D22
`
`(~T~MyC~Kpylon~ee Ae~CTBRe ~a~]oKo6a~aM~Ha ria pOeT
`onyxo~ ~aaaoro at~a ~ao~x (capRoMa Kyp Payca, ~6pceap~o~a
`~W-~, cap~oua 4~ ~ CCK ~p~c, Kap~m!oua repe~a, tapioca 180 n
`~ocap~oua ~me~) ~-~ oc~a~aenne aeqeOnoro ~eficTmm ~teKO~OpS~X
`Boonyxonesmx npe~,apaToB npH COBMeCTHOM HX np~MeseHHH c BHTaMMtlOM,
`Bte, OTMeqaeMB~e B paHHHX Hccae~oBaH~IRX, o6ycnoBne~ flKTHBII~IM
`.CHHTe3OM er0 K0~epMO~TOB B opraHHSMe 2K}IBOTH~X. O~e~Ka ~yHK~HO~anB-
`aO~ pOaH M~HaKO6anaMHHa--O~HOFO H3 KO~SaaMH]{OBBIX KO~epMeHTOB
`B np01Leccax po~a HOpMaabtt~x ~ onyxoaeB~X K~eTOK--~pHBneKaeT llaH-
`6onsmee BHHMaHHe.
`~THaK06aaaMHH KBaHeTC~ Ko~epM~HTOM MeTHOHH~ICHHTeTaSHO~ peaK-
`. ~MH--KaIOqeBoFo 3BeHa, onpe~e~;o~eFo cH[leprx3~ Ae~BHK KO6anaM[~I-
`HOB ~ coe~HeH~ ~OSHeBO~ KHCaOTBI B npo~eccax Kae~qtlofi npon~epa-
`~HH. Oco6as 3H~IMMOCT~ MeTHaKO~aaaMHHS AaH SKTHBa~HH DTO~ (~epMetIT-
`B0~ CHCTeMSI oTMeqeHa B pesynsTaTe Hay~eHM~ HapymeHHOZ0 OOMeHa
`
`Ba}IIiOA ~MTOCTaTHqeCKO~ TepanH]~ ~pH o~pe~eBHslX BapMaHTax OCTpOFO
`~e~Kosa, RpOTeKaIO~HX C B~COKO~ KoH~eSTpa~e~ MeTSaKO6anaMHHa
`RpOBH, nO~TB ep~ana CHe~H~HqHOCTB eFo ~£~CTB~H B opI’aHHSMe
`(H. B. ~ScH~eBa H coaBT., 1969). B Hac’zoa~ee BpeM~ ycTaaoBaet-la
`aKTHBHSH poab Me’rKaKO6aaaMSHfl B npo~eccax npoaH@epa~n KaeTOK KpO-
`BeTBopKo~ TKaBH ~0pOBbIX ~HBOTHSIX. ~0~ BOa~e~gTBH~M
`a~a B ceneseHKe M~e~ Bo3pacTa~T qHCnO Kae’roK, CHI{Te3Hpylo~Hx
`~, EX MHTOTHKecKa~ SKTHBHOCTb H BenH~I~a npon~epaT~nl-[o~o nyaa
`(0, ~. FoaeHKo , coasT.). O6Hapyx~ellO Sl-laqHTen~Hoe yBea~iqeH~m
`T~ pSSBHTHS FeMo6~aCTO3OB y Mbl~e~ npH KOM6HHnpoEaHI-IOM BBeAeHHH
`meT~aKo6aaaMHBa c SH~0FeHHBIMH 6naCTOMOPeHaMH. BS~(HblM MOMeHTOM
`MexaH~sMa CTMMy~MpyIO~erO ~e~CTBE~ Ko6aaaMtiHOB gBa~eTCA I.IX
`~Mpylo~ee BJfH~HHe Ha flKTHBHOCT5 MeTHOIIHHGHHTeTflSb[. B KynbTypax HOp-
`MansHMX ~eTOM MaeKonl{Tal0~[X ~ OnyXoaeBblX KneTOK qenoseKa 8KTHB-
`"OCTb MeTMOIIHHCHHTeTaSbI SaMeTWO Bo3pacTa~ c yBenHWeHHeM co~ep~ca-
`
`H COaBT.). OnyxoaeBme K~fiTI~tl paaHOrO THna, o~HaKo, OT~}]qIIbI OT HOp-
`MaJI~HBIX HO CBOe~ C~OC06EOCTH HO~ BOa~OfiCTBHeM KO6a~MHHOB yCB~II-
`BaTB 6HOCKHTe3 MeTHOHIiHfl, HeO6XOKAMBIfi npH HIITOHC~IBHOM pocTe (Hal-
`pern H coaBT.; Che]10 H Berfino). C~acaTe.~SH~lfi ~yT5 C HOMO~hI0 KO-
`6aaaMMHSaBHCHMO~ MeTI~IOI-tHT~CHHTeTa3bI, o~ecneqHBaR yBe~HqeHHe BHyTpH-
`KneTOt]IIOP0 nyna TeTpaFH~po~o~Heso~ KHCnOT~] H~aBHCHMO OT ~)OJlaTpe-
`~yKTBSH0~ CHCTeM51, npe~fiTaBa~eT, H0-BB~HMOMy, OCHOBHO~ Mexa;¢HSM pa3-
`BMTHK yCTO~KHBOfiTH ne~KOat~ix K~eTOK K MeTOTpeKcaTy (~TX)
`(H. B. M~e~xm~a; Sauer ~ Jaenieke).
`B CBR3H c ~THM pean~a BO3M0)KHOCTb yc~neH~ HpOTHBOOHyXOBeBOFO
`s~Oe~a ~aHHOFO meTa6On~Ta nyTeM eFo KOM6~HHpOBaHHOrO
`72
`
`Sandoz Inc.
`Exhibit 1026-0001
`
`Teva – Fresenius
`Exhibit 1026-00001
`
`

