CZ83195A3

CZ83195A3 - Process for preparing a preparation based on egg 1-o-alkyl-2-acyl-phosphatidylaminoethanols exhibiting dietic-preventive and threapeutical activity

Info

Publication number: CZ83195A3
Application number: CZ95831A
Authority: CZ
Inventors: Jindrich Rndr Drsc Kara; Vlastimil Rndr Csc Liebl; Alois Doc Ing Drsc Novacek; Ludmila Rndr Phmr Bejsovcova
Original assignee: Kara Jindrich Drsc; Liebl Vlastimil Csc; Novacek Alois; Bejsovcova Ludmila Phmr
Priority date: 1995-04-03
Filing date: 1995-04-03
Publication date: 1996-10-16
Also published as: CZ282139B6

Abstract

This method of producing a preparation with a dietetic-preventive and therapeutic effect based on egg 1-0-alkyl-2-acyl-phosphatidyl ethanolamines which consists of N-acylation of separated egg phosphatidylethanolamine phospholipids individually or mixed with phosphatidylenoline by a palmitic acid in the presence of carbonyldiimidazole and pyridine in a toluene solution at a temperature of 40 to 45 degrees C while producing N-acylated phosphatidylethanolamines which contain 1-0-octadecyl-2-oleolyl-SN-glycero-3-phospho-(N-palmitoyl)ethanolamine which has selective anti-tumour effects. N-butanol can be preferably used for extracting egg phospholipids from the yolks and after concentrating the extract in a vacuum at 45 degrees C phospholipids are precipitated in an excess of acetone at -5 degrees C. The preparation is applied perorally and is quite non-toxic for the organism even during long-term daily administration as a dietetic preparation.

Description

~ é r. C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C ^ ^ výroby výroby výroby výroby výroby. _. " l.! " IlUfcl — 1.-. " i i lil ifc. and the therapeutic effect of and the base of the egg-1-ω-2-acv-1-phenylene-1-tert-butylamine compound; ťV-831 95 fržihlérser.c: 3 «—4 ^ —ί-99 > - Cfclast Techniques: 73: < “I - .Ό V - Ξ G < τΞ > O = < Lt < - ^ 3 o __ o < o w > BACKGROUND OF THE INVENTION The present invention relates to a process for the production of a novel preparation with a diethetic-preventive and therapeutic effect based on natural ether-phospholipids.

[alkyl-] phospholipids.

The preparation was isolated from the biological material (seed eggs) in which the active ether-phospholipid, 1-alkyl-1-aoyl-phosphutide 1 s * d is present. and η c 1 ami n.

A semisynthetic method has been developed which employs the isolation of isolated egg phospholipids with quaternary palmitate in the presence of carbocryldiimidazole to give a new ether-phospholipplide. ionad-o-o-ocr-iodine-m-Ir-kk-ad-ekyd-or-i-o1e cyi-s n-glye e rc- 3-phthalic-N-almi-thyloxy), in general, general plasma- (β-3-yl) ethanolamm (ΡΓ'λΒ), which is not toxic to the normal, physiological cells of the human body and selectively the cell membrane of the tumor cells. Bar and cell, patent no.

Ether-phospholipids. they have been the subject of intensive study in the last ten years, especially with the discovery of their antitumor effect In plant sources, alkyl phospholipids have not been detected, except that they are. animal and plant phospholizides source of saturated and unsaturated fatty acids and phosphorus. Phospholipids are one of the foundations of the organism. They are therefore a supplement to rational nutrition. Background Art:

Fractional and ranioprotective effects: viral ether-lipids have been discovered in 1953 by 3rohult, CLcl.d., lol, 1484, 1998). it has been found that alkylglycerols contained in the liver oil of the Greenland shark! It has a clinically proven protective effect against leukopenia caused by self-inflammation and inhibits the growth of experimental tumors (Hallgren B.: ect Lipide, Biochetrioioal et al. Eiomedical Aspecte (tfangcld KK, Paltauf F., ed.) p.261- 275) Academic Press, No * York 1983 *

