Title of Invention	NOVEL ORALLY ADMINISTRABLE ANTI-DIABETIC COMPOUNDS, PROCESS FOR PRODUCING THEM AND THERAPEUTIC COMPOSITIONS CONTAINING THE SAME .
Abstract	A novel class of orally and parenterally administrate, mono- and bi-cyclic peptides having pronounced anti-diabetic activity, acid salts and metal ion derivatives

Full Text

The present invention relates to a class of novel orally administrale anti-diabetic compounds, process for producing them and therapeutic compositions containing the same. More particularly, this invention pertains to a new class of compounds effective against type I or IDDM (Juvenile onset) form of diabetes and administrable via oral route in the form of therapeutic composition. The subject invention also discloses a process for preparing the aformentioned novel class of compounds. The incidence of diabetes has recently been on the rise all the world over, particularly in developing countries including India, thanks to a new trend of food habits and lack of physical exercise among people, particularly youngsters' preference for 'fastfood'. The most common cause of diabetes is chronic pancreatitis, often associated with nutritional and toxic factors. Also, in specific instances, diabetes results from point mutations in the insulin gene. 'Diabetes mellitus' is a group of syndromes characterized by hyperglycemia, glycosuria, negative nitrogen imbalance and at times, Ketonemia. Treating patients afflicted with diabetes (i.e. Diabetes mellitus) is becoming an increasingly importtant medical challenge, not only in terms of growing patient population, but also in terms of effectiveness of the emerging areas of therapy. Present-day therapeutic classification refers to two major types of diabetes, namely, type I or IDDM (juvenile onset) and type II or NIDDM (maturity onset), the former probably being an autoimmune disorder, whereas in the latter event, there is no loss or moderate reduction in ß-cell mass. But the ß-cells secrete substantial quantities of insulin which, however, is not adequately utilized to control the glucose levels in the body. Previously administration of extracts of naturally occurring plants, shrubs or fruits (like black berry seeds) was resorted to in controlling blood sugar level in diabetic people. This line of treatment is still being practised extensively in India by Ayurvedic doctors and there is very little or no side effects among patients even after prolonged intake of the medicaments. However, their action is also slow and a judicious selection of dosage plays an important role. -2- SRD/DS/FILE-1/PATENT With the advent of chemo-therapeutic drugs, use was made of benzene-sulfonyl urea and derivatives thereof in treating diabetes. Drugs like "Rastinon" (Hoechst), "Diabenese" (Pfizer), "Glyphos" (Schering) are being prescribed even today, but patients seem to grow resistant to these drugs leaving no other option but to administer insulin in injectable form through i.v. or i.m. routes. Administration of insulin often becomes a painful procedure, particularly for children of tender age. Presently treatment of diabetes is aimed at preventing development of metabolic abnormalities experienced by diabetics. Now-a-days attention is being paid on the role of peptides in the treatment of diabetes. Production of pure insulin by using r-DNA technogy is very expensive and the synthetic routes for arriving at insulin are also equally expensive to permit complete synthesis of insulin on a commercial scale. It is known that amino acid and peptides form an integral part of human body's inventory of drugs which maintain and control various functions vital to human life and health. If their biological activity is properly studied and understood, their functions and properties may be improved upon by synthesizing the analogues of peptides in the laboratory. These synthetic peptides have been found to be an extremely promising and viable proposition for being used-as a new generation of medicine for tackling diabetes. Synthesis of peptides, both linear chain and cyclic, has been made by adopting and selecting procedures to minimize or even to eliminate substantially the side products or contaminants which might cause undesirable side effects. Cyclic peptides have surprisingly been found to be more potent in combatting diabetes. Cyclic peptides have a number of advantages over linear peptides such as, for instance, they are (i) more conformationally constrained, (ii) resistant to protease digestions, (iii) enhanced metabolic stability and (iv) increased biological activity. These properties render the cyclic peptides more acceptable as a preferred antidiabetic drug compared to insulin, and administrable through both oral and parenteral routes, preferably by the former. Moreover, cyclic peptide molecules SRD/DS/FILE-1/PATENT -3- enjoy greater stability due mainly to resonance which impart higher storage stability to the therapeutic formulations prepared thereform. The principal object of this invention is to provide a novel class of orally and parenterally administrable, mono- and bi-cyclic peptides having pronounced anti-diabetic activity, acid salts and metal ion derivatives thereof. A further object of this invention is to provide novel cyclic peptides and metal ion derivatives thereof effective against IDDM (juvenile onset) variety of diabetes. A still further object of this invention is to provide a process for producing a novel class of orally and parenterally administrable, mono- and bi-cyclic peptides having pronounced anti-diabetic activity. Another object of this invention is to provide a therapeutic composition effective against Diabetes mellitus administrable through both oral and parenteral routes, preferably the former. A few typical members of the mono- and bi-cyclic peptide of this invention administrable by oral and parenteral routes showing pronounced anti-diabetic activity have been shown hereafter in a tabular form. Metal ion and acid salt derivatives of this new class of compounds are also included within the purview of the present invention, some of which are