Title of Invention

"NOVEL PEPTIDE ANALOG OF SUBSTANCE P CONTAINING αα-DIALKYLATED AMINO ACIDS"

Abstract The present invention provides a novel peptide analog of Substance P of the following general formula X-D-Arg-Pro-Lys-Pro-D-Phe-Gln-D-Trp-Phe-D-Trp-Leu-R-NH2 wherein X is acetyl or straight, branched, or cyclic alkonyl group from 3-18 carbon atoms, or is deleted; R is Aib or Deg or Dpg or Ac5c or Ac6c; or a hydrolysable carboxy protecting group or pharmaceutically acceptable salts.
Full Text FIELD OF INVENTION
The present invention provides novel peptide analogs of substance P useful in the treatment of cancer The present invention encompasses novel synthetic peptide analogs that are antagonists to Substance P, substance P like peptides and related peptides and are useful for the treatment of cancer The invention particularly relates to the design and synthesis of the novel substance P antagonist analogs incorporating alpha , alpha -dialkylated amino acids in a site specific manner The invention encompasses methods for the generation of these peptides, compositions containing these peptides and pharmacological applications of these peptides specifically in the treatment and prevention of cancer BACKGROUND OF THE INVENTION
Substance P is one of the main members of the tachykinin family The tachykinins are mammalian regulatory peptides and are present in the central and peripheral nervous systems, and in gut endocrine cells Substance P was the first gut neuropeptide to be discovered It is a 11 residue neuropeptide of the following sequence Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH sub 2 (SEQ ID NO 1) Substance P regulates gastrointestinal motility, increases blood flow in the gut, stimulates secretion of the pancreas, salivary glands, small intestines and inhibits acid secretion In the central nervous system, tachykinins play a role in the sensory nervous pathways and in motor control. (Dockray, G J, 1994, 401 Gut peptides Biochemistry and Physiology, Raven Press Ltd, New York)
Substance P role in cancer has been well recognized particularly in small cell lung cancer Small cell lung cancer (SCLC) cell growth is sustained by multiple autocrine and paracrine growth loops involving neuropeptides In the search for novel antiproliferative agents for small cell lung cancer the first substance P antagonist to be studied was [D-Arg1, D-Pro2, D-Trp7'9, Leu11] substance P (Antagonist A) Substance P is structurally unrelated to bombesin/GRP and has no bombesin/GRP antagonist activity, antagonist A was found to block the secretory effects of bombesin on a pancreatic preparation, diminish bombesin/GRP binding to its receptor and inhibit mitogenesis in Swiss 3T3 cells (Jensen et al, 1984, Nature, 309, 61-63, Zachary and Rozengurt,
1986, Biochem Biophys Res Commun., 137,135-141) However it did not affect mitogenesis induced by polypeptide growth factors, such as platelet-derived growth factor and epidermal growth factor Among other congeners of substance P tested for bombesin/GRP antagonism, two compounds with inhibitory activity were identified antagonist D, [D-Argl, D-Phe5,D-Trp7,9,Leu11] substance P, and antagonist G [Arg6;D-Trp7'9,MePhe8] substance P(6-ll) Antagonist D was shown to be five fold more potent than antagonist A in preventing the cellular effects of bombesin/GRP and vasopressin in mouse 3T3 cells and in inhibiting the growth of SCLC cells in serum-free medium Overall, in Swiss 3T3 cells both antagonists were demonstrated to have in common the ability not only to inhibit the effects of bombesin/GRP but also the effects of other neuropeptides, including vasopressin, bradykinin, endothelin, and substance P This is also reflected by data showing that antagonist G is ten fold less potent than antagonist D in blocking bombesin/GRP mediated mitogenesis in Swiss 3T3 cells and it is almost as potent as antagonist D in inhibiting SCLC proliferation in vitro Antagonist D inhibited proliferation of H-510 and H-69 SCLC cells in liquid culture and in semi-solid media (IC50=5uM) Colony formation stimulated by multiple neuropeptides including vasopressin and bradykinin was also blocked by Antagonist D [Seckl, M J et al, 1997, Can Res,57(l) 51-4] In addition, antagonist G showed inhibition of SCLC xenografts in vivo [Woll PJ and Rozengurt,E, 1990, Can Res, 50(13)3968-73, Everard,M.J, et al,1993, Eur J Cancer, 29A(10) 1450-3, Br J Cancer,1992, 65(3)388-92] Reeve,JG and Bleehen, NM [Biochem Biophys Res Commun, 1994, 199(3) 1313-19] found that treatment of lung tumour cells with Antagonist D caused a concentration-dependent loss of cell viability which was accompanied by the onset of apoptosis as defined by cytological criteria and DNA fragmentation