Further connections between tumor biology and ether-lipids have been reported by F.Snyders and co-authored in the US by the discovery of high concentrations of ether-phospholipids in membranes. tumor cells and the absence of enzyme in tumor cells, cleaving the ether linkage in an ether-ffolipid (O-alkylglycerol-monooxygenase) molecule (Socdsma JF, Fiar.tadcsi C., Snyder F., Cancer Res., 3C, 309-311, 1970; Snyder F. Ether Lipids Chemistry and Biology, Academic Press, New York 1972). In 1967, the synthesis of 4-alkyl-lysyl-lecithin analogue was realized (Arnold D, Vtzen KU, Westphal 0, Liebigs Ann. Chem., 709, 234, 1967). in the synthetic lysophosphatidyl-choline analogue, 1-O-octadecyl-2-Hiethoxy-rac-glycero-3-phosphocholine (preparation of ET-IS-CCH 2), the presence of 1-O-alkyl ether group in the phospholipid molecule was shown to work is a prerequisite for the antitumor effect of ether phospholipid (Eerdel WE, Elut, 44, 71-73, 1932; Eerdel W * E, Eausert WRE, Fink U, Rastetter J. and Munder PG, Anticar. 1, 3, 45-352, 1981; Eerdel WE, Ar.dreesen R, f, f PG, In Phospr.clipids and Cellul Regulatic vol.II, et 41-73, (edit.JFKuo), CEC Press.Inc Florida 1985 Other synthetic derivatives of alkyl-lysophosphatidylcholine with antitumor activity were presented on the First International Symposium " Ether Lipids in Oncclc ", held in Gottingen (Germany) in 1936 (Lipids, 22, (No.11), p737-924, 1987). In particular, a synthetic thioether derivative of choline phosphatyl-1, BM 41.440 (iodine), ie 1-hexadecylmercapto-2-methoxymethylalkyl-rac-glycro-3-phosphocholine, is particularly useful. Feroral administration of this thioether phospholipid to patients for 12-15 weeks resulted in the removal of tumor metastases from the liver in six patients (Hermann DBJ et al., Lipids 22,962-966, 1967; Hermann DEJ et al., Eeitrage zur Gnkologie, vol. 37 (Eckhardt S. 'Holzner JH, Nagel Ga, editors), p. 224, Karger, Easel 1989) ·

This very interesting field of application of ether-phospholipid has hitherto been limited to synthetic analogues of alkyl-lysophosphatidyl cboline. Recently, however, they have published results suggesting that natural ether phospholipids derived from 10-alkyl-2-acyl-phenylethylethanolamine or plasoanyl-CE-acylJethanolacine (PNAE isolated from both avian and mammalian ischer tissues have very selective and significant antitumoral properties) have also been reported. and are non-toxic to normal physiological cells (Kara J., Borovicka M., Liebl V., · / ·

Smolíková. J. (Jbik K., Neopiasrna, 33, 187-205, 1936; Kára J., Liebl V., Pelztauer, Z .: HigMights of Modem Eiocheaistry Proceedings of the I4th International Cycle of Liochemistry, Prague, Czechia, 10-15 Ody 1988, Vel.2, pp. 1459-1474, 1989. 'Non-toxic ether phospholipid metal with selective protease activity, fungal- (Γ-acyl) ethanamine (iλ £), was isola from chicken ischemic tissue embryos from anti-cancer biopreparation cACPLCcrude anricancer phospholipids), This lyophilized, yellow powder prepared from egg yolks containing ischenic chicken embryos, Bioprepurét cACPL was produced in 1988-1990 for clinical trials (manufacturer of EIOIKTER as, Prague) according to our patent (J. Kár et al., 5s * patent no. 276477, 1939). At. long-term oral administration of cACPL (2 grams of cACFL 3 times daily for at least 2 months) significantly improved the clinical condition in animals with tumor metastasis. in the liver, at the same time with the analgesic effect, so that patients did not experience pain, it was possible to stop the daily doses of morphine.

