Title of Invention	"RADIOLABELED VASOACTIVE INTESTINAL PEPTIDE ANALOGS FOR DIAGNOSIS AND THERAPY"
Abstract	Novel radiolabeled peptide analogs of vasoactive intestinal peptide with application for diagnosis and therapy in a mammalian living system are disclosed. The analogs of the present invention have excellent use as imaging and scintigraphic agents These analogs comprise of a synthetic receptor-binding peptide analog of vasoactive intestinal peptide (VIP) radiolabelled with technicium Tc-99m wherein said peptide analog of vasoactive intestinal peptide has the sequence: His-Ser-Asp-Xxx-Val-4-Cl-D-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Leu-Ala-Val-Lys-Lys-Tyr-Leu -Asn-Ser-Tle-Leu-Asn-NH2 (SEQ TD NO:1). Where Xxx = Ala or Aib or Deg or Ac5c

Title of Invention

"RADIOLABELED VASOACTIVE INTESTINAL PEPTIDE ANALOGS FOR DIAGNOSIS AND THERAPY"

Abstract

Novel radiolabeled peptide analogs of vasoactive intestinal peptide with application for diagnosis and therapy in a mammalian living system are disclosed. The analogs of the present invention have excellent use as imaging and scintigraphic agents These analogs comprise of a synthetic receptor-binding peptide analog of vasoactive intestinal peptide (VIP) radiolabelled with technicium Tc-99m wherein said peptide analog of vasoactive intestinal peptide has the sequence: His-Ser-Asp-Xxx-Val-4-Cl-D-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Leu-Ala-Val-Lys-Lys-Tyr-Leu -Asn-Ser-Tle-Leu-Asn-NH2 (SEQ TD NO:1). Where Xxx = Ala or Aib or Deg or Ac5c