effective as antidiabetic substance. According to another aspect of this invention there is provided a process for producing a novel orally and parenterally administrable mono- and bi-cyclic peptides having pronounced anti-diabetic activity, which comprises the following steps : (a) protecting the amino group of an a-amino acid by using phthalic anhydride or phthaloyl halide as the protecting agent ; (b) protecting the carboxyl group of the amino acid derivative obtained from step (a) by passing HC1 gas through a suspension of the compound in anhydrous ethanol under stirring followed by neutralization with aqueous alkali solution, if need be, and cooling in an ice bath until crystals appear ; (c) reacting the compounds from step (b) with chlorophosphate ester reagent -4- SRD/DS/FILE-1/PATENT prepared by adding anhydrous ethanol to PC15 to produce an anhydride type reactive moiety ; (d) treating the said anhydride type moiety with a second carboxyl protected amino acid in presence of a tertiary amine under dry conditions to produce a condensation product ; (e) cleaving the protecting phthaloyl group by reacting the condensation product from step (d) dissolved in ethanol with hydrazine hydrate under reflux on steam bath, followed by cooling and addition of cone. HC1; (f) converting the hydrazide compound from step (e) into the corresponding azide by reacting with NaNO2 and cone. HC1 at low temperature ; (g) cyclizing the azide derivative by pouring the azide solution into a cold, dilute solution of NaHCO3 followed by neutralization under dilute conditions, if needed or called for, (h) isolating the desired cyclized product from the reaction medium by adding water, boiling and filtering while hot, followed by cooling to produce fine crystals of the desired product and, if desired, (i) converting the products into their salts or metal ion derivatives by methods known per se. The end product thus obtained was characterized by determining the melting point, Rf value from HPLC and spectral data like IR, NMR, etc. Usually a dipeptide is formed first, followed by formation of a tripeptide using a similar sequence of reaction. The linear tripeptide in its ester form is converted sequentially into a hydrazide and an azide, which in turn is subjected to cyclization by reaction with dilute NaHCO3 solution. It is to be noted that the nature and constitution of the end product will depend on the nature of the a-amino acid selected as the starting material. The principal criterion of the cyclic tripeptide will be not to cleave or disrupt the dithio bond of insulin produced within human system, or in special cases, supplied externally. The actual course of synthesis of cyclic peptides may be outlined as under giving the sequential performance of the constituent steps : SRD/DS/FILE-1/PATENT -5- (a) protection of NH2- group of selected a-amino by phthalic anhydride ; (b) protection of the - COOH group of the amino acid ; (c) converting the phthaloyl amino acid into an anhydride type of reactive moiety ; (d) treating the moiety with a second carboxyl protected amino acid in presence of a trialkyl amine [e.g. N(Et)3] under absolute dry conditions ; (e) treating the condensation product from step (d) with hydrazine hydrate/ HC1 to cleave the phthaloyl group ; (f) cyclization of tripeptide azide (prepared by diazotization of tripeptide hydrazide) by adding the azide solution in a solution of NaHCO3 followed by neutralization under dilute conditions and (g) isolating and crystallizing the corresponding cyclic peptides from the filtrate. Preparation of a representative dipeptide "Glycine - leucine" may be outlined by the flow-sheet diagram below : Activation Phthalic anhydride + glycine > Phthaloyl glycine > Anhydride type of compound CPE (Phosphoric acid [ PCl5 + EtOH ] diester derivative ) Coupled with Isoleucine ethylester, HCl&NEt, Deprotection of Acid hydrolysis iso amino group H2O/H+ Glycine-leucine NH2-NH2/HC1 CO2H H2N-CH2-CO-NH-CH-C2H5 ! CH3 ( Dipeptide ). -6- SRD/DS/FILE-1/PATENT A few novel cyclic peptides prepared in accordance with this invention are mentioned below outlining their characteristics in a tabular form wherein the individual compounds have been identified by using code numbers SSSI (TDI), to SSS VII (TD VII), respectively, for the sake of convenience and brevity - SL.NO. CODE NO. CHEMICAL FORMULA (MOLECULAR FORMULA) . M.W (Mol.wt) M.P (Melting point 3 : 1 Rf CHCl2: CH2OH Solubility Nature Crystal Spectra 1. SSS-I (TD-I) C12H18AN4O4S2 346 216-218°C 0.69 Boiling water/ CHCl2/Acetone White shiny I.R./ NMR 2. SSS-II (TDI-II) C13H23O3N3S1 301 229-232°C 0.76 chloroform/ dichloro ethane/Acetone White amorphous compound I.R. / NMR 3. SSS-III (TDI-III) 544 290-295°C 0.51 Methanol/ acetone boiling water White neddle shaped crystal I.R./ NMR 4. SSS-IV (TDI-IV) C15H27O3N3S1 329 215-218°C 0.41 Acetone/ CHCl3 methanol White amorphous compound I.R./ NMR 5. SSS-V (TDI-V) C16H24N6O6Sa 460 280-282°C 0.53 Chloroform/ acetone methanol White amorphous cmpound I.R./ NMR 6. SSS-VI (TDI-VI) C12H18N4O4S2Zn 411 260-262°C 0.18 Chloroform water White neddle shaped crystal 7. SSS-VII (TDI-VII) C13H23O3N3SZn 366 248-2 52°C 0.22 Chloroform water White neddle shaped crystal SRD/DS/FILE-1/PATENT -7- From the foregoing disclosure it may be seen that by following the synthetic route enunciated before, one may arrive at small chain mono- and bi-cyclic peptides, their mineral acid salts and metal ion derivatives such as hydrochlorides, or sulphates and sodio, potassium or zinc salts, etc. Peptide synthesis may be achieved by adopting liquid phase method with chlorophosphate ester, triethylamine being used as the condensing agent. Phthaloyl halide may be used for protecting amino group, the carboxyl may be protected by forming methyl or ethyl ester and cyclizatioe may be brought about by forming hydroxides and azides as the successive intermediate stages. The novel cyclic mono- and di-peptides may be formulated into orally or parentally administrable forms by using fillers selected from lactose, starch, calcium phosphate, magnesium