`
`/~ a T e p H a n st M e’r o K t~. NceJ,e~o,~aaml nposeKe~b, tm ~.t~amax ~m.tCs C~BL. CBA.
`BALB/c H rH6p]{~ax BDF,/C~vBLx DBA(2), Macco~
`
`~I~ CC~P. ~pOTHSOOnyxo~lena~ flKTI.IBHOCTB a~a~oros MeTH~KO6a~a~HHa
`penun, eM~X ~efiKoaax L-1210
`~eaut (Ca-755), paKe me~K~
`~[b~ Bm~pa~IH B Ka~IeCTBe O~HOBIIOFO O~KT~ H~0~HH~ ~O~tI~Hbl~ O~yXOn~, H~ KOTO-
`
`Mt,~me~ L-1210 ~ La, c BmCOKI.IM HpOaH~epaTMBHBIM
`
`~eTHa~¢o6a~amH~ (CH~Cbl) ~ ~OTopxo~opMa~HaKO6aaaM~s (GFsG[Cb[) no~eK~ mo
`
`(E. ~. TaqKosa n coasT.). XaopnaaaoxaT
`
`l0 MKP/KF 2 pasa Ha ~¢ypc aeqettaa c WI~TepBa~oM 96
`
`~ ~Hefi. ~l~OXO paCTBOpHM~ x~opnaa~;o~a~
`
`dmpMt,l cLederle~ tlCH0~b3OBflaH B
`n II~I~HX HCC~£~OBaltH~X ~KTHBIIDCTb KO6adIflMHHOBMX npo~sBO/il.lMX HS~telt8 H£ TOZb-
`
`KO ~pll I(OM6HHIIpOBSHIIOM npl.IMel.lenHH
`
`(NSC-176319) :
`
`N
`
`0
`
`~) CH~- ~Be
`
`73
`
`Sandoz Inc.
`Exhibit 1026-0002
`
`Teva – Fresenius
`Exhibit 1026-00002
`
`

`
`.~,oa a ripe-
`
`npenapa’~a, % ~ ~<sHTpo..lO
`
`I0
`10
`I0
`
`tO
`
`++74 180
`+20
`
`+126
`
`+21
`+65
`+23
`
`+~7
`
`+33
`
`2-fl ~ 6.fl
`
`Ca-755 Cv~BL
`BDF:~
`AKAq’OJ-I:
`C~MKH
`
`74
`
`Sandoz Inc.
`Exhibit 1026-0003
`
`Teva – Fresenius
`Exhibit 1026-00003
`
`

`
`13pwtusoonyxoJmsoe ~e~cl"i~He aBa~oro~ MeTHJ~KO~aJIa.MHH~t .
`
`Tara ttlt a 2
`
`K l¢O~]TpO?I IO
`
`Ca-755
`
`PLIIM-5
`AKATOJI
`
`(CF~C[Cbl)
`
`](oMnRe~c ~pR-
`X~OpM~T~KO~a-
`
`(MetCb, PdCI~)
`
`250+250
`250-}-250
`250-[-250
`
`25~0~0250
`250+250
`5OO
`250+250
`
`2~6-fi
`
`30
`
`0
`
`90
`13
`80
`
`0
`
`-t-8
`38
`o
`
`59
`16
`23
`
`0
`
`0
`0
`
`20
`o
`
`o
`
`54
`16
`0
`
`5O
`
`10
`o
`0
`
`~]~JI;[ IIOHHMaHHR BO3MO~KHOrO MexaHH3Ma ~e~CTBH~ aHaJXOrOB MeTH~-
`Ko6a~aMM][a B opraH11aMe ~(HBOTH~IX 5~ ocyMeCTBaeH
`aHflnn3 pOCTa TeX 2Ke onyxones~x HITaMMOB HpH HSOJIHpOBaHHOM BnH~l-IHH
`H~rnfuTopa Me~HoH~CHHTeTaabI~ X~HO~m~OB0rO npotlsso~oro-- ~ ero
`co,leTfl}IHoro BosAe~[CTB~ C ~TX. TopMo)~en~e pocTa Ca-755, P~-5 ~
`AI<ATO~ ys~n~Banocb s sas,cHMOCT~[ OT KOIiReHTpa~H ffpe~apaTa.
`Ha~6oaee 9~eKTHBHO npenapaT BOS~e~CTBOBfln Ha Ca-75~. ~p~
`qeI~M ~O3bl OT ~ ~O ~5 MF/KF TOpMOYKel-Me pocTa onyxoaH BospaCTanO COOT-
`BeTCTBeHHO ~O 40 H 960/o. O~HaKo C yBeJ[HqelIHeM AOSBI npenapaTa saMeTHO
`BoapacTaaa t~ ero TOKCHqHOCTb. Sanp~Mep, npn [UTaMMaX
`L-J210 s La Ha~6oJ~ee OHTPIMa~BHO~ A030~, ~O Ha~HM ~aSI-ll~IM,
`~O3fl ]0 MF/KF, np~, KOTOpO~ B 8--4 pasa yBeaHnHBaaac5
`}[OCTB )KHSHH )KHBOTHbIX. ~pH yMeHBmeHHH ~OSbI B~fiKT BO3~e~CTBH~
`napaTa Ha MNmeii C ,,efiKO3aMM ~Mn cy~ecTBeHH0 }IH~(e. ~pH
`o~yxoasx B HflHIMX IlCCneAoBaHHsX l.le 65[30 OTMeqeH0 3HaqHTe~SIf0ro yBe-
`atiqesz~ npO~Oa>KHTCabHOCTH ~HSHH MNmefi. ~pl{ COtIeTaHHOM BBe~e~HH
`npenapaTa c MTX AaHce ~ Maaofi ~oae (5 MP/Kr) l-iad~lo~a~acb
`~eKTa. qTO llO~TBepY~a~o yBeaHqe~[e ’~pMO)KeH~(cid:128)~ pOCTa OnyXO~
`
`nOCae nepeBHBKH onyxonll) H e~(eAI.IeBHoM BBe~eI.I~H npenapa-ros B Teye-
`she 5 CyT (5 Mr/~r XHHOanHOB0ro npoHSB0~*-*O~; 2 Mr/KF ~TX)
`Tbl 6~aH cme 60;lee,XCMOHc’rpaTnSHm (Ca-755), HO npM CyMMapHo~ BOa-
`~fi~CTBHH yBealtqlIBa,lflCb TflIOKe H 06MaS "l’OI~CHtlgOC’I’b (Ta6n. 5).
`~BeaHqeHHe TOpMOX<eHH~ potTa OffyXO~ n np0~O,.)KHTea~I-IO~TH }KH@HM -
`~¢HBOTH~X OTMetleHO npz KOM6HHHpOBaHHOM BO3~eIrlCTBHH xaopHaaaoAaTa
`MeT~axo6anaMm.ia }t xmlonln-mBoro npOHSBOK~oro (NSC-176319; ’ra6n. 6).
`~qIiTmSa~ ycHaeH}le ~eflCTBH$1 ~TX npH ero KOM6MIIHpoBaHHOM HCHO~SSO-
`BaHHH c al.lanOraMH MeTHnKO6aaaM}IHa [.I MIIFH6HTOpOM MeTHOHHIiCHH’reTa-
`SBI, Mm Ocy~eCTBHnH KOM6t{}H~poBaBHoe ~leqeHHe M]~imefi e Ca-755 c np~-
`MeHeHHeM BCeX 3 HHt’H6HTOpOB: ~TX, XIIHO~HHOBOF0 npoHsso~l-/oro ~1 Ha}I-
`6o,~ee flKTHBIiOPO ana,,iora KO6a.naMHHOBOPO KO@epMeHTa ~ xd, opnaana~aTa
`a<eTt, s~o6aaaMHHa (cM. "raSa.
`B pesyn~TaTe KOM6~{HHpODaIiHOrO npHMeHeHHS HHFI-16HTOpOB ~THOI-IHH-
`CHHTeTa~5] H ~HrH~po~oaa’rpeAyKTa3M silaqHTe~I~}io ycHdIHBfl~OC5 HpOTHB0-
`onyxoaeBoe ~efiCTSHe, oco6eHuo ~ OTAaaeHtI5Ie Cp0KH Hoc~e OKoHqaHH~
`qeHH~. B 9THX yC~OBH~X qepe3 ~ He~ noc;m OKO~tqa~HS ~e~eM{a npenapa-
`TOB TOpMomesHe pocTa onyxonM COCTaBn~n0 85%, B TO BpeMa KaH B rpyn-
`nax Mmme~I, ~onyyaBmHx Ka~K~oe HS ncc~e&yeMsix coe~H}teH~fi
`BaHHO HnH KOM~HHa~H~O MS 2 Bpenapa,~B, B STn CpOKH yrHeTeKHe pOCTa
`
`76
`
`Sandoz Inc.
`Exhibit 1026-0004
`
`Teva – Fresenius
`Exhibit 1026-00004
`
`