Short-chain SP antagonist viz pHOPA-D-Trp-Phe-D-Trp-Leu-Leu-NH2 (Analog R) (SEQ ID NO 2) and its analogs were studied by the Hungarian group and were found to inhibit the proliferation of H69 SCLC cells both in vitro and in xenografts in vivo in nude mice [Int J Cancer, 1995,60(1) 82-7] In a further extension of the work,the C-terminal peptide bond was replaced by a methylene-amino (pseudopeptide ) bond Substance P analogues D-MePhe-D-Trp-Phe-D-Trp-Leu(psi)-(CH2NH)-Leu-NH2) Analog 6(SEQ ED NO 3) and D-MePhe-D-Trp-Phe-D-Trp-Leu-MPA Analog 7(SEQ ID NO 4), inhibited SCLC proliferation more effectively than Analog R (6 IC50=2u,M, 7 IC50=5[iM and R IC50=10uM) Moreover, 6 inhibited the
respiratory activity of SK-MES 1 epithelial type of lung carcinoma cells in proliferating but not in the quiescent state suggesting that the anti-proliferative effect of these compounds is not due to simple cytotoxicity and these short chain SP analogues may be promising candidates as therapeutic agents in the treatment of SCLC [Nyeki, O, et al, 1998, J Pept Sci, 4(8) 486-95] Antagonist D and its role in cancer has been described in the US patent 5,434,132 and WO88/07551
The present invention describes the preparation and use of peptide analogs of substance P especially the Antagonist D of substance P using constrained amino acids and their use thereof for cancer therapy, alone, or in combination or as an adjunct to cancer chemotherapy
The design of conformationally constrained bioactive peptide derivatives has been one of the 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 α,α-dialkylated aminoacids, a-aminoisobutyric acid (Aib) or a,oc-dimethyl glycine has been shown to induce P-turn or helical conformation when incorporated in a peptide sequence (Prasad and Balaram, 1984, CRC Crit Rev Biochem 16, 307-347, Karle and Balaram, 1990 Biochemistry,29, 6747-6756) 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 ct,a-dialkylated amino acids such as 1 -aminocyclopentane carboxylic acid (Ac5c), 1-aminocyclohexane carboxylic acid (Ac6c), as 1-aminocycloheptane carboxylic acid (Ac7c) and as 1-aminocyclooctane carboxylic acid (Ac8c) have also been shown to induce folded conformation (Prasad,S et al, 1995 Bioploymers,35, 11-20, Karle,I L , et a/, 1995, J Amer
Chem Soc, 117, 9632-9637) a,a-Dialkylated amino acids have been used in the design of highly potent chemotactic peptide analogs (Prasad, S et al, 1996 Int J Peptide Protein Res 48,312-318 The present invention exploits the conformational properties of such a, a-dialkylated amino acids for the design of biologically active peptide derivatives of substance P with specific anticancer activity Furthermore in the prior art it has been shown that lipophilazation of bioactive peptides improves the stability, bioavailability and 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 )
The present invention also includes synthesized peptide derivatives having N-terminal alkanoyl groups from 2-18 carbon atoms with improved anticancer activity
Abbreviations and Symbols
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 in this application
Aib = a- Aminoisobutyric acid
Deg = ct,a- Di-ethyl glycine
Dpg = a,a- Di-n-propyl glycine
Ac5c =1- Aminocyclopentane caboxylic acid
Ac6c = 1 - Aminocyclohexane caboxylic acid
BOP - Benzotriazole-l-yl-oxy-tris-(dimethylamino)-phosphonium hexofluorophospate
PyBOP -Benzotriazole-1-yl-oxy-tris-pyrrolidino -phosphonium hexofluorophospate
HBTU -0-Benzotriazole-N,N,N',N'-tetramethyl-uronium-hexofluoro-phosphate
TBTU-2-(lH-Benzotriazole-lyl)-l,l,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
SUMMARY OF THE INVENTION
The present invention comprises of polypeptides of the following general formula (I),
X- D-Arg-Pro-Lys-Pro-D-Phe-Gln-D-Trp-Phe-D-Trp-Leu-R- NH2
wherein,
X is Acetyl or straight,branched,or cyclic alkanoyl group from 3-18 carbon atoms, or is deleted,
R= Aib or Deg or Dpg or Ac5c or Ac6c,