stearate, talc, steatite, microfined silica and polymeric cellulose "AVICEL", binders are selected from starch paste and carboxy methyl cellulose (CMC) and non-aqueous adjuvant is ethyl cellulose. The main emphasis of the present invention is to identify a specific zone or portion of the insulin molecule, which are important areas for binding to the receptor. As pointed out earlier in this specification, the cyclic peptides are more conformationally constrained, more stable and readily pass through the cell membrane, thereby showing better biological activity. The invention will be further amplified by the Examples given hereafter narrating the synthesis of a typical cyclic peptide of this invention and pharmacological studies conducted on rabbits with induced diabetes, which has shown quite encouraging results. The examples are given by way of illustration and the invention is not confined to the specific embodiments given therein. -8- SRD/DS/FILE-1/PATENT A. SYNTHESIS OF CYCLIC PEPTIDE 1. PREPARATION OF INTERMEDIATES 1.1 Preparation of chlorophosphate ester (CPE) reagent Phosphorous pentachloride (12.5, 0.06 ml) was taken in a clean and dry RBF, which was cooled at 0-5°C. To it anhydrous ethanol (35 ml) was added dropwise over a period of 10 minutes with constant strring. After the complete addition of ethanol, mixture was strried further for half an hour. The CPE reagent thus prepared was stored in a tightly closed container. Caution - Special care should be taken while handling phosphorous pentachloride and exposure to eyes and hands should be avoided as far as possible. 1.2 Amino protection of a-amino acid a. Phthaloyl Alanine A mixture of purified Phthalic anhydride (2.2 g, 0.015 mol) and Alanine (1.125 g, 0.015 mol) was heated in a sand bath at 160-180°C. -9- SRD/DS/FILE-1/PATENT Within 10-15 min, the mixture melted, heating was continued for another 15 minutes. The reaction, mixture was cooled; the residue was dissolved in ethanol and filtered off. Water was added to the clear filtrate till turbid and the solution was cooled at 0°C for 2-3 hours (preferably overnight) Fine needle shaped crystals of Phthaloyl Alanine separated were filtered and dried. Theoretical yield = 3.075 g Practical yield = 2.5 g Percent yield = 81.3% b. Phthaloyl Isoleucine A mixture of purified Phthalic anhydride (2.2 g, 0.015 mol) and Isoleucine (1.965 g, 0.015.mol) was heated in a sand bath at 160-180°C. Within 10-15 min, the mixture melted, heating was continued for another 15 minutes. The reaction, mixture was cooled; the residue was dissolved in ethanol and filtered off. Water was added to the clear filtrate till turbid and the solution was cooled at 0°C for 2-3 hours (preferably overnight). Fine needle shaped crystals of Phthaloyl Isoleucine separated were filtered and dried. Theoretical yield =4.215 g Practical yield = 3.6 g Percent yield = 85% -10- c. Phthaloyl Glycine A mixture of purified Phthalic anhydride (2.2 g, 0.015 mol) and Glycine (1.125 g, 0.015 mol) was heated in a sand bath at 160-180°C. Within 10-15 minutes, the mixture melted, heating was continued for another 15, minutes. The reaction, mixture was cooled; the residue was. dissolved in ethanol and filtered off. Water was added to the clear filtrate till turbid and the solution was cooled at 0°C for 2-3 hours (preferably overnight). Fine needle shaped crystals of Phthaloyl Glycine separated were filtered and dried. Theoretical yield = 3.075g Practical yield = 2.8g Percent yield = 91.06% 6.1.3 Carboxyl protection of a-amino acid a. Alanine ethyl ester Alanine (1.7g, 0.02 mol) was suspended in anhydrous ethanol (11.5 ml, 0.02 ml) in flat RBE filled with an inlet for passing HC1 gas into the reaction mixture. The flask was kept in an ice bath and the assembly was arranged for stirring on a magnetic stirrer. The stirring was started and steady stream of dry HC1 gas was passed into the suspension through a gas inlet until Alanine dissolves. The cooling bath was removed and the flask was fitted with a reflux condenser. The mixture was boiled for 10 minutes with the aid of hot plate of magnetic stirrer and also stirred while refluxing. Then it was neutralized with cold dilute aqueous NaOH solution. -11- After that the flask was cooled in an ice bath with stirring until' crystals appear. The crystals were collected by filtration and recrystallized with absolute ethanol. Theoretical Yield = 2,34g Practical Yield = 2.15g Percent yield = 91,88% b. Cystine Dimethyl Ester Cystine (4.80g, 0.02 mol) was suspended in anhydrous ethanol (11.5 ml, 0.02 ml) in flat RBF filled with an inlet for passing HC1 gas into the reaction mixture. The flask was kept in an ice bath and the assembly was arranged for stirring on a magnetic stirrer. -The stirring was started and steady stream of dry HC1 gas was passed into the suspension through. a gas inlet until cystine dissolves. The cooling bath was removed and the flask was fitted with a reflux condenser. The mixture was boiled for 10 minutes with the aid of hot plate of magnetic stirrer and also stirred while refluxing. After 10 minutes, the flask was cooled in an ice bath with stirring until crystals appear. The crystals were collected by filtration and recrystallized with absolute ethanol. Theoretical yield = 5.36g Practical yield = 3.49g Percent yield = 65.11% -12- c. Isoleutine Ethyl Ester Isdleucine (2.62g, 0.02 mol) was suspended in anhydrous ethanol (11.5 ml 0.02 ml) in flat RBF filled with an inlet for passing HCl gas'into the reaction mixture. The flask was kept in an ice bath and the assembly was arranged for stirring on a magnetic stirrer. The stirring was started and steady stream of dry HCl gas was passed into the suspension through a gas inlet until Isoleucine dissolves. The cooling bath was removed and the flask was fitted with a reflux condenser. The mixture was boiled for 30 minutes with the aid of hot plate of magnetic stirrer and also stirred while refluxing. Then it was neutralized with cold dilute aqueous sodium hydroxide solution. After that the flask was cooled in an ice bath with stirring until crystals appear. The crystals