Full Text	Radiolabeled vasoactive intestinal peptide analogs for diagnosis and radiotherapy FIELD OF THE INVENTION; The present invention encompasses radiolabeled peptide analogs of vasoactive intestinal peptide (VIP), labeled with Tc-99m, useful for imaging target sites within mammalian living systems The invention particularly provides radiolabeled-VIP derivatives that bind selectively to the VIP receptor on target cells. Specifically, the invention relates to the radiolabeling of VIP-receptor specific agents and their subsequent use for radiodiagnostic and radiotherapeutic purposes The invention encompasses methods for radiolabeling these peptide reagents with Tc-99m etc. and the use of these aforesaid derivatives as scintigraphic imaging agents. The present invention also encompasses the use of these radiolabeled peptides as anti-neoplastic agents for specific radiotherapy in cancer. BACKGROUND OF THE INVENTION: Vasoactive intestinal peptide is a 28-amino acid neuropeptide, which was first isolated from the porcine intestine (Said and Mutt, 1970). It bears extensive homology to secretin, PHI and glucagon. The amino acid sequence for VIP is : His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-I,eu-Asn-Ser-Ile-Leu-Asn-NH2 VIP is known to exhibit a wide variety of biological activities such as the autocrine, endocrine and paracrine functions in living organisms (Said, 1984). In the gastrointestinal tract, it has been known to stimulate pancreatic and biliary secretions, hepatic glycogenesis as well as the secretion of insulin and glucagon (Kerrins and Said, 1972 ; Domschke et al., 1977). In the nervous system it acts as a neurotransmitter and neuromodulator, regulating the release and secretion of several key hormones (Said, 1984). In recent years, attention has been focussed on the function of VIP in certain areas of the CNS as well its role in the progression and control of neoplastic disease (Reubi, 1995). The importance of peptide growth factors and regulatory hormones in the etiology and pathogenesis in several carcinomas has long been recognized. Data from epidemiological and endocrinological studies suggest that neuropeptides like VIP which are responsible for the normal growth of tissues like the pancreas can also cause conditions for their neoplastic transformation (Sporn el al, 1980). Several lines of evidence indicate that VIP acts as a growth factor and plays a dominant autocrine and paracrine role in the sustained proliferation of cancer cells (Said, 1984). The stimulatory effect of VIP on tumor growth can be mediated directly by its receptors on cell membranes or indirectly by potentiation of the activities of other growth factors in tumor cells (Scholar et al, 1991). The synergistic effect of VIP and related pituitary adenylate cyclase activating polypeptide (PACAP) in glioblastomas is an illustration to the above fact (Moody el al., 1996) The multiple physiological and pharmacological activities of VIP are mediated by high affinity G-protein coupled transmembrane receptors on target cells (Reubi, 1995). VIP receptors are coupled to cellular effector systems via adenylyl cyclase activity (Xia et al., 1996). The VTP receptor, found to be highly overexpressed in neoplastic cells, is thought to be one of the biomarkers in human cancers (Reubi, 1995). High affinity VIP receptors have been localized and characterized in neoplastic cells of most breast carcinomas, breast and prostate cancer metastases, ovarian, colonic and pancreatic adenocarcinomas, endometrial and squamous cell carcinomas, non small cell lung cancer, lymphomas, glioblastomas, astrocytomas, meningiomas and tumors of mesenchymal origin. Amongst, neuroendocrine tumors all differentiated and non-differntiated gastroenteropancreatic tumors, pheochromocytomas, small-cell lung cancers, neuroblastomas, pituitary adenomas as well tumors associated with hypersecretory states like Verner-Morrison syndrome were found to overexpress receptors for vasoactive intestinal peptide (Reubi, 1995, 1996, 1999; Tang et al, 1997a & b; Moody et al, 1998a &b; Waschek et al, 1995; Oka et al, 1998)). These findings suggest that new approaches for the diagnosis and treatment of these cancers may be based on functional manipulation of VIP activity, using synthetic peptide analogs of the same. Historically, the somatostatin analog '"in-DTPA-fD-Phe^-octreotide is the only radiopeptide, which has obtained regulatory approval in USA and Europe (Lamberts et al, 1995). Radiolabeled VTP has been shown to visualize majority of gastropancreatic adenocarcinomas, neuroendocrine tumors, as well as insulinomas (which are often missed by radiolabeled octreotide) (Behr et al, 1999). VIP-receptor scinitigraphy offers certain advantages over radioimaging involving somatostatin receptors. The presence of high affinity receptors for VIP have been demonstrated in a larger number of human tumors, relative to the somatostatin receptors. Secondly, the density of VIP receptors on tumors has been found to be greater than somatostatin (Behr el al, 1999). Therefore, the VIP-receptor scan is more sensitive and convenient in localizing tumors and their metastatic spread as compared to somatostatin The applications of this technique are manifold. It has been used for the sensitive detection of VIP-receptor positive tumors. This includes primary carcinoids, cancers of the gastrointestinal tract as well as distant metastases (Reubi, 1995, 1996). It can also be used to target cytotoxic radionuclides specifically to the tumor site. It predicts the VIP-receptor status of the patient and thereby the response of the patient towards radiotherapy by radiolabeled VIP analogs. Lastly, such radiolabeled peptides have been successfully used in radioguided surgery (Lamberts et al, 1995). 123I-VIP, I25I-VIP and their derivatives have been extensively used for imaging pancreatic adenocarcinomas, endocrine tumors of the gastrointestinal origin, mesenchymal tumors as well secondary tumor metastatic sites, in patients (Jiang et al, 1997; Virgolini et al, 1996, 1998; Raderer et al, 1998 ; Moody et al., 1998; Kurtaran et al, 1997; Pallella et al, 1999). Radioiodinated VIP and its derivatives have been also used to assess the binding affinity of peptides for VDP-receptors on tumor cells in vitro. The biodistribution, safety and absorbed dose of the aforesaid radioiodinated peptide derivatives have also been studied earlier (Virgolini et al, 1995). US Patent No. 5,849,261, granted to Dean etal, on December 15, 1998 describes the applications of radiolabeled vasoactive intestinal peptide (VIP) for diagnosis and therapy. In particular, this US Patent discloses a method for preparing a radiopharmaceutical agent, comprising of native vasoactive intestinal (VIP) peptide attached to a radionucleide like technetium or rhenium via a chelating moiety. The radiopharmaceutical when labeled with technetium or rhenium via a chelating moiety has a VIP binding affinity which is not less than about one tenth the affinity of radioiodinated native VIP for the said receptor. However, there is still a need for improved synthetic analog of VIP as radiopharmaceuticals, which are easy to generate and are capable of being employed with higher sensitivity and specificity in terms of their radio-imaging properties. This invention describes the preparation and use. of peptide analogs of VIP using constrained amino acids. 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 § , \\i angles, within the molecule, thereby stabilizing a desired peptide conformation. The prototypic member of a,a-dialkylated aminoacids, a-aminoisobutyric acid (Aib) or a5a-dimethylglycine has been shown to induce p-turn or helical conformation when incorporated in a peptide sequence (Prasad and Balaram, 1984, Karle and Balaram, 1990). The conformational properties of the higher homologs of a,a-dialkylated amino acids such as di-ethylglycine (Deg), di-n-propylglycine (Dpg), 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), as 1-aminocycloheptane carboxylic acid (Ac7c) and as 1-aminocyclooctane carboxylic acid (Ac8c) have also been shown to induce folded conformation (Prasad et al., 1995 ; Karle et al., 1995). a,a-dialkylated amino acids have been used in the design of highly potent chemotactic peptide analogs (Prasad et al., 1996) However, the applicants are not aware of any prior art for the synthesis of novel peptide analogs, encompassed in the present invention, particularly the synthesis of such VIP peptide analogs, containing a, a-dialkylated amino acids, by solid phase peptide synthesis methodology. Moreover, the use of such constrained amino acids for the design of peptides possessing anti-neoplastic activity is also unknown in any previous prior art. The present invention exploits the conformational properties of such a, a-dialkylated amino acids for the design of biologically active peptide derivatives, taking VIP as the model system under consideration. REFERENCES: Behr T.M. etal. Q. J. Nucl. Med., 43, 268-280,1999. Domschke, S. etal. Gastroenterology, 73, 478-480, 1977. Jiang S. etal. Cancer Res., 57, 1475-1480,1997 Karle, I.L. etal (1995) J. Amer. Chem. Soc. 777, 9632-9637. Karle, I.L. and Balaram, P. (1990) Biochemistry 29, 6747-6756. Kerrins, C and Said, S.I. Proc Soc. Exp Biol. Med., 142, 1014-1017, 1972 Kurtaran A. et al. J Nucl. Med., 38, 880-881, 1997 Lamberts, S.W.J. et al In Somatostatin and its Receptors, Ciba Found. Symp, 190, 222-239, 1995 Moody, T.W. el al Peptides, 19 (3), 1998a Moody, T.W. etal Ann. N.Y. Acad. Sci., 865, 290-296. 1998b. Oka, H. et al. (1998) Am. J. Pathol., 153 (6), 1787-1796, 1998. Pallella, V.R. el al J. Nucl. Med., 40(2), 352-360, 1999 Prasad, B.V.V and Balaram, P. (1984) CRC Crit. Rev. Biochem. 16, 307-347. Prasad, S etal. (1995) Biopolymers 35, 11-20 Prasad, S et al. (1996) Int. J. Peptide Protein Res. 48, 312-318. Reubi, J.C, J. Nucl. Med., 36 (10), 1995. Reubi, J.C. etal. Int. J. Cancer, 81 (3), 1999. Reubi, J.C. etal. Cancer Res., 56 (8), 1922-1931, 1996. Raderer, M. etal. J. Nucl. Med., 39 (9), 1570-1575, 1998 Said, S. I. and Mutt, V. Science, 169, 1217-1218,1970 Said, S.I. Peptides, 5, 143-150, 1984 Sporn, M.B., and Todaro, G.J. N. Engl. J. Med., 303, 378-379,1980. Tang, C. et al, Gut, 40 (2), 267-271, 1997a. Tang, C. etal, Br. J. Cancer, 75 (10)1467-1473, 1997b. Virgolini, I. etal. J. Nucl. Med., 36(10), 1732-1739, 1995. Virgolini, I. etal. Nucl. Med. Biol., 23 (6), 685-692, 1996. Virgolini, I. etal. J. Nucl. Med., 39 (9), 1998. Waschek, J A. et al. Cancer Lett., 92 (2), 1995 Xia, M. etal, J Clin. Immunol., 16 (1), 21-30,1996 SUMMARY OF THE INVENTION: The present invention discloses for the first time the use of certain novel VIP analogs to determine the binding affinities of these peptides for their cognate receptors on cancer cells. The VTP peptide analog of the present invention, which is a VIP receptor antagonist has the sequence His-Ser-Asp-Xxx-Val-4-Cl-D-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Leu-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2 (SEQ ID NO: 1) where Xxx = Ala or Aib or Deg or Ac5c Abbreviation: Aib- a- Aminoisobutyric acid Deg- a , a- Diethylglycine Ac5c- 1-Amino Cyclopentane Carboxylic acid The applicants have found that the VIP analogs discussed in the present invention have greater affinity for its cognate receptors on tumor cells as compared to native VIP, which in turn lead to better radio-imaging and radio-therapeutic efficacy of the radiopharmaceutical of the present invention. While not wishing to be bound by theory, the applicants believe that the improved efficacy of the radiopharmaceuticals of the present invention are due to the nature of the VIP analogs themselves, which have receptor bound conformations caused by the incorporation of the unusual amino acids. The chelating groups are attached at a novel site in the VTP analogs, i.e., to the N-terminal amino acid residue of the VIP analogs. As any reader skilled in the art will appreciate, the previous prior art encompasses radiopharmaceutical agents in, which the chelating agents are attached to the C-terminal end of the VIP (Dean et al., U.S. Patent No 5,849,261). While conventional chelating agents are within the scope of the present invention, the applicants have, for the first time, employed certain novel MAG3 derivatives as chelating agents. The present invention also envisages use of certain aminocaproic acid (Acp) and aminohexanoic acid (Aha) as spacer groups for the first time between a chelating moiety and VIP analog. The present invention therefore envisages a novel radiopharmacetical comprising of an hitherto unknown as VIP analog covalently linked to MAG3 derivatives via aminocaproic acid (Acp) or aminohexanoic acid (Aha). The radiopharmaceutical molecule so obtained is novel per se and has not been reported in any prior art known to the applicants and exhibits an improved radioscintigraphic and radiotherapeutic efficacy as compared to such radiolabed reagents known in the prior art. Accordingly, the present invention provides novel radiolabeled peptide analogs of vasocative intestinal peptide useful for imaging target sites within a mammalian living system, comprising of a synthetic receptor-binding peptide analog of vasoactive intestinal peptide (VIP) radiolabeled with Tc-99m, wherein said peptide analogs of vasoactive intestinal peptide has the sequence: His-Ser-Asp-Xxx-Val-4-Cl-D-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leii-Arg-Lys-Gln-Leu-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2 (SEQ ID NO: 1) Where Xxx = Ala or Aib or Deg or Ac5c In a preferred embodiment of the present invention, the radionucleide Tc-99m is bound to a chelating moeity, which is attached to the N-terminal amino acid residue of the said VIP analog. The labeling of peptides by Tc-99m has been accomplished in the present invention, by several strategies. In the present invention radioimaging of tumors has been done by 1) Direct labeling of Tc-99m to the peptide analogs. 2) Attachment of chelating groups to the peptide and subsequent radiolabeling by Tc-99m. 3) Incorporation of Tc-99m to chelator moiety covalently linked to the peptide via the spacer group. 4) Use of a variety of chelator moieties to the system (3). It is important to note that in the above cases, 2, 3 and 4, the chelator and spacer groups are incorporated site-specifically at a position which does not affect the specific binding properties of the peptide to the VIP receptor on tumor cells in vitro and in vivo. Ideally the chelating group is hydrophobic in nature. In a preferred embodiment, the chelating groups are based on peptides such as mercaptoacetyltriglycine (MAG 3) and its derivatives such as SH CH2-CO-Gly-Gly-Gly Cys-Gly-Aib-Ala Cys-Gly-Gly-Aib Gly-Gly-Ala-Aib Cys-Aib-Gly-Gly Cys-Ala-Gly-Aib Gly-Gly-Gly-Aib Gly-Gly-Aib-Ala The methods involved in the synthesis, purification, characterization and radiolabeling of these peptides are illustrated in detail in the following section containing examples. The following section also includes biological data relating to the imaging efficacy and dosimetry of the aforesaid radiolabeled peptides These examples have been furnished for illustrating and providing insight into the invention and should not be construed as limiting. DETAILED DESCRIPTION OF THE INVENTION The VIP receptor antagonist His-Ser-Asp-Xxx-Val-4-Cl-D-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Leu-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Tle-Leu-Asn-NH2(SEQ TD NO: 1) Where Xxx = Ala or Aib or Deg or Ac5c has been shown in our previous studies to be selectively binding to VIP receptors on cancer cells. Several lines of evidence suggest that VIP can also function as a growth factor mediating the sustained proliferation of cancer cells, by an autocrine or paracrine mechanism. This has led to intense research focussed on the design and biological activity of VIP antagonists. The anti-proliferative activity of the aforesaid VIP antagonist has been previously demonstrated in a number of experimental models of pancreatic, prostate, mammary and lung cancer, suggesting its high anti-neoplastic therapeutic potential. The present invention relates to the use of the above peptide, process for its production, pharmaceutical preparation for its use as a diagonstic, imaging as well as a radiotherapeutic agent in vivo. The present invention also includes several strategies involved in the generation of the above mentioned radiopharmaceutical. These have been described in detail in the "Examples" section, however, a few illustrations are also detailed below: A preferred embodiment of the invention involves the direct radiolabeling of the above mentioned VIP antagonist directly to Tc-99m. Tc-99m forms a co-ordinate covalent linkage with certain specific amino acid residues of the peptide. The formation of a stable Tc-peptide bond is one of the major advantages for its use for imaging purposes. The attachment of Tc-99m to the peptide involves the reaction of pertechnate (a salt of Tc-99m) to the peptide, in the presence of a reducing agent like stannous chloride. The radiolabeled peptides are separated from the unincorporated Tc-99m as described in example and used for radioscintigraphy. Another embodiment of the present invention includes the attachment of certain chelating groups to the VIP analogue. According to the invention, the chelating moiety may be attached directly or indirectly to the peptide, eg by means of a spacer or a bridging group to the amino terminus of the VIP analog The present invention encompasses compounds of both these categories The present invention relates to physiologically acceptable chelating groups, capable of complexing a detectable element. According to one preferred embodiment of the invention, the chelating group has substantial hydrophobic character. Examples of chelating groups include eg iminodicarboxylic groups, polyaminopolycarboxylic groups, e.g. those derived from non-cyclic ligands e.g. ethylene diamine tetra acetic acid (EDTA), diethylene triamine pentaacetic acid (DTPA), ethylene glycol-0,0'-bis (2-aminoethyl)-N,N,N',N'-tetraacetic acid (EOTA), N,N'-bis(hydroxybenzyl)ethylenediamine-N,N'-diacetic acid (HBED) and triethylenetriaminehexaacetic acid (TTHA). The chelating groups derived from macrocyclic ligands include, e.g. l,4,7,10-tetra-azacyclododecane-N,N',N",N"'-tetra acetic acid (DOTA). l,4,7,10-tetra-azacyclotridecane-l,4,7,10-tetra acetic acid (TTTRA) 1,4,8,ll-tetra-azacyclotetradecane-N,N',]SP',N"'-tetra acetic acid (TETA) 1,4,8,11-tetra-azacyclotetradecane (TETRA) Aryl chelating moieties e.g. hydrazinonicotinamide (HYNIC) The present invention also encompasses chelating .groups based on peptides eg preferred derivatives of mercaptoacetyltriglycine (MAG 3) which are not previously known be employed as chealting agents in this field. These MAG 3 chelating agents include SH-CHrCO-Gly-Gly-Gly Cys-Gly-Aib-Ala Cys-Gly-Gly-Aib Gly-Gly-Ala-Aib Cys-Aib-Gly-Gly Cys-AJa-Gly-Aib Gly-Gly-Gly-Aib Gly-Gly-Aib-Ala The invention also envisages preferred spacer / bridging groups like those derived from aminoacids such as aminocaproic acid, aminohexanoic acid etc. Thus according to the present invention there is also provided a novel radiolabeled peptide analog of vasocative intestinal peptide useful for imaging target sites within a mammalian living system, comprising of a synthetic receptor-binding peptide analog of vasoactive intestinal peptide (VIP) radiolabed with Tc-99m, wherein said peptide analog of vasoactive intestinal peptide has the sequence: His-Ser-Asp-Xxx-Val-4-Cl-D-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Leu-Ala-Val-Lys-Lys-Tyr-Leu -Asn-Ser-Ile-Leu-Asn-NH2 (SEQ ID NO: 1) where Xxx = Ala or Aib or Deg or Ac5c, the radionuclide Tc-99m being bound to a chelating moeity, which is attached to the N-terminal amino acid residue of the said VIP analog. Preferably, the chelating groups are selected from MAGS peptide derivatives selected from the group consisting of SH-CH2-CO-Gly-Gly-Gly, Cys-Gly-Aib-Ala, Cys-Gly-Gly-Aib, Gly-Gly-Ala-Aib, Cys-Aib-Gly-Gly, Cys-Ala-Gly-Aib, Gly-Gly-Gly-Aib and Gly-Gly-Aib-Ala. The invention involves also envisages preferred spacer / bridging groups like those derived from amino acids, e.g. aminocaproic acid, aminobutyric acid, aminohexanoic acid etc and with the specific MAG 3 chelators described above, the present invention envisage a radiolabeled vasoactive intestinal peptide which is novel per se and which exhibits improved efficacy in terms of imaging, diagnosis, and therapy. This novel peptide reagent comprises of 33 amino acids - 28 from VIP analog, 1 from spacer and 4 from chelating moeity attached to radiolabeled technitium to provide a novel and hitherto unknown radiotherapeutic and radioscintigraphic agent. All the radiolabeled chelated peptides retain their affinity for VIP receptors on cancer cells. The radiolabeled VIP derivatives of the present invention exhibit pharmacological activity and therefore are useful as either imaging agent for visualization of VIP-receptor positive tumors and metastases or as a radiotherapeutic agent for the treatment of such tumors in vivo by specifically targeting the cytotoxic radionucleide selectively to the tumor site in mammalian living systems EXAMPLES : Solid phase peotide