V2?atky z odborn? publikace RNDr.Jind°icha K?ry DrSc. "ether fosfolipid PNAE proti n?dorov2m bu?k?m: Prevence a terapie metast?z", vydan? firmou AREKO v kv?tnu 2000 ku p°?le?itosti mezin?rodn?ho onkologick?ho sympozia, konan?ho ve dnech 5. - 6.kv?tna 2000 v Praze - Pr?honic?ch pod z??titou Ligy proti rakovin? Praha.

2.1. Ether-fosfolipidy s protin?dorov2m ·?inkem inhibuj? aktivitu protein kin?zy C a indukuj? selektivn? destrukci membr?n n?dorov2ch bun?k.

Protin?dorov? ·?inky ether-fosfolipid? byly pops?ny v °ad? publikac? (21-35). Nejv?ce byl studov?n syntetick2 analog lysofosfatidylcholinu (lyso-lecithinu), 1-0-oktadecyl-2-methoxy-glycero-3-fosfocholin (ET-18-OCH3) (21-28). Tento prepar?t selektivn? ni?? n?dorov? bu?ky a jeho ·?inek se projevuje destrukc? bun??n2ch membr?n n?dorov2ch bun?k, jak bylo prok?z?no elektronovou mikroskopi? (24,25,26). Prepar?t ET-18-OCH3 (Edelfosfin) zvy?uje rovn?? imunologickou obranu organismu, aktivuje protin?dorovou aktivitu makrof?g a m? v2znamn2 ·?inek proti n?dorov2m metast?z?m v experiment?ln?ch modelech (21-24,48). Podobn? protin?dorov? ·?inky m? tak? dal?? syntetick2 analog lysolecithinu, thioether 1-hexadecylmercapto-2-methoxy-methyl-rac-glycero-3-fosfocholin, (BM 41,440, Ilmofosin) (28). Byla p°ipravena i dal?? syntetick? analoga fosfatidylcholinu s protin?dorov2m ·?inkem (27,32). Klinick? zkou?ky prok?zaly therapeutick2 ·?inek t?chto prepar?t? (27,28,50), ale tak? vedlej?? toxick? ·?inky na organismus, omezuj?c? jejich klinick? vyu?it?.

Objev nov?ho alkyl-fosfolipidu se selektivn?m protin?dorov2m ·?inkem (29), 1-0-alkyl-2-acyl-sn-glycero-3-fosfo-(N-acyl)-ethanolaminu (PNAE), kter2 je p°?rodn?m deriv?tem fosfatidylethanol-aminu a nem? ??dn? vedlej?? toxick? ·?inky na organismus p°i peror?ln?m a tak? parenter?ln?m pod?n? (29-35), otev?r? novou perspektivu klinick?ho vyu?it? tohoto netoxick?ho ether-fosfolipidu, zvl??t? v prevenci metast?z lidsk2ch n?dor? do r?zn2ch org?n?, obzvl??t? metast?z do jater (33,35). Na rozd?l od syntetick2ch prepar?t? (ET-18-OCH3, BM 41,440), kter? p°edstavuj? sm?s racemick2ch stereoisomer?, m? prepar?t PNAE (i PNAE(s)) p°?rodn? stereo-konfiguraci a neobsahuje ve struktu°e sv? molekuly ??dn? nep°irozen? skupiny. Proto PNAE a jeho metabolity nejsou toxick? pro organismus a PNAE je proto mimo°?dn? vhodn2 pro dlouhodobou peror?ln? aplikaci a prevenci lidsk2ch n?dorov2ch metast?z (33, 35). V2voj a p°?prava semisyntetick?ho ether-fosfolipidu PNAE(s) byly pops?ny (30, 34, 35), protin?dorov2 ·?inek PNAE(s) in vitro a in vivo (30,31) farmakokinetika a metabolismus in vivo (33) byly publikov?ny (viz d?le v ??sti 5).

2.2. Indukce apoptozy v n?dorov2ch bu?k?ch p?soben?m ether-fosfolipid?
Apoptoza je forma bun??n? smrti charakterizovan? morfologick2mi a biochemick2mi zm?nami, jako nap°. kondenzace chromatinu, fragmentace DNA zp?soben? aktivac? endonukle?zy (57,58,69). Ether-fosfolipidy indukuj? apoptozu pouze v n?dorov2ch bu?k?ch, nikoliv v?ak v norm?ln?ch fyziologick2ch bu?k?ch (58). V bu?ce existuj? regula?n? geny, jejich? exprese m??e zabr?nit apoptick? smrti bu?ky (nap°. mitochondri?ln? protein Bc1-2 (58, 71, 72). Pravd?podobn? v?echny protin?dorov? ether-fosfolipidy indukuj? apoptozu v n?dorov2ch bu?k?ch. M. Modolell a spolupracovn?ci (58) na z?klad? sv2ch experiment? uzav?raj?, ?e indukce apoptozy ether-fosfolipidem ET-18-OCH3 zahrnuje t°i hlavn? stupn?:
a) vazbu ether-fosfolipidu na povrchu bun??n? membr?ny;
b) inkorporace ether-fosfolipidu do bu?ky;
c) spu?t?n? signaliza?n?ho mechanismu pro apoptozu, kter2 m??e b2t zru?en zv2?enou koncentrac? b?lkoviny Bc1-2. Jde tedy o parametry, kter? mohou b2t r?zn? v r?zn2ch typech n?dorov2ch bun?k. To vysv?tluje tak? r?znou citlivost n?dorov2ch bun?k k cytotoxick?mu ·?inku ether-fosfolipid?.

5.1. P°?rodn? netoxick2 ether-fosfolipid PNAE (plasmanyl-(N-acyl)-ethanolamin), jeho chemick? struktura, protin?dorov2 ·?inek in vitro a in vivo a jeho mechanismus ·?inku.
Nov2 fosfolipid byl identifikov?n jako 1-0-alkyl-2-acyl-sn-glycero-3-fosfo-(N-acyl)-ethanol-amin, neboli plasmanyl-(N-acyl)-ethanolamin (PNAE) (29).

Hlavn? molekul?rn? species PNAE m? strukturu 1-0-oktadecyl-2-oleoyl-sn-glycero-3-fosfo-(N-palmitoyl)-ethanolamin (29,30).

R-oktadecyl; R+-CO-oleoyl; R++-CO-palmitoyl

N?mi navr?enou strukturu PNAE potvrdili tak? prof. Dr. H. K. Mangold a spolupracovn?ci (103), kte°? p°ipravili prepar?ty PNAE s pou?it?m syntetick2ch alkyl-ether? glycerolu a biotechnologie tk??ov2ch kultur rostlinn2ch bun?k. Touto stereospecifickou semisynt?zou p°ipravili deriv?ty s r?znou d?lkou 1-0-alkylov?ho °et?zce (1-0-tetradecyl, 1-0-hexadecyl a 1-0-(Z)-9-octadecenyl)-PNAE, kter? maj? podobnou protin?dorovou aktivitu jako n?? semisyntetick2 alkyl-fosfolipid PNAE(s) (103). Tyto v2sledky potvrzuj? spr?vnost chemick? struktury PNAE (29,30).

Protin?dorov? ·?innost ?ist?ho alkyl-fosfolipidu PNAE byla testov?na v tk??ov2ch kultur?ch lidsk2ch n?dorov2ch bun?k (linie Hep-2 a T24) kvantitativn?m m?°en?m inkorporace radioaktivn?ho thymidinu-3H do bun??n? DNA p°i stoupaj?c?ch mikrogramov2ch koncentrac?ch PNAE v 1 ml media kultury (29).

Alkyl-fosfolipid PNAE p?sob? selektivn? cytolyticky na lidsk? n?dorov? bu?ky linie Hep-2 a T24 (lidsk2 n?dor mo?ov?ho m?ch2°e) v tk??ov2ch kultur?ch p°i koncentraci 50 mikrogram? PNAE/ml a inhibuje proliferaci t?chto n?dorov2ch bun?k ji? p°i koncentraci 2 mikrogramy PNAE/ml, av?ak neinhibuje biosynt?zu DNA v norm?ln?ch bu?k?ch p°i 50ti n?sobn? v?t?? koncentraci v tk??ov? kultu°e lidsk2ch fibroblast? (29,30) (obr.2). Tento obdivuhodn? selektivn? ·?inek PNAE pouze proti n?dorov2m bu?k?m indikuje vynikaj?c? therapeutick2 index PNAE a jeho netoxi?nost pro organismus a tak? r?st bun?k lidsk?ho karcinomu rekta resistentn?ch k fluorouracilu na bezthymov2ch hol2ch my??ch byl signifikantn? potla?en p°i s.c. d?vk?ch 4,5 mg PNAE/my? po dobu 21 dn? (P.Pou?kov? a sp.-100).

Podobn? jako syntetick? alkyl-fosfolipidy, analoga lysofosfatidylcholinu (24-26), alkyl-fosfolipid PNAE zp?sobuje selektivn? destrukci bun??n2ch membr?n lidsk2ch n?dorov2ch bun?k (30,35). Ve spolupr?ci s Dr.Z. Pelcbauerem (+stav makromolekul?rn? chemie +SAV, Praha), byl s pou?it?m rastrovac? elektronov? mikroskopie prok?z?n destruk?n? ·?inek alkyl-fosfolipidu PNAE na bun??n? membr?ny lidsk2ch n?dorov2ch bun?k Hep-2 za 24 - 48 hodin inkubace t?chto bun?k v tk??ov2ch kultur?ch v p°?tomnosti 50 mg PNAE/ml (30,35).

Bu?ky HEp-2 a LEP byly inkubov?ny v kultiva?n?ch mikromisk?ch v m?diu obsahuj?c?m uveden? koncentrace alkyl-fosfolipidu PNAE po dobu 48 hodin. Po odstran?n? m?dia s PNAE byla m?°ena inkorporace thymidinu-6-3H do DNK bun?k HEp-2 a bun?k LEP. Proliferace n?dorov2ch bun?k HEp-2 byla zastavena p°i koncentraci PNAE 12,5 mg/ml, zat?mco r?st norm?ln?ch fibroblast? LEP nebyl zastaven alkyl-fosfolipidem PNAE ani p°i koncentraci 10? vy??? (125 mg/ml).

1 inkorporace thymidinu-6-3H do DNK n?dorov2ch bun?k HEp-2.

2 inkorporace thymidinu-6-3H do DNK fibroblast? LEP.

Selektivn? p?soben? alkyl-fosfolipidu PNAE na membr?ny n?dorov2ch bun?k bylo potvrzeno tak? Dr. A. Kotykem (Fysiologick2 ·stav +SAV, Praha), kter2 se spolupracovn?ky experiment?ln? potvrdil zv2?enou permeabilitu bun??n2ch membr?n n?dorov2ch bun?k vystaven2ch ·?inku alkyl-fosfolipidu PNAE. Difuze 2-deoxy-D-glukozy zna?en? radioisotopem 3H membr?nami t?chto bun?k se zna?n? zv2?ila ve srovn?n? s kontroln?mi n?dorov2mi bu?kami, kter? nebyly inkubov?ny v p°?tomnosti PNAE. Permeabilita bun??n2ch membr?n norm?ln?ch fyziologick2ch bun?k nebyla za stejn2ch podm?nek inkubace s PNAE zm?n?na (101,102).

V2znamnou roli v protin?dorov? a antimetast?zov? ·?innosti PNAE m??e hr?t tak? inhibi?n? ·?inek PNAE na enzymovou aktivitu protein kin?zy C, kter2 byl experiment?ln? prok?z?n (39). Vzhledem k v2znamu protein kin?zy C pro metastatickou kapacitu n?dorov2ch bun?k (11,18,19), m??e m?t dlouhodob? syst?mov? p?soben? alkyl-fosfolipidu PNAE na protein kin?zu C n?dorov2ch bun?k in vivo v2znamn2 preventivn? ·?inek proti disseminaci n?dorov2ch bun?k v organismu a proti vzniku metast?z zvl??t? v j?trech, kde se PNAE p°i opakovan? aplikaci nejv?ce kumuluje (33).

5.3. Nov? pr?myslov? technologie v2roby prepar?tu OVOSAN obsahuj?c?ho PNAE.

V posledn? dob? byla vypracov?na nov? technologie pr?myslov? v2roby prepar?tu OVOSAN, kter2 obsahuje vysok? procento ether-fosfolipid? PNAE. Prepar?t OVOSAN ve form? ?elatinov2ch kapsl? obsahuje suspenzi vaje?n2ch fosfolipid? s 30% PNAE a 60% vaje?n?ho lecithinu (fosfatidylcholinu) v rostlinn?m oleji.

5.4. Mo?nost prevence metast?z do jater u pacient? po operaci kolorekt?ln?ho adenokarcinomu dlouhodobou peror?ln? aplikac? prepar?tu obsahuj?c?ho PNAE.