were collected by filtration and recrystallized with absolute ethanol. Theoretical yield = 3.18g Practical yield = 2.40g Percent yield =73.47% d, Cysteine ethyl ester Cysteine (2,42g, 0.02 mol) was suspended in anhydrous ethanol (11.5 ml, 0.02 ml) in flat RBF filled with an inlet for passing HCl gas into the reaction mixture. The flask was kept in an ice bath and the assembly was arranged for stirring on a magnetic stirrer. The stirring was started and steady stream of dry HCl gas was passed into the suspension through a gas inlet until cysteine dissolves. The cooling bath was removed and the -13- flask was fitted with a reflux condenser! The mixture was boiled for .30 minutes with the aid of hot plate of magnetic stirrer and also stirred while refluxing. Then it was neutralized with cold -dilute aqueous sodium hydroxide solution. After that the flask was cooled in' an ice bath with stirring until crystals appear. The crystals were collected by filtration and recrystailized with absolute ethanol. Theoretical yield = 2.98gg Practical yield - 2.50 g Percent yield = 83.89% e. Methionine Methyl Ester Methionine (2.984g, 0.02 mol) was suspended in anhydrous ethanol (11.5 ml, 0.02 ml) in flat RBF filled with an inlet for passing HG1 gas into the reaction mixture. The flask was kept in an ice bath and the assembly was arranged for stirring on a magnetic stirrer. The stirring was started and steady stream of dry HCl gas was passed into the suspension through a gas inlet until Methionine dissolves. The cooling bath was removed and the flask was fitted with a reflux condenser. The mixture was boiled for 30 minutes with the aid of hot plate of magnetic stirrer and also stirred while refluxing. Then it was neutralized with cold dilute aqueous sodium hydroxide solution. After that the flask was cooled in an ice bath with stirring until crystals appear. The crystals were collected by filtration and recrystallized with absolute ethanol. Theoretical yield = 3:26g -14- Practical yield = 2.12g Percent yield = 65,03% f. Glycine ethyl ester Glycine (1.5g, 0,02 mol) was suspended in anhydrous ethanol(11.5 ml, 0.02 ml) in flat RBF filled with an inlet for passing HC1 gas into the reaction mixture. The flask was kept in an ice bath and the assembly was arranged for stirring on a magnetic stirrer. The stirring was started and steady stream of dry HC1 gas was passed into the suspension through a gas inlet until Glycine dissolves. The cooling bath was removed and the flask was fitted with a reflux condenser. The mixture was boiled for 30 minutes with the aid of hot plate of magnetic stirrer and also stirred while refluxing. After 10 minutes, the flask was cooled in an ice bath with stirring until crystals appear. The crystals were collected by filtration and-recrystallized with absolute ethanol. Theoretical yield = 2,06 g Practical yield =1.7 g Percent yield =82.5% 2 PEPTIDE SYNTHESIS 2.1 Preparation of Compound SSS I Step 1 : Preparation of Phthaloyl Ala-Cys Dimethyl ester (condensation) The CPE reagent was added to Phthaloyl Alanine (2.05 g, 0,01 mol) and stirred it to a clear solution. Then Cystine (2.68 g, 0.01 mol) Dimethyl -15- ester was added to the mixture and again stirred to a clear solution {3 hours). To this mixture triethylamine was added to pH-7 maintaining the reaction temperature between 0-5°C. The mixture was then left at 0°C for 6 hours. The product was crystallized from dichloroethane, filtered, washed with solvent ether and dried. Theoretical yield = 4.73 g Practical yield = 4.30 g Percent yield = 90.90% Step 2 : Preparation of Phthaloyl Ala-Cys (Deprotection of the carboxyl group) Phthaloyl Ala-Cys-dimethyl ester (2.365 g, 0.005 mol) was dissolved in 80 ml of 0,2 N HC1 and 15 ml of acetone. It was then refluxed for 30 minutes, cooled and crystallized from dichloroethane. Fine needle shaped crystals of Phthaloyl Ala-Cys separated were filtered and dried. Theoretical yield = 2.225 g Practical yield = 1.98 g Percent yield = 89.18% Step 3 : Preparation of Phthaloyl (Ala)2 - Cystine ethyl ester (condensation) The CPE reagent was added to phthaloyl Ala-Cys (1.33 g, 0.003 mol) and stirred it to a clear solution. Then Alanine ethyl ester (0.351 g, 0.003 mol) was added to the mixture and again stirred to a clear solution (3 hours). To this mixture triethylamine was added to maintain the pH-7 and reaction temperature was maintained between 0-5°C. The mixture -16- was then left at O0C for 6 hours. The product was crystallized from dichloroethane, filtered, washed with solvent ether and dried. Theoretical yield = 1.686 g Practical yield = 1.4 g Percent yield = 83.03% Step 4 : Preparation of(Ala)2 - Cystine ethyl ester (Deprotection of the, amino group) Phthaloyl (Ala)2 Cystine ethyl ester (1.4 g, 0.003 mol) was dissolved in ethanol (25 ml), hydrazine hydrate (0.1 ml, 70%) was added and the mixture was refluxed on steam bath for 2 hours. The reaction mixture was cooled, acidified with concentrated HC1 and again heated on waterbath at 50°C for 1 hour it was then left overnight. Fine crystals of Phthaloyl hydrazide separated was filtered off. The filterate was neutralized with pyridine to liberate the free peptide which was held back in solution. Removal of solvent and pyridine was carried out under vacuum distillation followed by crystallization from dichloroethane, gave the tripeptide ester. Theoretical yield = 1.338 g Practical yield = 1.24g Percent yield = 93.98% -17- Step 5 : Preparation of (Ala)2 Cystine hydrazide Hydrazine hydrate (1.4 ml) was added to a solution of (Ala)2 Cys-ethyl ester (1.24 g, 0.003 mol} in methanol (50 ml) and the mixture was refluxed for 1 hour then allowed to stand for 1 hour at room temperature. The precipitate was filtered off. This product was combined with a second crop obtained from the concentrated mother liquor and recrystallized. Theoretical yield. = 1.752 g Practical yield = 1.5g Percent yield =85.61% Step 6 : Preparation of Compound SSS I A solution of (Ala)2 - Cystine hydrazide (1.46 g, 0.0025 mol) in 48.4 ml of 0.2083 N HC1 was treated with 0.173 g of sodium nitrite in 2 ml of water at 0°C.Cyclization was initiated by pouring the azide solution into 500 ml of NaHCO3 solution and the mixture was kept for 48 hrs. Following adjustment to pH5, the solution was concentrated to approx. 50 ml. Acetone (350 ml) was added and the colourless precipitate obtained was collected. The precipitate was added to 25 ml of water, boiled and filtered while hot to remove the polymeric material. On cooling fine crystals of Compound SSS I separated were filtered and dried. Theoretical yield = 0.5825 g Practical yield = 0.521 g Percent yield = 89.44% The cyclic peptide obtained as the final product was tested for purity by thin layer chromatography (TLC) using Silica gel G as adsorbent and n-butanol : H - Ac : water (5:1:4) used as eluant. IR spectra (using KBr pellet method), NMR and other spetral characterization was also conducted in this regard. -18- Example 2 B. PHARMACOLOGICAL INVESTIGATION Earlier studies of the pharmacological activities of various amino acid and peptides synthesized along with their metal ion-derivatives, have been reported to be quite promising results. They have shown significant anti-inflammatory, heptoprotective, anti-fungal and anti-diabetic activity. Here we have mainly concentrated on Insulin Dependent Diabetes Mellitus (IDDM) and its treatment with small chain cyclic peptides, Here, in this project we have investigated the antidiabetic activity of the synthesized peptides on alloxanised rabbits. PRINCIPLE The protein free blood filtrate is heated with alkaline copper sulphate solution. The cuprous oxide thus formed is treated with phosphomolybdic acid solution a blue colour being obtained is compared with that of a standard. Alloxan Induced Diabetes in Rabbits In 1943, Dunn, Sheehan and McLetchie at the University of Glasgow reported that alloxan can produce specific pancreatic islet necrosis in rabbits. Alloxan proved to be a suitable compound for inducing experimental diabetes in animals.This alloxan diabetes in animals presented like diabetes mellitus in man, with the typical symptoms such as body weight loss, polydipsia, polyuria, glycosuria, ketouria, hyperglycaemia and ketonemia. -19- The difficulties encountered with in vivo experiments with alloxan are the instability of alloxan under physiological conditions100 and its thiol reactivity; in addition, other factors such as diet, age and animal species affect the dose of alloxan necessary to induce a diabetic state in animals. This has made it virtually impossible to establish a clear relationship between the dose of alloxan and its effective concentration in the pancreas, which induces the effects on the function and the morphology of the pancreatic B-cells. Mechanism of induction of Diabetes by Alloxan 1. The favored site for the action of alloxan was not a sulfhydryl enzyme in the pancreatic B cell, but rather a presumptive gluco-receptor in the pancreatic B cell membrane.105 But experimental support for the existence of such a plasma membrane receptor remained circumstantial within the last few years, the concept that glucokinase, a glucose phosphorylating enzyme with low affinity is of central importance for the normal function of the pancreatic B cell. Glucokinase is the enzyme in the pancreatic B cell and also in the liver, which is apparently most sensitive to inhibition by alloxan and may be the primary target for alloxan in the pancreatic B cell toxic action of the agent.106 Inhibition of the pancreatic B cell glucokinase due to an oxidation of SH groups in the sugar binding site of the enzyme. -20- 2. The selective toxicity of alloxan to the pancreatic B-cell results from the coincidence of two distinct and independent features : a. rapid accumulation of the drug into islet cells, b. and, their exquisite sensitivity towards peroxides.. Antidiabetic Studies The antidiabetic activity of the prepared test drugs was studied on alloxanised rabbits by measuring the decrease in blood glucose level.107 3. MATERIALS AND METHODS I. Animals Inbreed albino rabbits of either sex, weighing 1.5-2 kg were used. Animals were kept in a galvanised iron cage, maintained at 25 ± 2°C in a well ventillated animal house under natural photoperiod conditions. They were provided with standard diet and water. Each experimental group were consisted of 3 animals each. Drugs Dose/Kg b wt Test drug Standard insulin 0.2 units Compound SSS I 50 mg I Compound SSS II 50 mg II Compound SSS III 50 mg III Compound SSS IV 50 mg IV Compound SSS V 50 mg V Compound SSS VI 50 mg VI Compound SSS VII 50 mg VII Suspension of test drugs I - VII were prepared using 0.5 percent sodium - CMC (low viscosity grade) to make the desired concentration for -21- subcutaneous and oral administration, 0.5% sodium CMC was used as vehicle control. II. Reagents used and its preparation a. Isotonic sodium sulphate - copper sulphate solution - 30 g of sodium sulphate, Na2SO4.10 H2O and 6g of copper sulphate CuSO4. 5H2O was dissolved in one litre of distilled water. b. Sodium Tungstate solution - 100 g of sodium tungstate was dissolved in one litre of distilled water. c. Alkaline tartarate solution, - 25 g of sodium bicarbonate was dissolved in 600 ml of distilled water in a one litre volumetric flask at room temperature and 20 g of anhydrous sodium carbonate was added to this solution and shaken vigorously. 