The first preparations of fertilized hen eggs (called " Cancero-lyt ") were empirically prepared by K. Borovicka and tested on experimental tumors as early as 1967. However, the chemical character of the anti-contraceptive component contained in the bio-preparation was not known at that time, we have launched another, research of antitumor effects " of this biopreparate. The result was the discovery of a new ether-phospholipid, which was identified as 1-O-octadecyl-2-octyl-sn-glycero-3-phospho (K-palmityl) ethanolanine. This novel ether-phospholipid acts selectively cytolytically on human tumor cells, y tissue cultures, and causes " destruction of the cell membranes of tumor cells as expressed by experimental evidence using scanning electron microscopy (Liebl V., Pelztauer Z .: Highlight by Plod Eiocheiristry, Voi * 2,1459-1474, 1939). The permeability of normal, physiological cell membranes was not altered under the same conditions.

The chemical structure of the ether-phospholipid FKAE was confirmed by eemisynthesis (Kára J., Liebl V., Ledr: V. V., Dsjšovcová L., Čs.patent 275396, (1937), and by German authors who used ether-f for partial synthesis. osf olipid PJAAE is a biotechnological method of metabolizing synthetic alkyl ethers of glycerol in plant cell tissue cultures (e.g., S.3., Weber K., Mangold H * K., FEbS Letters 26: 2, 104-106, 1990).

In the experimental study, an important first finding was that the in vivo antitumor activity of Pih-iL in experimental tumor models is increased by calot ion. Humoricidal effect of FLaSis) applied intraperitoneally to C37B1 / 10 with sarcoma, is significantly enhanced by the simultaneous oral administration of caicous gluconate in rye water (Kára J.Koňova- / * lova AaL., Krasnova p. Liebl 7, Bejsova La, Neoplasma 4G, 113-217, 1993).

Summary of the Invention:

In order to increase the yield of antitumor-ether-fciipid PNA3 (s), we have developed a new semisynthetic method which is the subject of the invention by Uara J., Liebl. 7., Dedkova 7, Lejsova Lt Ss, patent 275396, 1987). The starting material is phospholipids, which are made from egg yolk of normal, hen eggs, from which the precursor, 1-O-alkyl-2-acyl-phosphatidylethanolamir was isolated. and by chemically N-acylating with palmitic acid using organic catalysts, the desired senisynthetic ether-phosphoric acid (s) was isolated with good yield. λ2 (s) ·

Chemical characteristics (infrared, chromatographic R, in various solvent systems) as well as antitumor efficacy of experimental mouse tumors as well as tissues of new human tumor cells. The properties of ether phospholipiae PRaSCs) with properties of natural ether phospholipid P1, which we previously isolated and published (J. Kara et al. , Necplasna, 3, 187-205, 1986),

Based on our experimental study of pharmacokinetics and ir.etabo.il smu. ether-phospholipid plasmanyl- (Rx ^ -cyalmitoyl) ethanolamine is mainly accumulated in the liver of the mouse at repeated intravenous doses (Kara J., 2imakova I, Ph, Sersbrjakova Α.A., Dedkova 7 , Zolotar et al., J.Cancer Rea. And Clir. Cr.cology, 120, 662-667 ', 1994), the use of semisyretic ether-foafolipid rhizis can be indicated to prevent tumor metastasis in the liver in patients operated on by colorectal carcinomas. Non-toxicity, selective antitumor effect, and ether-phospholipid farxakokinetics P1 and E (s) for this peroralpine prophylactic use. ,. C. -J Γ yy products in the clinic ideal. Prevention of human rays is one of the main tasks of ankolo.Aie.

In comparison with the bicarbonate of chCPL prepared according to our patent no. 276477 from fertilized hen eggs, the first preparation has the advantage of: (1) providing an ethereal etheric ether; it is siardurdic and defined and is of the order of magnitude higher than the content of rihi2 natural ether-phospholipid in the cACFL phospho-preparation. 2 / Prepared not contaminated by bacteria. In this technology, the hen's eggs do not leach at 37cC at all and therefore there is no risk of proliferation of bacterial microspheres. Dried egg yolks,. which; For example, when the perordline form of the formulation is used, it is descended / sterilized by pasteurisation. The effectiveness of the anti-cancer agent, the purity and aging of the oral preparation indicate its dietetic-preventive use in the prevention of tumor metastases to the liver, especially in patients p <*; colorectal cancer. This non-toxic product. - dc-m allows its systemic action in the body to prevent the emergence of human tumors in other organs (lungs, rzozekj nodes, urinary bladder). The preparation can also be used in combination with immunotherapy and chemotherapy.

Applicability of the Invention for Production;

The invention can be relatively easily implemented in industrial production. The whole technological process is simple, waste-free and hygienically safe from the point of view of hygiene, it is clear from the example, which does not limit the method according to the invention. Example preparation preparation; 1, Extraction of total lipids. from frozen egg yolks of hens (eg product of EGMCC as, Benešov u Prahy) can be advantageously carried out using n-butanol. This solvent is more suitable for manufacturing purposes than for the conventional chloroform / methanol mixture (Fclcha) for hygienic and environmental reasons. The precipitated vials are filtered off and the phospholipids are separated by 0 for neutral lipids after de-distillation of n-butar.clu in a rotary vacuum evaporator at 45 ° C to 50 ° C) by aspiration of phospholipids in excess of acetone.

On temperature -1 ° WC to +5µ · 2. For separation and isolation of phospholipids PS (fc & fatidylethanolaraine) and PC (phosphatidylchclir) it is advantageous to use a chromatographic separation on a layer of silica gel (in preparative nucleation) by washing. n-butanol with an increasing gradient of H., C in butanol (c 5 H, C 1 H 2 O to 1 C H 2 O in n-butanpyl). From the silica gel layer, first, the neutral lipid residues are eluted with a trace of cholesterol n-butanolec, then the phosphatidylethanclamines (?) In the gradient of 56 [mu] O to 11%. , C in r-zut anol and fostalide, which is more strongly bound by silica gel, " a " In this case, the tc1c1r1 * 1 · 1i1i1i1 in 9-1: • in) tanlu to this 31i1i p Λv4. ^ - η r 'G u 2 2 ^ rologicky e yenerac i Γ- - ZU * živ-'. Ϋ ί Vajec.n. ý lezithin v 'h ούη'ίτ in V / p,' p ^ '2 C. 1' i * Γ- ''cited;'j' gt > jw " % > (c) .beta.-alkyl-.beta.-acyl-.alpha., .alpha.-acyl-.alpha.-acyl-Fi, and 1-, 2-di-acyl-FF, .alpha. acid; palmitium in the presence of pyridine and in a toluene solution at 50 ° C for 15 hours. The M-acylae foatidletlethancl < a """ is run under the above conditions with a yield of 95% to 100% of the thiocarboxylic acid product. after evaporation of the toluene and pyridine in a vacuum evaporator, the introduction of: - the evaporator liquid end point in dc; rivers of evil = read at -5 ^ d pc parts of constant agitation. auctioned K-: cyl-? f f; sť: ii_ id.y se. o let i Γ * 1 r- ^ 1. 'Uv * V - s' r O, λ ~ 1 ^ - - " it Τ' r < 7 - o. v ace -cr.u at t ° rxcte - ι, ρ u 1.::.r are isolated by centrifugation with some filtration at a lower temp: t-5 ° 1) and the evaporation is canceled. of those tkl acetone in a vacuum rotary cdz determine. se nos nos nos nos nos nos nos nos nos nos nos nos nos nos nos nos nos nos nos nos c c c c c c c c c c c c c qureli7- " * ^ af oř clvbd e rové vet yp Ikohclu (diet ckrcmate ,, ramu] - * 0 u * rq ^ S TL 4 £ 1 Γ ^ t α ^ V Λ V Γ S t · 7 4? i ^ i > r 0 o λ ^ p > q? C 1. ^ N '''wL u / nizsc: eror 1 nif cr - -?' L · 'p reyl-ΡΞ n c. 2 U. V id 1 -i Λ te te U te £ d 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 mu mu mu mu mu mu mu mu mu mu áním áním áním : iv- ri var e irymi i_o-tky: ster i_u-i spravevamu dc upper air) r: hu jzvs; e f,, ^ V 1; "In C 4" by heat: vb 0 1: m: m: i - diisulfonate and python - n - butanol n - butanol, and soda - free with acetone Ink mentioned in bel. .) u leads; i in bed4 3 í .4 " 1 V - - ~ L »1 * κ. i ~ zl CJ J- - 1 " - from olefin after evaporation of toluene and cyridir. by precipitation in excess of ace at r-0. Example 1) The obtained N-acyl-β and phosphoric acid suspension can be purified in the presence of a peroral preparation and suspended in an oil with yelow. ultrasonic water bath. : foliridd in plant

Claims

; n 'j o v o r.srok v .ser vyrccy ;; " dietotleuo-p. j uh e tiier-fcsfclipiJi v, tag;.,; the following: tyl -f i_ravr: r. a novel ether phosphate which, in particular, is a minor inferior scholar and a tulle character is characterized by the addition of -S-oieoyl-sn-lycerol-j-phospho- (1-palladium) ethers. .olmir, ct pl and many i - (N - a cy 1) and h - nc 1 anin, from: rakurseru 1 -ok ^ .: scy 1 - 2- cleoy 1- 1 -rn-ylycero-1 In this case, the use of egg yolk is not affected by the presence of diisocyanates in the form of yarns in the yeast. Sa saiir.C '£ c7a oř oř st st .. .. .... * * * * * * / Ar ar ar ar ar ar ar ar ar , í = and-ví né né uj uj uj uj uj uj uj uj uj uj uj uj. ή ή W W W W W na na na na na ce vys vys vys vys vys vys vys from -15 ° C dc · + -5 ° 0;

2. A method according to claim 1, characterized in that I-octyl-1-cylic acid is a yellow egg yolk of a non-valuable nature: the egg is glued together, and the egg yolk is then mixed. ', 1. q - i ^ λ; '' I r · o xi - τ ^, / 'i - - i ^ li J' - * 1 .. - -7 .. --1. 'Jlxťi. . . ΓΓ J.. . L. ... ^ ^ -L „^ C onu - ώ j. - j * 'extrj.:v1 -1 V-Λ • doo = t > co + cr routa oo ^ r dn - n azc id 11 cr.j oh: h.: ncra í ^ ucne civilu foezatidvle below izc-uji preparativn ^ Ί · 'r- V' - 1 i 'water Íiradiert cd χί HnC The term " H " is used to denote the term " - 1 " it is re-active. and pouring: latices, i.e., 1-C-octadecyl-t-cycloyl-ss-glycerol; nlepic.cn: at 1: 1 or zcurcb genus, for the peronal, knife 4, it is characterized in that it re-prepares the drug F:. injection or suppository. u 1 you from η. and by re-preparing Fern'i.i.2 (n) in f οηζ. I want a limit on phosphate and phosphate. is not isolated by f isi dyl-, s re acylu.je is: rome kyre l in.su palm.it ovcu for -et'i ^ nolaizine, (obrazeni v ('í-pal.T.itop 1-fcsfaticyletrn ^ ciarninovd The fraction of fecalipids is converted by DC to the final l-phrv, which is also targeted by the malfunctioning yeasts from the normal ileum and also by the skin dermal tumors.