ENTEROGENIN PHARMACEUTICAL FORM

Abstract: This patent relates to the obtaining of two pharmaceutical forms - pulvis and ampules of the veterinary medical preparation "Enterogenin". The bioactive ingredient of the preparation is isolated by fractionation of pig small intestinal mucosa cell, extraction with acetic acid, sedimentation with ethyl alcohol, ion-exchange chromatography and diaflo ultrafiltration in the range of 0.5 - 2 kDa. The bioactivity of the preparation is related to two nucleopeptides: guanosine tripeptide and adenosine heptapeptide. The basic criterion for the biological action of "Enterogenin" pulvis and ampules is the activation of biosynthesis of nucleic acids and specific proteins in the muscles, liver, intestines and spleen.

Description: I 10 ENTEROGENIN PHARMACEUTICAL FORM is The invention relates to the preparation 'Εnterogenin - pulvis and ampules", the method for its isolation on an industrial scale, production of the pharmaceutical forms and investigation of the pharmacological effects of the preparation. The active component of "Enterogenin - pulvis and ampules" is a bioactive substance isolated from intestinal mucosa. Data have been reported about substances present 20 in the intestinal mucosa that inhibit cell proliferation (1,2,3). There are no reports in the literature concerning isolation of substances that exert stimulating effect on morphogenesis. We have patented a method for isolation of biostimulating substance from intestinal mucosa of warm-blooded animals (4,5). This method hascertain disadvantages - complex technologic process which renders relatively 25 expensive the production of the substance, and incompleteness of the method to the stage of producing final pharmacologically tested forms. This invention has as its objects to: 1) refine the said method for isolation of the bioactive component of the preparation Εnterogenin - pulvis and ampules"; 2) develop pharmaceutical forms of the preparation "Enterogenin - pulvis and ampules"; 3) Test the pharmacological action of the preparation "Enterogenin - pulvis and ampules" in the veterinary and zootechnical practice after experimental investigation of the effects of the bioactive component. The first object is accomplished by method for isolation of a stimulator of biosynthetic processes from pig intestinal mucosa. Principally, the production scheme comprises the following steps: 1) isolation of a specific cellular mass from D animal intestinal mucosa which is discarded as a waste product in the casing cleaning shops of meat packing plants; 2) obtaining an alcoholic acid extract after settling the high molecular weight polymers; at this stage any contamination associated with the initial biomass is prevented; 3) ion-exchange fractionation in batch procedures; 4) ultrafiltration of the eluent obtained from the fractionation. The technological scheme is described in Example 1.1. Compared with the method described in Patent BG 49927 MPK A61K37/02 the improvements are as follows: the reagents are used in smaller amounts; some of the stages of the basic technologic scheme are changed and their number reduced; the parameters of the ultrafiltration are changed and further specified. As a result, two instead of one bioactive nucleopeptides, named by us "stimulating enterocytogenins", have been isolated. The second object is achieved by preparation of two pharmaceutical forms: 1) packets of enterosolvent granules "Enterogenin pulvis" for peroral administration; 2) vials of "Enterogenin ampules" containing the purified bioactive preparation in a depot form. Stored at a temperature of 2 to 6°C the pharmaceutical forms can preserve their action for three years. The analysis of the above mentioned pharmaceutical forms has been officially approved by the Laboratory for Veterinary Preparation Control with the National Office of Veterinary Medicine (6). The analysis comprises the reactions for chemical and biological identification of the biologically active nucleopeptides in "Enterogenin". Additionally, an analysis of the vehicle components of the pharmaceutical forms are given. The third object is achieved by laboratory and clinical experiments and introductions in veterinary practice. The bioactive nucleopeptides contained in "Enterogenin - pulvis and ampules" act at a molecular level; the proliferative cycle of the physiologically regenerating intestinal mucosa cells is shortened which results in an increase of the resorptive surface of small intestines; in non- proliferating cells they stimulate the synthesis of specific proteins: actomyosin in the striated muscles, detoxicating proteins in the liver, immunogenic proteins in the spleen, and digestive enzymes in the gastrointestinal tract. In smooth muscle cells- these nucleopeptides induce depolarisation by stimulating the Ca 2+ -influx (7). In small experimental animals the daily use of the preparation increases their body mass by more than 200%. Application of "Enterogenin" for 20 days in pig and cattle breeding farms gives as a result a long-term stimulating effect followed up for six months. The effect manifests itself in an increase of the daily weight growth by 125 