18g of potassium oxalate was dissolved separately in 150 ml of distilled water and was added gradually to the above solution. Then 12 g of sodium potassium, tartarate was dissolved separately, in 100 ml of distilled water and added to the above solution. The final volume was made up to one litre with distilled water. d. Phosphomolybdic acid reagent - 35 g of molybdic acid and 5 g of sodium tungstate was dissolved in 200 ml of 10% sodium hydroxide solution, now 200 ml of distilled water was added to this solution. It was then diluted to about 350 ml with distilled water and transferred with washing to a 500 ml volumetric flask, diluting 125 -22- ml of phosphoric acid (specific gravity 1.95 g/ml) was added and mixed. The final volume was made to 500 ml with distilled water. e. Stock standard glucose solution - 100 mg of glucose AR was dissolved in 100 ml of Isotonic sodium sulphate - Copper, sulphate solution. Ethylene Diamine Tetracetic acid solution (1-5%)- 1 to 5 g of EDTA was dissolved in 100 ml of distilled water. g. Sodium CMC suspension - 5 g of sodium CMC was dissolved in 100 ml of distilled water. h. Alloxan solution - 1 g of alloxan was dissolved in 20 ml of distilled water. III. Induction of Hyperglycaemia Alloxan was used to induce diabetes in experimental animals. Alloxan is a. urea derivative which causes selective necrosis of the pancreatic islet ß-cells. For all animals, single dose of alloxan of 150. mg/kg was given as a 5% w/v in distilled water, injected intravenously. The animals were fasted 36 hours before injection. A rest period of 7 days for rabbits was allowed during which the animals have free access to food and water. After 7 days, rabbits with moderate diabetes having hyperglycaemia (blood glucose 200-250 mg/100 ml) were divided into 9 groups of 3 animals each. -23- 4. EXPERIMENTAL PROTOCOL Before administration of test drugs, the blood glucose levels were determined for all the nine groups of 3 animals each as mentioned below. The experimental protocol was same during the oral administration of test drug I-VII. Group I Group II Group III Group IV Received Sodium CMC (volume equivalent to test drugs / kg) via SC route after attainment of permanent hyperglycaemia. Received the standard drug insulin (0.2 units / kg) via SC route after attainment of permanent hyperglycaemia. Received the test drug I (50 mg/kg) via SC route after attainment of permanent hyperglycacinia. Received the test drug II (50 mg/kg) via SC route after attainment of permanent hyperglycaemia. Group V Received the test drug III (50 mg/kg) via SC route after attainment of permanent hyperglycaemia. Group VI Received the test drug IV (50 mg/kg) via SC route after attainment of permanent hyperglycaemia. Group VII Received the test drug V (50 mg/kg) via SC route after attainment of permanent hyperglycaemia. Group VIII Group IX Received the test drug VI (50 mg/kg) via SC route after attainment of permanent hyperglycaemia. Received the test drug VII (50 mg/kg) via SC route after attainment of permanent hyperglycaemia. 3. Sampling Method The blood sample was withdrawn with the help of tuberculin syringe from the marginal car vein of the rabbit. 1 ml of blood was withdrawn each time and was transferred immediately to the vial containing few drops of EDTA aqueous solution. It was plugged immediately using rubber closures to be used for the estimation of blood glucose level. -24- b. Analysis of Blood, samples 0,1 ml of blood was pipetted in 3,8 ml of Isotonic sodium sulphate -Copper sulphate solution and the solution was mixed thoroughly. Now 0.1 ml of sodium tungstate solution was added and, mixed, the solution was centrifuged at 1000 rpm for 10 min. 1 ml of supernatant fluid was taken in a hard glass test tube and to this 1ml of alkaline tartrate was added. The tubes then plugged lightly with cotton wool and heated by keeping in a boiling waterbath for 10 min, The tubes were cooled and 3 ml phosphomolybdic acid reagent and 3 ml distilled water was added, the solution was mixed well and . measured the. extinction in spectropho to meter at 630 nm. At the same time, 1 ml of working standard (stock standard glucose solution) and blank (1 ml of isotonic sodium sulphate - copper sulphate solution) were taken in two separate tubes and proceeded as mentioned in the above procedure. The blood glucose level was calculated using the formula-Blood Sugar (mg%) = 5. RESULTS AND DISCUSSION The objective of the present work was to test the antidiabetic activity of the synthesized small chain cyclic peptides using alloxanised rabbits. -25- The antidiabetic activity of the hydrochloride salt and metal ion complex of a cyclic peptide was also tested. The test drug has been given by subcutaneous as well as by oral route.The results were very encouraging in both the cases.In the subcutaneous route, the T.D.- I, II, IV and VI showed significant antidiabetic activity compared to standard drug insulin at a dose of 0.2 unit / kg body weight. In Fig. 1.. the curve for the standard drug has shown a sudden fall in the mean blood glucose level, which may lead to hypoglycemic state, whereas the test drugs has shown a gradual decrease in the mean blood glucose level, when compare to standard. Percentage reduction in blood glucose level in case of standard drug Insulin was found to be 65.86%, whereas T.D-IV showed a percentage reduction in blood glucose level of 60.82% and T.D - I, II, VI showed a percentage reduction in blood glucose level of 60.34%, 60.16% and 58.33% respectively. In the oral route; the T.D.- I, II and VII showed significant antidiabetic activity compared to standard drug insulin at a dose of 0.2 unit / kg body weight. In Fig. 3 the curve for the standard drug has shown a sudden fall in the mean blood glucose level, which may lead to hypoglycemic state, whereas the test drugs has shown a gradual decrease in the mean blood glucose level, when compare to standard. Percentage reduction in blood glucose level in case of standard drug Insulin was found to be 60.58%, whereas T.D-VII showed a percentage -26- reduction in blood glucose level of 52.45% and T.D - I and II showed a percentage reduction in blood glucose level of 55.52%,50.40% respectively. . Data Treatment The anti-diabetic activity of the test drugs was studied 'using alloxanised rabbits with percentage reduction in blood glucose level as the index. The results have been shown in table no§ 1 and 2. The graphical representation which shows the mean blood glucose level after administration of standard and test drugs in mg/100 ml have been shown in Fig. 1 (s.c) and in Fig.6.2.2(oral) and the mean% reduction in blood glucose level after administration of standard and drugs in Fig: 3 (s.c) and in Fig. 4 Statistical Treatment The results obtained were subjected to student's t-test for control vs standard and test drugs. ANOVA (Analysis of variance) was also