Abstract:
This patent relates to the obtaining of two pharmaceutical forms--pulvis and ampules of the veterinary medical preparation &#34;Enterogenin&#34;. 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 &#34;Enterogenin&#34; pulvis and ampules is the activation of biosynthesis of nucleic acids and specific proteins in the muscles, liver, intestines and spleen.

Description:
This is a 371 of PCT/BG95/00009 filed Oct. 9,1995. 
    
    
     BACKGROUND OF THE INVENTION 
     The invention relates to the preparation &#34;ENTEROGENIN--pulvis and ampules&#34;, 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 &#34;ENTEROGENIN--pulvis and ampules&#34; is a bioactive substance isolated from intestinal mucosa. Data have been reported about substances present 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 has certain disadvantages--complex technologic process which renders relatively expensive the production of the substance, and incompleteness of the method to the stage of producing final pharmacologically tested forms. 
     SUMMARY OF THE INVENTION 
     This invention has as its objects to: 1) refine the said method for isolation of the bioactive component of the preparation &#34;ENTEROGENIN--pulvis and ampules&#34;; 2) develop pharmaceutical forms of the preparation &#34;ENTEROGENIN--pulvis and ampules&#34;; 3) Test the pharmacological action of the preparation &#34;ENTEROGENIN--pulvis and ampules&#34; in the veterinary and zootechnical practice after experimental investigation of the effects of the bioactive component. 
    
    
     BRIEF DESCRIPTION OF THE FIGURE 
     FIG. 1 is a flowchart describing the isolation of stimulating enterogenin. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     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 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 &#34;stimulating enterocytogenins&#34;, have been isolated. 
     The second object is achieved by preparation of two pharmaceutical forms: 1) packets of enterosolvent granules &#34;ENTEROGENIN pulvis&#34; for peroral administration; 2) vials of &#34;ENTEROGENIN ampules&#34; 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 &#34;ENTEROGENIN&#34;. 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 &#34;Enterogenin--pulvis and ampules&#34; 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 depolarization 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 &#34;ENTEROGENIN&#34; 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, &#34;ENTEROGENIN&#34; stimulates the biosynthesis of DNA and proteins increasing their body weight by 118%. &#34;ENTEROGENIN&#34; 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. &#34;ENTEROGENIN&#34; is neither toxic, teratogenic, embryotoxic, nor genotoxic which was proved by tests in the National Oncologic Center and the National Institute of Drug Administration. 
     The invention will now be 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 alkalized 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 &#34;ENTEROGENIN--pulvis and ampules&#34; 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 &#34;finger print&#34; 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 analysis: spectrophotometric determination of A 260  against a standard 0.01 mmol adenine sulfate; calculations: ##EQU1## f) the biologic activity is determined by measuring the incorporation of  3  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 &#34;ENTEROGENIN&#34; (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 &#34;ENTEROGENIN pulvis and ampules&#34; is the investigation of the biosynthesis of DNA, RNA and proteins by the incorporation of the corresponding isotropically marked precursors. The mean activation is greater than 200% (Table 1). Long-term morphological changes occur in the animals repeatedly treated with &#34;ENTEROGENIN&#34; (Table 2). The resorptive 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. Morphological 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 &#34;ENTEROGENIN&#34; in two models of experimental ulcers (reserpin 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 &#34;ENTEROGENIN&#34; showed protective and reparative effects. 
     
                       TABLE 1______________________________________Effect of &#34;ENTEROGENIN&#34; on the biosynthesis of DNA(incorporation of .sup.3 H-thymidinen), RNA (incorporation of .sup.14 C-uridine), and proteins (incorporation of .sup.3 H-leucine) given in percent in relation to the control animalswithout &#34;Enterogenin&#34;!          IncorporationAnimal  Body organ   Thymidinen Uridine                                 Leucine______________________________________Rats    Duodenum     221   Jejunum      190   Middle part of small                284   intestines   Ileum        337   Hepatocyte nuclei                161Mice    Duodenum     163   Jejunum      302        360   Middle part of small                212              212   intestines   Ileum        153        160   Muscle                        177Chickens   White muscle 103              104   Middle part of small                242              265   intestine   Red muscle   118              193   Liver        183              331______________________________________ 
    
     
                       TABLE 2______________________________________Morphologic changes after multiple treatmentwith &#34;ENTEROGENIN&#34;.  given in percent in relationto the control animals without &#34;ENTEROGENIN&#34;!Parameter    Body organ   Animal  % of changes______________________________________Body weight (g)        Total body weight                     mice    212        Liver        rats    159        Small intestines                     rats    126        Heart        rats    135        Spleen       rats    112        Kidney       rats    120Cellularity    Duodenum   rats    144(×10.sup.5 /l/1 μm.sup.2)        Jejunum      rats    173Intestinal mucosa        Middle part of                     rats    167        small intestines        Ileum        rats    135        Duodenum     mice    134        Jejunum      mice    163        Middle part of                     mice    173        small intestines        Ileum        mice    139Number of intestinal villi        Middle part of                     mice    133(on microscopic cut)        small intestinesLength of an intestinal        Middle part of                     mice    125villus (on microscopic        small intestinescut/μm)Area of a single muscle        Musculus     pigs    191fibre (μm.sup.2)        gastrocnemius        Diaphragm    pigs    279        Musculus obliquus                     pigs    252Diameter of a single        Musculus     pigs    154muscle fibre (μm in        gastrocnemiuselectron microscopy)        Diaphragm    pigs    170        Musculus obliquus                     pigs    157Thickness of the Z line        Musculus     pigs    300(μm. electron        gastrocnemiusmicroscopy)______________________________________ 
    
     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, Mar. 16, 1992, 1-3 (Bulgarian). 
     5. Roussev GK, Triffonov B, Petrov M, Boshev H. A method for isolation of substances with morphogenic activity. Invention patent 37396 MPK-A 61K35/38, Vol 6, Jun. 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 &#34;ENTEROGENIN&#34; 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.