or a hydrolyzable carboxy protecting group or R is hydrolyzable carboxy protecting group, or apharmaceutically acceptable salt of the peptide
A hydrolyzable carboxy protecting are those groups which on hydrolysis converts to carboxylic group such as -CONH2, -COOMe, etc
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
It also comprises the fragment of the above peptides having the general formula,
D-Phe-Gln-D-Trp-Phe-D-Trp-Leu-R-NH2
wherein ,
R= Aib or Deg or Dpg or Ac5c or Ac6c;
or R is a hydrolyzable carboxy protecting group or a pharmaceutically acceptable salt of the peptide
Salts encompassed within the term "pharmaceutically acceptable salt" refer to nontoxic salts of the
compounds of this invention Representative salts and esters include the following
Acetate, ascorbate, benzoate, citrate,oxalate, stearate, trifluoroacetate,
succinates,tartarate,lactate,fumarate, gluconate, glutamate,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
The preferred novel analogs of substance P of the present invention are as follows
D-Arg-Pro-Lys-Pro-D-Phe-Gln-D-Trp-Phe-D-Trp-Leu-Ac5c-NH2(SEQ ID NO 5) Acetyl-D-Arg-Pro-Lys-Pro-D-Phe-Gln-D-Trp-Phe-D-Trp-Leu-Ac5c-NH2(SEQ ID NO 6) ButanoyI-D-Arg-Pro-Lys-Pro-D-Phe-Gln-D-Trp-Phe-D-Trp-Leu-Ac5c-NH2(SEQ ID NO 7) Octanoyl-D-Arg-Pro-Lys-Pro-D-Phe-Gln-D-Trp-Phe-D-Trp-Leu-Ac5c-NH2(SEQ ID NO 8) Lauroyl-D-Arg-Pro-Lys-Pro-D-Phe-Gln-D-Trp-Phe-D-Trp-Leu-Ac5c-NH2(SEQ ID NO 9) D-Arg-Pro-Lys-Pro-D-Phe-Gln-D-Trp-Phe-D-Trp-Leu-Ac6c-NH2(SEQ ID NO 10) Butanoyl-D-Arg-Pro-Lys-Pro-D-Phe-Gln-D-Trp-Phe-D-Trp-Leu-Ac6c-NH2(SEQ ID NO 11) Octanoyl-D-Arg-Pro-Lys-Pro-D-Phe-Gln-D-Trp-Phe-D-Trp-Leu-Ac6c-NH2(SEQIDNO 12) D-Arg-Pro-Lys-Pro-D-Phe-Gln-D-Trp-Phe-D-Trp-Leu-Aib-NH2(SEQ ID NO 13) D-Arg-Pro-Lys-Pro-D-Phe-Gln-D-Trp-Phe-D-Trp-Leu-Deg-NH2(SEQ ID NO 14) D-Arg-Pro-Lys-Pro-D-Phe-Gln-D-Trp-Phe-D-Trp-Leu-Dpg-NH2(SEQ ID NO 15) D-Phe-Gln-D-Trp-Phe-D-Trp-Leu-Aib-NH2(SEQ ID NO 16) D-Phe-Gln-D-Trp-Phe-D-Trp-Leu-Ac5c-NH2(SEQ ID NO 17) D-Phe-Gln-D-Trp-Phe-D-Trp-Leu-Ac6c-NH2(SEQ ID NO 18) D-Phe-Gln-D-Trp-Phe-D-Trp-Leu-Deg-NH2(SEQ ID NO 19) D-Phe-Gln-D-Trp-Phe-D-Trp-Leu-Dpg-NH2(SEQ ID NO 20)
The invention also includes pharmaceutical compositions comprising the polypeptides and the process for preparation of the peptides These peptides possess antagonist properties again substance P and substance P- like peptides and are useful in the treatment and prevention of cancer
DETAILED DESCRIPTION OF THE INVENTION
The novel peptide analogs of substance P embodied in the present invention contain amino acids, namely a, a-dialkylated amino acids, which have been known to induce highly specific constraints in the peptide backbone The a, a-dialkylated amino acids, used in the present invention are synthesized from the corresponding ketones In a preferred embodiment of the invention, the ketones are first converted into the corresponding hydantoins which are hydrolyzed to yield the aforesaid amino acids In a preferred embodiment of the present invention, sulphuric acid or hydrochloric acid or strong base such as NaOH has been employed as the hydrolyzing agent
Synthesis of Peptides:
The novel peptides in the present invention have been generated by a using solid phase techniques or by a combination of solution phase procedures and solid phase techniques or by fragment condensation Although these methods for the chemical synthesis of polypeptides are well known in the art (Stewart and Young, 1969, Solid Phase Peptide Synthesis,W H Freeman Co )
In a preferred embodiment of the present invention the peptides were synthesized using the Fmoc strategy, on a semi automatic peptide synthesizer (CS Bio, Model 536), using optimum side chain protection The peptides were assembled from C-terminus 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 mmol amino acid per gram resin
The N-terminal amino group was protected by 9-flourenylmethoxycarbonyl (Fmoc) group 2,2,5,7,8-pentamethyl-chroman-6-sulfonyl (Pmc) or 2,2,4,7,-pentamethyl-dihydrobenzenofuran-5-sufonyl (Pbf) were the preferred protecting groups for the guandino group of Arginine The tryptophan residue was either left unprotected or used with Boc protection The side chain amino group of Lysine was protected using Boc group preferrably