`
`75
`31
`65
`
`3!
`3O
`
`4O
`0
`0
`
`0
`0
`16
`
`43
`29
`19
`
`i
`
`40
`-~-8o
`4O
`
`40
`30
`44
`
`55
`
`41
`19
`
`67
`
`6"/"
`
`43
`27
`
`8
`9
`
`74
`~21
`
`+Ioo
`48
`
`75
`20
`lO
`
`65
`12
`45
`
`88
`20
`69
`
`2-~ ~ 6-~
`
`2-R ~ 6-~
`
`8I
`
`87
`
`2-f~ ~ 6-~
`2-~ r~ 6-f~
`2-Ii ~ 6-~
`
`+22O
`90
`
`58
`75
`
`hITX-FNSC-176319
`NSC-17~319
`MTX
`
`MTX-FNSC-176319
`NSC-176319
`/dTX
`
`MTX~NSC-176319
`NSC-176319
`MTX
`
`(BALB/c)
`AKATO~I
`
`~ITX+/~e/Cb].
`
`MTX-FCF~ CICbl
`CF~CICbl
`MTX
`PdCI,
`
`Ca-755
`
`MTX÷MetCbl.
`
`Me~Cbl.PdCl~
`MTX
`
`MeiCbl.PdCla
`MTX
`PdC]~
`
`Te6~a3-
`
`[IPo~’~moonyxo.,ie~me ,Aellc’re~e eo~mra~ee hiTX e aeaaoro-, MeT~’P{o6aaaMeea
`
`~0~ I)De~eH:a~f
`
`Lr’jp enapaw
`
`Teva – Fresenius
`Exhibit 1026-00005
`
`

`
`2O
`
`8
`23
`0
`0
`14
`
`4
`13
`
`0
`
`95
`
`88
`
`58
`58
`
`7
`51
`
`!
`00
`
`99
`
`99
`9O
`75
`37
`99
`
`2-fi ~ 6-fi
`
`2-~ H 6-f~
`
`ao~e
`m~o, a MTX-~cp~ 20
`MeiCbl-PdCl~ ~oA~ o~osp~
`5+~0+10 (NSC-17~19
`20 ~ aoeze NSG17~Ig)
`b+lO (MTX BBO~ ~p~
`20 ~a ~oc~e MetCbI.PdCI~
`250+10 (MTX
`~+250 (sso~.a~ o~o~pe~maao)
`
`250
`5
`10
`
`bITX
`N SC-176319÷ MeiCal. PdCIs+
`
`NSC-176319+MTX
`
`MetCBI.PdCIs+]~TX
`NSC-176319+MetCbI’PdCI~
`MetCbl.PdCl~
`NSC-176~19
`MTX
`
`~oaa ~penapa~o~, Mr/m-
`
`~penapa~
`
`~e~m~e ua pocT Ca-755 ~e~ MTX, NSC.176319 a KOMUZeKca ~p~aop~aeTl~.~Ko6a.~aMega c na.~aase~
`
`Ta6a~x~a 6
`
`5/8
`0/6
`I/6
`
`76
`12
`
`8--12
`
`H~spem~So)
`2~5 (~o~cz oa-
`
`5
`2
`
`176319
`biTX+NSC-
`NSC-176~[g
`MTX
`
`:~H~OTH~X ~ rpyn-
`
`~rixr
`
`ou~oJ~IL ~ x KO~ITpO-
`Top~o~eu~e pocTa
`
`Teva – Fresenius
`Exhibit 1026-00006
`
`

