ANTITUMOR AGENTS ISOLATED FROM INTESTINAL MUCOSA, A METHOD FOR THEIR ISOLATION AND THEIR APPLICATION

Abstract: This patent relates to the obtaining of two antitumor agents (IEG1 and IEG2) tested under experimental conditions. These bioproducts are isolated through fractionation of pig intestinal mucosa cells, extraction with acidic acid, precipitation with ethyl alcohol, ultrafiltration, lyophilization and gel filtration. IEG1 is a peptide comprising 33 amino acid residues bound to 2 guanile residues with a molecular weight of 4450 +/- 180 Da; IEG2 is a guanosile-hexapeptide with molecular weight of 950 +/- 120 Da. The two agents suppress the DNA synthesis. IEG1 shows antitumor effect on three levels: "in vitro" on malignant cell lines, in combination "in vitro-in vivo", and "in vivo".

Description: ANTITUMOR AGENTS ISOLATED FROM INTESTINAL MUCOSA, A METHOD FOR THEIR ISOLATION AND THEIR APPLICATION This invention relates to a method for isolation of two agents with cytostatic action from pig intestinal mucosa which were shown experimentally to exert an inhibitory effect on cell pro¬ liferation. Our previous studies [1] have shown that cell dif¬ ferentiation causes the emergence of specific morphogenic stimulators and inhibitors generally referred to as ontogenins; in particular, stimulating (SEG) and inhibiting (IEG) enterocytogenins were isolated from the physiologically regenerating mucosa cells [2]. In later studies we have reported data indicating that there are substances of chalonic type in the colonic mucosa which inhibit specifically the cell proliferation in the colon [3]. The objects of the present invention are: 1. To specify, under industrial conditions, the method for producing the two inhibiting enterocytogenins (IEGi and IEG 2 ). 2. To identify these enterocytogenins chemically. 3. To test in experimental animals the effect of IEGi and IEG 2 on the morphogenic and biosynthetic processes. 4. To test in vitro IEGi in normal and malignant cellular cul- tures 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 isolat¬ ing inhibiting enterocytogenins from pig intestinal mucosa. The production scheme under industrial conditions includes: 1. Fractionation of waste cellular mass from intestinal mucosa to a specific cellular type; 2. Extraction of high molecular polymers with 2% acetic acid and subsequent precipitation by alcohol; 3. Separation and after filtration the light fraction is ultrafiltered through a DDS filter (10 kDa); 4. Lyophilization of the ultrafiltrate and subsequent separation through molecular sifts. The second object is achieved by obtaining highly purified preparation under laboratory conditions using FPLC and HPLC methods. The conducted quality chemical and spectral analy¬ sis identifies IEGi and IEG 2 as nucleopeptides; the amino acid content was determined by an amino acid analyser. Laboratory experiments were carried out to accomplish the third object concerning the effect of preparations on biosynthetic processes which was assessed by the incorporation of H -thy- 14 midine (DNA-synthesis) and C -uridine (RNA synthesis) in various regions of the intestines of the mice tested. The prepa¬ ration's morphogenic effect was assessed by the enterocytic cellularity and DNA concentration in the same regions. The fourth object is accomplished by studying the effect of IEGi on cellular cultures of 6 normal and 7 malignant cell lines in vitro, on a transplantable solid tumor in vitro-in vivo and on one tumor only in vivo. The invention will be described by way of the following examples: Example 1. Object 1. (Fig. 1) The cellular mass removed at casing stripping roller No 2 on a Biterling machine in the cas- ing cleaning wards of slaughter houses is collected and con¬ centrated acetic acid is added, at constant stirring, to achieve a final concentration of 2% acetic acid relative to the whole mass. The mass so