synthesis An analog of the present invention can be made by exclusively solid phase techniques, by partial solid phase / solution phase techniques and fragment condensation. Methods for chemical synthesis of polypeptides is well known in art, and in this regard, reference is made, by way of illustration, to the following literature: Stewart and Young, Solid Phase Peptide Synthesis (W. H. Freeman and Co., 1969), Atherton and Shepherd etc. Preferred, semi-automated, stepwise solid phase methods for synthesis of peptides of the invention are provided in the examples below. Example 1: Preparation of VIP analogs Peptides were synthesized using preferably, Fmoc (9-fluorenyl methoxy carbonyl) solid- phase methodology, on CS Bio (Model 536) Peptide Synthesizer (CS Bio Co., San Carlos, California, U.S. A.). Sequential assembly of a peptide analog was conducted from the carboxy- terminus, by loading of a Fmoc protected amino acid to a solid- phase resin, to the amino terminus. This was proceeded by subsequent removal of the Fmoc protecting group of the amino acid and a stepwise, sequential addition of Fmoc protected amino acids in repetitive cycles to obtain an intermediate protected peptide resin. For peptides that were amidated at the carboxy-terminus, Rink Amide resin was employed, and the loading of the first Fmoc protected amino acid was affected via an amide bond formation with the solid support, mediated by diisopropylcarbodiimide and HOBt. Substitution levels for automated synthesis were preferably between 0.2 and 0.8 mmole amino acid per gram resin. Steps in the synthesis of VIP analogs encompassed in the present invention, employed the following protocol:(Table Removed) The 9-fluorenyl methoxy carbonyl (Fmoc) group was used for the protection of the a-amino group of all amino acids employed in the syntheses. However, other protecting groups known in the art for a-amino group may be employed successfully. Side chain functional groups were protected as follows: Trityl (trt) and t-butyloxycarbonyl (Boc) were the preferred protecting groups for the imidazole group of Histidine. Hydroxyl groups of serine, threonine and tyrosine were protected by t- butyl (t-Bu) groups. Pmc (2,2,5,7,8-pentamethyl-chroman-6-sulfonyl) and Pbf (2,2,4,6,7-pentamethyldihydro benzofuran-5-sulfonyl) were the preferred protecting groups for the guanido group in Arginine. Trityl protection was used for asparagine and glutamine. Tryptophan was either used with Boc protection or unprotected. The lysine side chain was Boc protected and aspartic acid and glutamic acid had t-butyl side chain protection. The resin employed for the synthesis of carboxy-ami dated analogs was 4-(2',4'-Dimethoxypheny1-Fmoc-aminomethyl)-phenoxyrnethyl- derivatized polystyrene 1% divinylbenzene (Rink Amide) resin (100-200 mesh), procured from Calbioichem-Novabiochem Corp., La Jolla, U.S.A., (0.47 milliequivalent NH. Sub. 2 /g resin). Typically, 2-8 equivalents of Fmoc protected amino acid per resin nitrogen equivalent were used. The activating reagents used for coupling of amino acids in the solid phase synthesis of peptides arc well known in the art. These include BOP, PyBOP, HBTU, TBTU, PyBOP, HOBt. Preferably, DCC or DIG / HOBt or HBTU/HOBT and DIEA couplings were carried out. Swelling of the resin was typically carried out in dichloromethane measuring to volumes 10-40ml /g resin. 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. 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). Any incomplete reactions were re-coupled using freshly prepared activated amino acids. After complete assembly of the analog, the ammo-terminal Fmoc group was removed using steps 1-6 of the above protocol and then the peptide- resin was washed with methanol and dried. The analogs 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® CIS (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 C18 LiChrospher®, WP-300 (300.Times.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. (a) Preparation of Fmoc-Asn(trt)- Resin A typical preparation of the Fmoc-Asn(trt)-resin was earned out using 0.5g of 4-(2',4'-Dimethoxyphenyl-Fmoc-aminomethyl) phenoxymethyl-derivatized polystyrene 1% divinylbenzene (Rink Amide ) resin ( 0.47 mM / g ) (100-200 mesh), procured from Calbiochem-Novabiochem Corp., La .lolla, U.S.A. The resin was first allowed to swell in methylene chloride (2. Times. 25ml for 10 min.). It was washed once in dimethylformamide for 1 min. All solvents in the automated protocol were in 20ml portions per addition. The Fmoc- protecting group on the resin was removed by following steps 3 to 7 of the synthesis protocol. Deprotection of the Fmoc group was checked by the presence of blue beads in a positive Kaiser test. For loading of the first amino acid on the free amino (N. H. sub. 2) group of the resin, the first amino acid, Fmoc-Asn(trt)-OH, was weighed in four 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, Phillipsburg, New Jersey, U.S.A.) and activated with DIG, 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 Arg, Asn, Gin and His. The coupling reaction was carried out for a period ranging from 1-3 hours. Loading of the first amino acid was complete when Kaiser test gave a negative result and there was adequate weight increase when the resin, with the first amino acid attached, was dried in vaccuo overnight and weighed. Example 2: (b) Synthesis of: His-Ser-Asp-Ala-Val-4-Cl-D-Phe-Thr-Asp-Asn-Tyr-Thi'-Arg-Leu-Arg-Lys-Gln- Leu-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NHj The synthesis of (4-Cl-D-Phe6,Leu17)-VIP, amidated at the carboxy- terminus, was initiated by using all of the resin loaded with Fmoc-Asn(trt)-OH as prepared in example (a) above. 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, DIG and HOBt were used. The amounts of components are summarized in the table below: GRAMS OF GRAMS OF CYCLE PROTECTED CYCLE PROTECTED AMINO ACID AMINO ACID(Table Removed) Upon completion of synthesis and removal of the N-termmal Fmoc protecting group (steps 1-6 of the synthesis cycle), the peptide- resin was washed twice with methanol, dried and weighed to obtain 0.649g. This was subjected to cleavage in a cleavage mixture consisting of trifluoroacetic acid and scavengers, crystalline phenol, ethanedithol, thioanisole and water for a period of 3-5 hours at room temperature with continuous stirring. The peptide was precipitated using cold dry ether to obtain ~ 330 mg of crude peptide. The crude peptide was purified on a Clg preperative reverse phase HPLC column (250. Times . 10) on a .gradient of acetonitrile and water in 0.1% TFA as described elsewhere. The prominent peaks were collected and lyophilized, reanalysed on analytical HPLC and subjected to mass analysis. The calculated mass was 3342.09 and the mass obtained was 3344.43 . The HPLC pure peptide was then subjected to bio-analysis. INCORPORATION OF SPACER/BRIDGING AND CHELATING GROUPS TO THE PEPTIDE DERIVATIVES The attachment of the spacer groups to the peptide derivatives were carried out on solid phase, using the Fmoc-derivatives of the spacer compounds. Subsequently, the chelating compounds were conjugated to the peptides also on solid phase. General methods for radiolabeling with Tc-99m Example 3: In forming a complex of radioactive technetium with the peptide of this invention, the technetium complex, preferably a salt of Tc-99m pertechnetate, was reacted with the peptide of this invention in the presence of a reducing agent; in a preferred embodiment, the reducing agent being stannous chloride. Img of a peptide was dissolved in 1ml of water or 0.9% normal saline. To 100\|.ig of freshly dissolved peptide added 8-15\|ig of stannous chloride dissolved in 10% acetic acid. Set pH to 5.5 with 0.5N NaHCC>3. Added ImCi of freshly eluted Tc-99m sodium pertechnetate to the peptide, allowed the reaction to proceed at room temperature for 15-45 minutes and then filtered through a 0.22(.im filter. The radiolabeled peptide was either used directly or purified on a Sep Pak CIS cartridge using 50%MeCN-water/0.1% TFA as eluant. The extent of Tc-99m peptide labeling achieved was determined by instant thin layer chromatpgraphy (ITLC). 5jal of the radiopharmaceutical was spotted at the base of silica get coated ITLC strips and chromatographed with acetone or normal saline. Under these conditions 99% of Tc-99m associated radioactivity remained at the origin (Rf = 0) in either solvent indicating that no significant concentration of free Tc-99m pertechnetate could be detected in the sample. Example 4: Alternatively, the peptide of the invention was reacted with technetium-99m in a reduced form. Example 5: In another alternative, both the peptide of the invention and technetium-99m were reacted with a reducing agent prior to being reacted with each other; preferred reducing agent being stannous chloride. Example 6: In forming a complex of radioactive technetium with the MAG3 chelated peptide of this invention, the technetium complex, preferably a salt of Tc-99m pertechnetate, was reacted with the peptide of this invention in the presence of a reducing agent; in a preferred embodiment, the reducing agent being stannous chloride, Img of the MAG3- peptide was dissolved in 1ml of water or 0.9% normal saline. To lOOng of freshly dissolved peptide added 8-15}.ig of stannous chloride dissolved in 10% acetic acid. Set pH to 5.5 with 0.5N NaHCC-3. Added ImCi of freshly eluted Tc-99m sodium pertechnetate to the peptide, allowed the reaction to proceed at room temperature for 15-45 minutes and then filtered through a 0.22j.im filter. The radio!abeled peptide was either used directly or purified on a Sep Pak CIS cartridge using 50%)MeCN-water/0.1% TFA as eluant. The extent of Tc-99m peptide labeling achieved was determined by instant thin layer chromatography (ITLC). 5^1 of the radiopharmaceutical was spotted at the base of silica gel coated ITLC strips and chromatqgraphed with acetone or normal saline. Under these conditions 99% of Tc-99m associated radioactivity remained at the origin (Rf = 0) in either solvent indicating that no significant concentration of free Tc-99m pertechnetate could be detected in the sample. Example 7: Alternatively, the MAG3-peptide complex of the invention was reacted with technetium-99m in a reduced form. Example 8: In another alternative, both the MAG3-peptide complex of the invention and technetium-99m were reacted with a reducing agent prior to being reacted with each other; preferred reducing agent being stannous chloride. In vitro biological assays Example 9: The peptide of the invention was assayed for biological activity in homogeneous competition binding assays using 12^I labeled peptide and in heterogeneous displacement assays using either 125I labeled VIP (10-28) fragment or 125I labeled VIP. The assays were performed on a variety of human tumor cell lines In the practice of these methods, the peptide was radioiodinated using the iodogen method. Briefly, 5(.ig of the peptide in 10[il of 50mM PBS (pH 7.5), an appropriate amount of the radioisotope and 500\|.ig -Img iodogen were incubated at room temperature for 15-30min with occasional mixing Radioiodinated peptide was purified from unincorporated radioactive iodine by purification on a Sep Pak Cis cartridge, essentially following the same procedure as described for technetium labeling Example 10: Receptor binding and competition assays were performed at 4-8°C. Briefly. 50,000 cells were plated per well of a 24 well plate and allowed to adhere overnight. Before the assay, the cells were washed twice with ice cold Binding buffer (25mM HRPFS, H)mM MgCl2 and 1% BSA in RPMI 1640 medium). The cells were incubated for 2-3hrs with an appropriate concentration (0.1-lOnM) of the !25j labeled peptide in the presence and absence of the cold ligand (InM-lOuM). After incubation, the cells were washed thrice with the Binding buffer to remove the unbound peptide. The cells were lysed and counts were measured in a Gamma counter. From a comparison of the extent of binding in the presence or absence of the unlabeled peptide, the dissociation constant (Kd) (TABLE I) and maximal binding (Bmax) (TABLE II) were calculated for the peptide. The following tumor cell lines were assayed using the above described binding competition assay: HT29 (human colorectal adenocarcinoma); PTC (human primary tumor cells adenocarcinoma); KB (human squamous cell carcinoma); 4451 (human squamous cell carcinoma); A549 (human lung carcinoma); LI32 cell line and HBL100 (human breast carcinoma). Cells were grown in RPMI 1640 supplemented with 10% fetal calf serum, glutamine and antibiotics using standard cell culture techniques (see Animal Cell Culture : A practical Approach, Freshney, ed, IRL press: Oxford, UK, 1992). TABLE I(Table Removed) Example 11: Displacement assays were performed at 4-8°C. Briefly, 50,000 cells were plated per well of a 24 well plate and allowed to adhere overnight. Before the assay, the cells were washed twice with ice cold Binding buffer (25mM HEPES, l0mM MgCl2 and 1% BSA in RPMI 1640 medium). The cells were incubated for 2-3hrs with an appropriate concentration (0.1-lOnM) of 12SI labeled VIP (10-28) fragment in the presence and absence of the cold ligand (InM-lOuM). The non specific binding was ascertained using 10\|iM of VIP. After incubation, the cells were washed thrice with the Binding buffer to remove the unbound peptide. The cells were lysed and counts were measured in a Gamma counter. l;rom a comparison of the extent of binding in the presence or absence of the unlabeled peptide, a concentration was determined corresponding to inhibition of I25I labeled VIP (10-28) fragment binding by 50% (termed ihe ICso) (TABLE III). TABLE III(Table Removed) The following tumor cell lines were assayed using the above described displacement assay: HT29 (human colorectal adenocarcinoma); PTC (human primary tumor cells adenocarcinoma); KB (human squamous cell carcinoma); 4451 (human squamous cell carcinoma); A549 (human lung carcinoma); LI32 cell line and HBL100 (human breast carcinoma). Cells were grown in RPMI 1640 supplemented with 10% fetal calf serum, glutamine and antibiotics using standard cell culture techniques (see Animal Cell Culture : A practical Approach, Freshney, ed, IRL press: Oxford, UK, 1992). Example 12: Displacement assays were performed at 4-8°C. Briefly, 50,000 cells were plated per well of a 24 well plate and allowed to adhere overnight. Before the assay, the cells were washed twice with ice cold Binding buffer (25mM HEPES, lOmM MgCl2 and 1% BSA in RPMI 1640 medium). The cells were incubated for 2-3hrs with an .appropriate -concentration (0.1-lOnM) of ^25j labeled VIP in the preside and absence of the cold ligand (lnM-10jiM). The non specific binding was ascertained using lO^iM of VIP. After incubation, the cells were washed thrice with the Binding buffer to remove the unbound peptide. The cells were lysed and counts were measured in a Gamma counter. From a comparison of the extent of binding in the presence or absence of the unlabeled peptide, a concentration was determined corresponding to inhibition of *25j labeled VIP binding by 50% . The different tumor cell lines were assayed using the above described displacement assay: HT29 (human colorectal adenocarcinoma); PTC (human primary tumor cells adenocarcinoma); KB (human squamous cell carcinoma); 4451 (human squamous cell carcinoma); A549 (human lung carcinoma); L132 cell line and HBL100 (human breast carcinoma). Cells were grown in RPMI 1640 supplemented with 10% fetal calf serum, glutamine and antibiotics using standard cell culture techniques (see Animal Cell Culture : A practical Approach, Freshney, ed, IRL press: Oxford, UK, 1992). The ligand was able to significantly displace the binding of radiolabelled VIP to the cell lines. These results demonstrate that the VIP analog provided by the invention is capable of specifically binding to VIP receptors in standard in vitro assays on a variety of human tumor cell types. Imaging of human tumors induced in nude mice Example 13: 99mTc labeled peptide of the invention was used to image tumors induced subcutaneously in the abdomen of NIH nu/nu nude mice. Following intravenous administration in human adenocarcinoma tumor bearing nude mice, images were taken at different time intervals post infection, using conventional gamma camera. A rapid blood clearance was observed with little accumulation in liver and kidney and , while tumor uptake was found to achieve significant levels as early as 15 min post injection. The major pathway of clearance for the labeled peptide of the invention is through the kidneys as shown by a significant activity in the bladder and urine. These results indicate that the VIP analogue of the present invention has utility as scintigraphic imaging agent for imaging tumor of adenocarcinoma origin in humans. Maximum binding was seen at 3 hours leading to greater accumulation of radioactivity in tumors in comparison to the normal visceral tissue. The results are shown in Figure 1 which clearly depicts that the accumulation of Tc-99m labeled VIP analog is high in tumor (indicated with arrow) as compared to the accumulation in viscera (unmarked dots) after 3 hours of injection. We claim: 1. A radiolabeled peptide analog of vasoactive intestinal peptide comprising a synthetic receptor-binding peptide analog of vasoactive intestinal peptide (VIP) radiolabelled with a radionucleotide Technicium Tc-99m, a chelating group of the kind such as herein described and optionally a spacer group of the kind such as herein described wherein said receptor-binding peptide analog of vasoactive intestinal peptide has the sequence: His-Ser-Asp-Xxx-Val-4Cl-D-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Leu-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2 (SEQ IDNO:1). wherein Xxx is Ala or Aib (a-aminoisobutric acid), Deg (a,a -diethylglycine) or Ac5c (1-aminocyclopentane carboxylic acid). 2. A radiolabeled peptide analog as claimed in claim 1 wherein the chelating moiety is selected from the group consisting of one or more of iminodicarboxylic groups or polyaminopolycarboxylic groups such as non cyclic ligands or macrocyclic ligands such as herein described and chelating groups based on peptides. 3. A radiolabeled peptide analog as claimed in claim 2 wherein the chelating groups derived from non-cyclic ligands are selected from ethylenediaminetetraaceticacid (EDTA), ethylenetriaminepentaaceticacid (DTPA), ethyleneglycol-O,O'-bis (2- aminoethyl)-N,N,N',N'-tetraaceticacid (EGTA), N,N'-bis (hydroxybenzyl) ethylene diamine-N,N'-diacetic acid (HBED) and triethylenetriaminehexaacetic acid (TTHA). 4. A radiolabeled peptide analog as claimed in claim 2 wherein the chelating groups derived from said macrocyclic ligands are selected from 1,4,7,10-tetra- azacyclododecane-N,N',N",M'"-tetraaceticacid (DOTA), 1,4,7,10-tetra- azacyclotridecane-l,4,7,10-tetra aceticacid (TITRA), 1,4,8,11-tetra- azacyclotetradecane-N, N',N",N'"-tetraaceticacid (TETA), 1,4,8,11-tetra- azacyclotetradecane (TETRA) and aryl chelating moieties e.g. hydrazinonicotinamide (HYNIC). 5. A radiolabeled peptide analog as claimed in claim 2 wherein said chelating groups based on peptides comprises of mercatoacetyltriglycine (MAG3) such as herein described. 6. A radiolabeled peptide analog as claimed in claim 5 wherein said MAG 3 chelating agents comprises of SH-CH2-CO-Gly-Gly-Gly, Cys,Gly-Aib-Ala, Cys-Gly-Gly-Aib, Gly-Gly-Ala-Aib, Cys-Aib-Gly-Gly, Cys-Ala-Gly-Aib, Gly-Gly-Gly-Aib and Gly- Gly-Aib-Ala. 7. A radiolabeled peptide analog as claimed in any preceding claim wherein said bridging group is either aminocaproic acid or aminohexanoic acid. 8. A radiolabeled peptide analog as claimed in any preceding claim wherein said the chelating groups are attached to N-terminal amino acid residue of said peptide analog. 9. The radiolabeled vasoactive peptide analogs as claimed in any preceding claim wherein said the chelating groups are attached to N-terminal amino acid residue of said peptide analogs. 10. The radiolabeled vasoactive peptide analogs as claimed in any preceding claim wherein said chelating groups are attached to the peptide group directly. 11. The radiolabled vasoactive peptide analogs as claimed in any one of claims 1 to 9 wherein said chelating groups are attached to the peptide through spacers. 12. The radiolabeled vasoactive peptide analogs as claimed in claim 11 wherein it contains 33 amino acids of which 28 are from the peptide, one is from the spacer and four are from the chelating groups. 13. A method for the manufacture of a radiolabeled peptide analog of vasoactive intestinal peptide as claimed in any preceding claim, which comprises coupling a chelating group of the kind such as herein described to an analog of vasoactive intestinal peptide, having the sequence His-Ser-Asp-Xxx-Val-4Cl-D-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gin-Leu- Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2 (SEQ ID NO:1). where Xxx=Ala or Aib or Deg or Ac5c, coupling said chelating group to a spacer group of the kind such as herein described, and reacting the analog so produced with a pertechnate consisting of a salt of Tc-99m, in the presence of a reducing agent such as herein described. 14. A method as claimed in claim 18 wherein said reducing agent is stannous chloride.

