ac5c; A3 = Pen or Cys; or a hydrolyzable carboxy protecting group; or pharmaceutically acceptable salt thereof.
Full Text FIELD OF INVENTION:
The present invention relates to somatostatin analog peptides and encompasses the novel peptides that are agonists to somatostatin and their potential use for the treatment of cancer. The invention particularly relates to the design and synthesis of the novel analogs of somatostatin incorporating a,a-dialkylated amino acids in a site specific manner. The invention encompasses methods for generation of these peptides, compositions containing the peptides and the pharmacological applications of these peptides especially in the treatment and prevention of cancer.
BACKGROUND OF THE INVENTION
Somatostatin (SST) is a widely distributed peptide occurring in two forms SST-14 (with 14 ammo acids) and SST-28 (with 28 amino acids). It was originally isolated from the hypothalamus and characterized by Guillemin et al. and is described in U.S.Pat. No. 3,904,594 (Sep. 9, 1975). Somatostatin is found in the gut, pancreas, the nervous system, various exocrine and endocrine glands through the body and in most organs. In normal subjects somatostatin has a broad spectrum of biological activities. It participates in a large number of biological processes where it has the role of an inhibitory factor. It inhibits the release of insulin, prolactin, glucagaon, gastrin, GH, Thyroid stimulating hormone, secretin and cholecystokinin. (S.Reichlm: Somatostatin, N.Eng. J. Med.,309, 1495 and 1556, 1983.)
The mechanism of action of somatostatin is mediated by high affinity membrane associated receptors. Five somatostatin receptors (SSTR1-5) are known. (Reisme, T;Bell,G.I; Endocrine reviews, 1995,16, 427-42 ) All five receptors are heterogeneously distributed and pharmacologically distinct. Somatostain receptors have been found to be over-expressed in a wide range of tumors, those arising in the brain (including meningioma, astrocytoma, neuroblastoma, hypophysical adenoma, paraganglioma, Merkel cell carcinoma, and gliomas), the digestive-pancreatic tract (including insulinoma, gluconoma, AUODoma, VIPoma, and colon carcinoma), lung, thyroid, mammary gland, prostate, lymphatic system (including both Hodgkin's and non-Hodgkin's lymphomas),and ovaries.
One of the most important effects of somatostatin are its growth-inhibiting ability and its capability to influence pathological cell growth It is well known that it exerts an inhibitory effect on the growth of cancer cells both directly and by its antagonizing action on growth factors associated with malignant growth [A V Serially ,Cancer Res.,48,6977,(1988), Taylor, et al Biochem , Biophys Res Commun , 153, 81 (1988)] It has been shown by recent investigations that somatostatin and some somatostatin analogues are capable of activating the tyrosine phosphatase enzyme which antagonizes the effect of tyrosine kinases playing a very important role in the tumorous transformation [A V Schally Cancer Res 48, 6977 (1988)] The importance of tyrosine kinases is supported by the fact that the majority of oncogenes code for tyrosine kinase and the major part of the growth factor receptors is tyrosine kinase [Yarden et al Ann Rev Biochem 57, 443 (1989)]
Native somatostatin has a very short or transient effect in vivo since it is rapidly inactivated by endo- and exopeptidases A large number of novel analogues have been synthesized in order to increase its plasma half life and biological activity Most of the active analogues contain a disulphide bond and a peptide chain shorter than the original one The first cyclic hexapeptide showing the whole effects of somatostatin was synthesized by Veber et al [Nature, 292, 55 (1981)] As a continuation newer and more effective cyclic hexa- and octapeptides have been synthesized which possess the whole spectrum of effects of somatostatin [Veber et al, Life Sci 34, 1371 (1984), Murphy et al , Biochem Biophys Res Commun 132, 922 (1985), Cai et al , Proc Natl Acad Sci USA 83, 1896(1986)]
In spite of the high rates of over expression of somatostatin receptors on a variety of tumors, somatostatin analogues have not gained widespread clinical application for the control of cancer Their current clinical application is primarily in the control of symptoms associated with metastatic carcinoid or VIP-secreting tumors The somatostatin analogues have a wide therapeutic index and seem to be free of major side effects Most of the side effects are gastrointestinal in nature and include minor nausea, bloating, diarrhea, constipation, or steatorrhea Part of the reason for the restricted clinical use may be due to the need for long-term maintenance therapy, the consequent high cost of such therapy, and the variable effects observed in clinical settings
Somatostatin analogues, preparation of such analogues, and uses for such analogues are known in the prior art Such analogues are used in the treatment of certain cancers and other conditions One commercially available product, octreotide, D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol (disulphide bridge between the Cys residue), manufactured by Sandoz, and sold under the trade name Sandostatin, is being used clinically to inhibit tumour growth and and as a diagnostic agent to detect somatostain receptor expressing tumours Of the five receptor sub-types, octreotide and other clinically used somatostatin analogs interact significantly with three of the receptor subtypes, SSTR2, SSTR3 and SSTR5 SSTR2 and SSTR5 have recently been reported to mediate anti-proliferative effects of somatostatin on tumour cell growth, and may therefore mediate the effects of octreotide in humans
