CS276477B6 - A method for preparing an anti-tumor biopreparation cACPL - Google Patents

Abstract

The solution to the method of preparing the antitumor biopreparation cAGPL consists in sterilizing the surface of bird eggshells with ten to twelve-day-old embryos with UV radiation and interrupting their growth and life by paraffinizing the shells with liquid paraffin, or in an atmosphere of pure nitrogen at 37 °C for 10 days.

Description

The present invention relates to a cACPL (crude anticancer phospholipid) biopreparation, the active ingredient of which is 10-alkyl-2-acyl-sn-glycero-3-phospho- (N-acyl) -ethanolamine and whose antitumor activity is known and has been described, for example, in publication 3.Kárá et al., Neoplasma 33, 187-205, 19 & 3. The method of preparation of this biopreparation was described in Czech. author's certificate No. 252177 and in Czechoslovakia. patent 145770.

A method of killing ten-day-old chicken embryos using chloroform or other reagents as described in MS. patent 145770 or in Czechoslovakia. author's certificate No. 252177 proved unsuitable for the preparation of cACPL preparations intended for oral use, for the possibility of contamination of preparations with chloraformam or other harmful substances. This disadvantage is eliminated by a new method for the preparation of the antitumor biopreparation preparation cACPL from bird eggs with killed 10- to 12-day-old chicken embryos according to the invention. interrupting embryo growth and forming ischemic embryonic tissue producing 1-O-alkyl-2-acyl-sn-glycero-3-phosphorus- (N-acyl) -ethanolamine during subsequent incubation, or using an incubation of eggs in pure nitrogen at 37 ° C.

Example 1 _

Preparation of cACPL biopreparation by paraffin method:

(a) Chicken eggs containing 10-day-old embryos shall first be disinfected on the surface by irradiation under an ultraviolet germicidal lamp. Irradiation of eggs under UV lamps is performed on plates on which the eggs are placed in a thermostat (hatchery). Tubular UV lamps are placed approximately 1 meter above the eggs in a sterile box. The eggs are irradiated on one side for 30 minutes, then inverted 180 ° and irradiated again for 30 minutes on the other side. This method of disinfection is sufficient to remove microorganisms from the surface of the shells. This preventive disinfection is necessary to prevent losses due to bacterial contamination of the egg contents during further processing.

b) The disinfected eggs (irradiated under UV lamps) are immersed briefly (for a few seconds) in molten paraffin (temperature 50 ° C) and the paraffin on the surface of the shells is immediately cooled in air at room temperature. Paraffin closes the vents in the eggshells, leading to the death of the embryo under anaerobic conditions, the cessation of blood circulation in the embryonic tissue, and the formation of ischemic embryonic tissue in which the synthesis of N-acylathanolamine glycerophospholipids is induced in this way. Biologically active alkyl phospholipid PNAE ftj. plasmanyl- (N-acyl) ethanolamine) is formed by enzymatic transacylation of the precursor 1-O-alkyl-2-acyl-phosphatidylethanolamine, which is present in the egg yolk, during the incubation of paraffinized embryonic eggs.

c) The paraffined eggs are immediately placed in a thermostat (hatchery) at 37 ° C and incubated at this temperature for 24 hours. The paraffin is then removed from the egg surface by scraping and the rollers are further incubated at 37 ° C in a hatchery for 10 days.

d) After a 10-day incubation, the eggs placed in the paper fences on a tip on the upper half of the shells are disinfected with an iodine alcohol solution (Ifó solution) and the shell is then opened with sterile scissors and tweezers (circular cutting of the shell over an air bubble). Dead embryos are removed with a tweezers and stored in a freezer at -15 ° C (they can be used to extract the alkyl phospholipid PNAE.

e) The liquid content of the eggs is filtered through a double layer of sterile (autoclaved) gauze into sterile glass beakers (content 1 to 2 liters). The filtrate containing the predominantly liquid yolk is dispensed in 200 ml portions into sterile NTS bottles (500 ml contents), the bottles are closed with screw caps and the contents are frozen on the inner cylindrical surface of the bottle by rotation in an alcohol bath freezer at -50 ° C. The cologne, the bottles are opened, the neck is transferred with a nylon sieve (sterile), placed in a lyophilization drum of said apparatus and the contents of the bottle are freeze-dried (lyophilized).

