MXPA97004462A - Institute agents isolated from the intestinal mucosa, a method for its isolation and its application - Google Patents

Abstract

This patent refers to the obtaining of two antitumor agents (IEG1 and IEG2) tested under experimental conditions. These bioproducts are isolated by means of fractionation of pig intestinal mucosal cells, extraction with acetic acid, precipitation with ethyl alcohol, ultrafiltration, lyophilization and gel filtration. IEG1 is a peptide comprising 33 amino acid residues linked to 2 guanyl residues with a molecular weight of 4450 + 180 Da, IEG2 is a haxapeptide guanosyl with a molecular weight of 950 + 120 Da. The two agents suppress DNA synthesis. IEG1 shows antitumor effect at three levels: "in vitro" or malignant cell lines, in combination "in vitro-in vivo", and "in viv"

Description

ISOLATED INSTITUTE AGENTS OF THE INTESTINAL MUCOSA. A METHOD FOR YOUR ISOLATION AND YOUR APPLICATION This invention relates to a method for isolating two agents with cytostatic action of intestinal gut mucosa that experimentally showed to exert an inhibitory effect on cell proliferation. Our previous studies (1) have shown that cell differentiation causes the emergence of specific morphogenic inhibitors and stimulators generally referred to as ontogenins; in particular, stimulating (SEG) and inhibitory enterocitogenins (IEG) were isolated from physiologically regenerative mucosal cells (2). In subsequent studies we have reported data indicating that there are substances of the calotic type in the colonic mucosa that specifically inhibit the proliferation of cells in the colon (3). The objects of the present invention are: 1. Specify, under industrial conditions, the method for producing the two inhibitory enterocytogenins (IEG, and IEG2). 2. Identify these enterocitogenins chemically. 3. Test in experimental animals the effect of IEG, and IEG2 on the morphogenic and biosynthetic processes. 4. Test in vitro IEG, in normal and malignant cell cultures and study its effect on transplantable experimental tumors in vitro - in vivo and only in vivo. The first object is achieved by providing a method for isolating inhibitory enterocytogenins from pig intestinal mucosa. The production scheme under industrial conditions includes: 1) Fractionation of cellular mass from waste of intestinal mucosa to a specific cell type; 2) Extraction of high molecular polymers with 2% acetic acid and subsequent precipitation by alcohol; 3) Separation and after filtering, the light fraction is ultrafiltered through a DDS filter (10 Da); and 4 lyophilization of the ultrafiltrate and subsequent separation through molecular examinations. The second object is achieved by obtaining a highly purified preparation under laboratory conditions using FPLC and HPLC methods. The spectral analysis and chemical quality conducted identifies IEG, and IEG2 as nucleopéptidos; the amino acid content was determined by an amino acid analyzer. Laboratory experiments were performed to obtain the third object referring to the effect of preparations on biosynthetic processes that were tested by the incorporation of H - tinidine (DNA synthesis) and C - uridine (RNA synthesis) in several regions of the intestines of the tested mice. The morphogenic effect of the preparations was tested by enterocytic cellularity and DNA concentration in the same regions. The fourth object is achieved by studying the effect of IEG [on cell cultures of six normal cell lines and seven malignant cell lines in vitro on a solid tumor transplantable in vitro - in vivo and on a tumor only in vivo. The invention will be described by means of the following examples: EXAMPLE 1 Object 1 (Figure 1) .- The cell mass removed by the number two roll ripper on a Biterling machine in the traces cover cleaning rooms is collected and concentrated acetic acid is added, under constant coagulation, to achieve a final concentration of 2% acetic acid in relation to the whole mass. The mass thus prepared is subjected to cell destruction on a disintegrator followed by precipitation of the polymers heated in a reactor with three volumes of 96 ° ethyl alcohol. The illuminated fraction is separated in a separator and then filtered; the pH is adjusted to 6.5 - 7.0 and ultrafiltered through DDS filters at 6000 - 1000 Da. The collected filtrate is lyophilized and can be stored at a temperature of -10 ° C, retaining its biological activity for 5 years. When working with this, 0.1 g of lyophilisate is dissolved in 0.5 ml of water and further purified by gelatin filtration on Sephadex G-25 (medium grade). Object 2 (Figures 2a and 2b) .- The molecular weight of the purified bioactive fractions in object 1 was determined on a silica-gel Sorbax column using an HPLC apparatus (Figure 2a),, IEG was found, which has a molecular weight of 4.450 plus or minus 180 Da and IEG2 950 plus or minus 120 Da. The ultraviolet spectral analysis revealed two absorption maxima: for IEG, at 220 nm and 248 nm and for IEG2 at 220 nm and 245 nm. The presence of guanosine in both IEG identified them as core peptide. The amino acid analyzer (Figure 2b) showed that IEG contained glutamine (13.33%) followed by lysine (10.95%), serine, threonine, asparagine, glycine, alanine, valine, isoleucine, leucine, histidine and arginine. Cictina, methionine. Tyrosine and phenylalanine were probably present in a mixed state (<1.5%, Table 1). IEG2 is a hexapeptide with the following amino acid sequence: Arg-Arg-Asp-Asp-His-Arg-NH2. FLOW DIAGRAM Insulation of IEGj ^ e IEG2 elution volume my Fig 2a. Gel filtration. (above) and HPLC purification of IEG, (following).