In a preferred embodiment of the invention, 2-8 equivalents of Fmoc protected amino acid per resin nitrogen equivalent were 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 in situ or added in the form of preactivated 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 was completed, the amino-terminal Fmoc group was removed and then the peptide-resin was washed with methanol and dried The peptides were then deprotected and cleaved from the resin support by treatment with trifluoroacetic acid, crystalline phenol, ethanedithiol, thioanisole and de-ionized water for 1 5 to 5 hours at room temperature The crude peptide was obtained by precipitation with cold dry ether, filtered , dissolved, and lyophilized
The resulting crude peptide was purified by preperative 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 a CI8 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 electron-spray mass spectroscopy
Preferred, semi-automated, stepwise solid phase methods for the synthesis of peptides of the invention are provided in the examples discussed in the subsequent section of the document
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,orintraocular injections
In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically 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
A peptide of the present invention can be made by exclusively solid phase techniques, by partial solid phase / solution phase techniques and fragment condensation Preferred, semi-automated, stepwise solid phase methods for synthesis of peptides of the invention are provided in the examples discussed in the subsequent section of this document
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
The steps involved m the synthesis of the Substance P analogs employed the following protocol TABLE I
(Table Removed)
Example: 1
First loading on Rink Amide Resin
A typical preparation of the Fmoc-Ac5c-Rink Amide Resin was carried out using 1 0 g of 4-(2',4'-
Dimethoxyphenyl-Fmoc-aminomethyl) phenoxymethyl-derivatized polystyrene 1%
divinylbenzene (Rink Amide ) resin (07 mM / g ) (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, along with a similar fold excess of HOBt, in the amino acid vessel of the peptide synthesizer These were dissolved in dimethylformamide (A.C S grade) (J T Baker,, New Jersey, USA) and activated with DIPCDI, just prior to the addition to the resin in the reaction vessel of the peptide synthesizer HOBt was added in all coupling reactions, especially in the case of glutamine and histidine 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 The loading efficiency was ascertained by the increase of weight of the resin after the addition of the amino acid
Example: 2
Synthesis of D-Phe-Gln-D-Trp-Phe-D-Trp-Leu-Ac5c-NH2 (SEQ ID NO 17)
The synthesis of peptide SEQ ID NO 17 was initiated by using resin loaded with Fmoc-Ac5c-OH as prepared in example 1 above on Ig scale This was subjected to stepwise deprotection and coupling steps as in steps 1-10 of the synthesis cycle In each coupling reaction, a four- fold excess of amino- acid, DIC and HOBt were used On completion of synthesis and removal of the N-terminal Fmoc protecting group (steps 1-6 of the synthesis cycle), the peptide- resin was washed twice with methanol, dried and weighed to obtain 1 649g This was subjected to cleavage in a
cleavage mixture consisting of trifluoroacetic acid and scavengers, crystalline phenol, thioanisole, 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 the crude peptide The crude 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, in the art The prominent peaks were 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= -1036 , Observed Mass= 1037 2 ) The pure peptide was then used for bioassays
Example :3
Synthesis ofD-Arg-Pro-Lys-Pro-D-Phe-Gln-D-Trp-Phe-D-Trp-Leu-Ac5c-NH2 (SEQ ID NO 5)
The synthesis was carried out as in the examples above using the appropriate amino acids It was further cleaved and purified as described in example 2 The purified peptide was further characterised by its mass analysis The calculated mass of the above peptide was ~1515 and the observed mass was 1514 29
Example:4
Synthesis of Butanoyl-D-Arg-Pro-Lys-Pro-D-Phe-Gln-D-Trp-Phe-D-Trp-Leu-Ac5c-NH2 (SEQ ID
NO 7)
The conjugation of the Butanoyl group at the n-terminal position was done on solid phase
