# Patent

## Title
Peptide and peptide analog protease inhibitors

## 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.

## Background
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 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].

The objects of the present invention are:

1. To specify, under industrial conditions, the method for producing the two inhibiting enterocytogenins (IEG.sub.1 and IEG.sub.2).

2. To identify these enterocytogenins chemically.

3. To test in experimental animals the effect of IEG.sub.1 and IEG.sub.2 on the morphogenic and biosynthetic processes.

4. To test in vitro IEG.sub.1 in normal and malignant cellular cultures and study its effect on transplantable experimental tumors in vitro-in vivo and only in vivo.

FIG. 1. shows a flow chart for the isolation of IEG.sub.1 and IEG.sub.2.

FIG. 2a illustrates an HPLC-purification of IEG.sub.1.

FIG. 2b shows the amino acid content of IEG.sub.1.

FIG. 3 illustrates a gel filtration of IEG.sub.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.sub.1 in FIG. 2a. The conducted quality chemical and spectral analysis identifies IEG.sub.1 and IEG.sub.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.sup.3 -thymidine (DNA-synthesis) and C.sup.14 -uridine (RNA synthesis) in various regions of the intestines of the mice tested. The preparation'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.sub.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:

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.degree. 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.sub.2 O and purified additionally by gel-filtration on SEPHADEX G-25 (medium grade).

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.sub.1 was found to have molecular weight of 4450.+-.180 Da, and IEG.sub.2 -950.+-.120 Da. UV spectral analysis revealed two absorption maxima: for IEG.sub.1 at 220 nm and 248 nm and for IEG.sub.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.sub.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 (<1.5%, Table 1). IEG.sub.2 is a hexapeptide with the following amino acid sequence: Arg-Arg-Asp-Asp-His-Arg-NH.sub.2. SEQ. ID. NO. 1

Computer analysis of the amino acid content of IEG.sub.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 76.8 216.6 10  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  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 __________________________________________________________________________

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.sub.1 for 7 hours resulted in reducing the number of erythrocytes by 24% on the average, and with IEG.sub.2 --by 20%. Both IEGs have a suppressive effect on DNA synthesis especially in the lower regions of intestines: IEG.sub.1 by 45%, IEG.sub.2 --by 48%. In previous studies [4] we proved that IEG.sub.1 elevates the cytosol level of Ca.sup.2+  in smooth muscle cells by using the intracellular cellular Ca.sup.2+  depots. In this way it switches on the chain of molecular mechanisms by which it acts on the smooth muscle motility. __________________________________________________________________________ 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 with control animals not treated with IEGs) Section of the Incorporation DNA IEG 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 intestines IEG.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 __________________________________________________________________________

Object 4 (Table 3)

IEG.sub.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.sub.1 1050 .mu.g/ml). The observed pathomorphologic changes correlate with the dose-dependent effect of IEG.sub.1. IEG.sub.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.sub.1 in the range of 4.times.10.sup.-3 g/kg to 3.5.times.10.sup.-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 <0.01). __________________________________________________________________________ Effect of IEG.sub.1 on cellular cultures IC.sub.50 (.mu.g/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

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...6 NH2 groupnal  OTHER INFORMATION: -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: - Arg Arg Asp Asp His Arg 1               5 __________________________________________________________________________

## Claims

1. A method for obtaining a compound having antitumor activity from pig intestinal mucosa comprising: inhibitory enterocytogenin-2 (IEG.sub.2) peptide having an amino acid sequence of SEQ. ID. NO.:1 bound to one guanine residue.

2. An IEG2 compound having antitumor activity obtained according to claim 1, wherein the IEG2 compound is a hexapeptide having an amino acid sequence of SEQ. ID. NO:1 bound to one guanine residue.