I I I I I I I I I I I I I I I I I I I LO or LO O LO * - CM CM Fig 2b. Content of amino acids of IEG, Computer analysis of the amino acid content Do not . NAME RT HEIGHT AREA n mo l% ng REASON 1 4.32 1438 31566 0.031 0.0 0.0 2 Asp 5.86 36209 645265 2.414 6.94 321.4 100.6 3 Thr 6.53 25623 468376 1.627 4.68 193.8 100.5 4 Ser 7.14 36698 624028 2.111 6.07 221.8 99.1 Glu 8.02 65067 1186968 4,633 13.32 681.5 99.1 6 8.74 1958 54995 0.054 0.0 0.0 7 Gly 10.61 46608 916917 3.274 9.42 245.8 98.4 8 Wing 11.57 32003 789776 3.089 8.89 275.2 100.8 9 Cys 12.29 3368 90129 0.319 0.92 76.8 216.6 Val 13.09 35429 562107 1.989 5.72 233.1 96.0 11 Met 14.32 3148 111300 0.525 1.51 78.3 163.7 12 He 16.61 10418 287121 1.306 3.76 171.4 99.1 13 Leu 17.73 20034 578395 2,683 7.72 352.7 100.7 14 Tyr 18.64 3282 58156 0.447 1.29 81.1 95.1 Phe 19.49 3879 68355 0.308 0.89 50.9 86.5 16 21.25 1137 12883 0.012 0.0 0.0 17 Lys 21.84 77180 1189568 3.805 10.95 556.3 99.7 18 22.48 8631 174505 0.174 0.0 0.0 19 NH3 23.17 38740 975264 0.528 42.9 101.0 His 24.13 13573 304124 0.982 2.82 152.5 105.1 21 25.62 1 42 48214 0.048 0.0 0.0 22 Arg 28.34 18289 512259 2.077 5.98 361.8 100.6 23 Pro 3.168 9.12 Total 483843 9690271 35.604 4097.3 Object 3 (Table 2) .- Each fraction obtained by purifying IEG (see object 1) was tested on mice; 3mg of the peptide content of the fraction was administered intraperitoneally simultaneously with the test reagent (isotope); then the animals were sacrificed and investigated 7 h. after. The treatment with IEG, for 7 h. It resulted in the reduction of the number of erythrocytes by 24% on the average and with IEG2 by 20%. Both IEG have a suppressive effect on DNA synthesis especially in the lower regions of the intestines: IEG, by 45% and IEG2 by 48%. In previous studies, we proved that IEG elevates the cytosol level of Ca + in smooth muscle cells using intracellular Ca + stores. In this way it commutes the chain of molecular mechanisms through which it acts on smooth muscle motility.

Table 2. Effect of IEG, and IEG2 On Biosynthetic and Morphological processes in the Intestines. 7 h. after the treatment of mice with IEG! and IEG2. (Data are presented in percentages compared to control animals not treated with IEG).

Incorporation Section of the H3-C1'- ConcentraCelulari-IEG Intestines thymidine uridine DNA tion Duodenum 74 75 66 82 Jejunum 94 67 84 95 IEGj ileum 57 68 69 81 Mid section 62 58 76 of the 52 intestines Duodenum 77 78 68 88 Jejunum 88 78 97 75 IEG, ileum 55 74 77 85 Medlia section of the 48 74 62 83 intestines Object 4 (Table 3) .- IEG has a strong antitumor effect on malignant cell cultures. It also has a good pronounced cytotoxic effect on Lewis lung carcinoma cells (LLCa) both in vitro and in vivo in the line of C57 BI mice that was tested by the combined in vitro-in vivo method and the so-called bioassay to determine the survival of the cellular fraction of LLCa. There is a dependence of marked concentration effect. The rate of inhibition of tumor development (IIPG) of the subcutaneous form of LLCa is 88.2% (ai an IEG concentration of 1050 μg / ml.) The observed pathomorphological changes correlate with the dose-dependent effect of IEG, IEGt exerts in vivo an effect on the development of an experimental solid transplantable tumor in Syrian golden hamsters (flat cell carcinoma IC-Sofia-line 7) .IITG in that experiment was 60.5% (therapeutic dose of IEG, on the scale of 4 x 10"g / kg to 3.5 x 10 g / kg) Inhibition of DNA synthesis in the tumor was -48% Mitosis was reduced to 21.9% compared to the same parameters in the tumors of untreated animals (P <0.01).