CS 27S 477 SS

f) The lyophilized contents of the bottles are homogenized by grinding in sterile porcelain shorts in a sterile box (room irradiated with UV lamps), or homogenized in a sterile porcelain ball mill. The yellow lyophilized powder (cACPL preparation) is filled into sterile NTS bottles and sealed airtight with sterile rubber stoppers with screw caps. Bottles that are stored in a cold room and are protected from light, preferably in a closed cabinet. Under these storage conditions, the biological activity of the preparation (antitumor and radioprotective effect) is maintained for at least three years. This long-term expiration of cACPL preparations was verified by testing the biological activity of stored preparations in lots 1933 - 1988.

Nitrogen modification "preparation of cACPL;

Said cACPL preparation procedure can be modified as follows. that instead of paraffining (point b)), eggs with 10-day-old embryos are placed in a nitrogen atmosphere (airtight box filled with nitrogen from a cylinder) and irradiated at 37 ° C in nitrogen (ie anaerobically) for 10 days. The further processing of the eggs is the same as in the "paraffin method" (points d to f). '’

The cACPL preparation prepared by the paraffin method has antitumor activity as described previously (3. Kára _et al., NEOPLASIA, 33, 187-205, 1003). Evidence of this is also the results of experimental research / obtained on a number of experimental mouse tumors. The following types of tumors have been studied: Ca-755 mammary adenocarcinoma, r3m-5 cervical tumor, Mc11 fibrooarcoma, Crocker sarcoma 8-180 and sarcoma. 37 (sseit form). The preparation was administered orally to the animals in physiological saline as a homogeneous suspension. A wide dose range (from 10 mg to 1000 mg / kg) was used with multiple administrations of cACPL over 10-15 days. Oako treatment effect criteria were used: inhibition of tumor growth (in percent) and increase in survival of treated animals compared to control (in percentage).

The cACPL biopreparation was found to inhibit the growth of solid tumors Ca-755, RSH-5, Kc-11 by 73 2, 95% and 67 2 and prolong the survival time of s37-37 sarcomas by 56 °.

Pharmacokinetic studies in the oral administration of the cACPL raySim BDF biopreparation with McII tumor at a dose of 300 mg / kg revealed that the tumor contained fractions of N-acyl-phosphatidylethanolamine phospholipids which contained the antitumor component plasmenyl- (N-acylJ-ethanolamine). . ·

Repeated administration of the cACPL biopreparation results in the accumulation of phospholipids in the tumor tissue and blood of mice.

Pharmacokinetics PNAE-C in the blood of mice after jednorázové'perorální application is described with a mathematical model, pharmacokinetic half-life of a peak in the blood has a half-life of 1.3 for reducing the concentration of ^C-4-PNAE blood for 126 hours. Repeated intravenous appli. Accumulation of C-PNAE was observed in the tumor, liver, lung, spleen and brain of animals, and C-PNAE and its metabolites were not excreted by the kidneys.

The cACPL biopreparation is not toxic to normal tissues and has no adverse effects on blood formation. ·

Claims (1)

PATENT CLAIMS J * · Method for preparing an antitumor biopreparation of cACPL from bird eggs with ten to twelve-day-old embryos by interrupting their growth and life, characterized in that the surface of the eggshells of these eggs is first sterilized for 30 minutes by UV radiation and rotated by 160 ° C for another 30 minutes. , then the growth and life of the embryos are stopped by paraffinizing the shells with liquid paraffin at 50 ° C or in an atmosphere of pure nitrogen, incubating them under anaerobic conditions, or in an atmosphere of pure nitrogen at 37 ° C for 10 days. ‘