LV10303B

LV10303B - Method for elimination of breast cancer cells

Info

Publication number: LV10303B
Application number: LVP-93-1060A
Authority: LV
Inventors: Halling Blaik Philippe; Witkowski Debra Ann
Original assignee: Fmc Corp
Priority date: 1988-12-12
Filing date: 1993-09-09
Publication date: 1995-08-20
Also published as: GR890100817A; NZ231658A; BR8907817A; WO1990006748A2; GB9110472D0; GB2247404A; AR243605A1; HUT58202A; IE893925L; SE505938C2; DE3991484T; SE9101807D0; OA09362A; DK110791A; FI912784A0; IL92622A; ES2051675T1; ES2051675T3; MC2171A1; EP0447491A1

Description

LV 10303

Cnoco6 y6HBaHHH κιβτοκ onyxojiH mojiohhoh Hiejie3bi,

φαρΜΗαθΒΤΗΜΘΟΚΙΙβ Κ0ΜΠ03ΙΠΙΗΗ, ΗΗΓΙίβΗΤΟρΜ, CIIOCOO ΠΟΒΜΙΙΙβΗΗίϊ COAepHCaHHH npOTOIĪOp(ļ)HpHHa IX, cnoco6 JIOKajIH3aUHH KJI6TOK onyxojiH, cnoco6 nojiyHeHHa προτοπορφΗρΗΒβ IX ΟΠΗΟΑΗΗΕ

Oahh H3 acneKTOB HacTOHiuero Η3ο6ρετεΗΗΗ Kacaeica φΟΤΟΛΗΗΒΜΗ^εεΚΟΓΟ Jie48HH5ī MOJIOHHOM >Kejie3bi c onyxojieBbiMH KJieTKaMH, KaK sto AeJiaeTca πρπ φοτοΛΗΗΰΜΗ^εεκοΜ ksjichchhh paKa. Χοροΐΐΐο H3B8CT8H ΜετΟΛ ΒΒε^εΗΗΗ ΠΡΟΗ3ΒΟΛΗΟΓΟ ΓεΜ3Τ0Π0ρφΗρΗΗ3 HJIH CM8CH ΠΟρφΗρΗΗΟΒ, HBJIHK)mHXCH npOH3BOflHbIMH ^aHHoro εοεΛΗΗεΗΗΗ, ΗΒπρΗΜερ, ΒΒε^εΗΜε np8napaTa “ΦοτοφρΗΗ II” (Photofrin II) b οπγχοπεΒΒίε κιιετκΗ mojiohhoh xcejie3bi c nocji8nyiomHM Βθ3ΛεήετΒΗεΜ Ha 3th κπετκΗ CBCTOBoro M3.iy^8HM^ onpe^ejieHHOM λληηβι βοληη c u8jibio Bbi3BaTb pa3pyuieHHe ιοιετοκ. ZIaHHOMy MeToay φοτοΛΗΗΒΜΗΜεεκοίίί τερ3ΠΗΜ nocBameHa εερκπ CTaT8H, cocTaBJiHK>mHx cneuHa/ibHbiH BbinycK xcypHajia Photochemistry and Photobiology, 1987, t. 46, Ns 5 (ΗΗχε npHHBTO C0Kpam8HH8 P&P 46-5) B 3Thx cTaTbax noKa3aHo, ητο φοτοΛΗΗ3ΜΗπεεκ3Η TepanHH Hanuia ωπροκοε πρΗΜεΗεΗΗε πρκ πεπεΗΜΗ pa3JiHHHbix paKOBbix 3a6oji8BaHHft (b t.h. paica δροΗΧΟΒ, Mo^eBoro ny3bipa, immeBOjļa, ϋεποίχ, kojkh, opraHOB rojioBbi h ΙΙΙβΗ, M03ra, TOJICTOH KHIIIKH, a TaiQK8 BHyTpHDia3HbIX H ΓΜΗεκοΛΟΓΗπεεκΗχ paKOBbix 3a6oJieBaHHft). ΥτΒερχ^ετεπ, hto 6hOXMMH48CKH8 3φφεΚΤΒΙ ynOMHHyTOM ΠΟρφΗρΗΗΟΒΟΗ 1 4)OToceHCH6HJiH3au;HH BKJiicmiOT o6pa30BaHHe nonepenHbix CBioett c npoTeMHaMH b MeM6paHax, nepoKCHjiHpoBaHHe λμπηλοβ, nojiaBjieHHe nepeHoca ΗεκοτορΒίχ BaxcHbix MeTabojiHTOB, JiH3Hc JiH30C0MajibHbix (Ņsosomal) h MHTOXOHHpHajibHbix obojioneK, nojiaBJieHHe p*ma 3H3HMOB h noBbiuieHHe ycB06HHH πορφπρΗΗΟΒ KJiCTKaMH (P&P 46-5, c, 695). ΠορφΗρΗΗΟΒΜίί npenapaT ββολητ KaK npaBHJio nyreM ΗΗΊ>εκυΗ& (HanpHMep, BHyTpHBeHHO H/IH B 6pK)UIHyK) nOJIOCTb): THĪlHHHail fl03a cociaBJiHeT 2 μγ npenapaia “ΦοτοφρπΗ” Ha 1 κγ Maccbi Tejia.

KpoMe τοΓό, πορφΗρΗΗΟΒΒΐίί npenapaT mojkho BīcnioHHTb b jmnocoMy h BBecTH HH^eKiiHefi (HanpHMep, b 6pioiiiHyio nojiocTb) KaK yxa3aHo, b nacTHOCTH, b CTaTbe CnaftKca (Spikes) h ap. “OoTO^HHaMHHeCKOe ΠΟΒβΛβΗΗβ ΠΟρφΗρΗΗΟΒ B MOJjeJIbHbDi ΚΛεΤΟΗΗΗΧ, TKaHeBbix h onyxojieBbix CHCTeMax“ (Photodynamic behavior of Porphyrins in Mod Celi, Tissue and Tumor Systems) b c6opHHKe “OoTO^HHaMHHecKaH TepanHH onyxojieii h npyrHX 3a6ojieBaHHH (Photodynamic Therapy of Tumor and other Diclases), BbimefluieM πολ pe^aKiiHen Mopn (Jori) h IleppHa (Perria): Flaip/a, Η3£. Libreria Progretto, 1985, c. 45-53, a Taoce b ynoMHHyroM Bbime cneuBbinycKe. - B cnenHajībHOH jiHTepaiype roBopHTca o tom, hto BBe^eHHe φθΤΟΠθρφΗρΗΗ3 B T3KHe KJieTKH, KaK, HanpHMep, 3pHTpOHHTbI HeJIOBeKa hjih murine kjictkh npn jieHKeMHH, aaeT 3aMeTHbiH φoτoceHCH6HJ^H3Hpyκ)mHH θφφεκτ; cm. οοοτεβτοτΒβΗΗο: X(a66ejiMeH (Doebleman) h Jipyrne Biochimica et Biophysica Acta, 1978, t. 511, c. 141-151; Keccejib (Kessel), Cancer Research, 1982, e. 42, N° 5, c. 1703-1706. B CTaTbe Keccejia yTBepx<jiaeTca, ητο H3 Bcex HcnbiTaHHbix φοτοοεΗθΗ6ΗΛΗ3Ηρ>τοπΐΗΧ peareHTOB φοτοπορφπρΗΗ HaHOojiee aKTHBHbiH. OjiHaKo, Korjia Kaccejib h jipyrne HccjiejioBaTejiH, HanpHMep , EepeH6ayM (Berenbaum) η coTp. ( cm: Br.C Cancer 1982, t. 45, c. 571-581) jiejiajiH ηητ^κιιηη φοτοπορφπρΗΗΒ jkhbothmm, 3aMeTHoro coflepJKaHHH φοτοπορφκρΗΗΒ b onyxojuix He 6biJio o6HapyxceHO, hto CBHjieTejibCTByeT o bo3moxchom ObicTpoM yHoce φοτοπορφπρπ^, 2 LV 10303 BBefleHHOro B OpraHH3M ΠΟΛΟδΗΒΙΜ 06pa30M, BCJie^CTBHH KpoBoo6pameHHa.

Taiace χοροπιο M3BecTH0 npHMeHeHne προη3βοληογο ΓεΜ3τοπορφΜρΗΗ3 η aHanornHHbix npenapaTOB ķjix HHaraocTHpoBaHHii h jioKajiH3auHM onyxojien, t3khx KaK paK MoneBoro ny3tipH hjih JiencHX (cm., cm., HanpHMep: P&P 46-5, c. 759). AKTHBHoe coeaHHeHHe, npHMeHHiomeecfl coniacHO οληομυ H3 acneKTOB HacToautero Η3θ6ρετεΗΗΗ &ιιη JieHeHHa kjictok b pacCMaTpHBaeMOM φοΤΟβΗΗΒΜΗΗεΟΚΟΗ TepanHH HJIH B H3BeCTHbIX MeToaax ūHamocTHpoBaHHH h jioKajiH3auHH onyxojieH, npejļCTaBJineT C060H He ΠΟρφκρΗΗ HJIH xce He TOJIbKO ΠΟρφΗρΗΗ. B OTJIHHHe OT npOCTO ΠΟρφΗρΗΗΒ HaHHOe COejIHHeHHe ΗΗΓΗβπργεΤ 3H3HMaTH4ecKoe npeBpameHHe φοτοπορφΗρΗΗΟτε^ b cTpyicrype heme ynoMHHyTbix κ/ιετοκ, Bbi3biBaa tqm caMbiM o6pa30BaHHe SHjjoreHHoro φOTOΠOpφHpHHa IX B 3ΤΗΧ KJieTK3X. ABTOpbl yCTaHOBHJIH, HTO TaKHe peareHTbi, Kax HeKOTopbie βηλμ rep6HUHHHbix coejiHHeHHH, KOTopbie noaaBJIHIOT 3H3HMaTHHeCKOe npeBpameHHe ΠΟρφΗρΗΗΟΓβΗΟΒ B XJIOpoφHJI b pacTHTejibHbix κπετκαχ, Taoce nonaBjmiOT 3H3HMaTHHecKoe npeBpameHHe πορφπρΗΗΟΓβ^ b dpyKType heme kjictok MOJionHoft )Kejie3bi. Αβτορμ οηητβιοτ: ynoMHHyToe HHTH6HpoBaHHe sthmh peareHTaMH, BeponTHo, BJinneT Ha cTaimio npeBpameHHH φ0Τ0Π0ρφπρΗΗ0ΓεΗ3 B φοΤΟΠΟρφπρΗΗ IX ΠΟΛ fleftCTBHeM 3H3HMa (φθΤΟΠθρφΗρΗΗΟΓεΗ-ΟΚεΗ/Ι333, EC 1.2.3.4) TaKHM o6pa30M, HTO φοτοπορφπρΗΗΟΓεΗ y*;e He npeTepneBaeT 3Η3ημηογο npeBpameHHH b φοΤΟΠΟρφπρΗΗ IX HOpMaJIbHblM ΠγΤβΜ, a BMeCTO 3Τ0Γ0 HCnMTbIBaeT BHyTpHKJieTOHHoe OKHCjieHHe (HanpHMep, b njia3Me) BHe HopMajībHoro 3H3HMaTHqecKoro nponecca (HanpHMep Bce oSojto^kh opraHejuibi), BCJieacTBHe nero φοτοπορφπρΗΗ IX HaKanjiHBaeTCH b o6jiacTHx, me oh HejiocTyneH jum HopMajībHoro npeBpameHHH b heme, h b pe3yjibTaTe npn o6jiyqeHHH ιοιετοκ cBeTOM HaKonHBiHHHCH TaKHM o6pa30M φοτοπορφπρΗΗ IX Bbi3biBaeT οφφεκτ pa3pymeHHH κπετοκ, nojjoSHbift 3 τομυ, κοτορΒίΗ Hadjno/iajicfl b cjiyHae onHcaHHoii Bbiuie φοτοΛΗΗΒΜΗΗβοκοίί TepannH. OTBe^aiomHe HacTOflnieMy H3o6peTeHHio aKTHBHbie coeAHHeHHH, noaaB^HIOmHe 3H3HMBI, JIBJIfllOTCfl ΟΠϋΙίΜφΗΗβΟΚΗΜΜ HHnidHTOpaMH φ0Τ0Π0ρφΜρΗΗ0Γ6Η-0Κ0ΗΛ33Η B TOM CMbICJie, MTO OHH He φ>ΉκηΗθΗΗρ>τοτ KaK oObiHHbie 3H3HMHbie ηλβι πολο6ηο peareHTaM, Bbi3biBaiomHM B:eHaiypMpoBaHMe hjih o6pa30BaHHe nonepeqHbix CBH3eif (HanpHMep, πολο6ηο peareHTaM Ha ocHOBe cyj^Hrmi;pHJia): ohh TaioKe He HBJIHIOTCH npeJKUe Bcero TaKHMH BJIHHIOIHHMH Ha yCJIOBHH OKHCJieHHH npenapaTaMH, KaK aKuenTopbi ajieKrpoHOB, h, cjienoBaTejibHo, aKTHBHbie coe^HHeHHa, npejmoTTHTejibHbie corjiacHO HacTOHmeMy H3o6peTeHHio, HMeiOT 6oJiee OTpHLiaTeJIbHblH OKHCJIHTejIbHO-BOCCTaHOBHTejIbHblH noTeHHna^, 4eM -500 mB h jia;>Ke eme OTpHLiaTeJIbHblH, a hmchho, paBHbifi -800 mB (H3MepeHHbiH, HanpHMep, ο6μηημμ μθτολομ β ποΛχοΛΗΐΐίεΜ λλη aaHHoro coeaHHeHHH pacTBopHTejiH: Μετο^οΜ HHKJIHHeCKOft BOJIbTaMeTpHH HJIH xce nOJiapon^HHeCKHM ΜβΤΟΛΟΜ). IlpezinoHTHTejibHo Taoce, ητο aKTHBHoe coejiHHeHHe He npejicTaBjmeT codoii TeTpanHppoji h, hto ero noKa3aTejib I50 jļjui φοτοπορφκρΗΗΟΓβΗ-0Kcima3bi cocTaBJineT MeHee ImkM, HanpHMep, MeHee 0,3 μκΜ: b nacTHocTH noKa3aTe;ib I50 cocTaBJineT npH6jiH3HTejibHo 0,1, 0,03 hjih 0,01 mkM Aaxce MeHee.

npejinoHTHTejibHO TaKoe HHrH6Hpyioinee 3H3HM aKTHBHoe coenHHeHHe, κοτοροε oTJiHHaeTcn noBbiineHHoft cnocodHocTbio pa3pymeHHH n/ia3MajieMMbi (Plasmalemma) pacTHTejibHbix npenapaTOB. OjļHH H3 ΜεΤΟΛΟΒ HCnblTaHHH HaHHOH CnoeoSHOCTH OnHCaH ΠΟΛ Ha3BaHHeM “3KcnepHMeHT no BbiMbiBaHHio” b CTaTbe Xo;uiHHra h riHTepca (Halling and Peters): Phant Physiology, 1987, t. 84, c. 1114-5. CorjiacHO 3TOMy HcnbrraHHio (6o;iee πο/ιροδΗο oho onncaHo HHxce b npHJio)KeHHH A) npejinoHTHTejibHbie peareHTbi noKa3biBaioT no KpaHHen Mepe 50%-Hoe o6mee BbiMbiBaHHe npH ypoBHe o6pa6oTKH IOOmkM, npezinoHTHTejibHO xce npn ypoBHe o6pa6oTKH ΙμκΜ h MeHee, HanpHMep, npH 100 hM. BbicoKoaKTHBHbie npenapaTbi, TaKne KaK 4 LV 10303 1-(4-ΧΛθρ-2-φΤ0ρ-5-ΠρθΠ3ρΓΗΛΟΚεΗφ6ΗΜΛ)-3-Μ6ΤΗΛ-4-ΛΜφτθρΜεΤΗΛ-Δ2-l,2,4-TpHa3o;iHH-5-oH hjih ,π^κτοφεΗ (cm. HHXce) ποκ33μβ3Κ)τ odiuee , BbiMbiBaHHe cBbirne 90% πρκ KOHueHTpauHH 100 hM. JīpyroH ΜβτοΛΗΚοίί onpenejieHHH cnocoSHOCTH npenapaxa pa3pymaTb njia3MajieMMy pacTHTejibHoro BemecTBa hbjthiotch t.h. “HcnbīTaHHe Ha HHrndHpoBaHHe no3ejieHeHHfl, Bbi3biBaeMoro cbctobbim o6^yHeHHeM”, noapodHee onncaHHoe HHace b ripHJio^ceHHH B. 3ia MeTOAHKa npe^Ha3HaqeHa ķjw onpeuejieHHH cnocodHocra npenapaTa noaaBJiHTb no3ejieHeHHe oTSejieHHoft b TeMHOTe KyjibTypbi MHKpoopraHH3MOB Chlamydomonas rein hardi, y-I (pa3HOBHn;HOCTb MHKpoopraHH3MoB aglae, KOTopbie pacTyr b τβΜΗοτε, He o6pa3ya χϋοροφΗΛΛ3. Πρπ 3T0M HMeeTCH b Buay, Ητο Macca aglae oTdejiHBaeTCBi djiaroaapa HajiHHHio b Hen οΒεχαιχ, Heno3e;ieHeBiiiHx κπετοκ, ho BHOBb o6pa3yeT ΧΛοροφκ/ι Ha cBeTy. MHorne H3 npeunoHTHTejibHbix βκτηβηηχ coeji,HHeHMft, npMMeHHiomMxcH coniacHO HacTOHiueMy H3o6peTeHHio, cnocodHbi noaaBJiHTb no3ejieHeHne, Bbi3BaHHoe ceeTOBbiM o6jiyqeHHeM, no KpaiiHen Mepe Ha 50% npH KOHueHTpauHH HcnoJib3yeMoro coenHHeHHH 10'5M, npeunoHTHTejibHee πρκ 10_6M h MeHee, HanpHMep, npn KOHueHTpauHH 10'7M. KpoMe τογο, sto coeuHHeHHe uojdkho 6bm> TaKHM, κοτοροβ b xoue yKa3aHHoro HcnbīTaHHH Ha HHradHpoBaHHe no3eneHeHHH o6pa3yeT BcnjibmaioiuHH Ha noBepxHOCTb npouyKT c MaKCHMyMOM cBeTonorjiomeHHH npn 405 hM, hto npeBbiuiaeT MaKCHMyM &ΠΗ ΧΛΟροφΗ/υΉ, COCTaBJIHIOIUHH Β UaHHOft CHCTeMe 669 hM: HanpHMep, BcnjibmaiomHiii npouyKT noKa3biBaeT npn 405 hM MaKCHMyMa npn 668 hM.

CpeUH HHrH5HpyiOmHX 3H3HMbI 3ΚΤΗΒΗΜΧ COeUHHeHHH, KOTOpbie moxcho Hcnojib30BaTb npH ocyiuecTBJieHHH HacTOHiuero H3o6peTeHHH, ecTb repdnuHAbi cjreuyioiuHx τηποβ (A-D). A. ApHJi-reTepouHKJiHHecKHe repdHUHUbi odiueft φopMyJIbI:

Ph - Nhet 5 rae Ph - 3aMemeHHbiH φεΗΐυι, npezinoHTHTejibHo 2,4-nH3aMemeHHbift φεΗΐυι, eme npe^noHTHTejibHee 2,4,5-Tpn3aMemeHHbiH φεΗΐυι, a Nhet - 5- hjih 6-HJieHHoe reTepouHicJiHHecKoe KOJibuo (c 1-4 aTOMaMH a30Ta b KOJibue) φορΜγ^Η: - N - C = O HJIH - N - C - OH HJIH - N - C - C1

\/ III I II

Q Q - C - R Q - C - R rne Q 03HaqaeT 3aMbīKaK)myio rpynny reTepouHKJiHHecKoro KOJibiia, a R -βολορολ hjih 3aMemaiomyio ero rpynny. JiaHHbiH KJiacc coe/iHHeHHH BKjimaeT TaioKe npenapaTbi, κοτορπε b CTaTbe BaKa6a«cH (Wakabaijashi) η λρ.(ομ: J. Pesticide Sci., 1986, τ. II, c. 635-460) onpejiejieHbi KaK KJiacc rep6HUHHOB Ha ocHOBe lihkjihh8ckhx ημηλοβ, npMMeM Ha c. I 3toh cTaTbH npeacTaBJieHbi cjie^joomHe coe^HHeHHH:

CoejiHHeHHe I npejicTaBJineT cočoh ΓερβΗΐίΗΛ Ha ocHOBe apHJi-0KCHjiHa30Jia, a HMeHHO 2-TpeT.-6yTHJi-4-(2,4-jiHXjiop-5- i Η3οηροποκοΗφεΗΗϋ)-Δ2 -l,3,4-0KcmiHa30JiHH-5-oH, JļpyrHe 2-ajiKHji-4— (2,4,5-TpH3aMemēHHbift φεΗκπ) )-Δ2 -l,3,4-0KCHn,Ha30JiHH-5-0Hbi, κοτορπε mojkho Hcnojib30BaTb corjiacHO HacTOHmeMy Η3θ6ρετεΗΗΐο, oxapaKT8pn30BaHbi, HanpHMep, b naT8HTax CIIIA Ns 3 385 862, 3 836 539, 3 876 413. 6 LV 10303

CoeaHHeHHe II npeacTaBaaeT coOoft repOnnHa Ha ocHOBe apHJī-TeTparHAponHaa30Jia, a ΗΜβΗΗο 3-ΧΛορ-2-(4-χπορ-2φτορ-5H3onponoKc^eHHa)-4,5,6,7-TeTparHapo-2H-HHaa3oaa.

CoeaHHeHHe III, ΙΥ η Y npeacTaBaaioT οοβοπ repSHUHabi Ha ocHOBe apHJi-TeTparaapc^TajiHMMaa. HpyrHe apHJi- Τετρ3ΠΙίΐρθφΤ3ΛΗΜΗΛΙ>Ι, KOTOpbie ΜΟΧ.ΗΟ HCn0JIb30BaTb coraacHO HacToameMy H3o6peTeHHio, oxapaicrepH30BaHbi, HanpHMep, b naTeHTax CIIIA Ne 4 431 822, 4 670 046, 4 670 042, 4 439 229, a Taoce b BbmoxeHHoii MexnyHapoaHOH 3aaBKe Ns WO 87/07062. B aByx nOCJieaHHX HCTOHHHKaX, ΚρΟΜβ ΤΟΓΟ, OnHCaHbl ApyTHe Nhet -KOJibiļa, Hcnojib30BaHHe κοτορπχ Taoce aonycTHMo. ripHMepbi TaKHX KOJieu npHBeūeHbi b 43CTH0CTH Ha c. 12-14 MexayHapoaHoft 33ηβκη No WO 87/07062, a Taoce b naTeHTe CIIIA N° 4 439 229 (co cTpoKH 25 Ha c. 4 no CTpoKy 20 Ha c. 5). ūpyrHMH πολχοληιηημη repāHHHaaMH rana P- Nhet-aBJiaioTca: apHji-TpHajiHHOHbi, TaKHe, hto onncaHbi b naTeHTax CLUA noa HOMēpaMH 4 318 731, 4 398 943, 4 404 019, 4 702 945, 4 705 557, 4 702 763, 4 761 174, a Taoce b MexayHapoaHbix 3aaBKax noa HOMēpaMH WO 85/01637, WO 85/04307, WO 87/00730, WO 87/03782, WO 86/04481 hWO 88/01133: apHji-TeTpa3oaHHOHbi, TaKHe, hto onHcaHbi b naTeHTe CIIIA noa HOMepoM 4 734 124, a Taoce b MexayHapoaHbix 3aaBKax noa HOMēpaMH N2N2 WO 85/01939 h WO 87/03873: apHa-TpHa3HHaHOHbi, TaKHe, hto onHcaHbi b naTeHTax CIIIA noa HOMēpaMH N2N2 4 755 217 h 4 766 233, a Taoce b MexayHapoaHOH 3a«BKe N2 WO 86/00072; apHa-raaaHTOHHbi, TaKHe, mto onHcaH b naTeHTe CIIIA noa HOMepoM 4 427 438; apHJi-ypa30jibi, TaKHe, hto onHcaH b naTeHTe CIIIA N2 4 452 981 h apHJi-reKcarHaponHpHaa3HHbi, TaKHe, hto onHcaH b naTeHTe CIIIA No 4 619 687. 7 B. ApHJi-reTepoitHioiHHecKHe ypeTaHbi, Taioie, hto oimcaH b naieHTe CUIA N° 4 521 242. C. ΓβρβΗΙίΜΛΜ Ha OCHOBe ΠρΟΟΤΟΓΟ φβΗΗΛΟΒΟΓΟ 3φπρΒ H3 TeX, 4T0 HMeiOT napa-rajio- hjih napa-HHTpo^eHOi«^eHHJioByK) οτργκτγργ, a HMeHHO, TaKHe, KaK cjie^jTomne HMeiomHecH b npo^a»ce npenapaTbi: 5-(2-chloro-4-(trifluormethyl) phenoxy)-2-nitro-N-methanesul- fonylbenzamide (fomesafen) 5-[2-ΧΛ0ρ-4-(τρΗφΤ0ρΜεΤΗΛ)-φ6Η0Κ0Η]- 2-HHTpo-N-MeTaHcy;^o6eH3aMim ("ΦθΜε33φεΗ”) sodium5-(chloro-4-(trifluoromrthyl) ph8noxy)-2-nitrob8nzoat8 (acifluorfbn) 5-[2-[2-ΧΛορ-4-(τρΜφτορΜετκΛ)-φ8ΗΟΚεΗ]-2-ΗΗΤρθ68Η303Τ HaTpMii (“Αΐ1Μφ;ψοροεφεΗ”) m8thyl 5-(2,4-dichloroph8noxy)-2- nitrobenzoat8 (bif8nox) Μετϋπ-5-(2,4-ΛΗΧΛορφεΗθκεΜ)-2-HHTpo6eH3oaT (“ΒκφεΗοκε”) 2-chloro-1 -(3-8thoxy-4-nitroph8noxy)-4-trifluormethylbenz8n8 (oxyfluorf8n) 2-ΧΛορ-1-(3-3τοκεΗ-4-ΗΗτροφεΗθκεΗ)-4-τρΗφτορΜετΗΛ6εΗ3θϋ (“ΟκεΗφ;ψορφεΗ”)

ApyrHMH ΠΟΛΧΟΑΗΙΙΙ,ΚΜΗ η HMeiOlHHMHCil B npoflaace repGHUHAaMH Ha OCHOBe npocTbix ληΦοημλοββιχ 3φκροΒ HBJiaiOTca cjie,n,yioiHHe COe^HHČHHH. “ΙΐΒΚΤΟφεΗ”; 1-(κ3ρ6θ3τοκεΗ)-3ΤΉΛ-5-(2-ΧΛορ-4-τρΜφτορΜετΗΛφεΗοκεΗ)-2- HHTpo68H30aT; “ΦΛΙΟΟρπίΙΟΚΟφβΗ”; (κ3ρ6θ3τοκεΗ)-ΜετΗΛ-5-(2-χπορ-4-τρΗφτορΜετΜΛφεΗθκεΜ)-2-HHTpo68H30aT; ' “ΦρΟΜβΟΟφβΗ”; 5-[2-ΧΛορ-4-(τρκφτορΜετκ.τι)-φεΗθκεΗ]-Ν-ΜετΗ/ι-^.ΓΐΒφθΗΡυι-2~ HHTpo6eH3aMRa; 8 LV 10303 “ΧϋοροΗΗτροφεΗ”: 2,4,6-Tpnxūop-(4-HHTpo<|)eHOKCH)-6eH30Ji; “ΧϋγΟρΟΠΗφεΗ” 2-ΗΗΤρθ-1 -(4-ΗΗΤρθφεΗΟΚ0Η)-4-ΤρΗφΤθρΜετΜϋ6βΗ3ΟΛ; “ΗΗΤροφεΗ”; 2,4-ΛΗΧΛθρ-Ι-(ΗΗΤρθφεΗΟΚΟΗ)-6εΗ3θ;ΐ; “ΧΛΟΜετοκαίφεΗ”; 4-(2,4·^ΗΧΛορφβΗοκεΗ)-2-Μετοκ<:Η-1-ΗΗτρο66Η3ο;ι.

KpOMe ΤΟΓΟ, eme ΟΛΗΗΜ ΠροΧΟΛΗΙΙίΗΜ npOCTbIM ΛΗφεΗΗΛΟΒΗΜ 3φκρθΜ £BJW8TCH OKCHM-O-aHCTaT 5-(2-Χϋθρ-4-ΤρΗφΤθρΜ6ΤΗΛφεΗΟΚΟΗ)-2-HHTpOaiI8TC^CHOHa. 1. ΓερδπΐίΗΛΗ Ha ochobc apH;i-nHpa3o;ioB, τ3κηχ, ητο oiracaHbi β naT8HTax C1IIA Ν2Ν2 4 563 210, 4 496 390 η 4 459 150, a TaioKC β ΒΒυιοχεΗΗοίί 339Βκε ΓepMaHHH Ν2 3 530 327 ΑΙ. Ε. Coe^HHBHHH φορΜγΛΒΐ:

ΓΛε Xb - KHCJiopoa hjih cepa, a Ph ΗΜεετ cmbicji, yKa3aHHbift ΒΒίπιε: HaHHbie εοεΛΗΗ8ΗΗΗ OriHCaHbl Β EBpOn8ŪCKOft ΓΉΤεΗΤΗΟίί 3a«BK8 ΠΟΛ N2 273 417, a TaioKe b Kaiajiore Dervent Abstracts (cm. N2 87 040 749). •ΠεπεΗΜε aKTHBHbIMH COCAHHCHHaMM, OTBCKaiOmHMH HacTOHmcMy Η30δρετεΗΜ10, ΜΟ)ΚΗΟ OCymeCTBJIHTb nOCpeHCTBOM HHIbeKIiHft KaK BHyrpMBeHHO, TaK Η B 6pi01iIHyK) nOJIOCTb. ΑΚΤΗΒΗΟε C08JJHH8HH8 MOKHO HCnojIb30BaTb Β ΒΗΛ8 CTepHJIbHOft ΚΟΜΠ03ΗΗΗΗ, npeaCTaBJIHIOIII.eii CC)60H pCar8HT, paCTBOpCHHblfi HJIH BiHCncprHpOBaHHblft Β φΒρΜΒΙ^εΒΤΗΗεοΚίί npHCMJI8MOM HOCHTejie, HanpHMep, Β ΒΟΛΗΟΜ H30TOHHHeCKOM pacTBopc,

TaKOM KaK co^eHa» Bo^a (0,9% NaCl) hjih tkc noĶcojiSHbiPi 6yφepHbI¾ padBop J1sui6čko (Dulbccca) c KOHucHTpaiiHCH nop^Ka 2,5 μγ/mji. Hjih

JKC aKTHBHOC COC^HH8HH8 MOXC8T 6bITb BKJH048HO B JIMIIOCOMaJIbHJTO cHCTCMy, TaKyio, ητο μοχηο noJiyHHTb β φοοφοΛΗΠΗΛΗοίί ορε/ι;ε aHajiorHHHo TOMy, KaK sto onHcaHO Ha c. 1659-1660 cnpaBOHHHKa 9

Remington’s Pharmaceutical Science, 17-e H3iiaHHe φκρΜΜ “MaK naOjmuiHHr K°” (Mack Publishing C°). HanpHMep, aHajiorMHHO peuerrrype, κοτοργιο ormcajiH ftopn h coTp. (cm.: Br. J.Cancer, 1983, t. 48, c. 307), mojkho pacTBopHTt 51,5 μγ ΛΗΠ3ΛΒΜΗτορυΐΛΗφοΰφητΗΛΗΛΧθΛΜΗ3 b 10 MJi 1 mM pacTBopa peareHTa B CMecM χΛοροφορΜ3 c ΜβτΗΛΟΒΗΜ cnnpTOM (οδΒβΜΗοε cooTHOUieHHe 9:1) c nocjieujTOLLiHM yaajieHHeM pacTBopHTejin (nocjie npejiBapHTejibHoro TmaTejibHoro nepeMeuiHBaHHH) noa BaicyyMOM πρκ 30°C, nojtyHan b pe3yjibTaTe TBepaoe BemecTBo, κοτοροε mojkho bhobb nepeBecTH bo B3BeuieHHoe cocTOHHHe b 10 μλ 0,01 Μ φοοφ3ΤΗθ ΟγφερΗΟΓΟ pacTBopa c BejipmHHOH pH=7,4, coaep>Kamero 150 mM NaCL, c nocjie,ayiomefi[ yjibTpa3ByKOBOM o6pa6oTKon 3aMyTHeHHoro pacTBopa npn 50°C b TeneHHe 30 MHH. MHrH6npyiomHe 3H3HMbi peareHTbi, κοτορΒίε mojkho ncnojib30BaTb npH ocymecTBJieHHH HacTonmero Η3θ6ρετεΗΗΗ, jionycKaeTCH coe^HHHTb HJIH CBH3bIBaTb C ΠΟΛΧΟΛΗΙΙίΗΜΗ MOHOKJIOHajIbHblMH aHTHTejiaMH, οπείίκφΜΗεοκΗ B03aeHCTByioiiiHMH Ha onyxojm (MABs), c HCĪIOJIb30BaHHeM ΤεΧΗΗΚΗ CBH3bIBaHHH, XOpOIiIO H3BeCTHOii cneuHajmcTaM β aaHHoii o6jiacTH b Ka^ecTBe cpeacTBa aocTaBKH peareHTa, HHni6Hpyiomero 3H3HMbi, k KOHKpeTHOMy ynacTKy, nopaxeHHOMy onyxojibio. MHrn6Hpyioin,He 3H3hmm aKTHBHbie coeaHHeHHH, npHMeHHiomHeca corjiacHo HacTOHmeMy H3o6peTeHHio, μοχηο TaioKe ββοληή» npocTbiM Ha3HaHeHHeM πγτεΜ rjioTaHHa, npeanoHTHTejibHo b pa36aBJieHHOM BHne BMecTe c φ3Μ3η6ΒΤΗ4εα<Η npHeMJieMbiM HocHTejieM, a Tarace nocpezicTBOM aoOaBJieHHH ηχ κ nnme, KaK 3to noKa3aHo HHJKe b IlpHMepe 6. )KejiaTejibHo, b ocoOchhocth npH Ha3HaHeHHH npenapaTa qepe3 rjioTaHHe, npHMeHHTb HHrH6HpyiomHe 3H3HMbi peareHTbi, npe^cTaBJiHiomHe co6oh pacTBOpHMbie b BOAe cojih hjih xce KHCJiOTHbie coe^HHeHHH, o6pa3yiomHe pacTBOpHMbie b Boae cojih narpm hjih k3jihh, το-ecTb TaKne, KaK cyj^aMHn τογο BHjia, κοτορωκ ormcaH b 10 LV 10303 Μβ^γΗαροΛΗΗΧ 3aHBKax Ne WO 87/037873 (HanpHMep, peareHT 6c b b npHBejieHHOM HHxce npHMepe 6), a Taoce N° WO 85/001939 WO 87/037873 hjih ero Bo^opacTBopHMaa co;ib, jih6o cooTBeTCTByiomafl Kap6oHOBaH KHCjiOTa hjih ee BonopacTBopHMaa cojib, HanpHMep, HaTpneBaa cojib, H3BecTHaa πολ Ha3BaHHeM “ΑηΗφϋγορφβΗ”. jļonycKaeTca BKJnoneHHe ηηγηΟηρ^τοπιηχ 3H3HMbi aKTHBHbix coeiiHHeHHH, Hcnojib3yeMbix corjiacHO HacToameMy H3o6peTeHHio, b o6biHHbie φ3ρΜ3ΐίεΒΤΗιΐ6οκΗ6 npenapaTbi, TaKHe KaK TaSjieTKH (HanpHMep, npeccoBaHHbie Ta6jieTKH, Ha KOTopbie μοχηο HaHecra čaxapHyio naciy m/hjih cnpon), CBenn, Kancyjibi (HanpHMep, TBepubie HcejiamHOBbie Kancyjibi), cycneH3HH, pacTBopbi, nopoimcH hjih aMnyjibi. B Τ3ΚΗΧ npenapaiax aKTHBHbift κοΜΠΟΗβΗτ MoxceT HaxoMTbcn b CMecn c φαρΜΒαβΒΤΗΗεΟΚΗ npHeMJieMbIM TBep/ibIM HJIH JfCHHKHM HOCHTeJieM, KOTopbift no xcejiaHHK) Μοχβτ 6biTb nHineBbiM nponyicroM. HanpHMep, 3TO Μοχετ 6biTb TBepjļbiii nponyKT, TaKOH xax KyKypy3HbiH KpaxMaji, hjih ΧΗΛΚΗΐί pa36aBHTejib, TaKoii KaK BOjja hjih nnmeBoe jih6o MHHepajībHoe MaCJIO, HJIH xe paCTBOpHTeJIb: ΛΗΜβΤΗΙΙΟ>'.1ΒφθΚΟΗΛ Η Τ.Π. Mojkho npHMeHHTb CMeCH HHrH6HpyiOmHX 3H3HMbI ΒΚΤΗΒΗΒΙΧ ΚΟΜΠΟΗβΗΤΟΒ, HanpHMep, cMecb aicrHBHoro KOMnoHeHTa 6c, yKa3aHHoro HHxe b npHMepe 6, c “ΑιΐΗφϊ^ορφεΗΟΜ”, b npH6jiH3HTejibH0 paBHbix KOJiHHecTBax. Ha3HaqaeMyio 503HpoBKy mojkho onpenejiHTb ο6ηηηημ 3KcnepHMeHTajibHbiM nyTeM, χοροπιο H3BecTHbiM cnennajinciaM b naHHOft oOjiacTH, no ΜβτοΛΗΚβ 3KcnepHMeHTa, onHcaHHož jsjia npenapaTa “ΦοτοφρΗΗ” (Photophrin) b cTaTbe ,ΰ^φερτΗ “OoTojiHHaMHnecKan Tepannn (Φ/1Τ) 3JioKaHecTBeHHbix onyxojieft’\ cm: CRC Critical Reviews b H3naHHH Oncology/Hematology, 1984, t. 2, Bbin. c. 83-116.

Hnxe c uejibio HJunocipHpoBaHHfl HacTomnero H3o6peTeHHH npHBeneHbi cjienyioiUHe npHMepbi. ΠρΗΜβρ 1

KjieTKH HeLa (jihhhh onyxoJieBbix κ/ιβτοκ nejioBeKa, οδωηΗΟ Hcnojib3yeMbix npn HccjienoBaHHH onyxojien, BbiBeneHHaa H3 6aHKa KyjibTyp American Type Culture Collection), HaxonHiiiHecH β φ83ε 11

JiorapmļjMHMecKoro pocTa (BbipauieHHbie npH 37°C b TeneHHe 5 cyT. b OHHOJIHTpOBfeIX φ3ΛΒΚΟΗΟΒΟΚΗΧ KOJl6aX AJĪH TKaHeBbIX KyjibTyp) noflBeprajiH npoMbiBKe b cojieBOM φοοφατΗΟΜ 6yφepHOM pacTBope (COBP). iļajiee κ hhm aoOaBJiHJin 0,25%-Hbift pacTBop TpnncHHa ,b 2 mji ΟΦΕΡ. Hepe3 HecKOJibKo mhhjt npenapaT οοτορωκΗΟ H3BJieKajiH H3 KOJi6bi h noMemajiH b cTaicaH, co^epxcamMH 100 mji 25 mM pacTBopa HEPES b “MHHHMajībHOM 3φκρΗ0Μ HOCHTejie” (Minimum Essential Medium) c jioOaBJieHHeM κ MEM 10-% (06. jhojih) HcaKTHBHpoBaHHoii TejmHbeii cbiBopoTKH. 1,1 mji 200mM pacTBopa rjijraMHHa b 0,85%-hom cojieBOM pacTBope, a Taoce 11 000 eaHHHij neHHiļHJiJiHHa Ha 11 000 μκγ cTpemoMnu;HHa riojiy4eHHyio b pe3yjibTaTe KJieToqHyio KyjibTypy, bhobb nepeBeji;eHHyK) bo B3BemeHHoe cocTOHHne, noMemajin nopiiHHMH no 5,0 mji b φaJIbK0H0Bcκne KOJi6bi juih TKaHeBbix KyjibTyp BMecraMocTbio 50 mji m (3a HCKjno4eHneM KOHTpojibHoft npo6bi) CMeuiHBajm c JieKapcTBeHHbiM aKTHBHbiM coenMHeHMeM, YKa3aHHbie nopnnn jjajiee BbmepjKMBajiH b TeMHOTe npn 37°C b Te^eHHe 3 cyTOK. riocjie βτογο KyjibTypy ιοιετοκ H3BJieKajiH h nojļBeprajiH sKCTparHpoBaHHio npn 3ejieH0M ceeie cjiejiyioiHHM o6pa30M.

KjieTKH nojiHHMajm co jiHa koji6 AoOaBJiCHHCM no 0,8 mji 0,25%-ηοβογο pacTBopa TpnncHHa β ΟΦΕΡ. Ilocjie HHKy6aimoHHoro BbipamnBaHHH β τβΜΗοτβ b TeneHiie 1 naca kjictkh h cpeay H3BJieKJiM H3 koji6, noMemajin b KpyrjioaoHHbie npoOnpKn ņjin ueHTpi^yrnpoBaHMii h no^Beprajin jiByM UHKJiaM 3aMopaxcHBanHH orraHBaHHH c uejibio pa3pymeHMH ιαιβτοκ m o6ecneqeHHH bo3mojkhocth 3KCTparnpoBaHHH.

HccjiejioBaHHe KJieT04Hbix B3Beceii nocjie aaHHoft pa3pymaiomeH onepauMH He bbihbhjio HenoBpexmeHHbix ιοιετοκ. 3aTeM b Kaxcayio npo6npKy .nočaBJinjin no 10 mji aiieTOHa co cBoftcTBaMH οοηοββημη (90% aueTOHa: 10% 0,1 Η NH OH), nocjie nero προδηρκΗ nonBeprajin ^eHτpHφyΓHpoBaHHIO juih ynajieHHfl npoTenHa n Kycom<:oB KJieTOK. Bo Bcnjibmaioinee Ha noBepxHocTb BemecTBo ^oOaBJUUiH no 50 mji βολμ, a 3aTeM no 0,5 mji HacbimeHHoro βοληογο pacTBopa NaCl. /ļajiee 12 LV 10303 peryjiHpoBajiH pH ķo BejiHHHHbi 6,8 BBeaeHHeM no KanjiHM 2M pacTBopa KH2PO4. 3aTeM 3anojiHHjin κολοηκυ rana C 8 Baker Prep. aueTOHO-BOflHblM 3KCTpaKT0M. KOJIOHKH npOCyiHHBaJIH H KaxayiO npOMLIBaJIH 2 MJI Bonbī. TeTpanHppojibi BbiMbiBajiH CMecbio CH40H:H20 npw cooTHomeHHH 90 : 10, Β3Ητοίί b oObeMHOM KOJiHHecTBe 2x1,5 mji. Ilocjie 3ΤΟΓΟ SKCTpaKTbl 6bIJIH ΠΟΛΒερΓΗγίΗ ΚΟΛΜΊεΟΤΒβΗΗΟΜγ OnpefleJieHHK) Ha cneKTpocļ)JiyopOMeTpe. B paccMaipHBaeMOM SKcnepHMeHTe 6bum Hcnojib30BaHbi cjie^jTomne jieicapcTBeHHbie aKTHBHbie coeflHHeHHH. A. 5-aMHHOJieByjiHHOBaa Kuc/iOTa, M3BecTHaa KaK npoMexcyT04Hbift προζιγκτ πρπ cHHTe3e TenpanHppojioB.

AicraBHoe coeanHeHHe A 6biJio nociaBJieHo b BHne CTepHJIH30BaHH0r0 φΗΛΒΤρΟΒΒΗΗβΜ 250 mM βοληογο pacTBopa (pH=6,5). AKTHBHbie coeflHHeHHH B, C, I η E 6biJiH nocTaBJieHbi b βηλθ 50 mM pacTBopa b aueTOHe. AueTOH aoOaBJiHJiH Taiace b KOHTpojibHyio npo6y, c TeM HTo6bi KOHueHTpauHH b Hei* cocTaBHJia 0,2% (o6.,ū;ojih). B icjieTOHHbie Ky;ibTypbi ββοληλη TaKHe KOJiHTecTBa yKa3aHHbix aKTHBHbix κομποηθητοβ, κοτορΗβ o6ecneHHBajm 6bi KOHneHTpauHH, npHBe^eHHbie HHXce b Ta6ji. 1. B stoh xce TaSjiHue npeTCTaBJieHbi KOJiHHecTBa nojiyHeHHoro npoTonop(ļ>HpHHa IX TeTpannppojia (Proto IX).

TaOjiHpa 1

HaKonjieHne TeTpanHppojia b o6pa6oTaHHbix KJieTKax HeLa AKTHBHblH ΚΟΜΠΟΗβΗΤ KOHUCHTpaUHH 3ΚΤΗΒΗ0Γ0 areHTa, mkM OjiyopecueHTHaa 3MHCCHH, 400\630 c KoJIHHeCTBO Proto IX, IIHKOMOJIH A 5000 41842 4,2 B 100 12921 1,3 C 100 33477 3,5 D 100 10551 U y 100 8967 0,9 KOHTpojibHaa npo6a 2795 0,3 13

C. A repoimiLObi corjiacHO φορινι>\ιιβ:

D. A repGminabi corjiacHO φορΜ)Μΐβ:

C=CH E. A repSiimtubi corjiacHO φοριν^Λβ: ο α 14 LV 10303 IIpHMep 2

KjieTKH HeLa b ΛΟΓαρΗφΜΗΗβεκοίίί φ83ε pocia BbipaniHBajiH b TeHeHHe 6 cyr. B inecTH ojiHOJiHTpoBbix Φ3λβκοηοβοκμχ KOJi6ax ajih TKaHeBbix KyjibTyp npn 37°C, nocjie nero npoMbiBajin φοοφ3ΤΗΗΜ δγφερΗΗΜ cojieBbiM pacTBopoM (ΦΕΟΡ). /ļajiee Tyzia £o6aBjifljin 0,25%-HblH paCTBOp TpHĪICHHa B ΟΦΒΡ H Hepe3 HeCKOJIbKO MHHyT KJieTKH ocTopoxHo M3BJieKajiH h noMemajiH b CTaKaHbi, coztep^KaiHHe no 110 mji 25 mM pacTBopa HEPES b MHHHMajībHofl^HpHOH cpejie (MEM) c aoēaBJieHHeM 10% (06. aojih) HeaKTHBHpOBaHHoii TejiflHbeft cbibopotkh, 1,1 mji 200 mM pacTBopa rjiyraMHHa b 0,85% cojieBOM pacTBope, a Taoce

I 11 000 ejiHHHLi neHHiiHJiHHa Ha 11 000 mkt cTpenTOMMijHHa.

KyjibTypy κϋβτοκ, BHOBb nepeBe^eHHyio bo B3BeineHHoe cocTOHHHe, noMemajin paBHbiMH nopuHHMH no 5,0 mji KajĶnan bo φaJIbκoHOBcκHe KOji6bi jyiH TKaHeBbixKyjibTyp BMecTHMocTbio no 50 mji. cojiepjKamHe rep6nmin, η npoBonHJiH HHKy6anHOHHyio o6pa6oTKy b TeMHOTe npn 37°C b TeHeHHe 4 cyroK πρηπεΜ aHajiorHHHO ΠρΗΜβργ 1 ΓερβΗΐι,Ηΐι; bbojīhjih b pa36aBJieHHbiH aueTOHOBbiH pacTBop. AneTOH Taoce noGaBJUiJiH h k KOHTpojibHbiM npo6aM ath oOecneneHHH b hhx KOHueHTpanHH aneTOHa 0,2% (06. aojih). KojuiHHecTBO repOmnina 6bUio tbkhm, hto6m oOecneHHTb KOHiieHTpaiiHH, yKa3aHHbie HHMce b TaOjiHue 2. Jļm o6pa6oTKH Hcnojib30BajiH cjieūyioinHe aKTHBHbie κομποηθητμ: B. Kax b npHMepe 1. C. KaK b ĪIpHMepe 1. F. ΓερδΗΐΐΗΛ φopMyJIbI.‘

15 3 ΠρΗΗβΜ KJieTKH, ΠρΗΛΗΠΙΠΗθ Κ flOHlļaM 4>aJIbKOHOBCKHX KOJl6 ocBoāoamajiH B;o6aBJieHHeM 0,5 mji 0,25%-ηογο pacTBopa TpnncHHa b COBP η nocjie BbmepacHBaHHii b Te^eHne HecmjibKHX MHHyr npH 37°C yKa3aHHyio KyjibTypy κπετοκ cjiHBajiH H3 koji6 h BoccoenHHHJiH c KJieTOHHoft cpefloft. /ļajiee οτ κβ)κλοη κΛβτοΗΗοίί cycneH3HH OTOnpajm paBHbie nopiiHH no 100 mici c nejibio onpejiejieHHH πλοτηοοτη ιοιετο^Ηοϋ KyjibTypbi. OcTaBiiiHecH 5,4 mji cycneH3HH KJieTOHHOH KyjibTypbi flajiee 6bum noaBepmyTbi yjibTpa3ByKOBOH o6pa6oTKe b TeneHHe 30 mhh λλη pa3pymeHHH κπετοκ. 3aieM b Kaxc«yio npo6npKy bbohhjih no 10 mji aiieTOHa c ochobhoh peaKuneH (90% aiieTOHa: 10% NH4OH), nocjie nero προβηρκΜ no/iBeprajm ηεΗτρηφ^ΗροΒΒΗηιο c iiejibio yo;ajieHHH npoTenHa h οβρΜΒΚοΒ KiieTOHHOM TKaHH. Bo BcnjibiBaiomee Ha noBepxH0CTb 1

BemecTBO noOaBJuuiH 5 mji bohm, a 3aTeM 0,5 mji HacbimeHHoro βοληογο pacTBopa NaCl. nocjie οτογο BejiHHHHy pH hobohhjih no 6,8 aoOaBJiflfl no KanjiaM 2 M pacTBop KH2PO4. /lajiee nponyKT SKCTparapoBajm H3 cpejibi BOjia/aiieTOH, 3arpy*:a« b KOJioHKy C8 Baxep P ep. KojioHKy cHa^ajia nojicynmBajiH, a 3aTeM 3 mji cmsch MenmoBoro cnHpTa c Bonoft (90:10). AHaiiorn^HO IlpHMepy 1 Bče ynoMHHyTbie onepaiinn SKCTparnpoBaHHH Omjih npoBejieHbi b ycjioBHHX, no cymecTBy nojiHocTbio HCKjnoqaiomHX B036y»aeHHe προτοπορφηρΗΗΒ IX πολ jiencTBHeM cBeTa, a hmchho, npn 3ejieHOM CBeTe hjih b TeMHOTe, HanpHMep, b cocynax c OejibiM ποκρΒίτηεΜ. 3aTeM Ha cπeκτpoφJIyopoMeτpe CPEX onpenejnuin napaMeipbi φ,τ^ορεαιβΗαΐΗ OKCTpaKTOB. KojiH^iecTBeHHbie noKa3aTejin HaKonjieHHfi προτοπορφΗρΗΗΒ (Proto IX) yciaHaBJiHBajiH npH πομοιιιη HaiiweHHbix 3apaHee κοθφφκιιεΗτοΒ 3aTyxaHHH. Pe3yjibTaTbi npejjcTaBJieHbi Ηκοκε b TaSjiHije 2. 16 LV 10303

Ta6jmua 2

Cpejmne noKa3aTejiH 3aMejĻjieHHH pocTa h HaKomieHHH Proto IX ΑΚΤΗΒΗΟΘ coeAHHeHHe KoHueHTpamifl αΚΤΗΒΗΟΓΟ KOMnoHeHTa 3aMezLieHne poda, % KOJI-BO Proto IX, IIHKOMOJIH OraocHTejibHoe cojiepjKaHHe Proto IX B 100 99 5,20 2364 F 100 106 2,71 1232 C 100 52 4,41 2005 C 10 -15* 0,57 259 C 1 -26* 0,19 86 Πρημθ*ι3Ηηλ:* OTpHutaiejibHoe 3HaneHHe noKa3aTejia “aaMejvieHHa pocTa” ΓΟΒΟρΜΤ 0 TOM, HTO pOCT BCe )Ke ΠρΟΗΟΧΟΛΗΤ ** CpejļHee 3HaHeHHe Ha 10 KJieTOK. *** B % no OTHoineHHio κ kohtpojilhom npo6e. ,]ļpyroft acneKT HacTonmero H3o6peTeHHa KacaeTca nojiyaeHHa ΠpOTOΠOpφHpHHa IX (HiDKe Proto IX). B COOTBeTCTBHH C 3THM ocym;ecTBJiiūiM ΓβτεροτροφΗΗβοκοβ BbipamHBaHHe MHKpoopraHH3MOB algae othochihhxch k KJiaccy 3BKapH0T0B, b TeMHOTe (hjih xce npn ocBemeHHH, He Bbi3biBaioineM B036yx«eHHa Proto IX) b cpene, co^epacamett onncaHHoe Bbirne aKTHBHoe coenHHeHHe, HHrH6Hpyiomee 3H3HMaTHMecKoe npeBpameHHe προτοπορφκρΗΗΟΓβΗΒ IX b Proto IX. Cpeay hjih MHKpoopraHH3MH algae, hjih xe η το h apyroe BMecTe najiee o6pa6aTbiBajiH c nejibio 3KCTparHpoBaHHa Proto IX h ero oTflejieHHn ot XJIOpoφHJUIa, KapoTeHOHHOB, oOpbiBKOM KJieTo<mbix TKaHeii h t. n. Pa3,nejieHHe mojkho ocymecTBJum> jhoOmm ο6βι*ιημμ cnocoOoM, HanpHMep, noepejļCTBOM >ΚΗΖΙΚθφΒ3ΗΟΜ XpOMOTOΓpaφHH, BKJIKma JKMJIKOCTHJTO Χρ0Μ0ΤΟΐρ3φΗΚ) C oGpaiiieHHeM φ33Η, HJIH XCe nOCpejJCTBOM pacTBopeHHH pacTBopHTejieM, a Taiotce ocajKaeHHeM Proto IX nyreM ero nepeeoaa b xeJiaTHoe coe/jHHeHHe c MeiajuioM, HanpHMep, xcejie30M, 17 iļHHKOM hjih MarHHeM c nocjieayioiitHM (no acejiaHHio) pereHepnpoBaHHeM Proto IX, KaK 3to flejiaeTCB no H3BecTHOM ΜβτοΛΗκβ o6pa6oTKM xejiaTa pa36aajieHHOH khcjiotom.

OnepaijHH Ha Bcex cTaj;HHX pa3^ejieHHH προβολητ npn ocBemeHHH, He BH3biBaiomeM B036yxcn;eHHfl Proto IX, HanpHMep, npn 3ejieH0M ceeTe, KaK 3to onHcaHO b IIpHJioxceHHH A, 03arjiaBJieHH0M ‘O6pa6oTKa b TeMHOTe”, hjih xce b nojmoft TeMHOTe. XejiaT xejie3a HeiiyBCTBHTejieH κ cBeiy, TaK hto pa6oTa Ha cBeiy npn HcnojiL30BaHHH 3τογο coejjHHeHHH B;onycTHMa b čojibmeft cTeneHH. MHKpoopraHH3Mw algae npejmoHTHTejiLHee BbipamHBaTb bbhjic cycneH3HH KJieTOHHOH Kyjn>Typbi npn TeMnepaiypax 15-30°C. KoHneHTpauHH HHrH6Hpyioinero βκγηβηογο coejiHHeHHH Μοχετ, HanpHMep, cocTaBJiHTb ot 10-5 λο 10-7 M. IIpHMepaMH npHeMJieMbix MHKpoopraHH3MOB algae hbjihiotch Scenedesmus sp/ Chlamydomonas reinhardi, Englena sp. Bumilleriopsis filipormis. IIpHMep 3

KyjibTypbi MHKpoopraHH3Ma Chlamydomonas reinhardi (masa pa3HOBHAHocTb) BbipanļHBajiH b pe3epByape H3 HepacaBeiometi cTajiH, BMeniaiomeM, HanpHMep, 300 ji.c Hcnojib30BaHHeM onHcaHHoii HHxe KyjibTHBaiiH0HH0H cpe^bi. K Kyjibiype jotoOaBJUUiH pacTBop i l-(Kap6o3TOKCH)-3THJi-5-(2-xjiop-4-TpH(ļ)TopMeTHJi<ļ>eHOKCH)-2-ΗΗΊΓρθ6εΗ303Τ3 (Τ.Η. “ΛΒΚΤΟφβΗ”) ΤβΧΗΗΊβΟΚΗΗ 4)eHHJI3<ļ>HpHblfi repČHiiHa) b aueTOHe c nejibio o6ecneqeHHa KOHneHTpanHH βκτηβηογο KOMnoHeHTa ΙΟ"5 Μ h kohchhoh KOHiļeHipanHH aneTOHa, paBHoii 0,1%. CMeCb OCTaBJIKJIH B nOKOe JUin BbipamHBaHHH B Heft MHKpOOpraHH3MOB npH 25°C Ha 4-7 cyT. npn octopojkhom MexaHHHecKOM nepeMeniHBaHHH m/hjim aapHpoBaHHH. npo^ojDKaji BbinoJiHBTb onepaiiHH b TeMHOTe, coaepKHMoe pe3epByapa 0T(j)HJibTp0BbiBajiH; φιυικη^τ no^BeprajiH aHajiH3y Ha npe^MeT o6HapyxceHH« b HeM προτοπορφΗρΗΗΒ IX. Οβ,ηη H3 ΜβτοΛΟΒ aHajiH3a c pejibio o6HapyxceHHa φοτοπορφκρΗΗ3 IX 3aKJiK)qajicH b Φηλβτροββηηη cojiepjKHMoro peaKiļHOHHoro pe3epByapa 18 LV 10303 (Hax(wimerocfl Bce sto BpeMfl b τεΜΗΟτε) h b no6aBJieHHH aiļeTOHa κ φρυιι>Ίρ3τγ. 3aTeM flaHHyio CMeci> noflBeprajiH SKCTparapoBaHMio neTpojieMHHM 3φκροΜ. Ilocjie 3Toro BOflHo-aLieTOHOBjao cpe^y no^BeprajiH SKCTparapoBaHHio λιότηλοβμμ 3φκροΜ, κοτορΒίή ποτομ ya;ajīHJiH Η3 ocamca BbinapHBaHHeM. 3τοτ oca^OK pacTBopium b MeTHJIOBOM CIIHpTe H nO^BepraJIH ΧρΟΜΒΤΟΓρΒφΗΗ C oSpaiJHeHHeM φΒ3ί>Ι c ItejIBIO I10JiyHeHHiI φθΤΟΠθρφΐφΗΗ3 IX. KpOMe ΤΟΓΟ, BO3MC0KHO 6lijio npMMeHeHHe η Μετο^κια* o6HapyxceHHH ααηηογο coeflHHeHHH, onHcaHHOM b npHM8pax 1 H 2.

CocTae cpe^bi COJIH MojiapHaa KOHueHTpauHa, x 10 M KOHuempaT % Mji KOHUēHTpaTa b 1 Jimpe (IļHTpaT Na) 6H20 1,7 10 5 Cjie^bi KaK yKa3aHO KaK yKa3aHO 10 MerajuioB HHxe ΗΗΒΚβ FeCl2. 6H20 0,37 1 1 CaCI2. 2H20 0,36 5,3 1 MgS04. 7H20 1,2 10 3 NH4NO3 3,7 10 3 KH2PO4 2,2 10 3 K2HPO4 1,7 10 3 CH CO Na 7,5 10 10

KoH«eHTpaT CMecH, coAepžKameii cjie^Bi MeTajiJia: μγ/μλ H3BO3 100 Z11SO4. 7H20 100 MnS04. 4H20 40 CoC12. 6H20 20 NaMoQ4 2HzO 20 C11SO4. 4 19

AnbTepHaTHBOH npHMeHeHHio MHKpoopraHH3MOB Chlamydomonas reinhardi aBJineTca npHMeHeHHe KyjibTypbi Scendesmus sp., BbipameHHoft Ha BbimeyKa3aHHOH cpe^e, b κοτοροή aueiaT HaTpHfl 3aMeHeH πιιοκο3οή. IIpHMep 4

ūaHHbiii npHMep aHajioraHeH npnMepy 3 3a HCKJimeHHeM τογο, HTO BMeCTO “JlaKTC^eHa” npHMCHHIOT 1-(4-ΧΛορ-2-φτορ-5-προ^ρΠ'υιοκοΗφ6Ηΐ'υι)-3-Μ6ΤΗ.π-4-ΛΗφτορΜ6ΤΗΛ-Δ2-1,2,4-TpHa30JIHH-5-0H. ΠρΗΜθρ 5

OnpejļejieHHe noKa3aTejia 150 ΠροτοπορφΗρΗΗΟΓεΗ IX (ΠροτοΓβΗ IX) nocTaBmMKOM npenapaTa Proto IX 6biJia φηρι^ “ΠορφΗρΗΗ npo^aKTc” (Porphyrin Products), r. JloraH, uit. K>ra, CIIIA. 3tot npenapaT BbmejimiH b hhctom Bitīte h onuca^H Oiocjiep h coTp. (T.P.Juester etall Plant Physiol 1981, t. 67, c, 246-249). Cbokum ΠροτοΓβΗ IX nojivHaiiH BOCCTaHOBJieHHeM Proto IX c HcnojTb30BaHHeM Na/H aMajībraMbi, KaK 3to ormcajiH ΙΕκεκοδο h ,ΙΕκβκοδο (N.J. Jacobs and J.M.Jacobs: Enzyme, 1982, t. 28, c. 206-219), padoTaBiime c onumeHHbiM Proto IX c KOHueHTpauneit 300 mkM.

PacTHTejibHbiH Maiepnaji

OrypeHHyio paccaya;y (Cacumis sativus L, copT Wiskonsin SMR 18), BbipamHBajm b 3aieMHeHHOH paccauHOH KaMepe Ha BepMHKyjiHTe, CMoneHHOM )κηλκμμ φκρΜεΗΗΗΜ yno6peHneM npH· 25°C H npH OTHOCHTejibHOH BJiajKHOCTH B03,n;yxa 80-90%. Paccan;y ocBemajtH npepblBHCTblM CBeTOM rt03HpOBaHHOH HHTeHCHBHOCTH 25 mkE./m2 c (PAR) ot ynpaB^aeMoro 3.rceKTpoHHbiM TaftMepoM HCTOHHHKa cBeTa Bright Stick φπρΜΗ “/ĻtceHepaji S^eKTpHK” H3 paeneia 1 MHHyTa ocBemeHHH b 20 LV 10303

TeqeHne 60-MHHyTHoro mīma. TaKHM o6pa30M 6bijia nojiyMeHa pacTHTejibHaH TKaHb, o6;iaaaiomaH cnocoSHocTbio ycKopeHHoro cnHT63a xjiopo4)HJUia npH cBe^eHMH κ MaHHMyMy KojinnecTB pe3epBHoro KpaxMa^a h ypoBHeft nepBOHaqajibHoro xjiopo<ļ)HJUia.

H30JlHp0BaHHe XJ10pOnJiaCT0B

BbmejīHIOmHeCH JUIOpOriJiaCTbl 6bIJIH H30JIHp0BaHbI MeTOJIOM, KOTopbift onncajiH Oiocjiep (T.P. Fuesler) η οοτρ. (cm; Plant Physiol., 1984, t.75,c.662-664) 3a MCKjimeHMeM τογο, hto Ha KOHeHHoft CTa^HH ohhctkm njiacmmubi ueHTpi^yrHpoBajiH «iepe3 40%-Hyio (o6.Ķom), a He nepe. 45%-Hyio npoKJiajiKy H3 MaTepnajia Perco II. 3aTeM xjioponjiacTbi 6biJiM BHOBb nepeBe^eHbi bo B3BemeHHoe cocTo^Hne b 6yφepHOM pacTBope npo6, cpiiepacameM 0,5 M MaHHHTOJia, 20 mkM TES, 10 mkM HEPES, 1 mkM 3THJienūHaMMHTeTpayKcycHOH kmcjtotk, 1 mkM MgCl2, 1% (Macc./oObeM) ajib6yMHHa 6bmbeii cbmopoTKH η 1 mkM AHTMoapHTpMTOJia πρΜ pH=7,7 η ητογοβομ coaepiKaHHH npoTeHHa 2 μγ/λ. ĪIpo6bI Πρ0Τ0Π0ρφΗρΗΗ0ΓεΗ-0Κ0ΗΛ33Μ

ΠροδΒΐ 6bijin BbiBefleHbi Taic, KaK sto ormcajm ,ΣΕκβκοδο h 7i)KeKo6c (J.M. Jacobs and NJ. Jacobs Arch. Biochem Biophys 1984, t. 229, c. 312-319). 06pa3iibi (no 0,2 mji) cycneH3HH xjioponjiacTOB 6biJiM noaBeprHyTbi npejiBapHTejibHoii HHKy6aiļMOHHoii o6pa6oTKe b TeMHOTe b TeHeHHe 15 mhh npH pa3JiHHHbix KOHU,eHTpauHHX a^HoφJIyopφeH-Meτ^UIa (AFM) hjih npH KOHpeHTpauHH aueTOHa 0,2% (06. jiojih) b cJiyHae KOHTpoJibHoii npo6w. 3aieM 50 mkji cBe>KenpHroTOBJieHHoro npenapaTa Protogen c KOHiieHTpauHeft okojio 15 hM bbo^hjih b cycneH3Mio c nejibio MHHiiHHpoBaHHii peaioiHH. 3aBepniajiacL· no^roTOBKa προβ BBezieHneM 2,75 mji φτοροΜετρΗΗεοκοΓο cpeacTBa, κοτοροε πρε,ιυιοχαυίΗ jjacefcobc h ϋχεκοδο (J.M. Jakobs and N.J. Jakobs Enzyme 1982, t. 28 c 206-219), coztepjKamero 1% (06. hojih) tbmh-20 (MOHOJiaypaT

no^HOKCM3THJieHcop6HTaHa), 50 mM Tpuc-HCl, η 1 mM 21 3THJiewi;HaMHHTeTpayKcycH0H khoiotm πρπ pH=8,5, npimeM ImM ūHTHoapHTpHTOJia 6buiH 3aMemeHbi 5mM rjijnraTHOHa. 3aTeM B3Becn aHajiH3HpoBajiH ΜετοΛΟΜ npaMoro HTeHHfl Ha οπεκτροφΛγοροΜβτρβ SPEX Fluorolog-2, ocHameHHOM jinueBbiM HHHHKaTopoM φΛγορβοΐίβΗυΗΗ 3aMyTHeHHbix 6HOJionmecKHX o6pa3uoB. KojiH<iecTBo o6pa30BaBiuerocH Proto IX Onpe^eJIHJIH no CTamiapTHOH KpHBOft Iļ^HKa 3aBMCHMOCTM Mexcay KOJiHHecTBeHHoii SMHccneft Proto IX npn 630 hm h B036yxcaeHHeM npH 400 hm, npHHeM jiSiHHd.ii KpHBaH 6biJia noJiyHeHa ηηοτογο Proto IX bo φ^οροΜβτρηΗεοκοϋ cpeae.

KojiHHecTBeHHoro onpenejieHHH cieneHH He3H3HMaTHqecKoro oKHCJieHHH £0 Proto IX b onncaHHOH Bbmie προβε πρηγοτοβηλη tohho TaKyio xce npo6y 3a HCKJiioHeHHeM τογο, hto bmccto cycneH3HH aKTHBHbix xnoponjiacTOB Mcnojib30Bajin cycneH3Hio, b κοτοροή xflopomiacTbi 6buin nepeee^eHbi b HeaKTHBHoe cocTOHHHe HarpeBaHneM npn 85°C b TeHeHne 15 mhh. BbinHTaHHe KojinnecTBa Proto IX, nojiyqeHHoro tbkhm o6pa30M, H3 ero KOJin^ecTBa, nojiyqeHHoro b npHcyrcTBHH ajcraBHbix xjioponjiacTOB Kax pa3 h flaeT kojihhcctbo Proto IX, κοτοροβ o6pa30Bajiocb 3H3HMaTHHecKHM nyreM. Pe3yjibTaTbi npeacTaBJieHbi η^φη^εοκΗ Ha pnc. 1, rae hSD-HaHMeHbinee 3Haqamee pa3;iHHHe (odmenpHHiiToe o6o3Ha*ļeHHe). Phc.1 noKa3biBaei, mo noKa3aTejib I50 (KOHiļeHTpanHH, o6ecne4HBaK>maH 50%-Hoe HHraOHpoBaHHe) cocTaBJiaeT MeHee 0,1 mkM, HanpHMep, okojio 0,03 mkM.

HcnbīTaHHe Ha BJiHHHHe 10 mkM AFM Ha 3H3HMaTHHecKoe npeBpameHHe Proto IX b Marae3HajibHbiH Proto IX b cycneH3HH XJioponJiacia (b npHcyrcTBHH ATP) He noKa3ajio HHKaKoro HHrH6Hpyioiiiero Bjnumm AFM Ha sto npeBpameHHe. ĪIpHMep 6

CorjiacHO £aHHOMy npHMepy HHrH6HpyiomHe 3H3HMbi aKTHBHbie KOMnoHeHTbi 6buin .uoēaBjieHbi b nHmy Mbimeft, HBjnnomHxcH HocHTejiHMH onyxojieH, Toraa Kax b nHiny HecKOjibKHx κοητρολβηηχ Mbiinefi HHKaKHX npenapaTOB He floāaBJiiuiH. ΓΙοα/ιε 3τογο Mbiinen 22 LV 10303 yMepiHBJIHJIH H M3BJieKajIH HX ΠΟΗΚΗ, KHIIieHHHKH, ΗΒ&ΠΟΗδΗΗΗΚΗ, neneHb h onyxojiH, noziBeprHyB hx aHajiH3y Ha coflepxcaHHe Proto IX. BbiJiH Hcnojib30BaHbi cjiejļyiomHe HHrn6HpyiomHe 3Η3ημβι aKTHBHbie coejiHHeHHfl: B HaCTHOCTM onblTbl npOBO^HJIM Ha Mbimax JIHHHH DBA/2 Ha, κοτορΜΜ b HyjieBOM jieHb b npaeoe ruie^o bbohhjih MUTberauHeM πολκοκηηη onyxojreBbift npenapaT SMT-F. /ļajiee b nepBbift ^eHb mbillih nojiyMajiH cneunajibHyK) nHmyipifl rpbi3yHOB: Purina Rodent Chow 5001 J Hasli 6e3 ao6aBOK. iļajiee Ha BTopbie-^ecaTbie cyrKH mmihh nojiyHajm Ty xce rmmy c flo6aBKoft HcnbiTyeMoro HHrn6npyio]nero 3H3hm βκτηβηογο COe^HHeHHH B KOJIHHeCTBe 2 npOMHJIJie (KOHTpO.lbHbie XCHBOTHbie nojiyHajiH Taicyio xe rwmy, ho 6e3 /io6aBOK). ,Iļo6aBKy bbohhjih b nmuy nyreM nepeMeiiiHBaHHH c He6ojibuiHM KOJinnecTBOM pacTBOpa HcnbiTyeMoro βκτμβηογο coeanHeHHii b aueTOHe. Jļajiee Mbiuieft yMepmBJi5uin Ha aecaTbie cyTKH 3a HCKJiiOHeHHeM μμιιιη, o6pa6oTaHHoft aKTHBHbiM coeaHHeHHeM, o6o3HaHeHHbiM KaK 6j: 3Ty Mbinib yMepmBJUUiH Ha ce^bMbie cyTKH. TKaHM χηβοτηηχ ocTaBJiium Ha ox.iaxaeHHe Ha HOHb npH 4°C, a 3aieM yaajiHJiH BJiary npoMaKaTejibHoft 6yMaroH h onpeAejuum Maccy KajKuoro o6pa3ua TKaHH. /lajiee H3 TKaHeft iotobmjih

23

24 LV 10303 roMoreHHyio cycneH3Hio b 5mji (b cjiyHae TKaHeft KHineHHHKa h πεπεΗΗ-10 mji) cMecu aueTOHa h 0,1 Η NH4OH, B3HTbix b oObcmhom cooTHoiueHHH 9:1. 3aTeM roMoreHHbie npenapaTbi 6buiH ΙίεΗΤρΗφγΓΗρ0Β3ΗΒΓ npH 1500 Γ Η BCrUIBIBUIHe Ha nOBepXHOCTI> BeiIļeCTBa 6buiM nojļBeprHyTbi aHajiH3y Ha cneKTpe<ļ>;iyopoMeTpe SPEX FA112. JJjiz Proto IX onTHMajībHbiMH «BJiaiOTCH B036yxaeHHe πρκ 400 hm h smhcchh npH AJiHHe BOJiHbi 630 hm. HaiconjieHHe Proto IX onpe^ejiHJiH no HHCJiy φ,π^ορβοηβΗΤΗΜΧ BcnbīuieK b ceKynay Ha 1 r TKaHH. Pe3yjibTaTbi npe^cTaBJieHbi ηηχθ b TaSjmne 3 (min Ka*aoro npenapaTa 3HūH&Hm HBJIHIOTCil epeflHHMH RJM napbl MblUieH 3a HCKJIIOHeHHeM HCnblTaHHM c HCnOJIb30BaHHeM aKTHBHbIX COeflHHeHHft 6f, 6h, 61 h 6j, Korfla mn HcnbrraHMH OpajiH TOJibKO o^Hy Mbiuib).

TaSjmua 3

Hhcjio <ļ)JiyopecueHTHb[x BcnbīuieK (χ 103"1 c) Ha 1 r TKaHH ajih npenapaTa Proto IX AKTHBHOe coeflHHeHHe Onyxojib ne<ieHb ΠΟΗΚΗ Haano leHHHKH KHUie>IHHK Macca onyxojieH, OTpearHpoBaBiueti Ha B03A6HCTBH6 ΜΓ 6a 1203 4291 10817 6572 5870** 143 6b 1520 1261 2441 2529 5577** 90 6c 14336 2739 16186 13790 9937** 157 6d 2006 1581 12865 443 5565** 294 6e 837 876 915 1331 2069** 165 6f 449 1170 661 5611 1157 69 166 803 606 1984 466 250 6h 298 914 585 2259 584 192 6i 932 1510 4014 36940 3454 20 6j 1251 538 315 491 572 11 KOHTpOJIb 239 536 494 1504 233 200

KoHTpOJIbH. 25 414** ΠρΗΜΘ»ΜΗΗΗ: * TaSjiHMHbie aaHHbie cjienyeT cHHTaTb c κοοφφκιιεΗτοΜ x 10. ** H3MepeHMH npoBeaeHbi Ha o6pa3uax, pa36aBJiCHHbix 3 q aueTOHa c ocHOBHbiMH cBoftcTBaMH Ha 1 h o6pa3ua. YKa3aHHbie ztaHHbic b Ta6Ji. 4 HHCJia cootbstctbjtot KOHIļCHTpaiJHflM Proto IX B COOTBeTCTByTOmHX ΤΚΒΗΗΧ. E^HHHUa H3MepeHHH: yjibTparpaMMbi (yr) Proto IX Ha 1 r τκβημ.

TaSjiHua 4 ' AKTHBHOe coeaHHeHHe Onyxojib IleHeHb ΠΟΗΚΙΙ HaanoHeHHHKH Khuichhhk 6a 11 39 99 60 54 6b 14 12 22 23 51 6c 131 25 148 126 91 6d 18 14 117 4 51 6e 8 8 8 12 19 6f 4 11 6 51 11 6g 2 7 6 18 4 6h 3 8 5 21 5 ; 6i 9 14 37 337 32 6j 11 5 3 4 5 KOHTpOJIb 2 5 5 14 2 4 B u,HTnpoBaHHoii Bbirne cTaTbe Λ^φερτΗ (Dougherty) npHBC/teHo 3Ha4eHHe 3,6 yr/r ķusi KOHUCHTpauHH πορφπρΗ^ β οπγχοΛΗ τογο >κε BHaa SMTF IIOCJIC HHbCKUHH 10 ΜΓ/ΚΓ ΓεΜ3Τ0Π0ρφΗρΜΗ3 MbirnaM toh >κε jihhhh DBA/2 Ha. Β λρ>τοη HayHHoii ny6jiHKauHH aBTopoB MoaH h οοτρ. (Moan etal: P&P 46-5, c/ 713-721,cm. npHM. Ha pHC. 2 Ha οτρ 716) yica3aHo, hto b cjiyHae onyxojieft THna CH/Tif KOHitcmpauMio 26 LV 10303 nopcfjHpnHa nopaaKa 12 yr/r, o6ecneqHBaeMyio b 6pioiiiHyK> nojiocTb MbiuiaM ληηηη DBA/H2 ΦοτοφρΗΗΒ II (Photofrin II) b KOJiH^ecTBe 25 μγ/κγ, ιιιπροκο npHMeHaiOT jajisi npiinaHHa qyBCTBHTe^bHOCTH K CBeTy πρπ φθΤΟΛΜΗ3ΜΗΗεθΚΟΜ JieneHHH H ^HarHocTHpoBaHHH paKOBbix 3a6o^eBaHHii.

Mmuih noe^ajiH cjie,qyioiHHe cyMMapHbie KO-imnecTBa πηιηη, coitepacaineH aKTHBHoe coe^HHeHne : 6a) 22 h 35r; 6) 37 h 35r; 6c) 25 h 22r; 6d) 41 h 32r ; 6e) 40 h 44r; 6f) 27r; 6g) 33 h 40r; 6h) 36r; 6i) lOr; 6j) 20r. ΠρΗ npoBeneHHH 3KcnepHMeHTOB} onHcaHHbix b ^aHHOM npHMepe 6, Ha ΧΗΒΟΤΗΒΙΧ, Ηβ HBJIHK)IHHXCfl HOCHTejIHMH 0nyX0Jieft, BbinOJIHHJiaCb HH^cecjieayiomaa nocjieaoBaTejibHocTb onepaijHft c Hcnojib30BaHHeM (ecjiM He oroBopeHO MHoe) aKTHBHoro coeaHHeHHB, κοτοροε b λβηηομ ΠρΗΜερε HMeeT o6o3Ha4eH.He 6c. а) B pe3y^bTaTe onpeaejieHHH noKa3aiejiH LD50 βκτηβηογο coejiHHeHHH oh OKa3ajicfl cBbiiue 2600 μγ/κγ ( το-ecTb, μγ βκτηβηογο coeAHHeHHH Ha I κγ Maccbi Tejia) jopia Kpbic, Koma stot noKa3aTejib onpeflejiHJiH 4epe3 14 flHeft nocjie ojjHOKpaTHoro noe^aHHa χκηβοτημμ nopuHH Kyicypy3Horo Macjia, co^epacamero 15% 3κτηβηογο coe/jHHeHHH, h cBbirne 700 μγ/κγ jum MbirneH, Konta aaHHbiH noKa3aTejib onpe/i;e;iH;iH 4epe3 14 ^Hen nocjie o^HOKparaoro noe^aHHH χηβοτηβιμ nopiļHH Macjia, cojiepacamero 5% aKTHBHoro coeztHHeHHa. б) ΠρΗ HCnbITaHHBX COCTaBOB JUM HHteKIļHft B 6pK)UIHyK) nOJIOCTb, He co/iep)KamHX εκτμβηογο coe^HHeHHH, 6biJia ycTaHOBJieHa cnocoOHocTb MblUieH nepeHOCHTb ΗΗΒεΚΗΗΗ npH Λ03ε 0,5 MJI CMeCH paBHbIX ΛΟΛβΗ ΛΗΜβτΗ^ΛΒφοκαΗ^ η βολή. B) ΠρΗ HCnbITaHHHX aKTHBHOrO COeflHHeHHa, ΒΒΟΛΗΜΟΓΟ HHteKUHeft b 6piomHyio nojiocTb b cocTaBe cMecn coaepacaiuefi 60% Kyxypy3Horo Macjia m 70% ziHMem/icyj^0KCH,āa, 6buio ycTaHOBJieHo, mto MbiniH cnoco6Hbi nepeHOCHTb J[03y 100 ΜΓ,ΚΓ. 27 r) Ha Mwuiax 6ηλμ npoBeaeHbi cjie,ayiomHe 3KcnepHMeHTbi: 1) eacejiHeBHoe cKapMJiHBaHHe λο3μ 50 μγ/κγ (c Mcnojib30BaHHeM 1%-ηογο pacTBopa aKTHBHoro coeaHHeHHfl b aueTOHe) Ha προτΗχεΗΗΗ 8 flHeH, 2) eaceiiHeBHoe BBeaeHHe ηητ>6κηηη b δριοιπΗγιο nojiocTb Λ03Η 50 μγ/κγ Ha npOTHJKeHHH 8 ΛΗβίί C HCnOJIb30BaHHeM 1%-HOMi aHCnepCHH aKTHBHoro coeaHHeHHH b MMHepajībHOM Macjie, npHroTOBJieHHoii paCTBOpeHHeM aKTHBHOrO COe£HHeHHfl B Kanjie ΛΗΜβΤΗΛΟγΛΒφοΚΟΗ^ H cMeuiHBaHHeM nojiyHeHHoro pacTBopa c MHHepajībHbiM MacjioM, 3) exeaHeBHoe MecTHoe HaHeceHne Ha Koacy ao3bi 50 μγ/κγ Ha

npOTJDKeHHH 8 ΛΗβίί C HdT0JIb30BaHHeM 1%-ΗΟΓΟ pacTBopa B AHMeTHJīcyjib4)OKCHa;e, 4) KopMJieHHe Ha npoTiDKeHHH 8 ΛΗείί cTaHjiapTHOž nHmeft c ZlOēaBKOH aKTHBHoro COeztHHeHHH B KOJTHMeCTBe 2 npOMHJIJie Oļ Maccbl riHIHH, 5) eaceflHeBHbie BHyTpHBeHHbie ΗΗΤ>εκυ,ΗΗ Λ03Μ 50 μγ/κγ Ha προτπχεΗΗΗ 4 ληοη c Hcnojib30BaHHeM 2%-ηογο pacTBopa b Π,ημ eTHJi cyji Βφο kc nae. 3aieM Mbiiueft, B3flTbix μη 3KcnepHMeHTOB, yMepmBJiHJiH, h onpe^ejiaiiH HaKon^eHHe Proto IX b ηχ τκβηλχ. B apyroM 3KcnepHMeHTe caMua Kpbicbi jihhhh Fisher 344 Ha npoTflaceHHH 24 hhch kopmhjih ΠΗΐηείί, coaepxameH 5 npoMHJUie aKTHBHoro coe^HHeHHH 6c, ποοπε qero jkhbothoc yMepmBJiajiH. B τκβηηχ 3toh Kpbicbi 6buio 3aMeqeHo noBbimeHHe ypoBHeft coaepacaHHH Proto IX ΠΟ CpaBHeHHIO C KOHTpOJIbHbIM JKHBOTHbIM, πρπΗβΜ 3aMeTHoe noBbimeHHe sthx ypoBHCH HaOjnoaajiocb b τκ3ηηχ ποηκη, KMLLiCHHHKa, xcejiyaKa η γολοβηογο M03ra, Toraa KaK b τκβηηχ Mbirnu; οτΜεπεΗο jiHiiib HeKOTopoe noBbimeHHe ypoBHH. Πρκ npoBeaeHHe onHcaHHbix Bbime 3KcnepHM6HTOB Ha Kpbicax h Mbimax χηβοτημχ co^epxcajiH πρκ CTaHaapTHOM pexcHMe ocBeiueHHa: 12 q ocBemeHHa h 12 q TeMHOTbi. 28 LV 10303 IīpHMep 7 Πρη npoBe^eHHH cepHH 3KcnepHMemoB corjiacHo IIpHMepaM 1 h 2 KyjibTypbi κπετοκ HeLa HHKy6HpoBanH b npHcyrcTBHH nepeHHCjieHHbix HiDKe jieKapcTBeHHbix βκτηβηβιχ coe^HHeHHH c KOHneHTpaiļHeH 100yM. ripHBeaeHHbie nocjieKaxcaoro o6o3HaqeHHii JieKapcTBeHHoro aKTHBHoro coe^HHeHHa noKa3aiejiH b %, noKa3biBaiOT yBejiHqeHHe KOJiHHecTBa Proto IX, o6pa3yiomerocH b npHcyrcTBHH aaHHoro JieKapcTBeHHoro aKTHBHoro coejļHHeHHH, no oTHouieHHK) κ TOMy KOJiHiiecTBy, κοτοροβ o6pa3yeTC« b κοητρολβηομ o6pa3ite b tom ace 3KcnepHM6HTe.

Hcnojib30BaHHbie aKTHBHbie coe^HHeHHH H3 ripHMepa 6 noKa3ajiH cjien;yK)mHe 3HaqeHH5i: 6a) 337%; 6b) 366%; 6c) 297%; 6e) 1210% 6d) 1200%; 6f) 280%; 6g) 326%; 6h) 431%; 6i) 544%; 6j) 469%. <DopMyjibi ūpyrax 3κτηβημχ coeOTHeHHH h cooTBeTCTByioiHHe nponeHTHbie 3HaHeHHfl ΛΠΗ ΗΗΧ npHBejreHbl HHJKe. I· IīpHMep 8 B .aaHHOM IIpHMepe HHrH6HpyiomHe 3H3HMbi aKTHBHoe coeaHHeHMe 6c H3 IIpHMepa 6 CKapMJiHBajiH KpbicaM c πρηβητημη onyxojWMH, nocjie qero onyxojieByio 30Hy o6jiyHajm cbctom, Bbi3biBan τεΜ caMbiM paccacbiBaHHe onyxojiH. B nacTHocTH TpeM KpbicaM jihhhh Spraque-Dawley Maccoft b cpe^HeM no 120 r Ha nepnoA b 6 AHeft 6hji Ha3HaneH paiļHOH πητβηηη c floSaBKOfi aKTHBHoro ΟΟβΛΗΗβΗΗΗ B KOJIHHeCTBS 2 npOMHŪJie. Ha TpeTbH CyTKH Ha npaByiO 38ΑΗΙΟΚ> KOHeHHOCTb KaiKAOŪ KpbICbl HMnJiaHTHpOBajIH xoHūpocapKOMy nocpe^cTBOM ηηέοκηηη 0,3 mji cycneH3HH onyxojieBbix KJieTOK (Imhjijihoh onyxojieBbix ιαιετοκ). Ha 6-e cyTKH npaByio 3aHHioio KOHeHHocTb Kaxcaoii[ Kpbicbi oOpHBajiH zuih ynajieHHH BOJiocHHoro noKpoBa h H3MepHJiH pa3Mepbi KajKUOH onyxojin. jdajiee oOjiacTb, nopaxceHHyio onyxoJibio, h oKpecTHbie ynacTKH κόχη o6jiy4ajiH HeTeruioBbiMH αο3βμη ceeTa c ΛΠΗΗΟίί BOJiHbi 630 ημ πρη iijiothocth noTOKa SHepran 270 JJik/kb.cm. Ha cjieAyioinHH jļeHb oKa3ajiocb, 4to aBe Kpbicbi b 3aMeTHoii 29 CTeneHH H36aBHjiHCb ot cbohx onyxojieii (το-ecTb, Macca onyxojw 6ojibine He npomynbiBajiacb, a y TpeTbeft οπγχοΛΒ nomu coBepiueHHO paccocanacb. Y Bcex Kpbic oicppicaBiiiHe onyxojib yHacTKH kojkhoh h MbimeMHOH TKaHeii cjienca no6e;iejin h Ka3ajiHCb KaK 6bi Ha6yxuiHMH, ho b 3HaHHTejibHO MeHbiueft CTeneHH, Heacejm sto MMeeT MecTO b φοτοΛΗΗΒΜΗΜβοκοίί TepariHH c Hcnojib30BaHMeM npenapaTa Photoprin II. 7a.

7b.

30 LV 10303 7c.

31 ΠρίυίΟΧβΗΗβ Α

HcnbīTaHHe Ha onpe^ejieHHe npoueHTHoro noKa3aTejia BbiMbiBarom B xoķq HcnbīTaHHH Ha onpeaejieHHe npou;eHTHoro noKa3aTejui BbiMbiBaHHH npHMeHHJiH ceMH^ojīH, coSpaHHbie H3 npopameHHon B TeMHOTe orypeqHoft paccaabi. Ha nepBOH cnumH Maccy ceMimojieft o6pa6aTbiBajiH b TeMHOTe βοληβιμ pacTBopoM (b npHcyrcTBHH βγφβρΗΟΓο coejļHHeHHa), co^epacaiitHM caxap c paaHoaKTHBHbiMH MeneHbiMH aTOMaMH. ,0,ajiee H3MepajiH KojinnecTBO caxapa, ycBoeHHoro ceMHflOJiHMH, no onHcaHHOH hick6 ΜετοβΗκε pacieTa. 3aTeM bck) Maccy ceMa^ojien flejiHJiH Ha ^Be πορηκΗ. OjHy H3 sthx nopHHH o6pa6aTbiBajiH b τεΜΗΟτε BOflHblM paCTBOpOM (B npHCJTCTBHH 6y<ļ)epHOrO COeflHHBHHfl), co^epMcamHM HcnbiTyeMoe coe^HHeHHe, a apyryio tohho TaK ace o6pa6aTbraajiH b τεΜΗΟτε τεΜ xce pacTBopoM, ho 6e3 HcnbrryeMoro coe^HHeHHH (KOHTpojibHaa npo6a). 3aMoqeHHbie b yKa3aHHbix βοληβιχ pacTBopax ceMHaojiH ^ajiee o6jiyHajm εεετοΜ b τεπεΗΗε 16 nacoB, nocjie Hero HX OT^eJIflJIH OT ΒΟΛΗΒΙΧ paCTBOpOB, B KOTOpbIX ΜβΤΟΛΟΜ H3MepeHHH h pacqeia onpeaejinjiH co^epxcaHHe BemecTBa c pa^HoaKTHBHbiMH ΜβΗβΗΗΜΗ aTOMaMH. rTojiyqeHHbie pe3yjibTaTbi 6mjih 3aTeM npeflCTaBJieHbi b BHfle npoiieHTHoro noKa3aiejiH BbiMbiBaHHH (ΠΠΒ), paccHHTMBaeMoro no φopMyJIe: nnB=(st - sc) /s γλβ S, St Sc - cooTBeTCTBeHHo kojih^ctbo pacnanoB (Ha o^Hy ceMimojno) BemecTBa c ρ^ηοκτηβηβιμη Me^eHMMH aTOMaMH: а) b 3TOM BemecTBe, ycBoeHHOM ceMHfloJfiīMH bo BpeMa hx nepBOHaHajībHOH o6pa6oTKH, б) b boji;hom pacTBOpe, coaepjKameM HcnbiTyeMoe εοεβΗΗεΗπε, nocjie o6jiyHeHHH εεετοΜ h B) B KOHTpOJIbHOM BOflHOM paCTBOpe ΠΟΟΙίε o6jiyHBHHfl CBeTOM.

Bojiee πολρο6ηο Hcnojib30BaHHbie MaTepnajībi, a Tarace ycjioBHa npoBe^eHHH ηοπβπήηηη Ha onpe^ejieHHe npou;eHTHoro noKa3aTejia BbiMbiBaHHH oxapaKTepH30BaHbi HHXce. 32 LV 10303

PacTHTejibHHft MaTepna/i B BepMHKyjiHTe, CMoqeHHOM MMeiomHMca b npoaaxe yn;o6peHHeM 9-45-15, 6buiH npopameHbi ceMeHa oiypu;a Cocumis salivus L., copT Uisconsin SMRjg, H3 κοτορΗΧ b HHKy6aiiHOHHOH KaMepe npH 25°C h OTHOCHTeJIbHOH BJia^KHOCTH B03,ayXa 80-90% BbipaiIiHBajIH B TeMHOTe paccajiy. CnycTH 5 cyr. c MOMeHTa BbiceBa η3-πολ 6jie,ņHbix, BbipameHHbix b TeMHOTe poctkob pacca^bi coOHpajm ceMimojiH h npoMHBajiH hx b 1,0 mM pacTBope CaCl2. Bce sth onepaiļHH npOBozuuiH πρκ 3ejieHOM CBeTe. ΕγφερκΜΗ pacTBop.

PacTBop co/tepjKHT 1 mM KC1, 1 mM CaCl2, 2,0 mM φοοφάτζ Ksuim h oTperyjiHpoBaH ķo pH=6,5 (KaK η πρκ ηοπολβ3οβ3ηηη NaOH).

Caxap C pajļHOaKTHBHbIMH ΜΘΜβΗΜΜΗ aTOMHMH 3Ta pa3HOBnn,HOCTb caxapa npeacTaBJiaeT co6oh 3-0-MeTHJi-3-[U-14C]-rjuoK03y co οπβυΗφΗΗβοκοίί aKTHBHocTbio 10,9 ΓΒκ/μΜ: nocTaBmnK; φκρΜ8 “AMepuisM Kopn.” (Amersham korp.)> Γ· Apjihhitoh xaftTC, uiT. Hjijihhohc, CIIIA.

IlepBOHa^ajībHaa o6pa6oTKa 180-230 uiTyK npoMbiTbix ceMjmojieft bbojihjih b iimpoK:oropjiyK) 3pjieHMeHepoBCKyio KOJi6y BMecTHMOCTbK) 250 mji c προΟκοΜ H3 BcneHeHHoro nojiHypeTaHa, cozi;epxamyK) 50 mji 6yφepHOΓo pacTBopa, Kyaa BioOaBJiiuiH Tarace caxap c pajiHoaicraBHbiMH MeqeHbiMH aTOMaMH b KOJiHMecTBe, očecneHHBaiomeM ero KOHueHTpaiļHio b pacTBope, paBHyio 600 hM. Koji6y nojiBeprajiH BCTpaxHBaHHio b TeMHOTe Ha BpamaioiiieMCH ycTpoiicTBe b Te^eHHe 24 η npH 125 mhh'1. 3aTeM ceMaaojiH BbirpyxcajiH Ha chto H3 HeftjioHa h noflBeprajin 3-KpaTHoft npOMbiBKe nopiļiuiMH 1m pacTBopa CaCl2 no 20 mji KaxcaaH. ycBoeHHe caxapa c pazuioaKTHBHbiMH MeqeHbiMH aTOMaMH onpe^ejiajm, pacTBopaq 3 o0pa3Lia no 5 ιιιτγκ ceMimojieft KajKjībiii b pacTBopHTejie TKaHeH (MapKa NCS, προη3βολοτβο 33 φκρΜΗ “AMepmaM Κορπ.”) η noflCHHTbiBajiH HToroBoe HCTomeHHe Ha cneicrpoMeTpe χαιακοοτΗοή cummuuimmi·

BlīJIO yCTaH0BJI6H0, 4T0 3TH 3KCnepHMeHTbI Ha HCTOUteHHe SKBHBajieHTHH aHajiorHqHOMy onpeaejieHHio, ocymecTBJuieMOMy nyreM OKHraHHH oro6paHHbix npo6 ceMimojieft b caMOOKHCJiHTejie. 06pa6oTKa c Hcnojfb30BaHHeM HcnbiTyeMoro οοθαμηθημη, a TaKHte KOHTpojibHaa o6pa6oTKa Πίτπ> ιιπγκ ceMHAOJieft ocTaBJiium iuiaBaTb HeoceBOH cropoHoft BBepx Ha noBepxHocTH 6yφepHOΓO pacTBopa, HajiHToro b KOJiHHecTBe 3 mji b ruiacTMaccoByio HaiiiKy Πβτρκ ^HaMeipoM 35 mm c KpbmiKOH. 3aTeM Ty^a bbo^hjih ncribnyeMoe coe^HHeHHe (b B*we ero pacTBopa b aueTOHe) b KojiH^ecTBe, o6ecneqHBaiomeM 3an,aHHyio KOHiieHTpaimio (ο κοτοροή cKa3aH0 Bbirne) HcnbiTyeMoro coe^HHeHHH b 6yφepHOM pacTBope. CoflepacaHHe aijeTOHa b 6yφepHOM pacTBope cocraBJisuio 0,1 % (06. λολη) KaK b KOHTpojibHOH npo6e, TaK h b pacTBope, coflepiKameM HcnbiTyeMoe coe^HHeHHe. jjajiee ruiaBaiomHe ceMaaojra b TeneHHe 16 nacoB coBepmajiH KpyroBoe flBHHceHHe b BonoBopoTe, o6pa3yioiii;eMCH b pe3yjibTaTe BCTpaxHBaHHH hsluiqk c nacTOToft 90 mhh-1 Ha noBepxHocra BpamaioinerocH BCTpaxHBaiomero ycnpo^TBa. 06jiy*ieHHe cbgtom

HauiKH ΠετρΗ c ceMflaojiflMH, iuiaBaiomHMH Ha noBepxHocTH pacTBOpOB, o6jiyqajiM b TeneHHe 16 qacoB cbctom neTbipex φ^ορεου^ΗΤΗΒΐχ JiaMn GE F20T12-CW πρκ 3a^aHHOH HHTeHCHBHOCTH 150 mkE/kb.m. c b cneKTpajībHoft oOjiacra φοτοοΗΗΤ633 (COOC), npHHeM ynOMHHyryK) HHTeHCHBHOCTb H3MepflUIH Ha nOBepXHOCTH ceM^nojieK, b to BpeMH KaK ηβιηκη nonpBeprajiHCb HenpepbiBHOMy BCTpHXHBaHHK). 34 LV 10303

IĪ3MepeHHe KOjnraecTBa HaxoAHmeroca b «hakocth Bemecrea c pa/moaKTHBHUMH MmeHbMH aTOMaMH

Bcio )κηλκοοτβ OT6HpajiH ot ceMimoJieft h 3aTeM onpe^ejiHJiH hhcjio pacnanoB Ha cneicrpoMeTpe xhakocthoh ci1hhthjmu;hh.

TeMHOTa

Bce bhhh o6pa6oTKH λο cTajiHH o6jiyHeHHH cbctom βηποληηλη b TeMHOTe HJIH npH 3e^eHOM CBeTe φΛγορβΟΙίβΗΤΗΟΓΟ HCTO^HHKa, TO eCTb npH CBete npoiue^ineM cKB03b 3ejieHbiii oTceicaioiiiHH macTMaccoBbift ΟΒβΤΟφΗΛΒΤρ, npOnyCKaiOmHH CBeT C flJIHHaMM BOJIH 450-600 HM.

IīpHjio^eHHe B

Culture Media Cpe^a jum KyjibTyp Ta6jimj;a 5

Cpe^a M

Cojih MojiapHOCTb KomiempaT mji KOHijempaTa Ha Iji iļHTpaT Cjiejļbi MeTajuioB CM. HHXe MgS04.7H20 1,2 x 10*3M 10% 3 NH4NO3 3,7 x 10'3M 10% 3 KH2P04 2,2 x 10-3M 10% 3 K2HP04 1,7 x 10‘3M 10% 3

KoHueHTpaT CMecH, co,nepJKameH cjie^bi Merajuia Stock of Trace Metals Mbcture

H3BO3 100 mg/L ZnS04«7H20 100 mg/L MnS04«4H20 40 mg/L C0C12*6H20 20 mg/lL NaMo04*2H20 20 mg/L CuS04 4 mg/L 35

Cpe#y A λλλ KyjibTyp γοτοβηλη, λο6ββληη k cpe^e M 10%-hmh βολήωϋ pacTBOp aijeTaTa HaTpHH c KOHiļeHTpauHeft 7,5 χ 10"3 M b KOJiHHecTBe 10 mji/ji.

Bce κομποηοητη ββοληλη b ληοτηληροβ3ΗΗυιο βολυ b πορΛΛκε, yKa3aHHOM Bbinie, ποατιε nero ocymecTBjiiuiH hx o6pa6oTKy b aBTOKJiaBe ΠΟΛ Λ&ΒΛΒΗΗεΜ 15 φγΗΤ/ΚΒ. ΛΚ)ΗΜ B TBHCHHe 15 MHH. 3anac KOHcepBHpoBaHHoro npenapaTa λλη BbipaumBaHHH Hyjibiyp πολ Λ6Η0ΤΒΗ6Μ CBēTa

3aK0HcepBHp0BaHHe κγΛΒτγρΗ κΛβτοκ y-l aKceHHHecKH ΠβρβΒΟΛΗΛΗ B ΧΗΛΚΗβ KyJIbiypbI Ha cpeae A ΗΛΗ Μ, ΠρβΛΠΟΗΤΗΤεΛΒΗΟ Ha ερεΛε A, b 3pjieHMeiiepoBCKHx Koji6ax, 3aKpbirbix ποΛΗγρετΛΗΟΒΜΜΗ npoOKaMH, npH 25°C b υολοβηηχ iļHKJia: 14 h ocBemeHHa h 10 h TeMHOTbl. KOHCepBHpOBaHHbie Ky^bTypbI ΠΟΛΒερΓΒΠΗ HHKy6HpOBaHHIO (6e3 ΛοποΛΗΗτεΛΒΗΟΓΟ aapnpoBaHHH) Ha BpamaiomHxca BcipaxHBaioiiiHX ycTpoftcTBax, pa6oTaK>mnx c qacTOTOH 125 mhh'1. HHTeHCHBHOCTb ocBemeHHH Ha ypoBHe cjioh KyjibTypbi cocTaBJiana 120 mkE/kb.m.c. (COOC). B 3thx υολοβηηχ KyjibTypbi η3χολητοη b pexoiMe ΠΟΛγΟΗΗΧρΟΗΗΟΓΟ pOCTa ΛΟ ΤβΧ ΠΟρ, ΠΟΚΛ OHH Ηβ nepefimyT ;B CTaHiļHOHapHyio φΛ3γ οοΛβρχΛίΐίγκ) (2-4) χ ΙΟ6 κπετοκ β 1 μλ.

BbipamHBaHHe KyjibTyp β otc^ctbhh cseTa (KyjibTypbi,

BbipamHBaeMbie β τβΜΗοτε)

KjieTKH, HaXO^mHeca Β CTaiļHOHapHOH φ33β KyjIbTHBHpOBaHHH H3 3aKOHcepBHpoBaHHOH κγΛΒτγρΒΐ, BbipaiueHHbie Ha CBeTe β KOJinnecTBe 7,5 μλ, nepeHOcmiH b 3pjieHMeftepoBCKyio KOJi6y, εοΛερχΛίΐίγκ) 750 μλ ερβΛΒΐ A. JOieTKH Π0ΛΒερΓ3ΛΗ HKy6HpoBaHHio b TeMHOTe πρκ aapupoBaHHH nepe3 3anry6rteHHyio a3paiiHOHHyio Tpy6xy npw TeMnepaType 25°C b TeneHHe 3-4 cjtok. 3a 3to BpeMH Amnan KynbTypa ιαιετοκ y-l λολ;<κη3 6bma πρετερπετΒ 7-8 ΚΛετοΗΗΒΐχ ΛεΛβΗΗίί, jrcpaHHBajī Becb βηλημβιη ΧΛΟροφΗΛΛ, npH KOHIļ8HTpaiļHH KJI8TOK (2-3) X 106 UIT/ΜΛ. 36 LV 10303

ΠρΗΓΟΤΟΕΙΙβΗΗβ KJieTOK fl/IH HCn0JIb30BaHHS B ΠροδΗΜΧ UCnblTRHHKK

KjieTKH OTČHpajiH m CBexenpnroTOBJieHHOH Kyjibiypw b

KojiHHecTBe 750 mji. BbipameHHOH b τβΜΗΟτε, nocpe^cTBOM ΠβΗΤρΗφ)ΤΗρθΒΒΗΗΗ H3 MaJIbIX o6opOTaX (OKOJIO 2000 MHH'1) ΠρΗ 20°C Β TeqeHHe npH6jiH3HTejibHO~5 mhh. 3th ιαιετκΗ octopojkho nepeBonHJiH orom» b cocTOJiHHe cycneH3HH b 50 mji cpejjbi A. B npo6e flaHHoii cycneH3HH (okojio 0,25 mji) onpejļejiflJiH πολβηχηοοπ, κκετοκ, a nocjie 3aĶperuieHHH 1%-hbim bozjhmm pacTBopoM rjijnrepaJībflerHiia-nOflCHHTblBajIH 4HCJI0 KJieTOK B 1 MJI. 06HHHO HHCJIO KJieTOK COCTaBJIfleT 5xl07 πιτ/mji. ΠορπΗΗ KyjibTypbi κπετοκ no 1 mji, cojļepjKaiiĻHe no 107 uit. KJieTOK, noMemajm b ncnbīTaTejibHbie cocyabi, HanpHMep b φ3ϋΒΚ0Η0ΒΒΐε TapejiKH c 24-mb jiyHKaMH, npejj;Ha3HaqeHHbie jļjm κιιετοπΗΜΧ KyjibTyp. Ha stoh crajiHH KJieTKH BecbMa nojļBjracHbi h oKpaineHbi b «ejiTbiū πβθτ.

HcnbiTyeMoe coenHHeHHe pacTBopajin b no,nxonain;eM pacTBOpHTejie, HanpHMep, b aneTOHe, othjiobom cnnpTe, ji;HMeTHJicyj^0KCH,ae hjīh b Bone (npennoHTHTejibHo b aneTOHe) ajih o6ecneneHHH KOHneHTpanHH, 1000-KpaTHo npeBbimaiomeH KOHeqHyio. B KajĶiijTo napy jiyHOK πρη πομοιπη MHKpomnpnna BMecTHMOCTbio 5 hjih 10 mkji ββολμη no 1 MKJi ynoMHHyroro HcnbiTyeMoro pacTBopa. Ha Kascnoft TapejiKe jļjih KJieToqHbix KyjibTyp totobhjih 4 KOHTpojibHbie jijthkh aHajiornHHo TOMy, KaK sto onncaHo Bbiuie, ho npn otcytctbhh HcnbiTyeMoro coejļHHOHHH. TapejiKH jļjui ιαιετοπΗπχ KyjibTyp HaKpbiBajin np03panHbiMH iuiacTMaccoBbiMH KpbiniKaMH m noMeinajiH Ha 13-16 q b KaMepy BbipainHBaHHH npn TeMnepaType 25°C n HHTeHCHBHocTH ocBemeHHH 70-90 mkE/kb.m.c. Ecjih conepJKHMoe ncnbiTaTejibHbix jiyHOK xcejiTejio, to ero H3BJieKajin TaK, KaK sto onncaHo Hioce b n. “Onemca pe3yjibTaTOB”. Ecjih xce conep>KHMoe HcnbiTaTejibHbix JiyHOK oKa3MBajiocb npo3paqHbiM hjih 6jienHO-3ejieHbiM, το ero nepen H3BJieqeHHeM noMeinajiH Ha BpaipaioineecH BCTpaxHBaioinee ycTpoficTBO, pa6oTaioinee c 37 MHH _Ι, Η Β TeqeHHe 2 Η o6jiyHajIH ΟΒβΤΟΜ ΓφΗ HHTeHCHBHOCTH OKOJIO 600 mkE/kb.m.c.

Ouemta pe3yjibTaT0B B Kax5yK) HcnbiTaTejibHyio jiyHKy bbo^hjih 250 mkji 10%-ηογο βοληογο pacTBopa AOjtenH jicyjibφaτa Haipna η 1 mji ΜβτκυιοΒΟΓο cirapTa, nocjie qero co^ep^HMoe jiyHKH TiitaTejibHO nepeMeuiHBajiH. rioji^eHHbie cMecH BbiflepjKHBajiH β τβΜΗοτε b Te^eHHe 3-4 q. TapejiKH c KJieTOHHbiMH KyjibTypaMH o6pa6aTbiBajiH Ha ΗβΗτρκφγτε Ha Majibix o6opOTax (okojio 2000 mhh'1) npH KOMHaTHoii TeMnepaType b TeneHHe 5 mhh. BcmībiBaiomee Ha noBepxHocTb BemecTBo chhm3jih h aHajiH3HpoBajiH Ha οπβκτροφοτοΜβιρε “EeKMaH” (Bekman), 35-Mo^ejib b HHTepBajie rjihh BOJIH 350-500 HM (Ha HajIHHHe ΠρΟΤΟΠΟρφΗρΗΗΒ IX, ΛΛΗ ΚΟΤΟρΟΓΟ B cHCTeMe c ynoM«HyTbiM pacTBopuTejieM xapaKrepeH πηκ πογλοιηβηηη χϋοροφΗΛΛ3 npn 668 hm), a Taioce npn 720 hm, .zļtch τογο ητο6η HCn0^30BaTb φΟΗΟΒΟε nOKa3aHHe MyTHOCTH. AHajiH3 aaHHbL\

JĻrra Ka^caoM jiyHKH BbinHTaHHeM 3HaHeHHH npn 720 hm H3 3HaHeHHH npH 668 hm nojiyMajiH noKa3aTejib no3ejieHeHHa. CoOTBeTCTByK>mHe ΒβΛΗΗΗΗΜ HBJIHIOTCH Cpe^HHMH ffjIX KaxgļOH KOHiļeHTpaiļHH HcnbiTyeMoro coeflHHeHHii, a Tarace κοΗτροjibhom npo6bi. ΠρορεΗΤΗΜΗ noKa3aTejib HHraOHpoBaHHH Haxo^HT no φορΜγϋβ: Q = 100 x (I - k), m;e κ - οτΗοιπβΗΗβ cpe,a;Hero 3HaqeHHH noKa3aie^H 03ejieHeHHH jduih ^aHHOH KOHKpeTHoft KOHneHTpaHHH κ cpejļHeMy 3HaieHHK) noKa3aTejiH no3ejieHeHHH KOHTpojibHOH προβΜ. 38 LV 10303

ΦΟΡΜΥΜ H30EPETEHHH 1. npHMeHeHHe ΟΠβΙίΗφΗΗβΟΚΟΓΟ COeflHHeHHH, HBJIHIOmerOCH ΗΗΓΗβΗΤΟρΟΜ 3H3HMaTHqeCKOrO IIpeBpaiHeHHH npOTOnOp$HpHHOreHa B ΠρΟΤΟΠΟρφΗρΗΗ IX ΠΟ^ B03^eMCTBHeM Πρ0Τ0Π0ρφΗρΗΗ0Γ6Η0ΚΟΜ33Μ iijm y6nB3HHH κτιβτοκ onyxojiH mojiohhoh x:ejie3hi, B03^eHCTBya Ha sth KJieTKH CBeTOM. 2. OapMaueBTHHecKag κομπο3ηιι,ηη jjji% Hcnojib30BaHroi corciacHO cnoco6y y6MBaHHH κιιβτοκ onyxojm mojiohhoh xejie3bi nyreM B03fleftCTBHH Ha 3TH KJieTKH CBeTOM B npHCyTCTBHH TeTpaiIHppOJia, CnOCOāHOrO K aKTHBHpOBaHHK) CBeTOM, npHHeM Κ0ΜΠ03Η11ΗΗ HBJIHeTCH cTepHJībHoft, omAUHatoiuflRCR meM, qTo conepjKHT coenHHeHHe HBJIHIOmeeCH αίβΙΙΗφΗΗβϋΚΗΜ ΗΗΓΗβΗΤΟρΟΜ 3H3HMaTHHeCKOTO npeBpameHHH ΠρΟΤΟΠΟρφκρΗΗΟΓεΗα B ΠρΟΤΟΠΟρφΗρΗΗ IX ΠΟΛ fleHCTBHeM ΠρΟΤΟΠΟρφΗρΗΗΟΓβΗΌΚΟΗΠ^Μ B yKa3aHHbIX KJieTKaX, H Bbi3biBaiomee TeM caMbiM o6pa30BaHHe προτοπορφΗρκΗΕ IX b sthx KJieTKax; npHHeM ynoMHHyroe coejiHHeHHe ΗΜβετ noKa3aTejib I50 flJifl προτοπορφΗρΗΗθΓβΗ-οκοΗΑ33Η, cocTaBJunomeft MeHee qeM 1 mkM, h Κ0ΜΠ03ΗΗΗΗ mKKe coaepxMT φ3ρΜ3ΐιεΒΤΗ^εοκΗ npHeMJieMyio cpeny- H0CHT8JIb. 39 3. OapMaiieBTH^ecKaa κομπο3ηιι;ηη jum. Hcnojn>30BaHHH cornacHO cnoco6y y6HBaHHH ιαιετοκ onyxojiH mojiohhoh xcoji03bi nyreM B03fleiicTBHH Ha 3th kjictkh cbētom b npHcyTCTBHH TOTpanHppoAa, t cnoeo6HOrO K aKTHBHpOBaHHK) CBeTOM, npHHeM Κ0ΜΠ03ΗΑΗ5Ι HBAAOTOfl CTepHJībHOH, omAunammjanca meM, hto οοΑερχαπτ εοεΑΗΗεΗΗε HBJIJnOmeeCH ΟΠείίΚφΗΗεεΚΗΜ ΗΗΓΗβΗΤΟρΟΜ 3H3HMaTHHOCKOTO npeBpameHHH προτοπορφκρΗΗΟΓεΗ b προτοπορφκρΗΗ IX ποα aefiCTBHeM ΠρθΤΟΠθρφΗρΗΗΟΓεΗ-ΟΚΟΗΛΒ3Η B yKa3aHHbix κπετκΒΧ, H BH3biBaiomee τεΜ caMbiM o6pa30BaHHe προτοπορφκρΗΗ IX b sthx KJieTKax; πρππεΜ ynoMHHyroe coe^HHeHHe He hbjihotch TeTpanHppojioM h oSjia^aeT OKHCJiHTejibHO-BOccTaHOBHTejibHbiM noTeHHHajiOM 6ojiee OTpHnaTejibHHM, HexcejiH-500 mB, η κομπο3ΗΠΗΗ oonepacHT Taic«o φΒρΜΒπεΒΤΗΗεεκπ npHeMJieMyio cpeay-HocHTe^b.

4. ΟπεπΗφΗΗεοκΗή ηηγη6ητορ 3H3HMaTHHocKoro npeBpaineHHH προτοπορφκρΗΗΟΓεΗ b προτοπορφκρΗΗ IX ΠΟΑ fleŽCTBHeM ΠρθΤΟΠθρφΗρΗΗΟΓεΗ-ΟΚΟΗΠΑ3ΒΙ Β KAOTKaX OnyXOJIH ΜΟΑΟΗΗΟή xcejie3bl, BbI3bIBaiOII];HH TeM OaMbIM 06pa30BaHH8 ΠρΟΤΟΠΟρφκρΗΗΒ IX Β 3ΤΗΧ KJT8TKax; πρκπεΜ ynoMHHyTbiH ηηγη6ητορ o6jia^aoT noKa3aTeji8M Ι50 ΠρθΤΟΠθρφΗρΗΗΟΓΟΗΟΚεΗΑ&3ΒΙ, COCTaBJIflK>lli;HM ΜεΗεε 1 mkM πρκ HCn0JIb30BaHHH Β ΛεΗεβΗΒΙΧ 110JUIX.

5. ΟπείίΗφΗ'ΤεεΚΗΗ ΗΗΓΗ6ηΤΟΡ 3Η3ΗΜ3ΤΗΠεεΚ0Γ0 np8BpaiH8HHH

Πρ0Τ0Π0ρφκρΗΗ0ΓεΗ3 Β ΠρΟΤΟΠΟρφκρΗΗ IX ΠΟΑ A0HCTBH8M Πρ0Τ0Π0ρφΗρΗΗ0ΓεΗ-0ΚεΗΑ33ΒΙ Β KJIOTKaX OnyXOJIH ΜΟΑΟΗΗΟ& )K0JI83bI, BbI3bIBaiOni;Hft ΤεΜ CaMbIM 06pa30BaHH8 ΠρΟΤΟΠΟρφκρΗΗΒ IX Β 3ΤΗΧ KJi8TKax; πρπτεΜ ynoMHHyrbiH ηηγη6ητορ o6jiaAa8T noKa3aTBJiBM I50 ajih προτοπορφΗρΗΗθΓεΗ-οκεΗΑ&3Η, cocTaBJiinonļHM ΜεΗεε 1 mkM πρκ HCn0Ab30BaHHH JUM φοΤΟΑΗΗΜΗΠεεΚΟΓΟ H3JienOHHH onyxoJiefi..

6. ΟπεΠΗφΗΗεεΚΗΗ ΗΗΓΗβΗΤΟρ 3Η3ΗΜ3ΤΗΗ6εΚΟΓΟ np8BpaUX8HHH

ΠρΟΤΟΠΟρφΗρΗΗΟΓεΗ Β ΠρΟΤΟΠΟρφΗρΗΗ IX ΠΟΑ AeilCTBHOM 40 LV 10303 προτοπορφΗρΗΗθΓβΗ-οκοΗΛδ3Η b K/ieTKax onyxojm MOJIOHHOH XCeJie3bI, BbI3bIBaK)mHH TeM CEMhlM HaKOIUieHHe ΓφΟΤΟΠΟρφΗρΗΗΒ IX B 3ΤΗΧ KJieTKax; npHneM ynoMHHyrbm ηηγηΟητορ He «BJineTcn TeTpanHppojioM h o6jia^aeT OKHCJiHTejibHO-BoccTaHOBHTejibHbiM noTeHiiHajioM, 6ojiee oTpMiļaTejibHbiM, Hexcejm-500 mB πρκ Hcnojib30BaHHH Aira Jieqe6Hbix uejieft. 7. ΟπβΙίΗφΗΗβΟΚΗή ΗΗΓΗβΗΤΟρ 3H3HMaTHHeCKOrO npeBpameHMJI ΠρθΤΟΠθρφΗρΗΗΟΓβΗ3 B ΠρΟΤΟΠΟρφΗρΗΗ IX ΠΟΑ AeKCTBHeM προτοπορφΗρΗΗθΓ6Η-οκοΗ,π;33Η b KJieTKax onyxoAH MOJIO^HOH xejie3bl, Bbi3biBaK>mMM τεΜ caMHM o6pa30BaHHe προτοπορφκρΗΗα IX b yKa3aHHbix κκετκβχ; npHneM ynoMHHyrbift ηηγη6ητορ He HBJiaeTCfl TeTpanHppojioM h o6jia^aeT oKHCjiHTejibHo-BoccTaHOBHTejibHbiM noTeHiinajioM, 6onee OTpnnaTe;ibHbiM, Hexejin-500 mB npu HCn0JIb30BaHHH ΛΛΗ φθΤΟΑΗΗ3ΜΗΗεθΚΟΓΟ H3Jie4HBaHHH OnyXOJieft. 8. IīpuMenenue cnein^HHecKoro coeAHHeHHfl, BBJiaiomerocH ΗΗΓΗδΗΤΟρΟΜ 3Η3ΗΜΒΤΗΗβΟΚΟΓΟ npeBpameHHfl ΠρΟΤΟΠΟρφΗρΗΗΟΓεΗΒ B ΠρΟΤΟΠΟρφΗρΗΗ IX ΠΟΑ Β03ΑθήθΤΒΗβΜ ΠρθΤΟΠθρφΗρΗΗΟΓ6ΗΟΚΟΗΛΗ3Η Μ Λ nOBbimeHHH CO^epacaHHH ΠρΟΤΟΠΟρφίφΗΗΑ IX Β KJieTOK3X onyxojra M0JI04H0H XeJie3bI.

9. npuMenenue οπβΐΗίφίΓΊβοκοΓΟ coejļMHeHHR, ABAAioiAerocH ΜΗΓΗδΗΤΟρΟΜ 3Η3ΗΜ3ΤΗΗεθΚΟΓΟ npeBpameHHil ΠρΟΤΟΠΟρφίφΗΗΟΓεΗΒ Β ΠρΟΤΟΠΟρφΗρΗΗ IX ΠΟΑ B03^eMCTBHeM ΠρθΤΟΠθρφΗρΗΗΟΓεΗΟΚΟΗΛΒ3Η AJia ΑΗΒΓΗΟΟΤΗΚΜ HJIH JIOKajIH3ai];HH KJieTOK OnyXOJIH MOJIOMHOH acejie3bl nOCJie o6jiy4eHHB CBeTOM, aKTHBH3HpyK)IHHM ΠΟρφκρΗΗ, Π03Β0ΛΗΒ AHaraocTHpOBaTb onyxojib no ero φΛγορβοπβΗΠΗΗ. 10. Cnoco6 nojiyneHHH προτοπορφκρΗΜ IX, omAUHatouņiūcn meM, hto BbipamHBaioT ajTcapHOTH'iecKHe MMKpoBOjjopocjin b TeMHOTe hjim xe npn 41 OCBemeHHH, He aKTHBHpyiOmeM ΠρΟΤΟΠΟρφίφΗΗ IX, B npHCyrCTBHH 3φφ6ΚΤΗΒΗΟΓΟ KOJIH^eCTBa apHJI-TpHa30JIHHOHa, apHJI-TeTpa30JIHHOHa HJIH apmi-TpHa3HWi;HOHa, Ka)K£bIH H3 ΚΟΤΟρΗΧ HBJIHeTCH ΗΗΓΗβκτορΟΜ 3H3HMa- ΠρθΤΟΠθρφΗρΗΗΟΓ6Η-ΟΚϋΜΛ33Μ. 11. Cnoco6 no n. 4, omAmaiouņiūca mm, hto MHKpoBOflopocjm othochtch κ po^y Chlamydomonas. 12. ΟπβΙΙΗφΗΜβόΚΗΜ ΗΗΓΗβΗΤΟρ Π0 JIK)60My 0«H0My H3 Π:Π. 4-7, omumaiouĻuiica mm, hto yKa3aHHHft ηηγη6ητορ npe^cTaBJineT co6ofi 1-(4-ΧΛθρ-2-φτορ-5-ΠρθΠ3ρπυΐΟΚ0Ηφ6ΗΗΛ)-3-Μ6ΤΗΛ-4-ΛΗφΤ0ρΜ0ΤΗ;ΐ-Δ2-1,2,4-TpHa30JMH-5-1. 42

Claims

LV 10303 IZGUDROJUMA FORMULA 1. Specifiska savienojuma, kas inhibē protoporfirinogēna fermentatīvo pārvēršanos par protoporfirīnu IX protoporfirinogēnoksidāzes iedarbībā, tādā veidā izveidojot šūnās protoporfirīnu IX, pielietojums piena dziedzera audzēja šūnu iznīcināšanai, iedarbojoties uz minētajām šūnām ar gaismu.
2. Farmaceitiskā kompozīcija sterilā formā, kas paredzēta piena dziedzera audzēja šūnu iznīcināšanai, apstrādājot minētās ar gaismu tetrapirola, kurš spēj aktivēties ar gaismu, klātienē, atšķiras ar to, ka tā satur savienojumu, kurš ir specifisks protoporfirinogēna fermentatīvās pārvēršanās par protoporfirīnu IX protoporfirinogēnoksidāzes iedarbībā inhibitors minētajās šūnās, tādā veidā minētajās šūnās izveidojot protoporfirīnu IX, pie kam minētajam savienojumam l50 protoporfirinogēnoksidāzes inhibēšanā ir mazāks par 1 μΜ; kā arī farmaceitiski pieņemamu vidi-nesēju. i·
3. Farmaceitiskā kompozīcija sterilā formā, kas paredzēta piena dziedzera audzēja šūnu iznīcināšanas paņēmienam, apstrādājot minētās ar gaismu tetrapirola, kurš spēj aktivēties ar gaismu, klātienē, atšķiras ar to, ka tā satur savienojumu, kurš ir specifisks protoporfirinogēna fermentatīvās pārvēršanās par protoporfirīnu IX protoporfirinogēnoksidāzes iedarbībā 1 inhibitors minētajās šūnās, tādā veidā minētajās šūnās izveidojot protoporfirīnu IX, pie kam minētas savienojums nav tetrapirols un tā redokspotenciāls ir negatīvāks par -500mV; kā arī farmaceitiski pieņemamu vidi-nesēju.
4. Inhibitors, kurš ir specifisks protoporfirinogēna fermentatīvās pārvēršanās par protoporfirīnu IX protoporfirinogēnoksidāzes iedarbībā, nomācējs minētajās šūnās, tādā veidā minētajās šūnās izveidojot protoporfirīnu IX, pie kam minētajam inhibitoram l50 protoporfirinogēnoksidāzes inhibēšanā ir mazāks par 1 μΜ, to izmantojot ārstnieciskos nolūkos.
5. Inhibitors, kurš ir specifisks protoporfirinogēna fermentatīvās pārvēršanās par protoporfirīnu IX protoporfirinogēnoksidāzes iedarbībā nomācējs minētajās šūnās, tādā veidā minētajās šūnās izveidojot protoporfirīnu IX, pie kam minētajam inhibitoram l50 protoporfirinogēnoksidāzes inhibēšanā ir mazāks par 1 μΜ, to izmantojot audzēju fotodinamiskai ārstēšanai.
6. Inhibitors, kurš ir specifisks protoporfirinogēna fermentatīvās pārvēršanās par protoporfirīnu IX protoporfirinogēnoksidāzes iedarbībā nomācējs minētajās šūnās, tādā veidā minētajās šūnās izveidojot protoporfirīnu IX, pie kam minētais savienojums nav tetrapirols un tā redokspotenciāls ir negatīvāks par -500 mV, to izmantojot ārstnieciskos nolūkos.
7. Inhibitors, kurš ir specifisks protoporfirinogēna fermentatīvās pārvēršanās par protoporfirīnu IX protoporfirinogēnoksidāzes iedarbībā nomācējs minētajās šūnās, tādā veidā minētajās šūnās izveidojot protoporfirīnu IX, pie kam minētais savienojums nav tetrapirols un tā redokspotenciāls ir negatīvāks par -500 mV, to izmantojot audzēju fotodinamiskai ārstēšanai. 2 LV 10303
8. Specifiska savienojuma, kas inhibē protoporfirinogēna fermentatīvo pārvēršanos par protoporfirīnu IX, protoporfirinogēnoksidāzes iedarbībā, tādā veidā izveidojot šūnās protoporfirīnu IX,pielietojums protoporfirīna IX satura paaugstināšanai piena dziedzera audzēja šūnās.
9. Specifiska savienojuma, kas inhibē protoporfirinogēna fermentatīvo pārvēršanos par protoporfirīnu IX, protoporfirinogēnoksidāzes iedarbībā, tādā veidā uzkrājot šūnās protoporfirīnu IX, pielietojums piena dziedzera audzēja šūnu diagnosticēšanai vai nomākšanai pēc to apstrādes ar gaismu, kas aktivē porfirīnu, dodot iespēju noteikt audzēju pēc tā fluorescences.
10. Paņēmiens protoporfirīna IX iegūšanai, kas atšķiras ar to, ka kultivē eukariotiskas mikroskopiskās aļģes tumsā vai tādā apgaismojumā, kas neaktivē protoporfirīnu IX efektīva daudzuma ariltetrazolīna, ariltetrazolinona vai ariltriazīndiona klātienē, katrs no kuriem ir fermenta protoporfirinogēnoksidāzes inhibitors.
11. Paņēmiens pēc punkta 10, kas atšķiras ar to, ka mikroskopiskās aļģes pieder Chiamydomonas dzimtai.
12. Specifiskais inhibitors pēc jebkura no punktiem 4-7, kas atšķiras ar to, ka minētais inhibitors ir 1 -(4-hlor-2-fluor-5-propargiloksifenil)-3 metil-4-difluormetil-A2-1,2,4-triazolīn-5-ons-1. ♦ 3