Table 3. Effect of IEG on Cell Cultures.

IC50 (μ / ml) Origin Line Test- Test- cells NR KBP NORMAL CELL LINES 123.9 187.5 3T3 Fibroblasts mouse amnionic FL * human 223.2 264.4 BHK Kidney * Golden Syrian Hamster < 300 < 300 CHO ovaries * hamster 213.2 256.4 MDC kidney dogs 192.6 247.4 Vero African green monkey kidney 156.7 197.3 LINES OF MALIGNANT CELLS L517 mouse lymphoma 192.8 212.6 8 Hep carcinoma of human 290.4 300.0 2 the larynx Hela human carcinoma 291.5 268.5 epitheloid in the uterus RD human rhabdomyosarc 196.15 176.3 orna embryonal Sp 2 myeloma ** mouse 206.2 226.2 Ag8 myeloma ** mouse 1998.9 234.8 METH myeloma ** mouse 212.3 246.5 ** lines of suspension cells * continuous lines REFERENCES. 1. Roussev GK. Programmed manipulation of embryonic de-velopment. Ontogenins. Soria, Nauka and Izkustvo, 1974, 1-301. 2. Roussev GK, Trifonov B, Petrov M, Boshev H. A method for isolation of substances with morphogenic activity. Invention patent 37396 MPK-A 61K35 / 38, Vol 6, June 14, 1985, 1-6. 3. Skraastad O, Reichelt KI. An endogenous colon mitosis inhibitor and dietary calcium inhibit the increased colonic cells prolif-eration induced by cholic acid. J Gastroent 23 (1988), 801-807. 4. Trifonov 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 (3)

R E I V I N D I C A C I O N S

1. - Antitumor agents isolated from the intestinal mucosa that have two nucleopéptidos, IEG, and IEG2, with molecular weights of 4.450 ± 180 Da and 950 ± 120 Da, respectively; IEG, is a polypeptide comprising 2Thr, 4Glu, 5Gly, 2Asp, 2Ser, 4Ala, 2Val, lile, ITri, 3Lys, lHis, lArg, 2Leu and 3Pro bound to 2 guanyly residues; IEG2 is a hexapeptide with the following amino acid sequence: Arg-Arg-Asp-Asp-His-Arg-NH2 linked to a guanine residue. 2.- Method to obtain antitumor agents from the intestinal mucosa of the pig according to clause 1, which includes fragmentation of intestinal mass of waste, extraction with acetic acid, precipitation with ethyl alcohol, ultrafiltration on the scale of 1-6 kDa, lyophilization and filtration with supplemental gelatin. 3.- Application of IEG, to suppress the development of carcinoma of experimental Lewis lungs by the combined method "in vitro - in vivo" at a dose of 1.050 μg / 1, as well as to suppress flat cell carcinoma IC- Sofia line 7 using the "in vivo" method at a dose of 4 x 10"g / kg body weight, experimental application of IEG, as an inhibitor of the development of cell cultures of normal and malignant cell lines and of IEG, and IEG2 as inhibitors of DNA biosynthesis in the intestines. R E S U E N This patent relates to the obtaining of two antitumor agents (IEG) and IEG2) tested under experimental conditions. These bioproducts are isolated by means of fractionation of pig intestinal mucosal cells, extraction with acetic acid, precipitation with ethyl alcohol, ultrafiltration, lyophilization and gel filtration. IEG is a peptide comprising 33 amino acid residues linked to 2 guanyl residues with a molecular weight of 4450 ± 180 Da; IEG2 is a guanosyl hexapidoptide with a molecular weight of 950 ± 120 Da. The two agents suppress DNA synthesis. IEG, shows anti-tumor effect in three levels: "in vitro" or malignant cell lines, in combination "in vitro-in vivo", and "in vivo".