Full Text

Radiolabeled vasoactive intestinal peptide analogs for diagnosis and radiotherapy
FIELD OF THE INVENTION;
The present invention encompasses radiolabeled peptide analogs of vasoactive intestinal peptide (VIP), labeled with Tc-99m, useful for imaging target sites within mammalian living systems The invention particularly provides radiolabeled-VIP derivatives that bind selectively to the VIP receptor on target cells. Specifically, the invention relates to the radiolabeling of VIP-receptor specific agents and their subsequent use for radiodiagnostic and radiotherapeutic purposes The invention encompasses methods for radiolabeling these peptide reagents with Tc-99m etc. and the use of these aforesaid derivatives as scintigraphic imaging agents. The present invention also encompasses the use of these radiolabeled peptides as anti-neoplastic agents for specific radiotherapy in cancer.
BACKGROUND OF THE INVENTION:
Vasoactive intestinal peptide is a 28-amino acid neuropeptide, which was first isolated from the porcine intestine (Said and Mutt, 1970). It bears extensive homology to secretin, PHI and glucagon. The amino acid sequence for VIP is :
His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-I,eu-Asn-Ser-Ile-Leu-Asn-NH2
VIP is known to exhibit a wide variety of biological activities such as the autocrine, endocrine and paracrine functions in living organisms (Said, 1984). In the gastrointestinal tract, it has been known to stimulate pancreatic and biliary secretions, hepatic glycogenesis as well as the secretion of insulin and glucagon (Kerrins and Said, 1972 ; Domschke et al., 1977). In the nervous system it acts as a neurotransmitter and neuromodulator, regulating the release and secretion of several key hormones (Said, 1984). In recent years, attention has been focussed on the function of VIP in certain areas of the CNS as well its role in the progression and control of neoplastic disease (Reubi, 1995).
The importance of peptide growth factors and regulatory hormones in the etiology and pathogenesis in several carcinomas has long been recognized. Data from epidemiological and