applied to the data of total percentage reduction of blood glucose level after administration of test drugs and standard in alloxanised rabbits. t-test The 't' value was calculated as follows - Where X1 = mean of 1st sample of n1 observation X2 = mean of 2nd sample of n2 observation -27- Where S X1i2 is sum of squares of observations in 1st sample S X1i is sum of observations in 1st sample S X2i2 is sum of squares of observations in 2nd sample S X2i is sum of observations in 2nd sample Degrees of freedom (DE) = n1 + n2 - 2 t value for (s.c) Control Vs Standard = 2.585 Control Vs T.D. I = 2.513 Control Vs T.D. II = 2.908 Control Vs T.D. III = 2.578 Control Vs T.D. IV = 2.530 Control Vs T.D. V = 2.674 Control Vs T.D. VI = 2.526 Control Vs T.D. VII = 2.560 In the t-table, for P The value of t calculated is greater than this in case of the standard drug Insulin and test drugs I-VII. Hence, it can be stated that there is a statistically significant difference between these drugs and the control. We can infer that these drugs possess appreciable amount of antidiabetic activity. The t-value for T.D. II,III, V, VII,significant difference with that of control along with the standard drug insulin indicating that T.D. II, III, V, and VII showed -28- maximum antidiabetic activity amongst all drugs tested in alloxanised rabbits. The analysis of variance was also performed. Total % reduction of blood glucose level after administration of Standard drug and test drugs in alloxanised rabbits (s.c) Animals STD TD-I TD-II - TD-III TD-IV TD-V TD-VI TD-VII A1 66.58 60.28 59.73 49.70 60.84 55.02 59.39 57.79 A2 65.50 61.02 60.52 47.66 61.82 54.62 58.55 58.25 A3 65.40 59.72 60.23 48.99 59.80 55.76 57.05 58.51 Table 6.7.1.9 - Anova Table Source of variation Degree of freedom Sum of squares Mean of squares Variance ratio (F) Between samples 7 SSC = 497.31 MSC = 71.04 113.84 Within samples 17 SSE = 10.62 MSE = 0.624 F value at P As the calculated value is much higher than the tabulated value, there is a highly significant difference between the standard and the test drugs I - VII. So, we can conclude by stating that some of the test drugs have shown very promising results in reducing the blood glucose leyel in alloxanised rabbits by S.C routes. t-values for(oral) Control Vs Standard = 1.998 -29- Control Vs T.D.I = 2.028 Control Vs T.D. II = 2.O42 Control Vs T.D. III = 2.059 Control Vs T.D. IV = 2.091 Control Vs T.D. V = 2.057 Control Vs T.D. VI = 2.127 Control Vs T.D. VII = 2.029 In the t-table,for P The value of t calculated is greater than, this in case of the standard drug insulin and test drugs I-VII. Hence, it can be stated that there is a statistically significant difference between these drugs and the control. We can infer that these drugs possess appreciable amount of antidiabetic activity. The t-value for T.D. II, III, V and VII significant difference with that of control along with the standard drug insulin indicating that T.D. II, III ,V and VII showed maximum antidiabetic activity amongst all drugs tested in alloxanised rabbits. The analysis of variance was also performed. The present invention offers a number of advantages i) Stability of the synthetic cyclic peptide is considerably more than insulin since insulin is a long-chain complex polypeptide having a plurality of dithio bridges. ii) Insulin is a costly drug, storing of which poses problems. On the other hand synthetic cyclic peptide is far less costly. -30- iii) The novel class of cyclic peptides can be made from wholly indigenous starting materials and equipments. Synthesis does not require any sophisticated equipment(s) or imported chemicals and consequently the products will be considerably cost-effective. iv) The drugs are administrate orally, thereby avoiding hazards of injection and attended possibilities of secondary infection. v) The cyclic peptides also show promise as a potential agent to combat intestinal inflammation. vi) The novel class of compounds of this invention shows synergistic action with human insulin, triggering the enzymes responsible for bringing about burning/consumption of glucose in the blood stream. While the invention has been described in detail and with reference to the specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without deviating or departing from the spirit and scope of the invention. Thus the disclosure contained herein includes within its ambit the obvious equivalents and substitutes as well. Having described the invention in detail with particular reference to the illustrative examples given above, it will now be more specifically defined by means of claims appended hereafter. SRD/DS/FILE-1/PATENT -31- Table No. 1 : Comparison of Mean Blood Glucose level after Administration of Standard and Test Drug I - VII in alioxanised rabbits (s.c) Drug Mean Blood Glucose level after administration of Standard and Test drug at different time intervals in mg/l00ml (±SEM) 0 1 2 4 6 8 10 12 24 Control 242.17 ±0.1308 244.66 ±0.2255 240.82 ±0.4182 241.72 ±0.20003 238.07 ±0.1235 242.36 ±0.3307 245.06 ±0.3284 243.55 ±0,2523 240,42 ±0.2114 Standard 241.13 ±0.0828 208.52 ±0.3588 196.64 ±0.3135 150.34 ±0.6670 95.50 ±0.6233 82.31 ±0.6233 85.84 ±0.5616 102.45 ±0.9182 123.27 ±1.1049 T.D. 1 246.08 ±1.2940 212.46 ±1.1164 150.98 ±1.6864 117.85 ±1.0834 97.59 ±1.2602 105,02 ±1.4826 113.82 , ±1.4559 . 124,93 ±1.6410 13.93 ±1.8434 T.D. II 243.35 ±1.6009 218.27 ±1.3378 197.76 ±0.8439 124.67 ±1.4158 96,94 ±0.8921 105.48 ±1.6993 116.85 ±1.4551 120,35 ±1,5899 128.59 +2.6676 T.D, III 249.92 ±2.0659 232.02 ±1.7114 207.31 ±1.2407 172.06 ±1.0268 143.46 ±0.8821 127.96 ±0,8029 151.80 ±0.9959 163.64 +1.3852 170.86 ±1.3252 T.D, IV 246.69 ±0.9994 226.62 ±0.5774 190.94 ±0.7-261 125.67 ±1.8174 . 