A wide variety of somatostatin analogues have been developed These include RC-160, a potent somatostatin analogue originally synthesized by a team at Tulane University headed by Andrew V Schally (Cai R Z, Szoke B, Lu E, Fu D, Redding T W and Schally A V. Synthesis and biological activity of highly potent octapeptide analogues of somatostatin Proc Natl Acad Sci USA, 83 1896-1900, 1986) In recent studies conducted by Schally, among others, the effectiveness of RC-160 in inhibiting the growth of human glioblastomas in vitro and in vivo has been demonstrated (Pinski J, Schally A V, Halmos G, Szepeshazi K and Groot K Somatostatin analogues and bombesin/gastrin-releasing peptide antagonist RC-3095 inhibit the growth of human glioblastomas in vitro and in vivo Cancer Res 54 5895-5901, 1994)
Recent patents that describe somatostatin analogs for treatment of cancer are following
US06025372 (Feb ,2000)
WO00006185A2 (Feb.,2000)
WO09922735A1 (May, 1999)
WO09845285A1 (Oct ,1998)
WO09844921A1 (Oct ,1998)
WO09844922A1 (Oct ,1998)
US05753618 (May, 1998)
US05597894 (Jan, 1997)
EP00344297B1 (May, 1994)
JP05124979A (May ,1993) US04904642 (Feb ,1990)
The aim of the present invention is to synthesize novel somatostatin analogs showing a more advantageous and more selective biological action in comparison to that of known compounds The invention is based on the use of a,a-dialkylated amino acids in the octapeptide analog of somatostatin at position 6 These amino acids are known for inducing conformational constraint. The design of conformationaily constrained bioactive peptide derivatives has been one of the most widely used approaches for the development of peptide-based therapeutic agents Non-standard amino acids with strong conformational preferences may be used to direct the course of polypeptide chain folding, by imposing local stereochemical constraints, in de novo approaches to peptide design The conformational characteristics of a, a-dialkylated amino acids have been well studied The incorporation of these amino acids restricts the rotation of angles, within the molecule, thereby stabilizing a desired peptide conformation The prototypic member of a,a-dialkylated aminoacids, a-aminoisobutyric acid (Aib) or α,α-dimethylglycine has been shown to induce p-turn or helical conformation when incorporated in a peptide sequence [Prasad, BVV and Balaram,P CRC Crit RevBiochem 16,307-347 (1984), Karle,IL and Balaram,P. Biochemistry 29, 6747-6756, (1990)] The conformational properties of the higher homologs of a,a-dialkylated amino acids such as di-ethylglycine (Deg), di-n-propylglycine (Dpg) and di-n-butylglycine (Dbg) as well as the cyclic side chain analogs of a,a-dialkylated amino acids such as 1-aminocyclopentane carboxylic acid (Ac5c), 1-aminocyclohexane carboxylic acid (Ac6c), 1-aminocycloheptane carboxylic acid (Ac7c) and 1-aminocyclooctane carboxylic acid (Ac8c) have also been shown to induce folded conformation [Prasad,S et al, Biopolymers 35, 11-20 (1995) , KarleJ L et al, J Amer Chem Soc 117,9632-9637(1995)1 α,α-Dialkylated amino acids have been used in the design of highly potent chemotactic peptide analogs (Prasad, S et al, Int J Peptide Protein Res 48, 312-318,(1996)]
The present invention exploits the conformational properties of a, a-dialkylated amino acids for the design of biologically active peptide derivatives, taking somatostatin as the model system under consideration The invention is directed to somatostatin analogs containing a, a-dialkylated
amino acids A further object of the invention is the synthesis of somatostatin analogs containing α,α-dialkylated amino acids The inventors have also synthesized peptide derivatives having N-terminal alkanoyl groups of from C2 to C18 carbon atoms which retains anti cancer activity In the present invention, we have also synthesized peptide derivatives having N-terminal alkanoyl groups from C2- C18 carbon atoms, which retain anticancer activity A still further object of the invention is the preparation of the analogs by solid phase peptide synthesis methodology It has been shown that lipophilazation of bioactive peptides improves their stability, bioavailability and the ability to permeate biomembranes (Dasgupta ,P et al, 1999, Pharmaceutical Res 16, 1047-1053; Gozes,I et al, 1996, Proc Natl Acad Sci USA, 93, 427-432)
Through the specification and claims, the following abbreviations are used with the following meanings
BOP - Benzotriazole - 1 - yl - oxy- tris - (dimethylamino) - phosphonium hexo fluoro phospate
PyBOP- -Benzotriazole-1 -yl-oxy-tris-pyrrolidino -phosphonium hexofluorophospate
HBTU-0-Benzotriazole-N,N,N',N'-tetramethyl-uronium-hexofluoro-phosphate
TBTU -2-(lH-Benzotriazole-1 yl)-1,1,3,3,-tetramethyluronium tetrafluoroborate
HOBt - 1 -Hydroxy Benzotriazole
DCC - Dicyclohexyl carbodiimide
DIPCDI - Diisopropyl carbodiimide
DIEA - Diisopropyl ethylamine
DMF - Dimethyl formamide
DCM - Dichloromethane
NMP - N-Methyl-2- pyrrolidinone
TFA - trifluoroacetic acid
In the formula (I) below and throughout the specification, the amino acids residues are designated by their standard abbreviations Amino acids denote L-configuration unless otherwise indicated by D or DL appearing before the symbol and separated from it by hyphen
The following abbreviations are used for uncommon amino acids Orn = Ornithine
Pen = Penicillamine
Aib = α- Aminoisobutyric acid
Deg = α,α- Di-ethyl glycine
Dpg = a,a- Di-n-propyl glycine
Ac5c =1- Aminocyclopentane caboxylic acid
SUMMARY OF THE INVENTION
The present invention comprises of polypeptides of the following general formula (I),
X-D-Phe-Cys-Tyr-D-Trp-Al -A2-A3 -Thr-NH2
wherein X is Acetyl or straight,branched,or cyclic alkonyl group from 4-18 carbon atoms,
or is deleted;
Al= OrnorLys,
A2- Aib or Deg or Dpg or Ac5c;
A3= Pen or Cys,or a hydrolyzable carboxy protecting group, or pharmaceutically acceptable salt of peptide
DETAILED DESCRIPTION OF THE INVENTION:
The present invention comprises of polypeptides of the following general formula (I),
X-D-Phe-Cys-Tyr-D-Trp-Al -A2-A3 -Thr-NH2
wherein X is Acetyl or straight,branched,or cyclic alkonyl group from 4-18 carbon atoms,
or is deleted,
Al= Orn or Lys,
A2=Aib or Deg or Dpg or Ac5c,
A3= Pen or Cys,or a hydrolyzable carboxy protecting group, or pharmaceutically acceptable salt of peptide
A hydrolyzable carboxy protecting are those groups which on hydrolysis converts to carboxylic group such as -CONH2, -COOMe, etc In the case of somatostatin analogs of this invention the carboxylic group is of the Thr amino acid
Preferably the alkyl portion of the alkanoyl group has 4 to 12 carbon atoms Preferred alkanoyl groups are acetyl, n-butanoyl, n-octanoyl, lauroyL, n-hexanoyl, isohexanoyl,cyclohexanoyl, cyclopentanoyl, n-heptanoyl, decanoyl, n-undecanoyl, and 3,7-dimethyloctanoyl
Salts encompassed within the term "pharmacetically acceptable salt" refer to nontoxic salts of the
compounds of this invention Representative salts and esters include the following
Acetate, ascorbate, benzoate, crtrateoxalate, stearate, trifluoroacetate,
succinates,tartarate3lactate,fumarate, gluconate, gfutamate,phosphate/diphosphate, valerate
Other salt include Ca,Li,Mg,Na, andK salts, salts of amino acids such as lysine or arginine; guanidine, ammonium,substituted ammonium salts or aluminium salts.
The salts are prepared by conventional methods
In the formula given above, there is a disulphide bond between the two Cys residues or between Cys and Pen residues to indicate cyclization, in all of the compounds of the invention there is such cyclization, but the Cys-Cys bond or Cys-Pen bond lines are omitted for convenience
The present invention also envisages pharmaceutical compositions comprising the polypeptides described above and processes for their preparation These peptides are agonist to somatostatin and somatostatin related peptides and are useful in the prevention treatment of malignant diseases
The preferred novel somatostatin analogs of the present invention are as follows D-Phe-Cys-Tyr-D-Trp-Ora -Deg-Pen-Thr-NH2 (SEQ ID 2) D-Phe-Cys-Tyr-D-Trp-Orn-Ac5c-Pen-Thr-NH2 (SEQ ID : 3) D-Phe-Cys-Tyr-D-Trp-Orn -Deg-Cys -Thr-NHa (SEQ ID 4) D-Phe-Cys-Tyr-D-Trp-Orn-Ac5c-Cys-Thr-NH2 (SEQ ID 5)
D-Phe-Cys-Tyr-D-Trp-Lys -Ac5c-Pen-Thr-NH2 (SEQ ID 6)
D-Phe-Cys-Tyr-D-Trp-Lys-Ac5c-Cys-Thr-NH2 (SEQ ID 7)
D-Phe-Cys-Tyr-D-Trp-Lys-Aib-Pen-Thr-NH2 (SEQ ID 8)
D-Phe-Cys-Tyr-D-Trp-Orn-Aib-Pen-Thr-NH2 (SEQ ID 9)
D-Phe-Cys-Tyr-D-Trp-Om-Aib-Cys-Thr-NH2 (SEQ ID 10)
D-Phe-Cys-Tyr-D-Trp-Lys-Deg-Cys-Thr-NH2 (SEQ ID 11)
D-Phe-Cys-Tyr-D-Trp-Lys-Deg-Pen-Thr-NH2 (SEQ ID 12)
D-Phe-Cys-Tyr-D-Trp-Orn-Dpg-Pen-Thr-NH2 (SEQ ID 13) D-Phe-Cys-Tyr-D-Trp-Orn -Dpg-Cys -Thr-NH2 (SEQ ID 14) Acetyl-D-Phe-Cys-Tyr-D-Trp-Orn-Deg-Pen-Thr-NH2 (SEQ ID 15) Butanoyl-D-Phe-Cys-Tyr-D-Trp-Orn -Deg-Pen-Thr-NH2 (SEQ TD 16) Octanoyl-D-Phe-Cys-Tyr-D-Trp-Orn-Deg-Pen-Thr-NH2 (SEQ ID 17)
The novel compounds of the present invention have important pharmacological applications They are potent anti-neoplastic agents and thereby possess therapeutic potential in a number of human cancers
Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose
The term " an effective amount" means the amount of the drug or pharmaceutical agent that will elicit the biological or medical response of a tissue system, animal or human that is being sought
Suitable routes of administration are those known in the art and include, oral, rectal, transdermal, vaginal, transmocosal, or intestinal administration, parenteral delivery, including intramuscular, subcutaneous, intradedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections
In addition to the active ingredients, these pharmaceutical compositions may contain suitable pparmaceutically acceptable carriers, excipients, diluents, solvents, flavorings, colorants etc The
preparations may be formulated in any form including but not limited to tablets, dragees, capsules, powders, syrups, suspensions, slurries, time release formulations, sustained release formulations, pills, granules, emulsions, patches, injections, solutions, liposomes and nanoparticles
The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition
Toxicity and therapeutic efficacy of the peptides of this invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals
Synthesis of peptides
The novel peptide analogs embodied in the present invention contain amino acids, namely α,α dialkylated amino acids, which are known to induce highly specific constraints in the peptide backbone The α,α dialkylated amino acids were synthesized from the appropriate ketones These ketones were first converted into their corresponding hydantoins, which on hydrolysis with strong acid or alkali gave the respective amino acids
The novel peptides of the present invention can be made by exclusively, solid phase techniques, by a combination of solution phase procedures and solid phase techniques, or, by fragment condensation These methods for the chemical synthesis of polypeptides are well known in the art (Stewart and Young, 1969) Preferred, semi-automated, stepwise solid phase methods for synthesis of peptides of the invention are provided in the examples discussed in a subsequent section of this document The applicants are also not aware of any prior art describing the synthesis of the novel somatostatin analogs incorporatingα,α-dialkylated amino acids, as encompassed in the present invention
In a preferred embodiment of the present invention the peptides were synthesized using Fmoc strategy, either manually, or on a semi- automatic peptide synthesizer (CS Bio, Model 536), using optimum side chain protection The peptides were assembled from Oterminus to N-terminus Peptides amidated at the carboxy-terminus were synthesized using the Rink Amide resin The loading of the first Fmoc protected amino acid was achieved via an amide bond formation with the solid support, mediated by Diisopropylcarbodiimide (DIPCDI) and HOBt Substitution levels for
automated synthesis were preferably between 0 2 and 0 8 mmole amino acid per gram resin The steps involved in the synthesis of the somatostatin analogs employed the following protocol
TABLE I
(Table Removed)
The resin employed for the synthesis of carboxy-terminal amidated peptide analogs was 4-(2', 4'-
Dimethoxyphenyl-Fmoc-aminomethyl)-phenoxymethyl-derivatized polystyrene 1 %
divinylbenzene (Rink Amide) resin (100-200 mesh), procured from Advanced Chemtech, Louisville, KY, U S A, (0 7 milli equivalent NH sub 2 /g resin)
In a particularly preferred embodiment of the present invention the following chemical moieties were used to protect reactive side chains of the peptides during the synthesis procedure
The N-terminal amino group was protected by 9-flourenylmethoxycarbonyl (Fmoc) group The tryptophan residue was either left unprotected or used with Boc protection The side chain amino
group of Lysine and Ornithine was protected using Boc group, preferrably Threonine and Tyrosine residues were used with t-Butyl (t-Bu) protection Trityl or Acetamidomethyl (Acm) were the preferred protecting groups for Cysteine and Penicillamine was preferrably protected with the Acetamidomethyl (Acm) group.
In a preferred embodiment of the invention, 2-8 equivalents of Fmoc protected amino acid per resin nitrogen equivalent was used The activating reagents used for coupling amino acids to the resin, in solid phase peptide synthesis, are well known in the art These include BOP, PyBOP, HBTU, TBTU, PyBOP, HOBt Preferably, DCC or DIPCDI / HOBt or HBTU/HOBT and DIEA were used as activating reagents in the coupling reactions The protected amino acids were either activated m situ or added in the form of pre-activated esters known in the art such as NHS esters, Opfp esters etc The coupling reaction was carried out in DMF, DCM or NMP or a mixture of these solvents and was monitored by Kaiser test [Kaiser et al, Anal Biochem, 34, 595-598 (1970)] In case of a positive Kaiser test, the appropriate amino acid was re-coupled using freshly prepared activated reagents
After the assembly of the peptide chain was completed, disulfide bond formation was either carried out on-resin, where the Acm side-chain protecting groups were removed using Iodine in Dimethylformamide, and the free thiol groups thus generated were oxidized simultaneously to yield the cyclized peptide This was followed by the removal of the amino- terminal Fmoc group using steps 1-6 of the above protocol The peptide-resin was then washed with methanol and dried Where post- cleavage disulphide formation was carried out, the N- terminal Fmoc group was removed and the peptide was cleaved from the resin support by treatment with a cleavage mixture consisting of trifluoroacetic acid, ethanedithiol and de-ionized water for lto 4 hours at room temperature Normally, the cleavage mixture also simultaneously removed the side-chain protecting groups, except for the side-chain protecting Acm groups that were not removed by the above procedure The crude peptide was obtained by precipitation with cold dry ether, filtered, dissolved, and lyophilized Where disulfide formation was carried out on the crude Acm-protected peptide, the Acm group was removed by any of the known methods such as using Thallium trifluoroacetate, iodine etc
The resulting crude peptide was purified by preparative high performance liquid chromatography (HPLC) using a LiChroCART® C18 (250 Times 10) reverse phase column (Merck, Darmstadt, Germany) on a Preparative HPLC system (Shimadzu Corporation, Japan) using a gradient of 0 1% TFA in acetonitrile and water The eluted fractions were reanalyzed on Analytical HPLC system (Shimadzu Corporation, Japan) using aC18 LiChrospher®, WP-300 (300 X 4) reverse- phase column Acetonitrile was evaporated and the fractions were lyophilized to obtain the pure peptide The identity of each peptide was confirmed by mass spectra
The present invention will be further described in detail with reference to the following examples, as will be appreciated by a person skilled in the art, is merely illustrative, and should not be construed as limiting Various other modifications of the invention will be possible without departing from the spirit and scope of the present invention
Example: 1
Synthesis of SEP ID NO 2 D-Phe-Cys-Tyr-D-Trp-Orn-Deg-Pen-Thr-NH2
First loading on Rink Amide Resin
A typical preparation of the Fmoc-Thr-Rink Amide Resin was carried out using 0 5g of 4-(2',4'-Dimethoxyphenyl- Fmoc- aminomethyl) phenoxymethyl- derivatized polystyrene 1% divinylbenzene (Rink Amide ) resin, (0.7 milliequivalent NH sub 2 /g resin), (100-200 mesh), procured from Advanced Chemtech, Louisville, KY, U.S.A.Swelling of the resin was typically carried out in dichloromethane measuring to volumes 10-40ml /g resin The resin was allowed to swell in methylene chloride (2 X 25 ml, for 10 min ) It was washed once in dimethylformamide (DMF) for 1 min All solvents in the protocol were added in 20 ml portions per cycle The Fmoc-protecting group on the resin was removed by following steps 3-7 in the protocol The deprotection of the Fmoc group was checked by the presence of blue beads in Kaiser test For loading of the first amino acid on the free amino (NH2) group of the resin, the first amino acid was weighed in three to six fold excess in the amino acid vessel of the peptide synthesizer This was dissolved in dimethylformamide (ACS grade) (J T Baker, Phillipsburg, New Jersey, US A ) and activated with DIPCDI, just prior to the addition to the resin in the reaction vessel of the
peptide synthesizer For difficult couplings, alternative BOP/DTEA, HBTU/DIEA couplings were carried out The coupling reaction was carried out for a period ranging from 1-3 hours The loading of the amino acid on the resin was confirmed by the presence of colorless beads in the Kaiser Test Recoupling was carried out for incomplete reactions The loading efficiency was ascertained by the increase of weight of the resin after the addition of the amino acid
The peptide sequence was assembled by subsequent deprotection and coupling cycles, as mentioned earlier in the protocol
The synthesis Qf SEQ.ID NO 1 was started on 0 5s scale Upon completion of synthesis and
removal of the N-terminaT Tmoc protecting group (steps l-o of the synthesis cycle), the peptide-
resin was washed twice with methanol, dried and weighed to obtain 0 694g This was subjected to
cleavage in a cleavage mixture consisting of trifluoroacetic acid and scavengers, ethanedithol and
water for a period of 1-4 hours at room temperature with continuous stirring The peptide was
precipitated using cold dry ether to obtain 172mg of the crude peptide
Disulfide bond formation in the crude peptide was carried out in Iodine in methanol (3X to 12X molar excess of iodine for 5 min to lhr) and excess of Iodine was removed with sodium thiosulfate or ascorbic acid or extraction with CCl4 after evaporation of methanol on rotavapour
The crude, cyclised peptide was purified on a C18 preparative reverse phase HPLC column (250X10) on a gradient system comprising of acetonitrile and water in 0 1% TFA as described previously. The prominent peak was collected and lyophilized, reanalysed on analytical HPLC and subjected to mass spectrometry There was a good agreement between the observed molecular weight and calculated molecular weight (Calculated mass = -1073 , Observed Mass= 1074.1 ) The pure peptide was then used for bioassay
Example :2
Synthesis of Analog SEQ ID NO 3 D-Phe-Cys-Tyr-D-Trp-Orn-Ac5c-Pen-Thr-NH2
On a 0 5g scale of resin, 0 69g of peptide-resin was obtained post- deprotection of the N-terminal Fmoc group After cleavage and lyophilization, 236 mg of the crude peptide was obtained Disulfide formation and purification steps were carried out as in the examples above The calculated mass of the pure peptide was —1071 and the observed mass was 1073 1
Example :3
Synthesis of analog SEP ID NO 4 D-Phe-Cys-Tyr-D-Trp-Orn-Deg-Cys-Thr-NFb
On 0 5g scale of resin, 0 632g of peptide-resin was obtained post-deprotection of the N-terminal Fmoc group After cleavage and lyophilization, 268 mg of the crude peptide was obtained Disulfide formation and purification steps were carried out as in the examples above The calculated mass of the pure peptide was -1045 and the observed mass was 1047 1
Example :4
Synthesis of Analog SEQ ID NO 5 D-Phe-Cys-Tyr-D-Trp-Orn-Ac5c-Cys-Thr-NH2
On 0 5g scale of resin, 0 685g of peptide-resin was obtained post-deprotection of the N-terminal Fmoc group After cleavage and lyophilization, 326 mg of the crude peptide was obtained Disulfide formation and purification steps were carried out as in the examples above The calculated mass of the pure peptide was —1043 and the observed mass was 1044 8
Example :5
Synthesis of Analog SEQ ID NO 6
D-Phe-Cys-Tyr-D-Trp-Lys-AcSc-Pen-Thr-NHa
On 0 5g scale of resin, 0 794g of peptide-resin was obtained post-deprotection of the N-terminal Fmoc group After cleavage and lyophilization, 288 mg of the crude peptide was obtained Disulfide formation and purification steps were carried out as in the examples above The calculated mass of the pure peptide was -4086 and the observed mass was 1087 2
Example :6
Synthesis of Analog SEQ ID NO 7 D-Phe-Cys-Tyr-D-Trp-Lys-Ac5c-Cys-Thr-NH2
On 0 5g scale of resin, 0.770g of peptide-resin was obtained post-deprotection of the N-terminal Fmoc group After cleavage and lyophilization, 268 mg of the crude peptide was obtained Disulfide formation and purification steps were carried out as in the examples above The calculated mass of the pure peptide was ~1058 and the observed mass was 1059 0
Example :7
Synthesis of Analog SEQ ID NO 9 D-Phe-Cys-Tyr-D-Trp-Orn-Aib-Pen-Thr-NH2
On 0 5g scale of resin, 0 747g of peptide-resin was obtained post-deprotection of the N-terminal Fmoc group After cleavage and lyophilization, 218 mg of the crude peptide was obtained Disulfide formation and purification steps were carried out as in the examples above The calculated mass of the pure peptide was ~1046 and the observed mass was 1047 2
Example :8
Synthesis of Analog SEQ ID NO 10 D-Phe-Cys-Tyr-D-Trp-Om-Aib-Cys-Thr-NFfc
On 0 5g scale of resin, 0 762g of peptide-resin was obtained post-deprotection of the N-terminal Fmoc group After cleavage and lyophilization, 325 mg of the crude peptide was obtained Disulfide formation and purification steps were carried out as in the examples above The calculated mass of the pure peptide was -1018 and the observed mass was 1019 4
Example :9
Synthesis of Analog SEQ ID NO 14.
D-Phe-Cys-Tyr-D-Trp-Orn-Dpg-Cys-Thr-NHs
On 0 5g scale of resin, 0 752g of peptide-resin was obtained post-deprotection of the N-terminal Fmoc group After cleavage and lyophilization, 339 mg of the crude peptide was obtained Disulfide formation and purification steps were carried out as in the examples above The calculated mass of the pure peptide was-1074 and the observed mass was
1075 0
Example: 10
Invitro cytotoxicity of synthesized peptide analogs
The cytotoxic effect peptide SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 6 and SEQ ID NO. 9 was studied by MTT assay which is based on the principle of uptake of MTT [3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide], a tetrazolium salt by the metabolically active cells where it is metabolized by active mitochondria into a blue colored formazan product which can be read spectrophotometrically Briefly, tumor cells PTC (primary human colon cancer ceil line), KB (Oral squamous), U87MG (Glioblastoma), HBL100 (Breast), HeP2 (Laryngeal), ECV304 (Endothelial), PA-1 (Ovary) and LI32 (Lung) were incubated with the peptide analogs for 48 hours at 37°C in a 96-well culture plate, followed by the addition of 100 ug MTT and further incubation of 1 hour The formazan crystals formed inside the cells were dissolved with a detergent comprising of 10% Sodium dodecyl sulfate and 0 01 N HC1 and optical density read on a multiscan ELISA reader The optical density was directly proportional to the number of proliferating and metabolically active cells Percent cytotoxicity of peptide analogs is shown in the following Table
SEQ ID NO 2
(Sequence Removed)
SEQ ID NO 3
(Sequence Removed)
SEQ ID NO 6
(Sequence Removed)
SEQIDNO 9
(Sequence Removed)
Example :11
In vivo antitumour activity of the novel synthesized Somatostatin analogs on primary tumor (colon) xenografted mice
The antitumor activity of SEQ ID NO 2 and SEQ ID NO 3 was studied in human colon adenocarcinoma (PTC) xenografts in nude mice PTC tumor xenografts were grown in Balb/c athymic mice by subcutaneous inoculation of a single cell suspension of PTC cells (15 X 106 cells/100 µL) The tumor bearing animals were divided into 3 groups of three animals each including one group comprising untreated control animals Treatment with novel somatostatin analogs was initiated when the average tumor volumes, as measured using a vernier caliper, were between 400 - 800 mm3 Solutions of SEQ ID NO 2 and SEQ ID NO 3 were prepared at a concentration of 85µg/ml and intravenously administered to the assigned group of tumor bearing animals at a dose of 8 5 µg/100 u,L twice a day so that the total dose of 17µg/day was administered to each animal The treatment was continued for a period of 10 days
The antitumor activity of the compounds was monitored by measuring tumor volumes every fourth day using the formula W*W*L*0 4 (W = smaller diameter, L = larger diameter) The percentage inhibition of tumor growth was calculated using the formula (1- tumor volume-treated /
tumor volume-control) * 100 Following table shows the tumor volumes of individual animals measured till day 21 post-inoculation Figure 1 shows the tumor kinetics till day 21 in the treated and untreated animals All three peptides showed a significant antitumor activity on PTC xenografts The percentage inhibition of tumor growth caused by SEQ ID NO 2 and SEQ ID NO 3 peptides as compared to controls on day 21 was 57 1% and 54 7% respectively
TABLE Tumor volumes (mm ) of individual tumor bearing animals of treated and control groups
(Table Removed)







We Claim:
1. A somatostatin analog peptide of the following general formula:
X-D-Phe-Cys-Tyr-D-Trp-Al-A2-A3-Thr-NH2
wherein X is Acetyl or straight, branched, or cyclic alkanoyl group from 3 to 18 carbon
atoms, or is deleted;
A1 = Orn or Lys;
A2 = Aib or Deg or Dpg or Ac5c;
A3 = Pen or Cys;
or a hydrolyzable carboxy protecting group; or pharmaceutically acceptable salt thereof.
2. A somatostatin analog peptide as claimed in claim 1, wherein X is deleted and Al=Orn, A2=Deg and A3=Pen; and the said compound is D-Phe-Cys-Tyr-D-Trp-Om-Deg-Pen-Thr~NH2 (SEQ ID NO:2)
3. A somatostatin analog peptide as claimed in claim 1, wherein X is deleted and Al=Orn, A2-Ac5c and A3=Pen; and the said compound is D-Phe-Cys-Tyr-D-Trp-Orn-Ac5c-Pen-Thr-NH2 (SEQ ID NO:3)
4. A somatostatin analog peptide as claimed in claim 1, wherein X is deleted and Al =Orn, A2=Deg and A3=Cys; and the said compound is D-Phe-Cys-Tyr-D-Trp-Orn-Deg-Cys-Thr-NH2 (SEQ ID NO:4)
5. A somatostatin analog peptide as claimed in claim 1, wherein X is deleted and Al=Orn, A2=Ac5c and A3=Cys; and the said compound is D-Phe-Cys-Tyr-D-Trp-Orn-Ac5c-Cys-Thr-NH2 (SEQ ID NO:5)
6. A somatostatin analog peptide as claimed in claim 1, wherein X is deleted and Al=Lys, A2=Ac5c and A3=Pen; and the said compound is D-Phe-Cys-Tyr-D-Trp-Lys-Ac5c-Pen-Thr-NH2 (SEQ ID NO:6)
7. A somatostatin analog peptide as claimed in claim 1, wherein X is deleted and Al=Lys, A2=Ac5c and A3=Cys; and the said compound is D-Phe-Cys-Tyr-D-Trp-Lys-Ac5c-Cys-Thr-NH2 (SEQ ID NO:7).
8. A somatostatin analog peptide as claimed in claim 1, wherein X is deleted and Al=Lys, A2=Aib and A3=Pen; and the said compound is D-Phe-Cys-Tyr-D-Trp-Lys-Aib-Pen-Thr-NH2 (SEQ ID NO:8).
9. A somatostatin analog peptide as claimed in claim 1, wherein X is deleted and Al=Orn,
A2=Aib and A3=Pen; and the said compound is D-Phe-Cys-Tyr-D-Trp-Orn-Aib-Pen-Thr-
NH2 (SEQ ID NO:9)
10. A somatostatin analog peptide as claimed in claim 1, wherein X is deleted and Al=Orn,
A2=Aib and A3=Cys; and the said compound is D-Phe-Cys-Tyr-D-Trp-Om-Aib-Cys-Thr-
NH2 (SEQ ID NO:10)
11. A somatostatin analog peptide as claimed in claim 1, wherein X is deleted and Al=Lys,
A2=Deg and A3=Cys, and the said compound is D-Phe-Cys-Tyr-D-Trp-Lys-Deg-Cys-Thr-
NH2(SEQIDNO:ll).
12. A somatostatin analog peptide as claimed in claim 1, wherein X is deleted and Al=Lys, A2=Deg and A3=Pen; and the said compound is D-Phe-Cys-Tyr-D-Trp-Lys-Deg-Pen-Thr-NH2 (SEQ ID NO: 12).
13. A somatostatin analog peptide as claimed in claim 1, wherein X is deleted and Al=Orn, A2=Dpg and A3=Pen; and the said compound is D-Phe-Cys-Tyr-D-Trp-Om-Dpg-Pen-Thr-NH2 (SEQ ID NO:13)
14. A somatostatin analog peptide as claimed in claim 1, wherein X is deleted and Al=Orn, A2=Dpg and A3=Cys; and the said compound is D-Phe-Cys-Tyr-D-Trp-Orn-Dpgc-Cys-Thr-NH2 (SEQ ID NO: 14).
15. A somatostatin analog peptide as claimed in claim 1, wherein X is Acetyl, Al=Om, A2=Deg and A3=Pen; and the said compound is Acetyl-D-Phe-Cys-tyr-D-Trp-Om-Deg-Pen-Thr-NH2 (SEQ ID NO: 15)
16. A somatostatin analog peptide as claimed in claim 1, wherein X is Butanoyl, Al-Orn, A2-Deg and A3=Pen; and the said compound is Butanoyl-D-Phe-Cys-Tyr-D-Trp-Orn-Deg-Pen-Thr-NH2 (SEQ ID NO: 16).
17. A somatostatin analog peptide as claimed in claim 1, wherein X is Octanoyl, Al-Orn, A2-Deg and A3=Pen; and the said compound is Octanoyl-D-Phe-Cys-Tyr-D-Trp-Orn-Deg-Pen-Thr-NH2 (SEQ ID NO:17).
18. A solid phase synthesis process for the preparation of a somatostatin analog peptide analog of general formula (I):
X-D-Phe-cys-Tyr-D-Trp-Al-A2-A3-Thr-NH,
wherein X is Acetyl or straight, branched, or cyclic alkanoyl group from 4 to 18 carbon
atoms, or is deleted;
A1 = Orn or Lys;
A2 = Aib or Deg or Dpg or Ac5c;
A3 = Pen or Cys;
which comprises sequentially loading the corresponding protected a- a-dialkylated amino
acids in sequential cycles to the amino terminus of a solid phase resin, couping the amino
acids in the presence of conventional solvents and reagents to assemble a peptide-resin
assembly, removing the protecting groups and cleaving the peptide from the resin to obtain
a crude peptide analog.
19. A process as claimed in claim 18, wherein said a- a-dialkylated amino acids are protected at
their a- amino groups by a 9-fluorenyl methoxy carbonyl (F moc) group.
20. A process as claimed in claim 18, wherein the coupling was carried out in the presence of
activated agents selected from the group consisting of BOP, PyBOP, HBTU, TBTU,
HOBt.
21. A process as claimed in claim 18, -wherein the coupling was carried out in the presence of a
solvent selected from the group consisting of DMF, DCM, NMP or any mixtures thereof
22. A process as claimed in claim 18 wherein said crude peptide is cleaved from said peptide-
resin assembly by treatment with trifluoroacetic acid, ethanedithiol and deionised water for
1 to 4 hours at room temperature.
23. A somatostatin analog peptide substantially as herein described with reference to the
foregoing examples.
24. A solid phase synthesis process for the preparation of a somatostatin peptide analog
substantially as herein described with reference to the foregoing examples.

Documents:

705-del-2000-Abstract-(20-12-2011).pdf

705-del-2000-abstract.pdf

705-del-2000-assignment.pdf

705-del-2000-Claims-(20-12-2011).pdf

705-del-2000-claims.pdf

705-del-2000-complete specification(granted).pdf

705-del-2000-Correspondence Others-(17-08-2011).pdf

705-DEL-2000-Correspondence Others-(29-06-2011).pdf

705-del-2000-Correspondence-Others-(20-12-2011).pdf

705-del-2000-correspondence-others.pdf

705-del-2000-correspondence-po.pdf

705-del-2000-description (complete).pdf

705-del-2000-form-1.pdf

705-del-2000-form-19.pdf

705-del-2000-form-2.pdf

705-del-2000-Form-3-(17-08-2011).pdf

705-del-2000-form-3.pdf

705-del-2000-form-6.pdf

705-del-2000-gpa.pdf

705-del-2000-petition-137.pdf

705-del-2000-petition-138.pdf


Patent Number 251903
Indian Patent Application Number 705/DEL/2000
PG Journal Number 16/2012
Publication Date 20-Apr-2012
Grant Date 16-Apr-2012
Date of Filing 31-Jul-2000
Name of Patentee DABUR PHARMA LIMITED
Applicant Address 3 FACTORY ROAD, ADJACENT TO SAFDARJUNG HOSPITAL, NEW DELHI-110029, INDIA.
Inventors:
# Inventor's Name Inventor's Address
1 BURMAN, ANAND C. 22, SITE IV, SAHIBABAD, GHAZIABAD 201 010, UTTAR PRADESH, INDIA
2 PRASAD, SUDHANAND 22, SITE IV, SAHIBABAD, GHAZIABAD 201 010, UTTAR PRADESH, INDIA
3 MUKHERJEE, RAMA 22, SITE IV, SAHIBABAD, GHAZIABAD 201 010, UTTAR PRADESH, INDIA
4 JAGGI, MANU 22, SITE IV, SAHIBABAD, GHAZIABAD 201 010, UTTAR PRADESH, INDIA
5 SINGH, ANU, T. 22, SITE IV, SAHIBABAD, GHAZIABAD 201 010, UTTAR PRADESH, INDIA
6 MATHUR, ARCHNA 22, SITE IV, SAHIBABAD, GHAZIABAD 201 010, UTTAR PRADESH, INDIA
PCT International Classification Number A61K 47/00
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA