Abstract:
This invention provides a method for isolating antitumor agents, inhibiting enterocytogenins, from pig intestinal mucosa. It further provides the antitumor agents, which are nucleopeptides. Additionally the invention provides methods for suppressing the growth of certain tumor cells comprising administering an inhibiting enterocytogenin to tumor cells.

Description:
BACKGROUND OF THE INVENTION 
     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. 
     BRIEF DESCRIPTION OF PRIOR ART 
     Our previous studies [1] have shown that cell differentiation 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]. 
     SUMMARY OF THE INVENTION 
     The objects of the present invention are: 
     1. To specify, under industrial conditions, the method for producing the two inhibiting enterocytogenins (IEG 1  and IEG 2 ). 
     2. To identify these enterocytogenins chemically. 
     3. To test in experimental animals the effect of IEG 1  and IEG 2  on the morphogenic and biosynthetic processes. 
     4. To test in vitro IEG 1  in normal and malignant cellular cultures and study its effect on transplantable experimental tumors in vitro-in vivo and only in vivo. 
    
    
     BRIEF DESCRIPTION OF THE DRAWING 
     FIG. 1. shows a flow chart for the isolation of IEG 1  and IEG 2 . 
     FIG. 2a illustrates an HPLC-purification of IEG 1 . 
     FIG. 2b shows the amino acid content of IEG 1 . 
     FIG. 3 illustrates a gel filtration of IEG 1  and the effect of various fractions on tumor growth. 
    
    
     The first object is achieved by providing a method for isolating inhibiting enterocytogenins from pig intestinal mucosa as described in FIG. 1. 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 as described for IEG 1  in FIG. 2a. The conducted quality chemical and spectral analysis identifies IEG 1  and IEG 2  as nucleopeptides; the amino acid content was determined by an amino acid analyser. 
     DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
     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 3  -thymidine (DNA-synthesis) and C 14  -uridine (RNA synthesis) in various regions of the intestines of the mice tested. The preparation&#39;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 IEG 1  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 casing cleaning wards of slaughter houses is collected and concentrated 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  O 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 silicagel SORBAX column using HPLC apparatus (FIG. 2a) IEG 1  was found to have molecular weight of 4450±180 Da, and IEG 2  -950±120 Da. UV spectral analysis revealed two absorption maxima: for IEG 1  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 IEG 1  contained glutamine (13.33%) followed by lysine (10.95%), serine, threonine, asparagine, glycine, alanine, valine, isoleucine, leucine, histidine, and arginine. Cyctine, methionine, tyrosine and phenylalanine were present probably in a mixed state (&lt;1.5%, Table 1). IEG 2  is a hexapeptide with the following amino acid sequence: Arg-Arg-Asp-Asp-His-Arg-NH 2 . SEQ. ID. NO. 1 
     Computer analysis of the amino acid content of IEG 1   
     
         __________________________________________________________________________No. NAME   RT  HEIGHT            AREA n mol                     %   ng  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                             216.610  Val 13.09       35429            562107                 1.989                     5.72                         233.1                             96.011  Met 14.32       3148 111300                 0.525                     1.51                         78.3                             163.712  Ile 16.61       10418            287121                 1.306                     3.76                         171.4                             99.113  Leu 17.73       20034            578395                 2.683                     7.72                         352.7                             100.714  Tyr 18.64       3282 58156                 0.447                     1.29                         81.1                             95.115  Phe 19.49       3879 68355                 0.308                     0.89                         50.9                             86.516      21.25       1137 12883                 0.012   0.0 0.017  Lys 21.84       77180            1189568                 3.805                     10.95                         556.3                             99.718      22.48       8631 174505                 0.174   0.0 0.019  NH3 23.17       38740            975264                 0.528   42.9                             101.020  His 24.13       13573            304124                 0.982                     2.82                         152.5                             105.121      25.62       1142 48214                 0.048   0.0 0.022  Arg 28.34       18289            512259                 2.077                     5.98                         361.8                             100.623  Pro               3.168                     9.12Total       483843            9690271                 35.604  4097.3__________________________________________________________________________ 
    
     EXAMPLE 1 
     Object 3 (Table 2) 
     Each fraction obtained in purifying IEG (see Object 1) was tested on mice. 3 mg of the peptide content of the fraction was administered intraperitoneally simultaneously with the testing reagent (isotope); the animals were then killed and investigated 7 hours afterwards. Treatment with IEG 1  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 especially in the lower regions of intestines: IEG 1  by 45%, IEG 2  --by 48%. In previous studies [4] we proved that IEG 1  elevates the cytosol level of Ca 2+   in smooth muscle cells by using the intracellular 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 IEG.sub.1 and IEG.sub.2 on the biosynthetic and mor-phologic processes in the intestines(7 hours after treatment of mice with IEF.sub.1 and IEG.sub.2)(The data are presented in percentages in comparison withcontrol animals not treated with IEGs)Section of the       Incorporation DNAIEG intestines       H.sup.3 -thymidine              C.sup.14 -uridine                     concentration                            Cellularity__________________________________________________________________________IEG.sub.1    Duodenum       74     75     66     82    Jejunum 94     67     84     95    Ileum   57     68     69     81    Middle section       52     62     58     76    of the    intestinesIEG.sub.2    Duodenum       77     78     68     88    Jejunum 88     78     97     75    Ileum   55     74     77     85    Middle section       48     74     62     83    of the    intestines__________________________________________________________________________ 
    
     EXAMPLE 1 
     Object 4 (Table 3) 
     IEG 1  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 inhibition index of tumour growth (IITG) of the subcutaneous form of LLCa is 88.2% (at a concentration of IEG 1  1050 μg/ml). The observed pathomorphologic changes correlate with the dose-dependent effect of IEG 1 . IEG 1  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 IEG 1  in the range of 4×10 -3  g/kg to 3.5×10 -4  g/kg. The inhibition of DNA synthesis in the tumour was -48%; the mitosis were reduced to 21.9% in comparison with the same parameters in the tumours of non-treated animals (P &lt;0.01). 
     
                                           TABLE 3__________________________________________________________________________Effect of IEG.sub.1 on cellular cultures                        IC.sub.50 (μg/ml)Cell line         Origin     NR-test                             KBP-test__________________________________________________________________________NORMAL CELL LINES3T3 fibroblasts   mice       123.9                             187.5FL  amnionic*     human      223.2                             264.4BHK kidney*       golden Syrian hamster                        &gt;300 &gt;300CHO ovarium*      hamster    213.2                             256.4MDC kidney        dogs       192.6                             247.4Vero    kidney        African green monkey                        156.7                             197.3MALIGNANT CELL LINESL 5178    lymphoma      mice       192.8                             212.6Hep 2    carcinoma of the larynx             human      290.4                             300.0Hela    epitheloid carcinoma in the             human      291.5                             268.5    uterusRD  embryonal rabdomyosarcoma             human      196.15                             276.3Sp 2    myeloma**     mice       206.2                             226.2Ag8 myeloma**     mice       1998.9                             234.8METH    myeloma**     mice       212.3                             246.5__________________________________________________________________________ **suspension cell lines *continuous lines 
    
     References 
     1. Roussev GK. Programmed manipulation of embryonic development. 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. Invention patent 37396 MPK-A 61K35/38, Vol 6, Jun. 14, 1985, 1-6. 
     3. 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 (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. 
     
         __________________________________________________________________________#             SEQUENCE LISTING- (1) GENERAL INFORMATION:-    (iii) NUMBER OF SEQUENCES: 1- (2) INFORMATION FOR SEQ ID NO:1:-      (i) SEQUENCE CHARACTERISTICS:#acids    (A) LENGTH: 6 amino     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear-     (ii) MOLECULE TYPE: peptide-     (ix) FEATURE:     (A) NAME/KEY: Other     (B) LOCATION: 6...6NH2 groupnal  OTHER INFORMATION:-     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:- Arg Arg Asp Asp His Arg 1               5__________________________________________________________________________