99.51 ±1.4945 96.63 ±0.7918 117.37 ±0.9489 130.49 ±0.8952 148;08 ±0,8707 T.D, V 243.48 ±0.4398 211,20 ±0.7709 166.22 ±0.5036 106.78 ±0.8303 109.23 ±0.8226 114.86 ±1.0486 125.56 ±1.4230 136.95 ±0.9681 148.79 ±1.0640 T.D. VI 238.06 ±1.0870 225.13 ±0.8038 194.37 ±0.8947 149.81 ±0.7377- 101.20 ±0.5877 99,20 ±1.2855 99.17 ±0.8786 103.18 ±0.5176 122.97 ±0.9114 T.D. VII 237.75 ±0.8719 216.66 ±0.6905 15667 +0.9943 99.41 ±0.7693 102.28 ±1.0686 108.95 ±0,7953 119.66 ±0.8769 135,98 ±0.7165 155.80 ±0.5162 -32- Table No.2 : Comparison of Mean Blood Glucose level after Administration of Standard and Test Drug I - VII in alloxanised rabbits (oral) Drug Mean Blood Glucose level after administration of Standard and Test drug at different time intervals in mg/100ml (+SEM) 0 1 2 4 6 8 10 12 24 Control 255.54 ±0.3064 253.14 ±0.4461 254.61 ±0.4182 252.53 ±0.3796 249.67 +0.4771 252,63 ±0.3235 252.77 ±0.3876 250.63 ±0.3158 249.60± 0.2871 Standard 253.73 ±0.3844 214,64 ±0.2996 194.48 +0.3381 188.70 ±0.3467 175.68 +0.4393 108.33 ±0.3374 99.98 ±0.3260 110.39 +0.3599 115.40 ±0.5405 T.D. I 252.22 ±0.6905 218.74 ±0.4959 185.65 +0.6025 148.113 ±0.6359 112.15 ±0.4530 119.44 ±0.3030 134.07 ±0.6085 137.22 ±0.3454 135.61 ±0.6557 T.D. II 251.13 ±0.4919 238.60 ±0.3476 222.27 ±0.5108 172.31 ±0.5082 148.69 ±0.4133 124.53 ±0.5316 131.61 ±0.6273 140.39 ±0.5176 151.51 ±0.3802 T.D. III 245.45 ±0.4895 222.40 +0.4440 192.62 ±0.4390 152.28 +0.4996 123.23 ±0.5737 127.72 ±0.4578 131.64 ±0.5453 135.59 ±0.3914 145.68 ±0.6312 T.D. IV 250.49 ±0.3149 224.58 ±0.3256 201.57 ±0.6730 184.54 ±0.3810 165.50 ±0.4917 144.97 +0.4089 150.60 ±0.2857 154.81 ±0.3913 165.69 ±0.4342 T.D. V 247.81 ±0.4741 218.54 ±0.3546 165.70 ±0.5154 149.35 ±0.4937 138.40 . ±0.4339 128.35 +0.3056 131.40 ±0.4509 135.61 ±0.3034 140.66 ±0.4942 T.D. VI 250.76 ±0.4228 236.38 ±0.3421 213.74 ±0.5324 196.40 ±0.6122 174.67 ±0.3347 155.67 ±0.4766 161.59 ±0.5642 172.66 ±0.5634 1.80.56 ±0.3587 T.D. VII 249.52 ±0.3986 238.60 ±0,4096 220.47 ±0.2897 198.69 ±0.3336 175.57 ±0.3090 160.58 ±0.3309 168.58 ±0.3795 170.53 ±0.3842. 172.68 ±0.5007 -33- We claim : 1. A novel class of orally and parenterally administrate, mono- and bi-cyclic peptides having pronounced anti-diabetic activity, acid salts and metal ion derivatives thereof. 2. A process for producing a novel orally administrable, mono- and bi-cyclic peptides having pronounced anti-diabetic activity, acid salts like hydrochlorides and sulphates, and metal ion derivatives like sodio, potassium and zinc salts thereof, which comprises the following steps : a) protecting the amino group of an a-amino acid by using phthalic anhydride or phthaloyl halide as the protecting agent ; b) protecting the carboxyl group of the amino acid derivative obtained from step (a) by passing HC1 gas through a suspension of the compound in anhydrous ethanol under stirring followed by neutralization with aqueous alkali solution, if need be, and cooling in an ice bath until crystals appear ; c) reacting the compounds from step (b) with chlorophosphate ester reagent prepared by adding anhydrous ethanol to PC15 to produce an anhydride type reactive moiety ; d) treating the said anhydride type moiety with a second carboxyl protected amino acid in presence of a tertiary amine under dry conditions to produce a condensation product ; e) cleaving the protecting phthaloyl group by reacting the condensation product from step (d) dissolved in ethanol with hydrazine hydrate under reflux on steam bath, followed by cooling and addition of cone. HC1 ; f) converting the hydrazide compound from step (e) into the corresponding azide by reacting with NaNO2 and cone. HC1 at low temperature ; -34- g) cyclizing the azide derivative by pouring the azide solution into a cold, dilute solution of NaHCO3 followed by neutralization under dilute conditions, if needed or called for, h) isolating the desired cyclized product from the reaction medium by adding water, boiling and filtering while hot, followed by cooling to produce fine crystals of the desired product and, if desired, i) converting the products into their salts or metal ion derivatives by methods known per se. 3. A process as claimed in Claim 2, wherein the tertiary amine used in step (d) is triethylamine. 4. A process as claimed in Claim 2, wherein deprotection of amino group with removal of phthaloyl group is carried out in step (e) by reacting the condensation product dissolved in ethanol with hydrazine hydrate under reflux on steam bath, cooling the mixture, acidifying the same with cone. HC1, reheating on water both at arround 50°C for 1 hour, allowing it to stand overnight, filtered to remove phthaloyl hydrazide, filtrate neutralized with pyridine, solvent and pyridine removed by vaccum distillation, followed by crystallization from dichloroethane giving rise to tripeptide ester. 5. A process as claimed in Claims 2 and 4, wherein the hydrazide derivative is converted into azide by treating a dilute HC1 solution of hydrazide with aqueous solution of NaNO2 at 0°C, followed by cyclization by pouring the azide solution into aqueous NaHCO3 solution and allowing to stand for 48 hrs, concentrating and adding acetone to the solution to obtain a crude crop of the desired product. 6. A process as claimed in Claims 2 and 5, wherein the crude crop is dissolved in water, filtered hot to remove polymeric impurities and then cooled to get the cyclic tripeptides. -35- 7. A process for producing a novel class of orally and parenterally administrable mono- and bi-cyclic peptides having pronounced anti-diabetic activity, acid salts like hydrochlorides and sulphates, and metal ion derivatives like sodio, potassium and zinc salts thereof, substantially as hereinbefore described with particular reference to the appended Examples. 8. A therapeutic composition in orally administrable form comprising 50 mg/Kg body weight in combination with fillers, diluents/adjuvants and binders such as herein described. 9. A composition as claimed in Claim 8, wherein fillers are selected from lactose, starch, calcium phosphate, magnesium stearate, talc, steatite, microfined silica and polymeric cellulose "AVICEL", binders are selected from strach paste and carboxymethyl cellulose (CMC) and non-aqueous adjuvant is ethyl cellulose. 10. A composition as claimed in Claim 8, wherein 0.5% by wt. of CMC is added to the composition for stabilizing the same. Dated this 11th day of February, 2005. -36-

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