prepared is subjected to cellular destruction on a disintegrator followed by precipitation of the polymers heated in a reactor with 3 volumes of 96% ethyl alcohol. The light fraction is separated in a separator and then filtered; its pH is adjusted to pH 6.5 - 7.0 and ultrafiltered through DDS filter to 6000- 1000 Da. The collected filtrate is lyophilized and can be stored at a temperature of - 10°C retaining its biologic activity for 5 years. When working with it 0.1 g of the lyophisate is dissolved in 0.5 ml H 2 0 and purified additionally by gel-filtration on Sephadex G-25 (medium grade). Example 1. Object 2. (Figs 2a and 2b) The molecular weight of the bioactive fractions purified in Object 1 was determined on a silica- gel Sorbax column using HPLC apparatus (Fig.2a) IEGi was found to have molecular weight of 4450 ± 180 Da, and IEG 2 - 950 ± 120 Da. UV spectral analysis revealed two absorption maxima: for IEGi at 220 nm and 248 nm and for IEG 2 at 220 nm and 245 nm. Presence of guanosine in both IEGs identified them as having nucleopeptide nature. The amino acid analyser (Fig. 2b) showed that IEGi con¬ tained glutamine (13.33%) followed by lysine (10.95%), serine, threo¬ nine, asparagine, glycine, alanine, valine, isoleucine, leucine, histi¬ dine, and arginine. Cyctine, methionine, tyrosine and phenylalanine were present probably in a mixed state (< 1.5%, Table 1). IEG 2 is a hexapeptide with the following amino acid sequence: Arg-Arg-Asp- Asρ-His-Arg-NH 2 . FLOW CHART ISOLATION OF IEGi AND IEG 2 PRODUCT PROCEDURE EQUIPMENT Biterling machine Concentrated acetic acid Desintegrator Heating reactor The same reactor as above Separator Solution of sodium hydroxide DDS ultrafiltration equipment Lyophilizer Sephadex G-25 column (medium grade) Freezer Fig. l elution volume (ml) Fig. 2a. Gel filtration (above) and HPLC-purification of IEG- (below) I I I I I I I I I I I I I I I I I I I Fig. 2b. Amino acid content of IEG T Table 1. Computer analysis of the amino acid content of IEGi No. NAME RT HEIGHT AREA n mol % n g RATIO 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 5 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 Ala 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 10 Val 13.09 35429 562107 1.989 5.72 233.1 96.0 1 1 Met 14.32 3148 111300 0.525 1.51 78.3 163.7 12 Ile 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 15 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 20 His 24.13 13573 304124 0.982 2.82 152.5 105.1 21 25.62 1142 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 Example 1. Object 3. (Table 2). Each fraction obtained in pu¬ rifying IEG (see Object 1) was tested on mice. 3 mg of the pep¬ tide content of the fraction was administered intraperitoneally simultaneously with the testing reagent (isotope); the animals were then killed and investigated 7 hours afterwards. Treat¬ ment with IEGi for 7 hours resulted in reducing the number of erythrocytes by 24% on the average, and with IEG 2 - by 20%. Both IEGs have a suppressive effect on DNA synthesis espe¬ cially in the lower regions of intestines: IEGi by 45%, IEG 2 - by 48%. In previous studies [4] we proved that IEGi elevates the cytosol level of Ca 2+ in smooth muscle cells by using the intra- cellular Ca 2+ depots. In this way it switches on the chain of molecular mechanisms by which it acts on the smooth muscle motility. Table 2. Effect of IEGi and IEG 2 on the biosynthetic and mor¬ phologic processes in the intestines (7 hours after treatment of mice with IEFi and IEG 2 ) (The data are presented in percentages in comparison with control animals not treated with IEGs) ττ - ,-, Section of the Incorporation DNA Cellularity intestines H -thymidine C -uridine concentration Duodenum 74 75 66 82 Jejunum 94 67 84 95 IEG j Ileum 57 68 69 81 Middle section of the 52 62 58 76 intestines Duodenum 77 78 68 88 Jejunum 88 78 97 75 IEG 2 Ileum 55 74 77 85 Middle