The above peptide sequence was synthesised on resin as described in example3 After the deprotection of D-Arg amino acid it was further coupled with Butanoic acid in DMF using DIPCDI and HOBT following the standard protocol The cleavage and purification was further carried out following the standard protocol as described in example 2 The final purified peptide was further analyzed by mass spectroscopy The calculated mass and observed was in good agreement (calculated mass= -1585, observed mass =1586 )
Example:S
Synthesis of Octanoyl-D-Arg-Pro-Lys-Pro-D-Phe-Gln-D-Trp-Phe-D-Trp-Leu-Ac5c-NH2 (SEQ ID NO 8)
The conjugation of the octanoyl group at the n-terminal position was done on solid phase
The above peptide sequence was synthesized on resin in a similar way as described in example 4 except octanoic acid is used in place of butanoic acid The final purified peptide was further analyzed by mass spectroscopy The calculated mass and observed was in good agreement (calculated mass=~ 1641, observed mass = 1642 2)
Example: 6
Synthesis of Acetyl-D-Arg-Pro-Lys-Pro-D-Phe-Gln-D-Trp-Phe-D-Trp-Leu-Ac5c-NH2 (SEQ ID NO 6)
The conjugation of the Acetyl group at the N-terminal position was done on solid phase using acetic anhydride in a similar way as described in example 4 The final purified peptide was further analyzed by mass spectroscopy The calculated mass and observed was in good agreement (calculated mass=~1557, observed mass =1558 5)
Example: 7
In vitro cytotoxic activity of the novel synthesized peptide analogs
The cytotoxic activity of each of the synthesised peptide was tested on six human tumor cell lines namely MCF7 (breast), U373 (glioblastoma), PTC, (colon), L132 (lung), Su 86 86 (pancreas),
and KB (oral) The tumor cells were collected at exponential growth phase and resuspended in medium (1 5 x 106 cells/ml in RPMI 1640 containing 10% FBS) 150ul of medium was added to the wells of a 96-well tissue culture plate (Nunc, Denmark) followed by 30ul of cell suspension The plate was left in incubator (37°C? 5% C02) overnight 20ul of the peptide (100 pM to 10 JJM concentration) was added to marked wells of the 96-well plate Each concentration was plated in triplicates 20ul of medium alone was added to control wells while wells without cells served as blanks A total volume of 200ul was ensured in each well and plate was left in incubator (37°C, 5% C02) After 72 hours of incubation an MTT assay was performed andpercentage cytotoxicity was calculated with respect to control cells Following Tables 2&3 show the cytotoxicity achieved in various cancer cell lines of different peptides
CYTOTOXIC ACTIVITY OF (SEQ .I.D. NO: 17) TABLE: 2
(Table Removed)
CYTOTOXIC ACTIVITY OF (SEP J.D. NO: 5)
TABLE 3
(Table Removed)
Example :8
In vitro cytotoxic activity of lipo conjugates of Substance P analogs
The cytotoxic effect of Hpo-conjugates of substance P analogs SEQ ID NO 6, SEQ ID NO 7 and SEQ ID NO 8, 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 - 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 jj.g 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
SEP ID NO:6 TABLE:4
(Table Removed)
TABLE:5
(Table Removed)
SEP ID NP:8
(Table Removed)
Example : 9
In vivo antitumour activity of the novel synthesized Substance P analogs on primary tumour(colon) xenografted mice
The experiment were carried out following the protocol described below and the experimental results are summarised in the table 4 and figure 1
Protocol 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 4 groups of three animals each including one group comprising untreated control animals Treatment with novel substance P peptides was initiated when the average tumor volumes, as measured using a vernier caliper, were between 400 - 800 mm3 SEQ 1 D NO 5, SEQ I D NO 7 and SEQ I D NO 8 peptides were prepared at a concentration of 42 5 µg/ml and intravenously administered to the assigned group of tumor bearing animals at a dose of 4 25 µg/100 µL twice a day so that the total dose of 8 5 g was administered to each animal The treatment was continued for a period of 10 days
Results
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 Table 7 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 5, SEQ.I.D.NO 7 and SEQ ID NO 8 as compared to controls on day 19 was 68 34%, 78 54% and 75 16% respectively
TABLE 7: Tumor volumes (mm3) of individual tumor bearing animals of treated and control groups
(Table Removed)