Na?e koncepce prevence metast?z dlouhodob2m pod?v?n?m netoxick?ho alkyl-
-fosfolipidu PNAE se p°ibli?uje strategii prevence metast?z americk2m prepar?tem carboxyamidotriazolem (CAI) navrhovan? E.C. Kohnovou a L.A. Loittou (95). V2hodou PNAE a na?eho ?esk?ho prepar?tu obsahuj?c?ho PNAE ve srovn?n? s CAI je naprost? absence vedlej??ch toxick2ch ·?ink?, kter? naopak se objevuj? u pacient? p°i dlouhodob?m peror?ln?m pod?v?n? syntetick?ho prepar?tu CAI (94). Vysok? selektivita tumoricidn?ho ·?inku PNAE je rovn?? p°ednost? tohoto p°?rodn?ho alkyl-fosfolipidu.

Prevence metast?z nepochybn? m??e d?t pacient?m daleko lep?? perspektivu p°e?it?, ne?li jak?koliv terapie ji? existuj?c?ch metast?z v j?trech. Je l?pe p°edch?zet, ne?li l??it.

Literatura - reference:

11. BLOBE G.C., OBEID L.M., HANNUM Y.A.: Regulation of protein kinase C and role in cancer biology. Cancer and Metastases Reviews, 13, 411-431, 1994.

18. KORCZAK B., WHALE C., KERBEL R.S.: Possible involment of Ca2+ mobilization and protein kinase C activation in the induction of spontaneous metastasis by mouse mammary adenocarcinoma cells. Cancer Res.,49,2597-2602,

18b. GOPALAKRISHNA R., BARSKY S.H.: Tumor promoter-induced membrane bound protein kinase C regulates

18b. GOPALAKRISHNA R., BARSKY S.H.: Tumor promoter-induced membrane bound protein kinase C regulates hematogenous metastasis. Proc.Natl.Acad.Sci. USA, 85, 612-616, 1988.

19. DUMONT J.A., JONES W.D., BITONTI A.J.: Inhibition of experimental metastasis and cell adhesion of B16F1 melanoma cells by inhibitors of protein kinase C. Cancer Res., 52, 1195-1200, 1992.

21. BERDEL W.E., BAUSERT W.R.E., FINK U., RSTETTER J., MUNDER P.G.: Anti-tumor action of alkyl-lysophospholipids. Anticancer Research, 1, 345-352, 1981.

22. BERDEL W.E. and MUNDER P.G.: Antineoplastic actions of ether lipids related to platelet-activating factor. (in: Platelet-activating factor and related lipid mediators, Editor F. Snyder, Plenum Publishing Corporation, pp.449-467, 1987.

23. BERDER W.E., ANDREESEN R., MURDER P.G.: Synthetic alkyl-phospholipid analogs: A new class of antitumor agents. (in: Phospholipids and Cellular Regulation, editor J.F. Kuo, CRC Press Inc., pp. 42-73, 1985.

24. ANDREESEN R.: Ether-phospholipids in the therapy of cancer. Progress in Biochemical Pharmacology, 22, 118-131, 1988, Karger, Basel.

25. NOSEDA A., WHITE J.G., GODWIN P.I., JEROME W.G. and MODEST E.J.: Membrane damage in leukemic cells induced by ether and ester lipids: An electron microscopic study. Experimental and Molecular Pathology, 50, 69-83, 1989.

26. BERDEL W.E., GREINER E., FINK U., ZENKER K.S., STAVROU D., TRAPPE A., FAHLBUSCH R., REICHERT A., RASTETTER J.: Cytotoxic effects of alkyl-lysophospholipids in human brain tumor cells. Oncology, 41, 140-145, 1984.

27. BERDEL W.E.: Membrane - interactive lipids as experimental anticancer agents. Brit. J. Cancer, 64, 208-211, 1991.

28. HERRMANN D.B.J., NEUMANN H.A., BERDEL W.E., HEIM M.E., FROM M., BOERNER D., BICKER U.: Phase I trial of the thioether phospholipid analogue BM 41,440 in cancer patients. Lipids, 22,962-966, 1987.

29. K+RA J., BOROVI+KA M., LIEBL V., SMOL-KOV+ J., UBIK K.: A novel nontoxic alkyl-phospholipid with selective antitumor activity, plasmanyl-(N-acyl)ethanolamine (PNAE), isolated from degenerating chick embryo tissues and from an anticancer biopreparation cACPL. Neoplasma, 33, 187-205, 1986.

30. K+RA J., LIEBL V., PELCBAUER Z.: Natural and semisynthetic ether phospholipids with selective antitumor activity; their chemical structure and mechanism of action leading to tumor cell membrane destruction. (in: Highlights of Modern Biochemistry, Prague, Czechoslovakia, July 10-15, 1988) vol.2, pp.1459-1474 1989, Editors: A. KOTYK, J. ?KODA, V.PA+ES, V.KOSTKA, VSP Utrecht, The Netherlands, 1989.

31. K+RA J., KONOVALOVA A.L., KRASNOVA M.A., LIEBL V., BEJ?OVCOV+ L.: New tumoricidal semisynthetic ether phospholipid, plasmanyl-(N-acyl)ethanolamine (PNAEs) and enhancement of its tumoricidal activity by calcium ions. Neoplasma, 40, 213-217, 1993.

32.LAZENBY C.M., THOMPSON M.G., HICKMAN J.A.: Elevation of leukemic cell
intracellular calcium by the ether lipid SRI 62-834. Cancer Res., 50, 3327-3330, 1990.

33.K+RA J., ZIMAKOVA N.I., SEREBRYAKOVA E.A., D+DKOV+ V., ZOLOTARYOV A.F.:
Pharmacokonetics and metabolism of a new antitumor semisynthetic ether phospholipid,
14C-labeled plasmanyl-(N-acyl)ethanolamine in mice bearing sarcoma Mc11. J. Cancer
Res. Clin. Oncol:, 120, 662-667, 1994.

34.K+RA J., LIEBL V., D+DKOV+ V., BEJ?OVCOV+ L.: Zp?sob semisyntetick? p°?pravy
alkyl-fosfoplipidu se selektivn?m protin?dorov2m ·?inkem. +s. patent ?. 275396, 1991.

35.K+RA J.: Ether-fosfolipidy v onkologii. Chemick? listy, 87, 58-63, 1993.

39.MIKHAEVICH I.S., GERASIMOVA G.K., K+RA J.: Inhibition of protein kinase C by
semisynthetic phospholipid plasmanyl-(N-acyl)ethanolamine, a nontoxic antitumor
preparation. Biochemistry International, 23, 215-220, 1991.

50.KHANAYKAR B., ULBRICH F., GATZEMEIER U., MEYER-SCHEICKERATH E.,
LORENZ J., SCHREML W., BRUGGER R., SCHICK H.D., von PAWEL J., NORDSTROM
R., DRINGS P.: Treatment of non-smoll cell lung cancer with alkyl-phospholipid
Edelfosine. Contrib. Oncol., 37, 224-235, 1989.

57.DIOMEDE L., COLOTTA F., PIOVANI B., RE F., MODEST E.J., and SALMONA M.:
Induction of apoptosis in human leukemic cells by the ether lipid 1-octadecyl-2-methyl-
-rac-glycero-3-phosphocholine. A possible basis for its selective action. Int.J.Cancer, 53,
124-130, 1993.

58.MOLLINEDO F., FERN+NDEZ-LUNA J.L., GAJATE C., MARTIN-MART-N B., BENITO
A., MERTINEZ-DALMAU R., MODOLELL M.: Selective induction of apoptosis in cancer
cells by the lipid ET-18-OCH3 (Edelfosine): Molecular structure requirements, cellular
uptake, and protection by Bc1-2 and Bc1-XL. Cancer Res., 57, 1320-1328, 1997.

69.WYLLIE A.H.: Glucocorticoid-induced thymocyte apoptosis is associated with
endogenous endonuclease activation. Nature, 284, 555-556, 1980.

100.POU+KOV+ P., ZADINOV+ M., NAVR+TIL L., BLEHOV+ Z.: Vliv plasmanyl-(N-
-acyl)ethanolaminu na r?st karcinomu recta xenotransplantovan?ho na athymick? nu/nu
my?i. +asopis l?ka°? ?esk2ch, 126, 1569-1571, 1987.

101.KOTYK A., K+RA J., BAUDY?OV+ M., KNOTKOV+ A., DRAHOTA Z.: Effect of alkyl-
-phospholipids on mammalian cell permeability. v knize: Molecular Aspects of Human
Diseases, (Gorrod J.W., Albamo O., Papa S editors), vol.1., pp. 41-43, Ellis Horwood
Ltd., Chichestr, 1989.

102.KOTYK A., K+RA J., LIEBL V., DRAHOTA Z.: Internat Conf. on Perspectives in
Molecular Approaches to Human Diseases, Bari, Italy,1987.

103. APTE S.S., WEBER N., MANGOLD H.K.: Biologically active ether lipids.
Biotransformation of rac-1(3)-0-alkylglycerols in cell suspension cultures of rape and
semisynthesis of 1-0-alkyl-2-palmitoyl-sn-glycero-3-phospho-(N-palmitoyl)
ethanolamines, potent antitumor agents. FEBS Letters, 265, 104-105, 1990.