endocrinological studies suggest that neuropeptides like VIP which are responsible for the normal growth of tissues like the pancreas can also cause conditions for their neoplastic transformation (Sporn el al, 1980). Several lines of evidence indicate that VIP acts as a growth factor and plays a dominant autocrine and paracrine role in the sustained proliferation of cancer cells (Said, 1984). The stimulatory effect of VIP on tumor growth can be mediated directly by its receptors on cell membranes or indirectly by potentiation of the activities of other growth factors in tumor cells (Scholar et al, 1991). The synergistic effect of VIP and related pituitary adenylate cyclase activating polypeptide (PACAP) in glioblastomas is an illustration to the above fact (Moody el al., 1996)
The multiple physiological and pharmacological activities of VIP are mediated by high affinity G-protein coupled transmembrane receptors on target cells (Reubi, 1995). VIP receptors are coupled to cellular effector systems via adenylyl cyclase activity (Xia et al., 1996). The VTP receptor, found to be highly overexpressed in neoplastic cells, is thought to be one of the biomarkers in human cancers (Reubi, 1995). High affinity VIP receptors have been localized and characterized in neoplastic cells of most breast carcinomas, breast and prostate cancer metastases, ovarian, colonic and pancreatic adenocarcinomas, endometrial and squamous cell carcinomas, non small cell lung cancer, lymphomas, glioblastomas, astrocytomas, meningiomas and tumors of mesenchymal origin. Amongst, neuroendocrine tumors all differentiated and non-differntiated gastroenteropancreatic tumors, pheochromocytomas, small-cell lung cancers, neuroblastomas, pituitary adenomas as well tumors associated with hypersecretory states like Verner-Morrison syndrome were found to overexpress receptors for vasoactive intestinal peptide (Reubi, 1995, 1996, 1999; Tang et al, 1997a & b; Moody et al, 1998a &b; Waschek et al, 1995; Oka et al, 1998)). These findings suggest that new approaches for the diagnosis and treatment of these cancers may be based on functional manipulation of VIP activity, using synthetic peptide analogs of the same.
Historically, the somatostatin analog '"in-DTPA-fD-Phe^-octreotide is the only radiopeptide, which has obtained regulatory approval in USA and Europe (Lamberts et al, 1995). Radiolabeled VTP has been shown to visualize majority of gastropancreatic adenocarcinomas, neuroendocrine tumors, as well as insulinomas (which are often missed by radiolabeled octreotide) (Behr et al, 1999). VIP-receptor scinitigraphy offers certain advantages over radioimaging involving

somatostatin receptors. The presence of high affinity receptors for VIP have been demonstrated in a larger number of human tumors, relative to the somatostatin receptors. Secondly, the density of VIP receptors on tumors has been found to be greater than somatostatin (Behr el al, 1999). Therefore, the VIP-receptor scan is more sensitive and convenient in localizing tumors and their metastatic spread as compared to somatostatin The applications of this technique are manifold. It has been used for the sensitive detection of VIP-receptor positive tumors. This includes primary carcinoids, cancers of the gastrointestinal tract as well as distant metastases (Reubi, 1995, 1996). It can also be used to target cytotoxic radionuclides specifically to the tumor site. It predicts the VIP-receptor status of the patient and thereby the response of the patient towards radiotherapy by radiolabeled VIP analogs. Lastly, such radiolabeled peptides have been successfully used in radioguided surgery (Lamberts et al, 1995).
123I-VIP, I25I-VIP and their derivatives have been extensively used for imaging pancreatic adenocarcinomas, endocrine tumors of the gastrointestinal origin, mesenchymal tumors as well
secondary tumor metastatic sites, in patients (Jiang et al, 1997; Virgolini et al, 1996, 1998; Raderer et al, 1998 ; Moody et al., 1998; Kurtaran et al, 1997; Pallella et al, 1999). Radioiodinated VIP and its derivatives have been also used to assess the binding affinity of peptides for VDP-receptors on tumor cells in vitro. The biodistribution, safety and absorbed dose of the aforesaid radioiodinated peptide derivatives have also been studied earlier (Virgolini et al, 1995).
US Patent No. 5,849,261, granted to Dean etal, on December 15, 1998 describes the applications of radiolabeled vasoactive intestinal peptide (VIP) for diagnosis and therapy. In particular, this US Patent discloses a method for preparing a radiopharmaceutical agent, comprising of native vasoactive intestinal (VIP) peptide attached to a radionucleide like technetium or rhenium via a chelating moiety. The radiopharmaceutical when labeled with technetium or rhenium via a chelating moiety has a VIP binding affinity which is not less than about one tenth the affinity of radioiodinated native VIP for the said receptor.
However, there is still a need for improved synthetic analog of VIP as radiopharmaceuticals, which are easy to generate and are capable of being employed with higher sensitivity and specificity in terms of their radio-imaging properties.

This invention describes the preparation and use. of peptide analogs of VIP using constrained amino acids. 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 § , \\i angles, within the molecule, thereby stabilizing a desired peptide conformation. The prototypic member of a,a-dialkylated aminoacids, a-aminoisobutyric acid (Aib) or a5a-dimethylglycine has been shown to induce p-turn or helical conformation when incorporated in a peptide sequence (Prasad and Balaram, 1984, Karle and Balaram, 1990). The conformational properties of the higher homologs of a,a-dialkylated amino acids such as di-ethylglycine (Deg), di-n-propylglycine (Dpg), 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), as 1-aminocycloheptane carboxylic acid (Ac7c) and as 1-aminocyclooctane carboxylic acid (Ac8c) have also been shown to induce folded conformation (Prasad et al., 1995 ; Karle et al., 1995). a,a-dialkylated amino acids have been used in the design of highly potent chemotactic peptide analogs (Prasad et al., 1996) However, the applicants are not aware of any prior art for the synthesis of novel peptide analogs, encompassed in the present invention, particularly the synthesis of such VIP peptide analogs, containing a, a-dialkylated amino acids, by solid phase peptide synthesis methodology. Moreover, the use of such constrained amino acids for the design of peptides possessing anti-neoplastic activity is also unknown in any previous prior art. The present invention exploits the conformational properties of such a, a-dialkylated amino acids for the design of biologically active peptide derivatives, taking VIP as the model system under consideration.
REFERENCES:
Behr T.M. etal. Q. J. Nucl. Med., 43, 268-280,1999. Domschke, S. etal. Gastroenterology, 73, 478-480, 1977. Jiang S. etal. Cancer Res., 57, 1475-1480,1997 Karle, I.L. etal (1995) J. Amer. Chem. Soc. 777, 9632-9637.
Karle, I.L. and Balaram, P. (1990) Biochemistry 29, 6747-6756.
Kerrins, C and Said, S.I. Proc Soc. Exp Biol. Med., 142, 1014-1017, 1972
Kurtaran A. et al. J Nucl. Med., 38, 880-881, 1997
Lamberts, S.W.J. et al In Somatostatin and its Receptors, Ciba Found. Symp, 190, 222-239, 1995
Moody, T.W. el al Peptides, 19 (3), 1998a
Moody, T.W. etal Ann. N.Y. Acad. Sci., 865, 290-296. 1998b.
Oka, H. et al. (1998) Am. J. Pathol., 153 (6), 1787-1796, 1998.
Pallella, V.R. el al J. Nucl. Med., 40(2), 352-360, 1999
Prasad, B.V.V and Balaram, P. (1984) CRC Crit. Rev. Biochem. 16, 307-347.
Prasad, S etal. (1995) Biopolymers 35, 11-20
Prasad, S et al. (1996) Int. J. Peptide Protein Res. 48, 312-318.
Reubi, J.C, J. Nucl. Med., 36 (10), 1995.
Reubi, J.C. etal. Int. J. Cancer, 81 (3), 1999.
Reubi, J.C. etal. Cancer Res., 56 (8), 1922-1931, 1996.
Raderer, M. etal. J. Nucl. Med., 39 (9), 1570-1575, 1998
Said, S. I. and Mutt, V. Science, 169, 1217-1218,1970
Said, S.I. Peptides, 5, 143-150, 1984
Sporn, M.B., and Todaro, G.J. N. Engl. J. Med., 303, 378-379,1980.
Tang, C. et al, Gut, 40 (2), 267-271, 1997a.
Tang, C. etal, Br. J. Cancer, 75 (10)1467-1473, 1997b.
Virgolini, I. etal. J. Nucl. Med., 36(10), 1732-1739, 1995.
Virgolini, I. etal. Nucl. Med. Biol., 23 (6), 685-692, 1996.
Virgolini, I. etal. J. Nucl. Med., 39 (9), 1998.
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SUMMARY OF THE INVENTION:
The present invention discloses for the first time the use of certain novel VIP analogs to determine the binding affinities of these peptides for their cognate receptors on cancer cells. The VTP peptide analog of the present invention, which is a VIP receptor antagonist has the sequence
His-Ser-Asp-Xxx-Val-4-Cl-D-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Leu-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2 (SEQ ID NO: 1)
where Xxx = Ala or Aib or Deg or Ac5c
Abbreviation:
Aib- a- Aminoisobutyric acid
Deg- a , a- Diethylglycine
Ac5c- 1-Amino Cyclopentane Carboxylic acid
The applicants have found that the VIP analogs discussed in the present invention have greater affinity for its cognate receptors on tumor cells as compared to native VIP, which in turn lead to better radio-imaging and radio-therapeutic efficacy of the radiopharmaceutical of the present invention. While not wishing to be bound by theory, the applicants believe that the improved efficacy of the radiopharmaceuticals of the present invention are due to the nature of the VIP analogs themselves, which have receptor bound conformations caused by the incorporation of the unusual amino acids. The chelating groups are attached at a novel site in the VTP analogs, i.e., to the N-terminal amino acid residue of the VIP analogs. As any reader skilled in the art will appreciate, the previous prior art encompasses radiopharmaceutical agents in, which the chelating agents are attached to the C-terminal end of the VIP (Dean et al., U.S. Patent No 5,849,261).
While conventional chelating agents are within the scope of the present invention, the applicants have, for the first time, employed certain novel MAG3 derivatives as chelating agents. The present invention also envisages use of certain aminocaproic acid (Acp) and aminohexanoic acid (Aha) as spacer groups for the first time between a chelating moiety and VIP analog. The present invention therefore envisages a novel radiopharmacetical comprising of an hitherto unknown as VIP analog covalently linked to MAG3 derivatives via aminocaproic acid (Acp) or aminohexanoic acid (Aha). The radiopharmaceutical molecule so obtained is novel per se and has not been reported in any prior art known to the applicants and exhibits an improved radioscintigraphic and radiotherapeutic efficacy as compared to such radiolabed reagents known in the prior art.
Accordingly, the present invention provides novel radiolabeled peptide analogs of vasocative intestinal peptide useful for imaging target sites within a mammalian living system, comprising of a synthetic receptor-binding peptide analog of vasoactive intestinal peptide (VIP) radiolabeled with Tc-99m, wherein said peptide analogs of vasoactive intestinal peptide has the sequence: His-Ser-Asp-Xxx-Val-4-Cl-D-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leii-Arg-Lys-Gln-Leu-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2 (SEQ ID NO: 1) Where Xxx = Ala or Aib or Deg or Ac5c
In a preferred embodiment of the present invention, the radionucleide Tc-99m is bound to a chelating moeity, which is attached to the N-terminal amino acid residue of the said VIP analog.
The labeling of peptides by Tc-99m has been accomplished in the present invention, by several strategies. In the present invention radioimaging of tumors has been done by
1) Direct labeling of Tc-99m to the peptide analogs.
2) Attachment of chelating groups to the peptide and subsequent radiolabeling by
Tc-99m.