WE CLAIM:
1. A novel peptide analog of Substance P of the following general formula
X-D-Arg-Pro-Lys-Pro-D-Phe-Gln-D-Trp-Phe-D-Trp-Leu-R-NH2
wherein
X is acetyl or straight, branched, or cyclic alkonyl group from 3-18 carbon atoms, or is
deleted;
R is Aib or Deg or Dpg or Ac5c or Ac6c;
or a hydrolysable carboxy protecting group or pharmaceutically acceptable salts of the
peptide as herein described.
2. A peptide as claimed in claim 1 of the following general formula
D-Trp-Phe-D-Trp-Leu-R-NH2
wherein R is Aib or Deg or Dpg or Ac5c or Ac6c;
or a hydrolysable carboxy protecting group or pharmaceutically acceptable salts of the
peptide.
3. The peptide as claimed in claim 1 wherein X is deleted and R is Ac5c; said peptide having
the formula
D-Arg-Pro-Lys-Pro-D-Phe-Gln-D-Trp-Phe-D-Trp-Leu-Ac5c-NH2 (SEQ. ID NO. 5) or a pharmaceutically acceptable salt thereof.
4. The peptide as claimed in claim 1 wherein X is Acetyl and R is Ac5c; said peptide having
the formula
Acetyl-D-Arg-Pro-Lys-Pro-D-Phe-Gln-D-Trp-Phe-D-Trp-Leu-Ac5c-NH2 (SEQ. ID NO. 6) or a pharmaceutically acceptable salt thereof.
5. The peptide as claimed in claim 1 wherein X is Butanoyl and R is Ac5c; said peptide
having the formula
Butanoyl-D-Arg-Pro-Lys-Pro-D-Phe-Gln-D-Trp-Phe-D-Trp-Leu-Ac5c-NH2 (SEQ. ID NO. 7) or a pharmaceutically acceptable salt thereof.
6. The peptide as claimed in claim 1 wherein X is Octanoyl and R is Ac5c; said peptide
having the formula
Octanoyl-D-Arg-Pro-Lys-Pro-D-Phe-Gln-D-Trp-Phe-D-Trp-Leu-Ac5c-NH2 (SEQ. ID NO. 8) or a pharmaceutically acceptable salt thereof.
7. The peptide as claimed in claim 1 wherein X is Lauroyl and R is Ac5c; said peptide
having the formula
Lauroyl-D-Arg-Pro-Lys-Pro-D-Phe-Gln-D-Trp-Phe-D-Trp-Leu-Ac5c-NH2 (SEQ. ID NO. 9) or a pharmaceutically acceptable salt thereof.
8. The peptide as claimed in claim 1 wherein X is deleted and R is Ac6c; said peptide having
the formula
D-Arg-Pro-Lys-Pro-D-Phe-Gln-D-Trp-Phe-D-Trp-Leu-Ac6c-NH2(SEQ. ID NO. 10)
or a pharmaceutically acceptable salt thereof.
9. The peptide as claimed in claim 1 wherein X is Butanoyl and R is Ac6c; said peptide
having the formula
Butanoyl-D-Arg-Pro-Lys-Pro-D-Phe-Gln-D-Trp-Phe-D-Trp-Leu-Ac6c-NH2 (SEQ. ID NO. 11) or a pharmaceutically acceptable salt thereof.
10. The peptide as claimed in claim 1 wherein X is Octanoyl and R is Ac6c; said peptide
having the formula
Octanoyl-D-Arg-Pro-Lys-Pro-D-Phe-Gln-D-Trp-Phe-D-Trp-Leu-Ac6c-NH2 (SEQ. ID NO. 12) or a pharmaceutically acceptable salt thereof.
11. The peptide as claimed in claim 1 wherein X is deleted and R is Aib; said peptide having
the formula
D-Arg-Pro-Lys-Pro-D-Phe-Gln-D-Trp-Phe-D-Trp-Leu-Aib-NH2(SEQ. ID NO. 13)
or a pharmaceutically acceptable salt thereof.
12. The peptide as claimed in claim 1 wherein X is deleted and R is Deg; said peptide having
the formula