g for pigs, and by 110 g for calves. In chickens, applied in the drinking water from day 3 to day 45 after hatching, "Enterogenin" stimulates the biosynthesis of DNA and proteins increasing their body weight by 118%. "Enterogenin" has also shown a therapeutic effect in animal models: in experimental stomach ulcers, after liver intoxication, in the prophylaxis of infectious diseases by stimulating the immunogenesis. "Enterogenin" is neither toxic, teratogenic, embryotoxic, nor genotoxic which was proved by tests in the National Oncologic Center and the Ntional Institute of Drug Administration. The invention will be now described by way of the following examples: Example 1. Task 1. (Fig. 1) On Biterling machine in the casing cleaning wards of the slaughter house the cellular enriched mass removed at casing stripping roller No 3 is collected and 20% cold acetic acid is added simultaneously, at constant stirring, to achieve final concentration of 2% acetic acid relative to the total volume. The processing of the biomass can either proceed further or the biomass can be stored in a freezer cooled to -40°C. If the latter action is opted for partial defrosting is required subsequently. Processing with a meat mincer follows. 95% ethyl alcohol (0.2 volumes of the total mass) is added to the minced material. Then the biomass is rapidly heated to 70°C and cooled immediately to room temperature. The liquid extract is removed by separation and filtration. The high molecular weight detritus is thrown away. The acidic pH is adjusted to pH 6.5-7.0. The alcohol and the lipid admixtures are removed in ether in a glass reactor. At a constant control of the salt concentration the concentrate is absorbed on DEAE cellulose and eluted by batch procedures using alkalised saline solution. The active fraction is purified through diaflo ultrafilters collecting the fraction in the range of 0.5 - 2.0 kDa. The second filtration is realised under sterile conditions. The concentration of nucleopeptides is measured which is necessary for the determination of the dosage of the pharmaceutical forms. Example 1. Task 2. The actual preparation of the pharmaceutical forms "Enterogenin - pulvis and ampules" is preceded by chemical and biological investigations designed to identify the nucleopeptides: a) ultraviolet absorption spectrum - max. 260 nm, min. 237 nm; A 260 /A 237 = 1.21 - 1.19; b) specific reaction for peptides - e.g. ninhydrin reaction of Reichelt; c) molecular weight determination: by means of Sephadex G-25 fine column precalibrated with peptides with a known molecular weight; by K av , the referent limits of the two active nucleopeptides are 1.1 - 1.3 kDa and 0.6 - 0.65 kDa, respectively; d) amino acid composition: by "finger print" analysis on paper chromatography or, more exactly, through quantitative determination on amino acid analyzer. Glycine and tryptophan are detected in the low molecular weight nucleopeptide; the other nucleopeptide contains arginine, serine, leucine, histidine, glycine and tyrosine; e) quantitative FLOW CHART ISOLATION OF STIMULATING ENTEROGENIN PRODUCT PROCEDURE EQUIPMENT Biterling machine 20% acetic acid Alternative procedure Meat mincer (homogenizer) Heating and cooling reactor Separator Solution of sodium hydroxide DEAE cellulose in a column "Diaphlo" membrane disks Fig. l analysis: spectrophotometric determination of A 260 against a standard 0.01 mmol adenine sulfate; calculations: A 260 sample Nucleopeptides% = 0.0125 A 260 standard f) the biologic activity is determined by measuring the incoφoration of H-leucine (injected intraperitoneally in a dose of 0.04 MBq per 20 g of mouse weight). Eighteen hours after introduction of an isotope (for the control group) and the isotope together with 0.02 mg of "Enterogenin" (for the experimental group) the incorporation of the isotope is measured in 50 mg of muscle tissue taken from the adductors of a rear leg of a mouse. Example 1. Task 3. A rapid criterion for biologic activity of the preparation "Enterogenin pulvis and ampules" is the investigation of the biosynthesis of DNA, RNA and proteins by the incorporation of the corresponding isotopilcally marked precursors. The mean activation is greater than 200% (Table 1). Long-term moφhological changes occur in the animals repeatedly treated with "Enterogenin" (Table 2). The resoφtive surface of the intestines expands which increases the effectiveness of the food 2.56-fold at less amount of food taken and raises the coefficient of protein effectivity - 2.5 times that of the controls. Moφhological changes also occur in the striated muscles - contractile muscle proteins incease relatively. The growth dynamics in calves improved by injecting them 6 times with the preparation, in pigs and fresh water fish by mixing the preparation daily for 20 days in the food, and in chickens by adding the preparation for 45 days in the drinking water. A pronounced therapeutic effect of "Enterogenin" in two models of experimental ulcers (reseφin and stress-histamin models) in rats has been obtained. After tetrachlormethane intoxication in rats we investigated the dynamics of the metabolic and recuperating processes in the hepatorenal syndrome. The preparation "Enterogenin" showed protective and reparative effects. Table 1. Effect of " Enterogenin" on the biosynthesis of DNA (incorporation of ^ H-timidine), RNA (incorporation of 14c- uridine), and proteins (incorporation of ^ H-leucine) [given in per cent in relation to the control animals without " Enterogenin"] Animal Body organ Incoφoration Timidine Uridine Leucine Duodenum 221 Jejunum 190 Rats Middle part of small intestines 284 Ileum 337 Hepatocyte nuclei 161 Duodenum 163 Jejunum 302 360 Mice Middle part of small intestines 212 212 Ileum 153 160 Muscle 177 White muscle 103 104 Middle part of small intestine 242 265 Chickens Red muscle 118 193 Liver 183 331 Table 2 Morphologic changes after multiple treatment with "Enterogenin". [given in per cent in relation to the control animals without "Enterogenin"] Parameter Body organ Animal % of changes Total body weight mice 212 Liver rats 159 Body weight (g) Small intestines rats 126 Heart rats 135 Spleen rats 112 Kidnev rats 120 Duodenum rats 144 Jejunum rats 173 Middle part of rats. 167 small intestines Cellularity (χl0 5 /l/l um 2 ) Ileum rats 135 Intestinal mucosa Duodenum mice 134 Jejunum mice 163 Middle part of mice 173 small intestines Ileum mice 139 Number of intestinal villi Middle part of mice 133 (on microscopic cut) small intestines Length of an intestinal villus Middle part of mice 125 (on microscopic cut/Mm) small intestines Musculus pigs 191 gastrocnemius Area of a single muscle fibre m") Diaphragm Pigs 279 Musculus obliquus pigs 252 Musculus pigs 154 gastrocnemius Diameter of a single muscle fibre Diaphragm Pigs 170 («m in electron microscopy) Musculus obliquus pigs 157 Thickness of the Z line Musculus Pigs 300 ( n. electron microscopy) gastrocnemius REFERENCES 1. Skraastad O, Reichelt KI. An endogenous colon mitosis inhibitor reduces proliferation of colon carcinoma cells (HT 29) in serum restricted medium. Virchows Arch B, 50, (1989), 393-390. 2. Skraastad O, Reichelt KI. An endogenous colon mitosis inhibitor and dietary calcium inhibit the increased colonic cells proliferation induced by cholic acid. J Gastroent 23 (1986), 801-807. 3. Triffonov B, Roussev GK, Argirov C, Draganov M, Kamberov B. Biological effects of a novel intestinal peptide - inhibiting enterocytogenin on cultured 3T3 mouse fibroblasts and L5178Y mouse lymphoma cells. Regulatory Peptides, 51 (1994), 111-119. 4. Triffonov B, Roussev GK, Boshev H, Tyanev G. A method for isolation of a biostimulating substance. Patent 49927, BG 49927 MPK A61K37/02, Vol 3, March 16, 1992, 1-3 (Bulgarian). 5. Roussev GK, Triffonov B, Petrov M, Boshev H. A method for isolation of substances with moφhogenic activity. Invention patent 37396 MPK-A 61K35/38, Vol 6, June 14, 1985, 1-6. 6. Certificate No XIII-33/10.02.1995, Ministry of Agriculture, National Veterinary Office for Licence for production and use of preparation "Enterogenin" in the veterinary practice . 7. Triffonov B, Kristev A, Zaprianov G, Lukanov J, Kostadinova I. Effects of a novel intestinal peptide (enterogenin) on the contractile and bioelectric activity of intestinal smooth muscle from the rat and the guinea-pig. J Gastrointest Motil 4, (1992), 193-199.

Claims: PATENT CLAIMS We claim: 1. Pharmaceutical forms of a stimulator of biosynthetic processes containing "Enterogenin ampules" - vials containing 0.0125% bioactive nucleopeptide in depot form with 0.2% sodium alginate in a physiologic solution, and "Enterogenin pulvis" containing 0.125% bioactive nucleopeptide sorbed on starch and granulated in an enterosolvent form with hydrated fat. 2. Method for obtaining the bioactive component of above mentioned forms in Claim No 1, comprising the steps of fractionation of waste intestinal biomass, extraction by acetic acid, sedimentation by using ethyl alcohol at low concentration, ion-exchange chromatography and ultrafiltration in the range of 0.5 - 2.0 kDa. Two bioactive nucleopeptides are isolated. 3. Application of the preparation "Enterogenin - pulvis and ampules" in the veterinary practice: a) for pigs - "Enterogenin" pulvis" in a dose of lOOg per 200 kg body weight daily, mixed with the forage food for 20 - 30 days; b) for ruminants - 0.5 ml of "Enterogenin ampules" per 100 kg of body weight 6 to 8 times intramuscularly every third day; c) for chickens - 0.5 ml of "Enterogenin ampules" per 10 kg of body weight daily for 45 days mixed with the drinking water; d) for fresh water fish - during the breeding period 100 g of "Enterogenin pulvis" mixed in 5 kg of the forage food.

Inventors: BOSHEV NIKOLA ATANASSOV, PETROV METODI STEFANOV, ROUSSEV JEORGE KONSTANTINOV, TRIFONOV BORISLAV BORISOV, BOSHEV, NIKOLA ATANASSOV, PETROV, METODI STEFANOV, ROUSSEV, JEORGE KONSTANTINOV, TRIFONOV, BORISLAV BORISOV
Applicants: ALEXANDROV CHRISTO ALEXANDROV, BOSHEV NIKOLA ATANASSOV, PETROV METODI STEFANOV, ROUSSEV JEORGE KONSTANTINOV, TRIFONOV BORISLAV BORISOV, ALEXANDROV, CHRISTO ALEXANDROV, BOSHEV, NIKOLA ATANASSOV, PETROV, METODI STEFANOV, ROUSSEV, JEORGE KONSTANTINOV, TRIFONOV, BORISLAV BORISOV
Application year: 1995
Main classification: A61K9
IPC codes: A61K9, A61K35, A61K38, A61P1, A61P3, A61P15, A61P37, A61P39