`
`Foae,]=tzo O. /]L, Mac1~meaa H. B,, Paymen,6ax M. O. ~ AP. -- Boap. Men.
`~nn, 1974, N~ 5, C. 549--554. -- ~S~a men a H. B. Xapa~rep~eraza o6Me~a eoeAa-
`
`~ e B a H. B., ~e.~ ~,~a r. ~., ~ o p.~ e ~. H. r~ ~p. ~ Hpo6~, remoter., 19~, N~ 4,
`c. R0--25. -- M~cnmesa H. B., roae~xo O. 2., Kysnextosa
`Bonp. ~e~. x~.~H, 1977, N~ 5, c. 622~628, ~ Ta~Kooa E. M,, Py~aKo~a H. H.,
`Msva~e~a H, B. ~ ~P’H~ Bnoopra~n,. x~an, 1976, ~ 4, c. 58~541. -- Bur-
`ke G. T., Mangum J. ., Brodie J. D. -- Biochemistry (Wash.), 1971, v. 10,
`p, 8079--3085. ~ Ch.ello P. I., Bertin.o J. R. -- Bio=hem. Pltnrmacol., 1975, v. 25,
`889--892, -- F ! o ~ d h H., U 11 b e r ~ S. -- InL J. Cancer, 1968, v. 3, p. 69~699.
`~.
`alparn B, C.,Clark B. R., Hardy D. N. et el.-- Prec. nat Acad. ScL USA, 1974,
`v, 71, p. t188~1186. -- Kamely D., Littlefield J. W., Erbe R. W. -- Ibid., 1973,
`v. 70, p, 258~2689. ~ M a n g u m J. H., B y r ~o n K., M u r r a y J. et el. -- Bt~beml-
`s%ry (Wash0, I969, v. 8, p. 84~--3499. -- Sauer H., Jaenieke L. -- Blur, 1974,
`Bd 28, S. 821--327. ~ Ttsman ~., Herb.art V. -- Bl~d, 1973, v. 41, p. 465--
`469. -- Wood Y. N.. Kennedy T. S., Wolfe R. S.
`1968, v. 7, p. 1707--1713.
`
`POSSIBILITY OF POTENTIATING THE ANTINEOPLASTIC ACTION OF FOLIC ACID
`ANTAGONIST BY N[ETHYLCOBALA/VflNIE ANALOGUES
`
`Z. P. So]’{/gna, IV. K Mg~ch~va, F. G. Arsenyan, A. ~. YurRevlch
`
`S u m m a r y. The effect oI m~hylcobalamine and its analogues (dffluoro-chloromc-
`thylcobalamine--CF~lCbl and methylcobalamine chloropalladate~MetCbl.DdCID on
`the growth of transplantable ,tumours in mica: adcnocarcinoma of the marnmary gland
`(~n-755), carcinoma of the uterine cm’vix (COC-5), carcinoma of the ~nt~atina (ACATOL}
`~as studied. The actl~ty of the cobalamine coenzyme analogues was investigated when
`used alone or combined with inhibltora of dchydrofolate reducfase and me/hyonlne syn-
`tlmlaae. The results of the experhnents tndieate a stimulating effect of methyleobalamine
`on the growth of transplantable solid tumourz in the animal organism. The antttumour
`actfv~ty of the methyleobalamino analogues studied was found to b~ higher in combined
`ap~licatlon wflh mothotraxate. The most effective ~nhibition o~ tum*ur growth and the
`longer survival of the animals were achieved in combined application of methyleobalamine
`with mothotrexate and methyonine synthetaae inhibitor, depending upon the ~eheme of
`administration.
`
`Sandoz Inc.
`Exhibit 1026-0007
`
`Teva – Fresenius
`Exhibit 1026-00007
`
`

`
`CO]J, EP~KAH H E
`
`The XL Session of the
`ral Meeting of the Academy
`of Medical Sciences of the
`USSR Held in NIoscow on
`Ap r i 1 4--7, [978 (The materials will
`be published in Nos. 1 and 2, 1979)
`Sidorenko, G. I.: On the Work of the
`Presidium of the Academy of Medical
`Sciences of the USSR in 1976--1977
`K o sya ko v, P. N.: Immunology in Cur-
`rent M.edieina
`Baroyan, O. V., Kaulen, D.R.: Cur-
`rent Views on the Ways of Immuno-
`logy Development (Preblems and
`Prospects)
`So[ovyov, V. D.: Aspeet~ of Antiviral
`Immunity
`Ado, A. D.: Current Problems of Ailer-
`ffic Reactions
`Lopukhin. Yu. M.: Primary Immtmo-
`deficiencies and Methods of Their Cor-
`rectt0n
`Petrov, R. V,: Theoretical Foundati-
`ons, Pre~ent State and’ Prospects
`Clinical Immtmology’
`Discussion on the Summary Report
`the Presidium oI the Academy of Me-
`dical Sciences of the USSR for 1976~
`1977 at the XI Session of the Academy
`el /V~edfcal Sciences of the USS.R Held
`on April, 4--7, 1978
`Sofyina, Z. P., Myasishcheva,
`N, V., Arsenyan, F. G., Yurke-
`~ i c h, A. M.: Possibility of Potentiat-
`ing the Antineoplastic Action ’of Folio
`Acid Antagonist by Methylcobalamine
`Analogues
`
`3
`
`3
`
`l 4
`
`21
`
`30
`
`34
`
`43
`
`55
`
`68
`
`72
`
`PE~EPATbI CTATER, OnYBJIHKOBAHHblX B 8TOM HOMEPE
`
`79
`
`Sandoz Inc.
`Exhibit 1026-0008
`
`Teva – Fresenius
`Exhibit 1026-00008
`
`

`
`615.277.3:577.164.161
`
`D22A
`
`Sofyina Z.P., Myasisheva N.V., Arsenyan F.G., Yurkevich A.M.
`
`POSSIBILITY TO INCREASE THE ANTITUMOR EFFECT OF FOLIC ACID ANTAGONIST
`WITH THE HELP OF METHYLCOBALAMINE ANALOGS
`
`The Science Center of Oncology, Academy of Medical Sciences, USSR, Moscow
`
`The stimulating effect of cyancobalamine on the proliferation of subinoculated tumors in different
`
`animals (chicken sarcoma of Raus, fibrosarcoma PW-2, sarcoma 45 and CCK of rats, Geren
`
`carcinoma, sarcoma 1180 and lymphosarcoma of mice) and the weakening effect of several
`
`antitumor medications combined with vitamin B12 that were noted in previous studies can be
`
`explained by the active biosynthesis of its enzymes in the body of animals. The evaluation of the
`
`functional role of methylcobalamine - one of the cobalamine coenzymes in the proliferation of
`
`normal and tumor cells - is of utmost importance.
`
`Methylcobalamine is a coenzyme in the methionine synthase reaction - the key reaction that
`
`determines the synergy of cobalamine and folic acid compounds action in cell proliferation. The
`
`particular importance of methylcobalamine for the activation of this enzyme complex has been
`
`noticed when studying the disturbed metabolism of cobalamines in human leucosis. The low
`
`effect of the combined cytostatic therapy in certain types of acute leucosis with the high
`
`concentration of methylcobalamine in blood has confirmed the specificity of the latter in a human
`
`body (Myasisheva N.V. et al., 1969). The active role of methylcobalamine in the proliferation of
`
`the cells of hematopoietic tissue in normal animals has been determined nowadays.
`
`Methylcobalamine causes an increase in the number of cells that synthesize DNA in the spleen
`
`of mice, its mitotic activity and the size of the proliferating pool (Golenko O.D. et al.). A significant
`
`increase of the hemoblastosis in mice after the administration of methylcobalamine with
`
`endogenous blastomogenes has been found. The important part of the stimulating action of
`
`methylcobalamine is in its inducing effect on methionine synthase. In the cultures of normal cells
`
`in mammals and tumor cells in humans the activity of methionine synthase greatly increases
`
`with the increase of the concentration of cobalamines in a cultured medium (Mangum et al.;
`
`Kamely et al.). However, different types of tumor cells differ from normal cells in their ability to
`
`increase methionine biosynthesis required in case of acute proliferation under the influence of
`
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`
`cobalamine (Halpern et al.; Chello and Bertino). Cobaiamine-dependent methionine synthase
`
`increases the intracellular pool of tetrahydrofolic acid irrespective of the folate reductase system
`
`and serves as a main mechanism of resistance of leukemia cells to methotrexate (MTX)
`
`(Myasisheva N.V; Sauer and Jaenicke).
`
`Thus, there is a possibility to increase the antitumor effect of a given metabolite by its
`
`combination with the antagonists of cobalamine coenzyme. Understanding the mechanism of
`
`action of cobalamines allows to explain the directed synthesis of methylcobalamine analogs and
`
`investigations of their potential as antitumor compounds.
`
`Difluorinechlorinemethylcobalamine and chlorinepalladate methylcobalamine were studied as
`
`chemotherapeutic agents. They displayed an in vitro activity when suppressing the proliferation
`
`of bacterial cells and DNA synthesis in the culture of embryonic human fibroblasts (Myasisheva
`
`N.V. et al, 1977).
`
`While developing the scheme for the combined effect, the main aspects of the physiological
`
`action of cobalamines have been taken into account: the control over the entry of folic acid
`
`compounds in cells; the production of folate coenzymes and intensity of cobalamine uptake by
`
`tumor cells (Burke et al.; Tisman and Herbert, Floodh and UIIberg). It was now possible to count
`
`on the selective action of the investigated compounds and their ability to decrease the activity of
`
`cobalamine-dependent enzyme in the organism. However, it was difficult to expect a significant
`
`effect from their isolated use. Therefore, we found it important to evaluate the antitumor effect of
`
`these compounds with the inhibition of dihydrofolatereductase via the administration of MTX.
`
`Materials and Methods.
`
`Studies were performed on mice from line CsTBL, CBA, BACB/c and hybrids BDF1/CsTBIx DBA
`
`(2), with weight 20-25 gr from the nursery of the Academy of Medical Sciences of the USSR. The
`
`antitumor activity of methylcobalamine analogs was studied on subinoculated leucosis L-1210,
`
`La and solid tumors: breast adenocarcinoma (Ca-755), cervical cancer (CC-5) and intestine
`
`adenocarcinoma (ACAI). We chose solid tumors to be the main object of our research, because
`
`it is easier to determine the stimulating effect of methylcobalamine on solid tumors than on
`
`model of leucosis in mice L-1210 and LAwith high proliferating pool and rather short life-span of
`
`animals.
`
`2
`
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`

`
`Methylcobalamine (CH3Cbl) and difluoridechloridemethylcobalamine (CFzCICbl) were
`
`synthesized according to the well-known method, altered in the separation phase (Tachkova
`
`E.M. et al.). Methylcobalamine chloridepallodate (MetCbI-PbCI3) was synthesized by the method
`
`of Chauser E.G Methylcobalamine was administered intramuscularly 10 mkg/kg twice during the
`
`treatment course with a 96 hour interval, CF2CICbl was administered daily subcutaneously 500
`
`mg/kg once a day or twice a day 250 mg/kg for 5 days. Methylcobalamine chloride palladate,
`
`which was poorly dissolved, was administered per os in 2% starch suspension once a day
`
`500mg/kg for5 days, or twice a day with a 96 hour interval.
`
`In our studies, the activity of cobalamine derivatives was determined not only in its combination
`
`with MTX, but also with quinoline derivative (NSC-170319).
`
`N
`
`~ --NH .... NH
`
`N CH~-~B~
`
`The drug was delivered from the National Institute of Cancer of the USA according to the
`
`agreement about cooperation between USSR and USA in chemotherapy of tumors research.
`
`According to the description presented by the American scientists, the drug is an inhibitor of
`
`methionine synthase. (Carter et al.) Quinoline derivative was administered intraperitoneally 5
`
`mg/kg daily or with a 96 hour interval, which is half of the safety dose for the chosen regime. The
`
`treatment began 48 hours after the tumor subinoculation. The effect was evaluated 24 hours
`
`after the end of the treatment and at different times throughout the animals’ lives. The
`
`percentage of the tumor proliferation suppression was chosen to be the criterion for
`
`effectiveness measured based on the specific volume and increase of duration in life of animals.
`
`In each experiment the control and experimental groups were composed in such a way that its
`
`numbers would assure a statistic significance at a minimal percentage of the tumor proliferation
`
`(50%) and an increase of the life-span in mice (25%). According to the mentioned criteria, the
`
`experimental groups contained 6-10 mice, and control groups contained 6-13 mice, depending
`
`on the tumor.
`
`3
`
`Sandoz Inc.
`Exhibit 1026-0011
`
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`Exhibit 1026-00011
`
`

`
`Results and Discussion
`
`Our studies revealed a stimulating effect of methylcobalamine on the proliferation of
`
`subinoculated tumors Ca-755, ACAI, and to a smaller degree on the proliferation of CC-
`
`5 (table 1). The main intensity of the tumor proliferation under the influence of
`
`methylcobalamine was seen in mice-hybrids BDF1 (180%) that were subinoculated with
`
`Ca-755 than that in mice of the clean line CsTBI. The stimulation of the proliferation of the
`
`tumor cells occurred during the administration of cobalamine; the main difference
`
`between the sizes of the tumors in animals of the experimental and control groups was
`
`determined immediately after the administration of the drug. Later the tumor growth in
`
`mice receiving methylcobalamine slowed down. In mice inoculated with ACAI the
`
`intensity of the tumor proliferation under the influence of methylcobalamine varied. The
`
`stimulating effect of the drug was more significant in male mice (table1).
`
`As it was expected, an isolated effect of methylcobalamine analogs decreased the proliferation
`
`of subinoculated tumors Ca-755, CC-5 to some degree and only immediately after the
`
`administration of the drugs (table 2).
`
`In comparison, the inhibiting activity was the highest with methylcobalamine chloride palladate.
`
`The effective suppression of the Ca-755 proliferation was more significant in mice-hybrids BDF1
`
`compared to mice CsTBI. As it was already mentioned, the stimulating effect of methylcobalamine
`
`was also the most significant in mice-hybrids BDFI. In this series of experiments the life-span of
`
`the BDF1 mice with breast adenocarcinoma had a 50% increase under the effect of CF2CICbl
`
`and methylcobalamine chloridepallodate (table 2). At the same time there was no effect of the
`
`proliferation in mice with ACAI. The regime of administration of the drug showed a significant
`
`difference in the effect of cobalamine derivatives (table 2). Thus, a single administration of a
`
`large dose (500 mg/kg) creates the dissociation of the drugs with the following formation of the
`
`active form that stimulates the growth of the tumor.
`
`According to our assumption, the combination of analogs of methylcobalamine and MTX shows
`
`an increase in its effect on the tumor (Ca-755, CC-5; table 3). The increase of the antitumor
`
`effect as a result of the combination of the drugs was seen immediately after the administration
`
`of the drugs and, especially, in the following period: when the effect of MTX decreased, there
`
`4
`
`Sandoz Inc.
`Exhibit 1026-0012
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`Teva – Fresenius
`Exhibit 1026-00012
`
`

`
`was still a high percentage of suppression of the tumor growth.
`
`Table 1
`Effect of methylcobalamine on the growth of some of the inoculated tumors
`
`Administered
`
`Tumor
`
`dose of the drug
`
`mg/kg
`
`Ca-744, CsTBL
`
`BDF1
`
`ACAI
`
`male
`
`female
`
`10
`
`10
`
`10
`
`10
`
`Time of drug
`
`Growth of the tumor after the
`
`administration after the
`
`administration of the drug, %
`
`subinoculation of tumor,
`
`compared to control group
`
`days
`
`2nd and 6t~
`
`2nd and 6th
`
`2nd and 6th
`
`1 day
`
`17 days
`
`14 days
`
`+74
`
`+180
`
`+20
`
`+21
`
`+65
`
`+23
`
`+23
`
`+10
`
`+31
`
`2~ and 6th
`
`+126
`
`+37
`
`+33
`
`Note: Here and in tables 2-5, + symbolizes stimulation of the tumor growth
`
`5
`
`Sandozlnc.
`Exhibit1026-0013
`
`Teva – Fresenius
`Exhibit 1026-00013
`
`

`
`Table 2
`
`Antitumor effect of the methylcobalamine analogs
`
`Tumor
`
`Drug
`
`Administered
`
`dose
`
`Time of
`
`Reduce of tumor
`
`administration
`
`growth,% compared
`
`Increase of
`
`of the drug
`
`to control
`
`after
`
`subinoculation
`
`1st
`
`2nd
`
`3r~
`
`of the tumor,
`
`day
`
`day
`
`day
`
`days
`
`mice life
`
`span, %
`
`compared
`
`to control
`
`Ca-755
`
`Chloridedifluoride-
`
`250+250
`
`2nd_sth
`
`! methylcobalamine
`
`250+250
`
`2"d_6th
`
`CC-5
`
`(CF2CICbl)
`
`250+250
`
`2o~_6~
`
`ACAI
`
`Ca-755
`
`Trichloride-
`
`250+250
`
`2nd_6th
`
`(BDF1)
`
`methylcobalamine i
`
`5O0
`
`2nd_6t"
`
`with
`
`250+250
`
`2nd_6th
`
`30
`
`43
`
`0
`
`9O
`
`13
`
`80
`
`+8
`
`38
`
`0
`
`59
`
`16
`
`23
`
`0
`
`0
`
`20
`
`0
`
`CC-5
`
`(MeCbI*PdCI3)
`
`500
`
`ACAI
`
`250+250
`
`2o~_6th
`
`2n~_6th
`
`+130 +15
`
`+18
`
`0
`
`0
`
`~0
`
`54
`
`16
`
`0
`
`5O
`
`10
`
`0
`
`0
`
`In order to understand a possible mechanism of the action of methylcobalamine analogs in the
`
`organism of animals we performed a comparative analysis of the same tumor growth under the
`
`effect of the isolated influence of methionine synthase inhibitor - quinoline derivative - and its
`
`combined effect with MTX. The suppression of the growth of Ca-755, CC-5 and ACAI increased
`
`depending on the concentration of the drug. The effect of the drug on Ca-755 was most
`
`noticeable. By increasing the dose from 5 to 15 mg/kg, the tumor growth decreased to 40% and
`
`96% correspondingly. However, an increase in the dose of the drug led to the increase in its
`
`toxicity. For example, the optimal dose for leucosis L-1210 and La was, according to our data, 10
`
`mg/kg and it is only allowed to increase 3-4 times in a life span. A decrease in the dose led to the
`
`decrease in its effect. The solid tumors did not show any difference in the life span of mice. The
`
`6
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`Sandoz Inc.
`Exhibit 1026-0014
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`

`
`combination of the drug with MTX even in a small dose (5 mg/kg) revealed a combined effect
`
`that was confirmed by the reduction of the tumor growth (table 4). A later start of the treatment
`
`(8th day after the subinoculation of the tumor) and daily administration of drugs for 5 days (5
`
`mg/kg of quionoline derivative; 2 mg/kg MTX) revealed more significant results (Ca-755), but the
`
`combination therapy led to the increase in general toxicity (table 5).
`
`The increase in the reduction of the tumor growth and life span of the animals was noticed when
`
`using the combination of methylcobalamine chloride palladate and quinoline derivative
`
`(NSCp176319, table 6). Considering the increase of MTX effect in case of its combination with
`
`methylcobalamine analogs and with inhibitor of methionine synthase we performed a combined
`
`treatment of mice with Ca-755 and all 3 inhibitors: MTX, quinoline derivative and the most active
`
`analog of cobalamine coenzyme - methylcobalamine chloride palladate (table 6).
`
`The combination of the inhibitors of methionine synthase and dihydrofolatereductase showed a
`
`significant increase in the antitumor effect, especially later after the termination of the treatment.
`
`Under these conditions the reduction of the tumor growth was 85% 2 weeks after the drug
`
`administration was stopped. At the same time, there was almost no inhibition of growth in the
`
`groups of mice that were administered just one drug or a combination of two drugs at a time. It
`
`should be noted that the level of toxicity has increased with the increase of the effectiveness. We
`
`also found out that the effect of combining the drugs for administration was different and
`
`depended on the sequence of the drugs and intervals between their administration. Thus, the
`
`simultaneous administration of NSC-176319 and MTX appeared to be less toxic than the
`
`administration of the same drugs with a 3 hour break. It, however, brought the same antitumor
`
`effect.
`
`Having said that, the results of our experimental research supports our thesis about a possible
`
`increase of the antitumor effect of MTX with the use of methylcobalamine analogs and
`
`methionine synthase inhibitor. The obtained results open a new approach to the treatment of
`
`tumors with the antagonists of the physiological regulator of folic acid metabolism compounds in
`
`the organism. We have established the antitumor activity of the antagonists of cobalamine
`
`coenzyme. However, the activity of known methylcobalamine analogs that block some metabolic
`
`pathways is not strong enough for a complete and longer reduction of the tumor growth. The
`
`antitumor effect of analogs of cobalamine coenzyme can be increased by means of its
`
`combination with MTX. The available experimental data shows necessity of investigation of the
`
`7
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`Sandoz Inc.
`Exhibit 1026-0015
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`

`
`effect of such combinations in clinic. Our main purpose nowadays is to develop an optimal
`
`regime for the combined treatment for tumors with the previously mentioned drugs, based on the
`
`thorough analysis of the mechanism of its combined effects in the organism.
`
`8
`
`Sandoz Inc.
`Exhibit 1026-0016
`
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`Exhibit 1026-00016
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`

`
`group)
`
`to control
`
`life-span (%
`
`Increase of
`
`(% to control group)
`
`administration after
`
`Dose of drug
`
`the inoculation of
`
`administered, mg/kg
`
`Suppression of the tumor growth
`
`Time of drug
`
`Drug
`
`Tumor
`
`Antitumor effect of MTX and analogs of methylcobalamine
`
`Table 3 (on top of page 76)
`
`67
`
`5
`
`67
`
`74
`
`+21
`
`45
`
`99
`
`97
`
`+5
`
`+67
`
`81
`
`87
`
`40
`
`0
`
`0
`
`0
`
`16
`
`40
`
`+80
`
`40
`
`65
`
`+100
`
`48
`
`0
`
`14
`
`+32
`
`75
`
`20
`
`10
`
`97
`
`+220
`
`90
`
`97
`
`58
`
`75
`
`14 days
`
`10 days
`
`1day 5days 7days
`
`9
`
`hours prior to MTX
`
`was administered 3
`
`2nd and 6th
`
`10+500, CF2CICbl
`
`MTX + CF2ClCbl
`
`2n~ and 6th
`
`2nd and 6th
`
`2nd and 6th
`
`2"~ and 6th
`
`2nd and 6th
`
`50O
`
`10
`
`!(simultaneously)
`
`CF3CICbl
`
`MTX
`
`PdCI3
`
`10+500
`
`MTX + MetCbl
`
`500
`
`MetCbl*PdCI3
`
`10
`
`(simultaneously)
`
`MTX
`
`PdCI3
`
`2nd_6th
`
`10+250+250
`
`MTX + MetCbl
`
`(hybrid)
`
`Ca-755
`
`(CBA)
`
`CC-5
`
`2nd and 6th
`
`2nd and 6th
`
`tumor, days
`
`250+250
`
`MetCbl, PbCI3
`
`(C~7BL)
`
`10
`
`MTX
`
`Ca-755
`
`Teva – Fresenius
`Exhibit 1026-00017
`
`

`
`10
`
`176319)
`
`min after NSC-
`
`administered 20
`
`75
`
`31
`
`65
`
`31
`
`30
`
`23
`
`43
`
`29
`
`19
`
`4O
`
`3O
`
`44
`
`84
`
`55
`
`74
`
`66
`
`41
`
`19
`
`43
`
`27
`
`53
`
`62
`
`8
`
`9
`
`65
`
`12
`
`45
`
`88
`
`20
`
`69
`
`81
`
`18
`
`46
`
`14-16 days
`
`10 days
`
`7-8 days
`
`5 days
`
`1 day
`
`2nd and 6th
`
`10+5 (MTX
`
`MTX+NSC-176319
`
`(BALB/c)
`
`2nd and 6th
`
`2nd and 6th
`
`2nd and 6th
`
`2nd and 6th
`
`2nd and 6th
`
`2nd and 6th
`
`2nd and 6th
`
`2nd and 6th
`
`days
`
`5
`
`10
`
`NSC-176319
`
`ACALI
`
`MTX
`
`(simultaneously)
`
`10+10
`
`MTX+NSC-176319
`
`10
`
`10
`
`! NSC-176319
`
`MTX
`
`CC-5 (CBA)
`
`(simultaneously)
`
`5+5
`
`MTX+NSC-176319
`
`NSC-176319
`
`(BDF1)
`
`5
`
`MTX
`
`CA-755
`
`tumor inoculation,
`
`administered
`
`administration after
`
`Dose of the drug
`
`Drug
`
`Tumor
`
`Suppression of the tumor growth, % to control group
`
`Time of drug
`
`Effect of Methotrexate and NSC 176319 on the tumor in mice
`Table 4 (on the bottom of page 76)
`
`Teva – Fresenius
`Exhibit 1026-00018
`
`

`
`11
`
`5/6
`
`0/6
`
`1/6
`
`79
`
`+8
`
`+13
`
`76
`
`12
`
`12
`
`8-12
`
`8-12
`
`8-12
`
`simultaneously
`
`MTX+NSC-176319 2+5
`
`5
`
`2
`
`NSC-716319
`
`MTX
`
`in the group (%)
`
`number of animals
`
`animals to the
`
`Ratio of dead
`
`3 days
`
`1 day
`
`Suppression of the tumor growth, % to the
`
`control group
`
`tumor, days
`inoculation of the
`
`of the drug after the
`Time of administration
`
`Dose of the Drug
`
`Drug
`
`Effect of combination of MTX and NSC 716319 on CA-755 in mice
`Table 5 (on top of page 77)
`
`Teva – Fresenius
`Exhibit 1026-00019
`
`

`
`12
`
`2O
`
`85
`
`95
`
`00
`
`and 6th
`
`after the others
`
`but MTX within 20 minutes
`
`MetCbl*PbCI3 simultaneously
`
`(NSC-176319 and
`
`MetCbI*PbCI3+MTX
`
`5+250+10
`
`716319)
`
`within 20 min after NSC-
`
`NSC-176319+
`
`44
`
`88
`
`99
`
`2n~l and 6th
`
`5+10 (MTX administered
`
`NSC-716319+MTX
`
`% to control group
`
`6days 14 days
`
`2 days
`
`of the tumor, days
`
`animals life span,
`
`Increase in
`
`growth, % to control group
`
`drug after the subinoculation
`
`Dose of the drug
`
`Suppression of the tumor
`
`Time of administration of the
`
`Effect of MTX, NSC716319 and trichloridemethylcobalamine with palladium on mice CA-755
`Table 6 (on the bottom of page 77)
`
`!250
`
`5
`
`10
`
`MetCbl*PbCl3
`
`NSC-716319
`
`MTX
`
`Drug
`
`8
`
`23
`
`0
`
`0
`
`14
`
`5
`
`4
`
`13
`
`+29
`
`0
`
`58
`
`58
`
`40
`
`7
`
`51
`
`99
`
`90
`
`75
`
`37
`
`~99
`
`2"d and 6th
`
`2nd and 6th
`
`2nd and 6th
`
`2nd and 6th
`
`MetCbl*PdCI3
`
`within 20 min after
`
`2nd and 6t~
`
`250+10 (MTX administered
`
`MetCbI*PbCI3+MTX
`
`5+250 simultaneously
`
`NSC-716319+MetCbI*PdCI3
`
`Teva – Fresenius
`Exhibit 1026-00020
`
`

`
`Patentanwaftskanzlei
`df-mp
`Fenf H0fe t Theatinemtr. I6
`
`80333 M0nchen
`
`TRANSLATOR’S VERIFICATION
`
`We, LINGO Language Services GmbH,
`85-89, 44137 Dort-
`mundtG;ermany, represented by Eric LINGO, managing
`hereby certify that the
`following translation that we have prepared, tOtalling 12 pages’, is a true and correct
`translation from Russian into English of a documem presented to us as a copy:
`
`AHA~IOI-AMH METH,qKOBA,rlAMHHA
`
`EN:
`
`POSSIBILi~ TO INCREASE THE ANTi~MOR EFFECT
`OF FOLIC ACID ANTAGONt~ WITH THE HELP OF
`METHYLCQ~M|NE ANAL~S
`
`Prate, date: Dortmund, 12 October" 2009
`
`Sandoz Inc.
`Exhibit 1026-0021
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`Teva – Fresenius
`Exhibit 1026-00021