D-Arg-Pro-Lys-Pro-D-Phe-GLn-D-Trp-Phe-D-Trp-Leu-Deg-NH2 (SEQ. ID NO. 14) or a pharmaceutically acceptable salt thereof.
13. The peptide as claimed in claim 1 wherein X is deleted and R is Dpg; said peptide having
the formula
D-Arg-Pro-Lys-Pro-D-Phe-Gln-D-Trp-Phe-D-Trp-Leu-Dpg-NH2(SEQ. ID NO. 15) or a pharmaceutically acceptable salt thereof.
14. The peptide as claimed in claim 2 wherein R is Aib; said peptide having the formula
D-Phe-Gln-D-Trp-Phe-D-Trp-Leu-Aib-NH2 (SEQ. ID NO. 16) or a pharmaceutically acceptable salt thereof.
15. The peptide as claimed in claim 2 wherein R is Ac5c; said peptide having the formula
D-Phe-Gln-D-Trp-Phe-D-Trp-Leu-Ac5c-NH2 (SEQ. ID NO. 17) or a pharmaceutically
acceptable salt thereof.
16. The peptide as claimed in claim 2 wherein R is Ac6c; said peptide having the formula
D-Phe-Gln-D-Trp-Phe-D-Trp-Leu-Ac6c-NH2 (SEQ. ID NO. 18) or a pharmaceutically
acceptable salt thereof.
17. The peptide as claimed in claim 2 wherein R is Deg; said peptide having the formula
D-Phe-Ghi-D-Trp-Phe-D-Trp-Leu-Deg-NH2 (SEQ. ID NO. 19) or a pharmaceutically
acceptable salt thereof.
18. The peptide as claimed in claim 2 wherein R is Dpg; said peptide having the formula
D-Phe-Gln-D-Trp-Phe-D-Trp-Leu-Dpg-NH2 (SEQ. ID NO. 20) or a pharmaceutically
acceptable salt thereof.
19. A solid phase peptide synthesis process for the preparation of the peptide analog of
formula (1) as claimed in any preceding claim, which comprises sequentially loading
protected a,a-dialkylated amino acids in sequential cycles to amino terminus of a solid
phase resin, coupling the amino acids to assemble a peptide-resin assembly, removing the
protecting groups and cleaving the peptide from resin to obtain said peptide.
20. A process as claimed in claim 19, wherein said a, a-dialkylated amino acids are protected
at their oc-amino groups by a 9-flurenyl methoxy carbonyl (Fmoc) group.
21. A process as claimed in claim 19 -wherein the coupling is carried out in the presence of
activated agents selected from the group consisting of BOP, PyBOP, HBTU, TBTU and
HOBt.
22. A process as claimed in claim 19 wherein the coupling was carried out in the presence of
a solvent selected from the group consisting of DMF, DCM, NMP or any mixture
thereof.
23. A process as claimed in claim 19 wherein said crude peptide is cleaved from said peptide-
resin assembly by treatment with trifluoroacetic acid, ethanediol and deionised water for
1.5 to 5 hours at room temperature.
24. A process as claimed in claim 19 wherein the a, a-dialkylated amino acid is prepared by
conversion of a ketone to a hydantoin and hydrolysis of said hydantoin.
25. Novel peptides substantially as described hereinbefore and with reference to the
foregoing examples.
26. A solid phase peptide synthesis process substantially as described in the foregoing
examples.

Documents:

704-del-2000-abstract.pdf

704-del-2000-assignment.pdf

704-del-2000-claims.pdf

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

704-del-2000-correspondence-others.pdf

704-del-2000-correspondence-po.pdf

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

704-del-2000-drawings.pdf

704-del-2000-form-1.pdf

704-del-2000-form-13.pdf

704-del-2000-form-19.pdf

704-del-2000-form-2.pdf

704-del-2000-form-3.pdf

704-del-2000-form-6.pdf

704-del-2000-gpa.pdf


Patent Number 235732
Indian Patent Application Number 704/DEL/2000
PG Journal Number 35/2009
Publication Date 28-Aug-2009
Grant Date 19-Aug-2009
Date of Filing 31-Jul-2000
Name of Patentee DABUR RESEARCH FOUNDATION
Applicant Address 22, SITE IV, SAHIBABAD, GHAZIABAD 201 010, U.P, INDIA
Inventors:
# Inventor's Name Inventor's Address
1 BURMAN, ANAND C. DABUR RESEARCH FOUNDATION, OF 22 SITE IV, SAHIBABAD, GHAZIABAD 201 010, U.P, INDIA
2 PRASAD, SUDHANAND DABUR RESEARCH FOUNDATION, OF 22 SITE IV, SAHIBABAD, GHAZIABAD 201 010, U.P, INDIA
3 JAGGI MANU DABUR RESEARCH FOUNDATION, OF 22 SITE IV, SAHIBABAD, GHAZIABAD 201 010, U.P, INDIA
4 SINGH ANUT. DABUR RESEARCH FOUNDATION, OF 22 SITE IV, SAHIBABAD, GHAZIABAD 201 010, U.P, INDIA
5 MUKHERJEE, RAMA DABUR RESEARCH FOUNDATION, OF 22 SITE IV, SAHIBABAD, GHAZIABAD 201 010, U.P, INDIA
PCT International Classification Number A61K 38/08
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA