Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – 6 to 7 amino acid residues in defined sequence
Patent
1997-10-02
2000-01-18
Celsa, Bennett
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
6 to 7 amino acid residues in defined sequence
530344, 514 2, 514 8, 514 17, 514 21, 424550, 424551, A61K 3800, C07K 700
Patent
active
060158786
DESCRIPTION:
BRIEF SUMMARY
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.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.
BRIEF DESCRIPTION OF THE DRAWING
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.
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.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:
EXAMPLE 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
REFERENCES:
Copy of Search Report from PCT/BG96/00003 New Enterocytic Peptides with Morphogenic Effect, Folia Medica, 37(4A): 30 (1995).
Biological Effects of a Novel Intestinal Peptide-Inhibiting Enterocytogenin on Cultured 3T3 Mouse Fibroblast and L51784 Mouse Lymphoma Cells, Regulatory Peptides, 51(2): 111-119 (1994).
Altered Bioelectrical and Mechanical Activites of Rat Gastric Smooth Muscle Preparations by Inhibiting Enterocytogenin, 61(2): 119-123 (1996).
English Translation of BG 49927, Mar. 30, 1992.
Boshev Nikola Atanassov
Roussev Jeorge Konstantinov
Trifonov Borislav Borisov
Alexandrov, Christo Alexandrov
Celsa Bennett
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