3) Incorporation of Tc-99m to chelator moiety covalently linked to the peptide via the spacer
group.
4) Use of a variety of chelator moieties to the system (3).
It is important to note that in the above cases, 2, 3 and 4, the chelator and spacer groups are incorporated site-specifically at a position which does not affect the specific binding properties of the peptide to the VIP receptor on tumor cells in vitro and in vivo.
Ideally the chelating group is hydrophobic in nature. In a preferred embodiment, the chelating groups are based on peptides such as mercaptoacetyltriglycine (MAG 3) and its derivatives such as
SH CH2-CO-Gly-Gly-Gly
Cys-Gly-Aib-Ala
Cys-Gly-Gly-Aib
Gly-Gly-Ala-Aib
Cys-Aib-Gly-Gly
Cys-Ala-Gly-Aib
Gly-Gly-Gly-Aib
Gly-Gly-Aib-Ala
The methods involved in the synthesis, purification, characterization and radiolabeling of these peptides are illustrated in detail in the following section containing examples. The following section also includes biological data relating to the imaging efficacy and dosimetry of the aforesaid radiolabeled peptides These examples have been furnished for illustrating and providing insight into the invention and should not be construed as limiting.
DETAILED DESCRIPTION OF THE INVENTION
The VIP receptor antagonist His-Ser-Asp-Xxx-Val-4-Cl-D-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Leu-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Tle-Leu-Asn-NH2(SEQ TD NO: 1) Where Xxx = Ala or Aib or Deg or Ac5c
has been shown in our previous studies to be selectively binding to VIP receptors on cancer cells. Several lines of evidence suggest that VIP can also function as a growth factor mediating the sustained proliferation of cancer cells, by an autocrine or paracrine mechanism. This has led to intense research focussed on the design and biological activity of VIP antagonists. The anti-proliferative activity of the aforesaid VIP antagonist has been previously demonstrated in a number of experimental models of pancreatic, prostate, mammary and lung cancer, suggesting its high anti-neoplastic therapeutic potential.
The present invention relates to the use of the above peptide, process for its production, pharmaceutical preparation for its use as a diagonstic, imaging as well as a radiotherapeutic agent in vivo. The present invention also includes several strategies involved in the generation of the above mentioned radiopharmaceutical. These have been described in detail in the "Examples" section, however, a few illustrations are also detailed below:
A preferred embodiment of the invention involves the direct radiolabeling of the above mentioned VIP antagonist directly to Tc-99m. Tc-99m forms a co-ordinate covalent linkage with certain specific amino acid residues of the peptide. The formation of a stable Tc-peptide bond is one of the major advantages for its use for imaging purposes. The attachment of Tc-99m to the peptide involves the reaction of pertechnate (a salt of Tc-99m) to the peptide, in the presence of a reducing
agent like stannous chloride. The radiolabeled peptides are separated from the unincorporated Tc-99m as described in example and used for radioscintigraphy.
Another embodiment of the present invention includes the attachment of certain chelating groups to the VIP analogue. According to the invention, the chelating moiety may be attached directly or indirectly to the peptide, eg by means of a spacer or a bridging group to the amino terminus of the VIP analog The present invention encompasses compounds of both these categories The present invention relates to physiologically acceptable chelating groups, capable of complexing a detectable element. According to one preferred embodiment of the invention, the chelating group has substantial hydrophobic character. Examples of chelating groups include eg iminodicarboxylic groups, polyaminopolycarboxylic groups, e.g. those derived from non-cyclic ligands e.g. ethylene diamine tetra acetic acid (EDTA), diethylene triamine pentaacetic acid (DTPA), ethylene glycol-0,0'-bis (2-aminoethyl)-N,N,N',N'-tetraacetic acid (EOTA), N,N'-bis(hydroxybenzyl)ethylenediamine-N,N'-diacetic acid (HBED) and triethylenetriaminehexaacetic acid (TTHA).
The chelating groups derived from macrocyclic ligands include,
e.g. l,4,7,10-tetra-azacyclododecane-N,N',N",N"'-tetra acetic acid (DOTA).
l,4,7,10-tetra-azacyclotridecane-l,4,7,10-tetra acetic acid (TTTRA)
1,4,8,ll-tetra-azacyclotetradecane-N,N',]SP',N"'-tetra acetic acid (TETA)
1,4,8,11-tetra-azacyclotetradecane (TETRA)
Aryl chelating moieties e.g. hydrazinonicotinamide (HYNIC)
The present invention also encompasses chelating .groups based on peptides eg preferred derivatives of mercaptoacetyltriglycine (MAG 3) which are not previously known be employed as chealting agents in this field. These MAG 3 chelating agents include
SH-CHrCO-Gly-Gly-Gly
Cys-Gly-Aib-Ala
Cys-Gly-Gly-Aib
Gly-Gly-Ala-Aib
Cys-Aib-Gly-Gly
Cys-AJa-Gly-Aib Gly-Gly-Gly-Aib Gly-Gly-Aib-Ala
The invention also envisages preferred spacer / bridging groups like those derived from aminoacids such as aminocaproic acid, aminohexanoic acid etc.
Thus according to the present invention there is also provided a novel radiolabeled peptide analog of vasocative intestinal peptide useful for imaging target sites within a mammalian living system, comprising of a synthetic receptor-binding peptide analog of vasoactive intestinal peptide (VIP) radiolabed with Tc-99m, wherein said peptide analog of vasoactive intestinal peptide has the
sequence:
His-Ser-Asp-Xxx-Val-4-Cl-D-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Leu-Ala-Val-Lys-Lys-Tyr-Leu -Asn-Ser-Ile-Leu-Asn-NH2 (SEQ ID NO: 1)
where Xxx = Ala or Aib or Deg or Ac5c, the radionuclide Tc-99m being bound to a chelating moeity, which is attached to the N-terminal amino acid residue of the said VIP analog.
Preferably, the chelating groups are selected from MAGS peptide derivatives selected from the
group consisting of SH-CH2-CO-Gly-Gly-Gly, Cys-Gly-Aib-Ala, Cys-Gly-Gly-Aib,
Gly-Gly-Ala-Aib, Cys-Aib-Gly-Gly, Cys-Ala-Gly-Aib, Gly-Gly-Gly-Aib and
Gly-Gly-Aib-Ala.
The invention involves also envisages preferred spacer / bridging groups like those derived from amino acids, e.g. aminocaproic acid, aminobutyric acid, aminohexanoic acid etc and with the specific MAG 3 chelators described above, the present invention envisage a radiolabeled vasoactive intestinal peptide which is novel per se and which exhibits improved efficacy in terms of imaging, diagnosis, and therapy.
This novel peptide reagent comprises of 33 amino acids - 28 from VIP analog, 1 from spacer and 4 from chelating moeity attached to radiolabeled technitium to provide a novel and hitherto unknown radiotherapeutic and radioscintigraphic agent.
All the radiolabeled chelated peptides retain their affinity for VIP receptors on cancer cells. The radiolabeled VIP derivatives of the present invention exhibit pharmacological activity and therefore are useful as either imaging agent for visualization of VIP-receptor positive tumors and metastases or as a radiotherapeutic agent for the treatment of such tumors in vivo by specifically targeting the cytotoxic radionucleide selectively to the tumor site in mammalian living systems
EXAMPLES :
Solid phase peotide synthesis
An analog of the present invention can be made by exclusively solid phase techniques, by partial solid phase / solution phase techniques and fragment condensation.
Methods for chemical synthesis of polypeptides is well known in art, and in this regard, reference is made, by way of illustration, to the following literature: Stewart and Young, Solid Phase Peptide Synthesis (W. H. Freeman and Co., 1969), Atherton and Shepherd etc. Preferred, semi-automated, stepwise solid phase methods for synthesis of peptides of the invention are provided in the examples below.
Example 1:
Preparation of VIP analogs
Peptides were synthesized using preferably, Fmoc (9-fluorenyl methoxy carbonyl) solid- phase methodology, on CS Bio (Model 536) Peptide Synthesizer (CS Bio Co., San Carlos, California, U.S. A.).
Sequential assembly of a peptide analog was conducted from the carboxy- terminus, by loading of a Fmoc protected amino acid to a solid- phase resin, to the amino terminus. This was proceeded by subsequent removal of the Fmoc protecting group of the amino acid and a stepwise, sequential addition of Fmoc protected amino acids in repetitive cycles to obtain an intermediate protected peptide resin.
For peptides that were amidated at the carboxy-terminus, Rink Amide resin was employed, and the loading of the first Fmoc protected amino acid was affected via an amide bond formation with
the solid support, mediated by diisopropylcarbodiimide and HOBt. Substitution levels for automated synthesis were preferably between 0.2 and 0.8 mmole amino acid per gram resin.
Steps in the synthesis of VIP analogs encompassed in the present invention, employed the following protocol:(Table Removed)
The 9-fluorenyl methoxy carbonyl (Fmoc) group was used for the protection of the a-amino group of all amino acids employed in the syntheses. However, other protecting groups known in the art for a-amino group may be employed successfully. Side chain functional groups were protected as follows: Trityl (trt) and t-butyloxycarbonyl (Boc) were the preferred protecting groups for the imidazole group of Histidine. Hydroxyl groups of serine, threonine and tyrosine were protected by t- butyl (t-Bu) groups. Pmc (2,2,5,7,8-pentamethyl-chroman-6-sulfonyl) and Pbf (2,2,4,6,7-pentamethyldihydro benzofuran-5-sulfonyl) were the preferred protecting groups for the guanido group in Arginine. Trityl protection was used for asparagine and glutamine. Tryptophan was either used with Boc protection or unprotected. The lysine side chain was Boc protected and aspartic acid and glutamic acid had t-butyl side chain protection.
The resin employed for the synthesis of carboxy-ami dated analogs was 4-(2',4'-Dimethoxypheny1-Fmoc-aminomethyl)-phenoxyrnethyl- derivatized polystyrene 1% divinylbenzene (Rink Amide) resin (100-200 mesh), procured from Calbioichem-Novabiochem Corp., La Jolla, U.S.A., (0.47 milliequivalent NH. Sub. 2 /g resin).
Typically, 2-8 equivalents of Fmoc protected amino acid per resin nitrogen equivalent were used. The activating reagents used for coupling of amino acids in the solid phase synthesis of peptides arc well known in the art. These include BOP, PyBOP, HBTU, TBTU, PyBOP, HOBt. Preferably, DCC or DIG / HOBt or HBTU/HOBT and DIEA couplings were carried out.
Swelling of the resin was typically carried out in dichloromethane measuring to volumes 10-40ml /g resin. 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.
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). Any incomplete reactions were re-coupled using freshly prepared activated amino acids.
After complete assembly of the analog, the ammo-terminal Fmoc group was removed using steps 1-6 of the above protocol and then the peptide- resin was washed with methanol and dried. The analogs 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® CIS (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 C18 LiChrospher®, WP-300 (300.Times.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.