section of the 48 74 62 83 intestines Example 1. Object 4 (Table 3). IEGi has a strong antitumor effect on malignant cellular cultures. It also has a well pronounced cytotoxic effect on Lewis lung carcinoma cells (LLCa) both in vitro and in vivo in mice line C57BI which was proved by means of the combined method in vitro-in vivo and so-called bioassay for determination of surviving tumour cellular fraction of LLCa. There is a marked concentration-effect dependence. The inhi¬ bition index of tumour growth (IITG) of the subcutaneous form of LLCa is 88.2% (at a concentration of IEGi 1050 /xg/ml). The observed pathomorphologic changes correlate with the dose- dependent effect of IEGi. IEGi exerts in vivo an effect on the development of an experimental transplantable solid tumour in golden Syrian hamsters (flat cellular carcinoma IC-Sofia-line 7). IITG in this experiment was 60.5% (therapeutic dose of IEGi -3 -4 in the range of 4x10 g/kg to 3.5x10 g/kg. The inhibition of DNA synthesis in the tumour was - 48%; the mitosis were re¬ duced to 21.9% in comparison with the same parameters in the tumours of non-treated animals (P < 0.01). Table 3. Effect of IEGi on cellular cultures IC 50 (μg/ml) Cell line Origin NR-test KBP-test NORMAL CELL LINES 3T3 fibroblasts mice 123.9 187.5 FL amnionic* human 223.2 264.4 BHK kidney* golden Syrian hamster > 300 > 300 CHO ovarium* hamster 213.2 256.4 MDC kidney dogs 192.6 247.4 Vero kidney African green monkey 156.7 197.3 MALIGNANT CELL LINES L 5178 ! lymphoma mice 192.8 212.6 Hep 2 carcinoma of the larynx human 290.4 300.0 Hela epitheloid carcinoma in the human 291.5 268.5 uterus RD embryonal rabdomyosarcoma human 196.15 276.3 Sp 2 myeloma** mice 206.2 226.2 Ag8 myeloma** mice 1998.9 234.8 METH myeloma** mice 212.3 246.5 *suspension cell lines continuous lines REFERENCES 1. Roussev GK. Programmed manipulation of embryonic de¬ velopment. Ontogenins. Sofia, Nauka y Izkustvo, 1974, 1-301. 2. Roussev GK, Trifonov B, Petrov M, Boshev H. A method for isolation of substances with morphogenic activity. Inven¬ tion 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: PATENT CLAIMS What we claim is: 1. Antitumor agents isolated from intestinal mucosa which are two nucleopeptides, IEGi and IEG 2 , with molecular weights of 4450 ± 180 Da and 950 ± 120 Da, respectively; IEGi s a pep¬ tide comprising 2 Thr, 4 Glu, 5 Gly, 2 Asp, 2 Ser, 4 Ala, 2 Val, 1 Ile, 1 Try, 3 Lys, 1 His, 1 Arg, 2 Leu and 3 Pro bound to 2 guanile residues; IEG is a hexapeptide with the following amino acid sequence: Arg-Arg-Asρ-Asp-His-Arg-NH 2 bound to one guanine residue. 2. A method for obtaining these antitumor agents from pig intestinal mucosa according to claim 1 which includes fractionation of waste intestinal mass, extraction with acidic acid, precipita¬ tion with ethyl alcohol, ultrafiltration in the range of 1 - 6 kDa lyophilization and supplementary gel-filtration. 3. Application of IEGi for suppression of the growth of ex¬ perimental Lewis lung carcinoma by the combined method "in vitro-in vivo" at a dose of 1050 μg/1 as well as for suppression of the flat cell carcinoma IC-Sofia-line 7 by the method "in vivo" -3 at a dose of 4x10 g/kg body weight. Experimental applica¬ tion of IEG as an inhibitor of the growth of cellular cultures from normal and malignant cell lines, and of IEGi and IEG 2 as inhibitors of the DNA biosynthesis in the intestines.

Inventors: BOSHEV NIKOLA ATANASOV, ROUSSEV GEORGE KONSTANTINOV, TRIFONOV BORISLAV BORISOV, BOSHEV, NIKOLA, ATANASOV, ROUSSEV, GEORGE, KONSTANTINOV, TRIFONOV, BORISLAV, BORISOV
Applicants: ALEXANDROV CHRISTO ALEXANDROV, BOSHEV NIKOLA ATANASOV, ROUSSEV GEORGE KONSTANTINOV, TRIFONOV BORISLAV BORISOV, ALEXANDROV, CHRISTO, ALEXANDROV, BOSHEV, NIKOLA, ATANASOV, ROUSSEV, GEORGE, KONSTANTINOV, TRIFONOV, BORISLAV, BORISOV
Application year: 1996
Main classification: C07K7
IPC codes: A61K38, C07K7, C07K14