(a) Preparation of Fmoc-Asn(trt)- Resin
A typical preparation of the Fmoc-Asn(trt)-resin was earned out using 0.5g of 4-(2',4'-Dimethoxyphenyl-Fmoc-aminomethyl) phenoxymethyl-derivatized polystyrene 1% divinylbenzene (Rink Amide ) resin ( 0.47 mM / g ) (100-200 mesh), procured from Calbiochem-Novabiochem Corp., La .lolla, U.S.A. The resin was first allowed to swell in methylene chloride (2. Times. 25ml for 10 min.). It was washed once in dimethylformamide for 1 min. All solvents in the automated protocol were in 20ml portions per addition. The Fmoc- protecting group on the resin was removed by following steps 3 to 7 of the synthesis protocol. Deprotection of the Fmoc group was checked by the presence of blue beads in a positive Kaiser test. For loading of the first amino acid on the free amino (N. H. sub. 2) group of the resin, the first amino acid, Fmoc-Asn(trt)-OH, was weighed in four 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, Phillipsburg, New Jersey, U.S.A.) and activated with DIG, 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 Arg, Asn, Gin and His. The coupling reaction was carried out for a period ranging from 1-3 hours. Loading of the first amino acid was complete when Kaiser test gave a negative result and there was adequate weight increase when the resin, with the first amino acid attached, was dried in vaccuo overnight and weighed.
Example 2:
(b) Synthesis of: His-Ser-Asp-Ala-Val-4-Cl-D-Phe-Thr-Asp-Asn-Tyr-Thi'-Arg-Leu-Arg-Lys-Gln-
Leu-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NHj
The synthesis of (4-Cl-D-Phe6,Leu17)-VIP, amidated at the carboxy- terminus, was initiated by using all of the resin loaded with Fmoc-Asn(trt)-OH as prepared in example (a) above. 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, DIG and HOBt were used. The amounts of components are summarized in the table below:
GRAMS OF GRAMS OF
CYCLE PROTECTED CYCLE PROTECTED
AMINO ACID AMINO ACID(Table Removed)
Upon completion of synthesis and removal of the N-termmal Fmoc protecting group (steps 1-6 of the synthesis cycle), the peptide- resin was washed twice with methanol, dried and weighed to obtain 0.649g. This was subjected to cleavage in a cleavage mixture consisting of trifluoroacetic acid and scavengers, crystalline phenol, ethanedithol, thioanisole and water for a period of 3-5 hours at room temperature with continuous stirring. The peptide was precipitated using cold dry ether to obtain ~ 330 mg of crude peptide. The crude peptide was purified on a Clg preperative reverse phase HPLC column (250. Times . 10) on a .gradient of acetonitrile and water in 0.1% TFA as described elsewhere. The prominent peaks were collected and lyophilized, reanalysed on analytical HPLC and subjected to mass analysis. The calculated mass was 3342.09 and the mass obtained was 3344.43 . The HPLC pure peptide was then subjected to bio-analysis.
INCORPORATION OF SPACER/BRIDGING AND CHELATING GROUPS TO THE PEPTIDE DERIVATIVES
The attachment of the spacer groups to the peptide derivatives were carried out on solid phase, using the Fmoc-derivatives of the spacer compounds. Subsequently, the chelating compounds were conjugated to the peptides also on solid phase.
General methods for radiolabeling with Tc-99m Example 3:
In forming a complex of radioactive technetium with the peptide of this invention, the technetium complex, preferably a salt of Tc-99m pertechnetate, was reacted with the peptide of this invention in the presence of a reducing agent; in a preferred embodiment, the reducing agent being stannous chloride. Img of a peptide was dissolved in 1ml of water or 0.9% normal saline. To 100|.ig of freshly dissolved peptide added 8-15|ig of stannous chloride dissolved in 10% acetic acid. Set pH to 5.5 with 0.5N NaHCC>3. Added ImCi of freshly eluted Tc-99m sodium pertechnetate to the peptide, allowed the reaction to proceed at room temperature for 15-45 minutes and then filtered through a 0.22(.im filter.
The radiolabeled peptide was either used directly or purified on a Sep Pak CIS cartridge using 50%MeCN-water/0.1% TFA as eluant. The extent of Tc-99m peptide labeling achieved was determined by instant thin layer chromatpgraphy (ITLC). 5jal of the radiopharmaceutical was spotted at the base of silica get coated ITLC strips and chromatographed with acetone or normal saline. Under these conditions 99% of Tc-99m associated radioactivity remained at the origin (Rf = 0) in either solvent indicating that no significant concentration of free Tc-99m pertechnetate could be detected in the sample.
Example 4:
Alternatively, the peptide of the invention was reacted with technetium-99m in a reduced form.
Example 5:
In another alternative, both the peptide of the invention and technetium-99m were reacted with a reducing agent prior to being reacted with each other; preferred reducing agent being stannous chloride.
Example 6:
In forming a complex of radioactive technetium with the MAG3 chelated peptide of this invention, the technetium complex, preferably a salt of Tc-99m pertechnetate, was reacted with the peptide of this invention in the presence of a reducing agent; in a preferred embodiment, the reducing
agent being stannous chloride, Img of the MAG3- peptide was dissolved in 1ml of water or 0.9% normal saline. To lOOng of freshly dissolved peptide added 8-15}.ig of stannous chloride dissolved in 10% acetic acid. Set pH to 5.5 with 0.5N NaHCC-3. Added ImCi of freshly eluted Tc-99m sodium pertechnetate to the peptide, allowed the reaction to proceed at room temperature for 15-45 minutes and then filtered through a 0.22j.im filter.
The radio!abeled peptide was either used directly or purified on a Sep Pak CIS cartridge using 50%)MeCN-water/0.1% TFA as eluant. The extent of Tc-99m peptide labeling achieved was determined by instant thin layer chromatography (ITLC). 5^1 of the radiopharmaceutical was spotted at the base of silica gel coated ITLC strips and chromatqgraphed with acetone or normal saline. Under these conditions 99% of Tc-99m associated radioactivity remained at the origin (Rf = 0) in either solvent indicating that no significant concentration of free Tc-99m pertechnetate could be detected in the sample.
Example 7:
Alternatively, the MAG3-peptide complex of the invention was reacted with technetium-99m in a reduced form.
Example 8:
In another alternative, both the MAG3-peptide complex of the invention and technetium-99m were reacted with a reducing agent prior to being reacted with each other; preferred reducing agent being stannous chloride.
In vitro biological assays Example 9:
The peptide of the invention was assayed for biological activity in homogeneous competition
binding assays using 12^I labeled peptide and in heterogeneous displacement assays using either 125I labeled VIP (10-28) fragment or 125I labeled VIP. The assays were performed on a variety of human tumor cell lines
In the practice of these methods, the peptide was radioiodinated using the iodogen method. Briefly, 5(.ig of the peptide in 10[il of 50mM PBS (pH 7.5), an appropriate amount of the
radioisotope and 500|.ig -Img iodogen were incubated at room temperature for 15-30min with occasional mixing Radioiodinated peptide was purified from unincorporated radioactive iodine by purification on a Sep Pak Cis cartridge, essentially following the same procedure as described for technetium labeling
Example 10:
Receptor binding and competition assays were performed at 4-8°C. Briefly. 50,000 cells were plated per well of a 24 well plate and allowed to adhere overnight. Before the assay, the cells were washed twice with ice cold Binding buffer (25mM HRPFS, H)mM MgCl2 and 1% BSA in RPMI 1640 medium). The cells were incubated for 2-3hrs with an appropriate concentration (0.1-lOnM)
of the !25j labeled peptide in the presence and absence of the cold ligand (InM-lOuM). After incubation, the cells were washed thrice with the Binding buffer to remove the unbound peptide. The cells were lysed and counts were measured in a Gamma counter. From a comparison of the extent of binding in the presence or absence of the unlabeled peptide, the dissociation constant (Kd) (TABLE I) and maximal binding (Bmax) (TABLE II) were calculated for the peptide.
The following tumor cell lines were assayed using the above described binding competition assay: HT29 (human colorectal adenocarcinoma); PTC (human primary tumor cells adenocarcinoma); KB (human squamous cell carcinoma); 4451 (human squamous cell carcinoma); A549 (human lung carcinoma); LI32 cell line and HBL100 (human breast carcinoma). Cells were grown in RPMI 1640 supplemented with 10% fetal calf serum, glutamine and antibiotics using standard cell culture techniques (see Animal Cell Culture : A practical Approach, Freshney, ed, IRL press: Oxford, UK, 1992).
TABLE I(Table Removed)
Example 11:
Displacement assays were performed at 4-8°C. Briefly, 50,000 cells were plated per well of a 24 well plate and allowed to adhere overnight. Before the assay, the cells were washed twice with ice cold Binding buffer (25mM HEPES, l0mM MgCl2 and 1% BSA in RPMI 1640 medium). The
cells were incubated for 2-3hrs with an appropriate concentration (0.1-lOnM) of 12SI labeled VIP (10-28) fragment in the presence and absence of the cold ligand (InM-lOuM). The non specific binding was ascertained using 10|iM of VIP. After incubation, the cells were washed thrice with
the Binding buffer to remove the unbound peptide. The cells were lysed and counts were measured in a Gamma counter. l;rom a comparison of the extent of binding in the presence or absence of the unlabeled peptide, a concentration was determined corresponding to inhibition of
I25I labeled VIP (10-28) fragment binding by 50% (termed ihe ICso) (TABLE III).
TABLE III(Table Removed)
The following tumor cell lines were assayed using the above described displacement assay: HT29 (human colorectal adenocarcinoma); PTC (human primary tumor cells adenocarcinoma); KB (human squamous cell carcinoma); 4451 (human squamous cell carcinoma); A549 (human lung carcinoma); LI32 cell line and HBL100 (human breast carcinoma). Cells were grown in RPMI 1640 supplemented with 10% fetal calf serum, glutamine and antibiotics using standard cell culture techniques (see Animal Cell Culture : A practical Approach, Freshney, ed, IRL press: Oxford, UK, 1992).
Example 12:
Displacement assays were performed at 4-8°C. Briefly, 50,000 cells were plated per well of a 24 well plate and allowed to adhere overnight. Before the assay, the cells were washed twice with ice cold Binding buffer (25mM HEPES, lOmM MgCl2 and 1% BSA in RPMI 1640 medium). The
cells were incubated for 2-3hrs with an .appropriate -concentration (0.1-lOnM) of ^25j labeled VIP in the preside and absence of the cold ligand (lnM-10jiM). The non specific binding was
ascertained using lO^iM of VIP. After incubation, the cells were washed thrice with the Binding buffer to remove the unbound peptide. The cells were lysed and counts were measured in a Gamma counter. From a comparison of the extent of binding in the presence or absence of the
unlabeled peptide, a concentration was determined corresponding to inhibition of *25j labeled VIP binding by 50% . The different tumor cell lines were assayed using the above described displacement assay: HT29 (human colorectal adenocarcinoma); PTC (human primary tumor cells adenocarcinoma); KB (human squamous cell carcinoma); 4451 (human squamous cell carcinoma); A549 (human lung carcinoma); L132 cell line and HBL100 (human breast carcinoma). Cells were grown in RPMI 1640 supplemented with 10% fetal calf serum, glutamine and antibiotics using standard cell culture techniques (see Animal Cell Culture : A practical Approach, Freshney, ed, IRL press: Oxford, UK, 1992). The ligand was able to significantly displace the binding of radiolabelled VIP to the cell lines.
These results demonstrate that the VIP analog provided by the invention is capable of specifically binding to VIP receptors in standard in vitro assays on a variety of human tumor cell types.
Imaging of human tumors induced in nude mice Example 13:
99mTc labeled peptide of the invention was used to image tumors induced subcutaneously in the abdomen of NIH nu/nu nude mice. Following intravenous administration in human adenocarcinoma tumor bearing nude mice, images were taken at different time intervals post infection, using conventional gamma camera. A rapid blood clearance was observed with little accumulation in liver and kidney and , while tumor uptake was found to achieve significant levels as early as 15 min post injection. The major pathway of clearance for the labeled peptide of the invention is through the kidneys as shown by a significant activity in the bladder and urine. These results indicate that the VIP analogue of the present invention has utility as scintigraphic imaging agent for imaging tumor of adenocarcinoma origin in humans. Maximum binding was seen at 3 hours leading to greater accumulation of radioactivity in tumors in comparison to the normal visceral tissue. The results are shown in Figure 1 which clearly depicts that the accumulation of Tc-99m labeled VIP analog is high in tumor (indicated with arrow) as compared to the accumulation in viscera (unmarked dots) after 3 hours of injection.

We claim:
1. A radiolabeled peptide analog of vasoactive intestinal peptide comprising a synthetic receptor-binding peptide analog of vasoactive intestinal peptide (VIP) radiolabelled with a radionucleotide Technicium Tc-99m, a chelating group of the kind such as herein described and optionally a spacer group of the kind such as herein described wherein said receptor-binding peptide analog of vasoactive intestinal peptide has the sequence:
His-Ser-Asp-Xxx-Val-4Cl-D-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Leu-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2 (SEQ IDNO:1). wherein Xxx is Ala or Aib (a-aminoisobutric acid), Deg (a,a -diethylglycine) or Ac5c (1-aminocyclopentane carboxylic acid).
2. A radiolabeled peptide analog as claimed in claim 1 wherein the chelating moiety is
selected from the group consisting of one or more of iminodicarboxylic groups or
polyaminopolycarboxylic groups such as non cyclic ligands or macrocyclic ligands
such as herein described and chelating groups based on peptides.
3. A radiolabeled peptide analog as claimed in claim 2 wherein the chelating groups
derived from non-cyclic ligands are selected from ethylenediaminetetraaceticacid
(EDTA), ethylenetriaminepentaaceticacid (DTPA), ethyleneglycol-O,O'-bis (2-
aminoethyl)-N,N,N',N'-tetraaceticacid (EGTA), N,N'-bis (hydroxybenzyl) ethylene
diamine-N,N'-diacetic acid (HBED) and triethylenetriaminehexaacetic acid (TTHA).
4. A radiolabeled peptide analog as claimed in claim 2 wherein the chelating groups
derived from said macrocyclic ligands are selected from 1,4,7,10-tetra-
azacyclododecane-N,N',N",M'"-tetraaceticacid (DOTA), 1,4,7,10-tetra-
azacyclotridecane-l,4,7,10-tetra aceticacid (TITRA), 1,4,8,11-tetra-
azacyclotetradecane-N, N',N",N'"-tetraaceticacid (TETA), 1,4,8,11-tetra-
azacyclotetradecane (TETRA) and aryl chelating moieties e.g. hydrazinonicotinamide
(HYNIC).
5. A radiolabeled peptide analog as claimed in claim 2 wherein said chelating groups
based on peptides comprises of mercatoacetyltriglycine (MAG3) such as herein
described.
6. A radiolabeled peptide analog as claimed in claim 5 wherein said MAG 3 chelating
agents comprises of SH-CH2-CO-Gly-Gly-Gly, Cys,Gly-Aib-Ala, Cys-Gly-Gly-Aib,
Gly-Gly-Ala-Aib, Cys-Aib-Gly-Gly, Cys-Ala-Gly-Aib, Gly-Gly-Gly-Aib and Gly-
Gly-Aib-Ala.
7. A radiolabeled peptide analog as claimed in any preceding claim wherein said
bridging group is either aminocaproic acid or aminohexanoic acid.
8. A radiolabeled peptide analog as claimed in any preceding claim wherein said the
chelating groups are attached to N-terminal amino acid residue of said peptide analog.
9. The radiolabeled vasoactive peptide analogs as claimed in any preceding claim
wherein said the chelating groups are attached to N-terminal amino acid residue of
said peptide analogs.
10. The radiolabeled vasoactive peptide analogs as claimed in any preceding claim
wherein said chelating groups are attached to the peptide group directly.
11. The radiolabled vasoactive peptide analogs as claimed in any one of claims 1 to 9
wherein said chelating groups are attached to the peptide through spacers.
12. The radiolabeled vasoactive peptide analogs as claimed in claim 11 wherein it
contains 33 amino acids of which 28 are from the peptide, one is from the spacer and
four are from the chelating groups.
13. A method for the manufacture of a radiolabeled peptide analog of vasoactive
intestinal peptide as claimed in any preceding claim, which comprises coupling a
chelating group of the kind such as herein described to an analog of vasoactive
intestinal peptide, having the sequence
His-Ser-Asp-Xxx-Val-4Cl-D-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gin-Leu-
Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2 (SEQ ID NO:1).
where Xxx=Ala or Aib or Deg or Ac5c,
coupling said chelating group to a spacer group of the kind such as herein described,
and reacting the analog so produced with a pertechnate consisting of a salt of Tc-99m,
in the presence of a reducing agent such as herein described.
14. A method as claimed in claim 18 wherein said reducing agent is stannous chloride.

Documents:

0137-del-2000-abstract.pdf

0137-del-2000-claims.pdf

0137-del-2000-correspondence-others.pdf

0137-del-2000-correspondence-po.pdf

0137-del-2000-description (complete).pdf

0137-del-2000-drawings.pdf

0137-del-2000-form-1.pdf

0137-del-2000-form-19.pdf

0137-del-2000-form-2.pdf

0137-del-2000-form-3.pdf

0137-del-2000-form-4.pdf

0137-del-2000-gpa.pdf

0137-del-2000-pct-210.pdf

0137-del-2000-petition-137.pdf

137-DEL-2000-Correspondence-Others (02-02-2010).pdf

137-DEL-2000-GPA-(02-02-2010).pdf

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Patent Number

213818

Indian Patent Application Number

137/DEL/2000

PG Journal Number

04/2008

Publication Date

25-Jan-2008

Grant Date

17-Jan-2008

Date of Filing

18-Feb-2000

Name of Patentee

DABUR RESEARCH FOUNDATION

Applicant Address

22, SITE IV, SAHIBABAD, GHAZIABAD 201 010, UTTER PRADESH INDA

Inventors:

#	Inventor's Name	Inventor's Address
1	MISHRA, ANIL K.	INSTITUTE OF NUCLEAR MEDICINE AND ALLIED SCIENCE, OF LUCKNOW ROAD, TIMARPUR, DELHI-110 054
2	BURMAN, ANAND C.	DABUR RESEARCH FOUNDATION, OF 22, SITE IV, SAHIBABAD, GHAZIABAD 201 010, UTTAR PRADESH
3	PRASAD SUDHANAND	DABUR RESEARCH FOUNDATION, OF 22, SITE IV, SAHIBABAD, GHAZIABAD 201 010, UTTAR PRADESH
4	MUKHERJEE, RAMA	DABUR RESEARCH FOUNDATION, OF 22, SITE IV, SAHIBABAD, GHAZIABAD 201 010, UTTAR PRADESH
5	DUTT, SARJANA	DABUR RESEARCH FOUNDATION, OF 22, SITE IV, SAHIBABAD, GHAZIABAD 201 010, UTTAR PRADESH
6	SHARMA, RAJAN	DABUR RESEARCH FOUNDATION, OF 22, SITE IV, SAHIBABAD, GHAZIABAD 201 010, UTTAR PRADESH
7	AHUJA, RINKU	DABUR RESEARCH FOUNDATION, OF 22, SITE IV, SAHIBABAD, GHAZIABAD 201 010, UTTAR PRADESH
8	MATHEW ,T. LAZAR.	INSTITUTE OF NUCLEAR MEDICINE AND ALLIED SCIENCE, OF LUCKNOW ROAD, TIMARPUR, DELHI-110 054

PCT International Classification Number

A61K 51/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA