CA2136091A1 - Chemotherapeutic drug combinations - Google Patents

Abstract

The invention relates to certain compositions and combinations of ATP depletion compounds and apoptosis inducing agents. The invention also relates to methods of treating antineoplastic disease by administering such combinations.

Description

WO 93J23014 X ~ Pcr/us93/o477 ~OT~ERAPEUTIC D~UG CO~qBIN~TlONS

Eield of ~she In~rention . .
~ he pres~nt in~e~tion relates to composition~
c~mpri~ing compound~ which deplete ~ellular energy. The invention al~o relatefi to combinations of ~uch compounds with compound~ which induce apopto~is. m~ invent~on a~80 relates to th~ u~e of: these compositions and combinatio~
in the treatment of anti-neoplastic diseaBe.

BackarQund o ~e Invention Adenosine trlphosphate, ATP, is the key energy source :i~ m~jor metabolic~pr~ce~es ~uch as ~io~ ~ thesi~, active tr~n~port~and~A:repair. Conseguently, if ATP production s 1:nhi~ited, con~umption~of exi ing ATP w~ll re~lt in an:energy d~efici~cy that would adversely af~ect the tional~:and~morphologic integrity o:~ the cell.

e~ods:~for;:s~l~c~ive impairment~of ~n~rgy metabolism have ~e~n propo~ed:~primarily involYing industio~ of , aeidD~is~in~tu~or:s~via:~ou tained hyperglycemia, opti~ally in c~n~unction~wi~h;acid-actiYat~d~prodrugs of metabolic poi80~ (NcCarty,~:Med ~ypothe~e~ 16:39 60 (1985~). Thi~
approach~is~cDntl;ngent upon particular tumor having metabolic differe~ces relative to normal tis~ue~
fiufficient t~ produea aeido~si~, and i~ furthermore :d~pend~nt:o~ tumor~mass being suf iciently~large (snd homogeneou~) 80~ that acidosis:remainE~lccalized. ~he enesgy~defic1ts thu~ produced are~ elieved to b@ du~ to acidosi~-i~duced~réduc~ions in tumor~lood ~low, which are further~ore depe~d~nt on the charac~eristi~s of va~eularization of a particular tumor. This approach to impairing tumor energy metaboli~m i~ thereore not SUB~TI~JTE SHEE~

WO93/2301 ~ 6~ ~ PCTtUS93/0477~ ~ 1 univer~ally, or even generally, applicable to treating a variety of fsrms of cancer.

Severa1 compound~ ~re known to impair cellular energy metaboli~m; these compounds are discu~ed below:

The nicotinamide antagoni t 6-aminonicotinamide (6-AN:) (John~on et -l.,;Science 122:834 (19S5)) i~
converted in ~iYo into: the nicotinamide adenine dlnucleotlde (~NAD)~analogs, 6-ANAD~and 6-ANAD~. TheBe competitive ana10~s of NAD and NADP cannot be reduced either chemically or enzymatically (Dietrich, Antineo~la6tic~ and_ImmunosuPPre6sive Aaents, ed. by A.C.
Sartorelli~and~D.G~ John~ (New York: Springer-Ver1ag~, pp.
S39~-542 (~1975),~ Dietrich~et al.~, J.~iol. Chem.
233:Y64-968~:~(l95~8~ and;~Dletrich et al, Cancer Res.
`18~:~1272-12:80 ~(~19SB~ and con eguent1y act a~ pote~t inhl~ tors:of~NAD-dependent dehydrogeDases utilized in l~oly8i6,; ~ln~the~oxidative portion of the pe~to:~e-pho~phate~pathway, and in mitochondr~al oxidative phosphorylation~(Dietrich,~ Antineo~la~tic ~nd mmuno6u~Dressive~:~Aaent, :ed.~by A.C.: Sartorelli and D.G.
John~(Nèw~YorX~ 8pringer-Verlag)::, pp. 539-542 (1975), Di~etrich;~t~al.,~J.~B101~.~ Chem.~ 233:964-96B~(195B), Dietri~ch:~et~ ancer;Res.~;l`B:l272-l280 (l9~8), Wood~ et 81, ~ioch~m. Z.;33B~:381-392 (1963:~, Kaufm~n et al., J.
Neurobiol. 5:391 (1974), Varne~, NCI Monograph No. 6, pp.
:::199-202~(19~8~ 0fori-Nkan~h et al.~, Z. Krebsforsch 77~:64-76~(1972),:~0fori-Nkansah et~al., Naunyn-Schmiedebergs:~Arch. Pharmaool. 272:156-168 (1972), Ke~ller et al.,~ oppe-Seyler'~ Z. Physiol. Chem.
353:1389-1400::~1972),~Herken et al~., Biochem. Blop~ys.
Res. Comm. 36:93-100~(1969), Coper et al., Bioch~m.
Biophys.:Acta ~(Amst.) 82:167-170~(;19643). 6-AN ha~
d~monstrated preclinical anti-cancer activity (Martin et ~ , ~ al., Cancer Rec.~17:600-604 (19~7), Hunting et al., Nature ~ ~ ~:: SlJBSrlTVTE SHEET
; ~;~ . `: :

~;-``` wo g3,23~l~ 2 1 3 6 0 9 ~ P~T/US93/0477~

Bioch~m. P~armacol. 3~:3999-4003 (1985)), but at olerated do8e8, did not have antitumor efficacy a~ a ~ingle agent in hu~an~ rter et al.,.Can~r Re~earch 21:31-37 (1961)).

~ .

The adeno~ine anal~g 6-methylmeraptopurine riboside (MMPR~ has b~en show~ to result in A~P and GTP depletion, lnhibitlon of macromolecular ~ynthesi~, a~d inhibition of tumor ~rowth:(Elio~, Fed. Proc. 26:898 903 (lg67), Elion, J. Am. Chem. Soc. 74:411 414 (lgS2), Shantz et al., Can~er Re~. 33:2867-2B71 (1973), Woods et al., ~ur. J. Cancer 14:765-770 ~197B), WarnicX et al., Cancer Re~.
33:1711-1715 (1973), Nel~on et al., Can~er Re~.
32:2034-2041~(1972)). In addition, the biofiynthe~i~ of NAD may be~inhibited:~y MMPR because NAD is ~ynthe~ized in he cell from ni~otinamide mononucleotide (~MN) and ATP by an; en~yme (Nl!~ ad~nyltranE~era~e ) tha~ bit~d by lo-~l~ (Akkinsos~ al., Nature 192:946-948 ~1961) ) .

The 6-AN and MMPR Co~oinatio~

MMPR~and 6-AN are~ynergistic if~ the tl~ing of their admi~ni~tratio~i~6 appropriate, be~auee the lowering of NAD
level~ by~NNPR fa~ors the competition of 6-ANAD with NAD, and~ er~by ~nhance~ the magnitude:of ATP depletion ~hat is achieved~by ei ~ er:drug alone. The c~mbination of 6-AN
and MMPR:pxoduces re~res ions of advanced murine breas~
tumor which cannot ~e o~talned with either drug alone (Mart~n, Nt. S~nai J. Med~52 426:-~34 tl935))~ I~
additio~ ~o its:antipurine action, MMPR, in hi~h dosage, reported t~ decr~afie pyrimidine ri~onucleot~de conc~ntration~ in ~ivo (Woods et al.:, Eur. J. ~ncer 14:765-770 (1978),~Grindey et al., Cancer Re~. 36:379-383 :
: (1976)~

~:
~:
:: SUBSTITUTE SHEEl 2 ~3 6~9 ~ 4 The pyrimidine antagonist N-(phosphonacetyl)-L-aspartic acid (PALA), in low non-toxic dosage, can lower pyrimidine level6 in vivo selectively in certain tumor5 (Martin et al., Cancer ReE.
43:2317-2321 (1983)~.
, ~

The Tri~.le Combination .
The~triple combination of PALA ~ MMPR ~ 6-.AN was evaluated against advanced solid tumors in ~ice (Martin, Biochemical modulation -- Perspectives and Objectives.
~ "
In: New~Avenues in DeveloPmental Cancer ChemotheraP~. Ed.
by Kenn~th R. Harrap~,:London, En~land ~1987)).
Preliminary biochemical data (Martin, Metaboli6m and Action~of~Anti-cancer druas. Ed. by Garth Powi~ and Rus~sell A.~Prough~(London: Taylor & Francis), pp. 91-140 l987)~ demon trated~sub~tantial depres6lon at 24 and 48 hour~s of NAD level ,~and of the ~our ribonucleoside tripho phate~ of~ pur~nes~and pyrimidines, including ATP~
The~three~drug combination::produced a marked antitumor effect which~was~not~obtained~wlth any~of the indi~idual agéDts or~any~c~o~biDation;of two of;the agents (Martin, Mt.~Sinai~3.~Med.~52:~426-434 (198S),~Martin, Biochemical modulation ~ Per6pectives~and~Obj-ctives.; In: Ne~
Avenues in Develo~mental Cancer ChemotheraDv. Ed. by K~nreth~R.~Harr~p,~London, ~ngl~nd~(1987)~, Q~ject~ of the ~In tion It is an obj~ect;of the~i~nvention to provide compositions~ which~deplete~celluliar energy.

It is a further object of the~invention to provide drug combinations~which are useful~in treating antineoplastic di~ea~e.

, ~: : : :
SUBSTITIJTE SHEEl ~ W093~23014 PCT/US93/0477~
21~6Q91 summar-r o~ ~_ Irn~eion .
: The ~ubject invention relates to chemotherapeutic drug combination~ ~nd their u~e in the treatment of antineopla~tic di ease. Such drug combinations comprise:
a) cellular energy depletion compounds, and b) at least one apoptosis inducing agent.

Advantageously, the cellular energy depletion compounds compri Be:

~: 1) an inhibitor of purine nucleotide biosynthe~is, ~: 2) a nicotinamide antagonist, and optionally 3) an inhibitor of pyrimidine nucleotide biosynthe~

Brief~De~criDtion of the Drawinq~

F~ ~ re~l shows changes in PCr/Pi and NTP/Pi after treatment~wi:th the~triple~drug:combination PALA, MMPR, and 6-AN.~ ~The~change~ PCr/Pi~and ~ /Pi at 10 hours i~
statist:ically~sig~iicant~:(P le~ss than 0~01, p les~ than 0.~02~ The change~in~:NTP/Pi between the lO:and 24 hour me~asure~ment:~i~s:~not si:gnificant. Pretreatment value~.were ;detèrminéd prior~to:~tho~administration of PAhA:loo and appe~ar:on~the~y~intercept. At Time ~0" NNPRlSo ~ 6- AN10 were;admi~ni~eered~ n = 7~ f~r eacb~time point.

Figure 2:shows ~results obtained in Example 2~ CD8Fl mice bearing Fpontaneous, auto~hthvnous breagt tumors re~ei~vod~;three~c~ourse~:of trea~ment on a ~ 10-11 day chedule with~Adria:alone at l} mg/kg, or with Adria at 6 ;mg/kg~administered 2% hr~ after PALA ~ MMPR ~ 6-AN (PALA
at lOO:mg/kg 17 hrs~before MMPR~at 1~0 mg/kg plu6 6-AN at 10 mg/kg), or wi~h the same regimen of PALA ~ MMPR + 6-AN
~:~ : wi~h~ut Adria. Tumors averaged 304 mgs at initiation of : therapy.

SUBSTITLITE SHEET

WO93~23014 PCT/US93/0477~ ~ , 6~9~ 6 Detaîled DescriPtion of the_Inv~ntion The ~ubject in~ent~on relates to drug combinations compri~ing cellular energy depletion compound~ and at lea~t one apoptosis inducing agent. The invention al~o relates to the u~e of ~uc~ drug combination~ i~ the treatment of antineoplastic di ease.

A~optosi~ Inducing Aqents In the ~ubject invention, apopto~is inducing agents are u~ed in conjunction with celluIar energy depletivn compounds.

In the physiological context, apopto~is iR a proce~
by which cells are removed from embryonic and developing omatic ti8~ue8, and~has~been implicated i~ terminal differentiat~on o myeloid cells, a~d in hormone-dependent ti~ue atrophy. It has al~o been documented ~n the :cytoxic T-cell~klll~ng o~ tum~r ceIls,-and in tumor ; regres~ion (Wyllie~et~al., Int. Rev. Cytology 68:2~1-306 (lgB0~ Cell death via necrosi~ is charact~rized by cell swelling, chromatin flocculation a~d di~ruption of c~ll integrity followed by cell lyEi~ (Wyllia et al., Int. Rev.
ytology 68:251-306 (1980)).

Cell death induced by anti-cancer agent~ may take t~e form of both apopto is and necrosis. Apsptoti~ cell death may be more apparent at low levels of he~;e agerlt~, while at:high :Ievel~ ~which therefore pOGe greater ri~;k of toxic 6ide- effect6) necrosi~: may oc~ur due to ~evere meta~lic insult. Hence, cell~ which are not killed direetly, but mere:ly injured by anti-cancer agent6, ~ay activate a genetically programmed "fiuicide" mechani~m (Lennon et al., ~ ~ ~ Cell Proli. 24:203-214 (1991)).

'~
SOBSl ITUTE ~;HEEr ~i 1WO93/23014 PCT/US93~477' 213~Q~

A larg~ number of agen~ have been demon~trated to induce apopto~i~ (apoptosis inducing a~ents), including:

Antimetabolite~

~methotrexate (Barry et al., Biochem. Pharmacology 40:2353-2362 ~1990) and Marks et al., Biochem. Pharmacol.
~2:18~9-1~67 (1990)~, :
5- luorodeoxyuridi~e (Barry et al., ~iochem.
: Pharma~olo~y 40:2353-2362 (1990) and Kyprianou et al., ~: Biochem. Biophys. Res. Communications 165:73-81 (1989)3, ~-flu~rouracil (FUra) (Barry et al., Biochem.
: : Pharmacolo~y 40:2353-2362 (1990) and Kyprianou et al., iochem. Biophys. Re~. Communicationc 165:73-81 (1989~, B-D-arabin3~uranosyl-cyto~ine ~Gu~ et al., : OOOO~a~cer R~ 7~1~743 tl991)), puromycin (Xaufmann, Can~er Res. *9:5870-5R78 :: ` :
(19:89)), trifluorothymidine~(Kyprianou et al., ~lochem.' Biophy0.~Res.~Communication 16S:73-81 (1989)) r ~ DNA Dam-g~gL~9'D~ `

`I ei~platin (Barry et al., Biochem. PharmacolQgy :~ 40:23~3-23~2 (1990~3, etoposide:~Barry et al., ~iochem. Pharmacology ; 40:23~3-2362 ~l990), Kaufma~, Cancer Re~. 49:5870-~878 (1989), Tanizawa et al., Exp. Cell. Re~. 185:237-~46 ~; : (1989) and Mark~ et al., Biochem. Pharmacolo~y - 42~18~9-1867 (l990~j, SU135T~TIJTE SHEET

W~93~23014 PCT/US~3/0477~
~3609~ 8 campto~eci~ (Kaufmann, Cancer Re~. 49:5870-587R
(1939)), cytoxan (Kaufmann, Cancer Res. 49:5870-5878 (1989) and Msrk# et al., Biochem. Pharmacology 42:1859-1867 (1990)), adriamycin (ad~ia) (Marks et al., Biochem.
Pharmacology 42:1859-1867 (1990)), teniposide (Kaufmann, Cancer Res. 49:5870-5878 ~ : (:19B9)), : ~ -podophyllotoxin ~Kaufmann, Cancer ~es. 49:5B70-5B78 (198g) ), aphi:docolin (Barry et al., B~o~hem. PharmaGology 40:2353-2362 (l990)~and~Martin et al., Cell Tissue Kinet.
23:S4~5-5~9:(1990):), :

^N-methyl-N'-nitr~-;N~'mitrosoguan~di~e (Barry et al., Biocbem~ Pharmacolo~y~;40.:2~353-2362 (1990)), nitr~gen~mu~tard~:tO;'Connor et al., Cancer Res.
5~ :6550-6557 ~I991))~
bleomycin~ uo et;al., Nature 271:83-94 (1978)), 1,3-bis(2-chloroethyl)-a-nltro~ourea (BCNU, ~erger et al.:, Cancer Res.~4 ::4382-43B6 ~1982)), methyl glyoxal-bi~-(guanylhydrazone) (NGBG, ~rune et ;: al.,:~Exp. Cell Re~.:l95(2):323-329 ~1991)), radiotherapy ~(Harters, Can~er Res. 52:883-890 (1992))-:
Mi~rotu~ule Modifier~
SU8Sl ITUlE SHEET
.

r ~ ?
;~'''W093/23014 ~1 3 B O 9 I PCT/US93/~77 colce~id (Kaufma~n, Can~er Res. ~9:~70-58~8 (1989)), vincri~tine (Martin et al., Cell Ti~sue Ki~et.
23:545-~59 ~1990)), :
taxol (Martin et al., Cell ~ ue Kinet. 23:545-559 990) and Lennon ~t al., Cell Prolif. 24:203-214 ~1991)) taxotere :

~ ~ ~Hormones ~
.
, dexamethasone (Barry et al., Biochem. Pharmacol~gy, 40:23~-2362 ~1990)~

retiDo~c~;~cid~;~P~acentini et~al., Eur. J. Cell ' Blol.,~54:~46-~S~ 9~

purin~rgi~ 2~,~receptor agoDists (Trepel et al., W0 "~ Pat'.~Appl. *~9116056-A) aom~to6tat~n~analog ~Pagli~cci et 4 ~ Endocrino~logy,~129:255~-2562 (1991~)~

luteinizing~ho~rmone relea~ing;f~ctor analog6 (Szende ; et~al.,~Cancer~Res.,~ 50:3716-3721 (1990)) : ::
e trogen~sblation (Kyp~ianou et al., Cancer Res., 51:1~62~-166 ~1991~

tumor necrosis~fa~tor~(Piguet et al., Am J. Pathol., 136~:~103-110~(1990))~

Nisc~llaneo_s ,: ~ : :

tumoricidal antibodie~ capable of inducing apoptoiis ' (Krammer, Pat. W0911044B-A) SU85mUI E SHEEr :: :

W093/23014 c~3609 ~ P~T/US93/0477~ ' ~cytotoxic T-cells (Ucker, ~atur~, 327:62-63 (1987)~

sodium azide go88yp01 ~lonidami~e rhodamine 123 The signifieance of the pre~eding 1ist is that these different agent6~ are~all capab1e of inducing the ~ume fina1 common mechanism~of cancer cell death, regardle~s of the diverse biochemica1 lesions i~itiated by each indlvldual agent.~ Current information indiGate~ that the ap~ptotic~iochemical;oa~cade can be acti~ted at different pointfi~in~different~ce1l t ~e~ (D`uva11 et al., Immuno10gy:~Today~ 7(4)~-:115-119 (1989)).

Th-~invention~provide drug c~mbination~ and methods imp~iri~ c~11ular:en-rgy and~nucleotide metab~lism, an~d:~:th~ereby~dramatically increaslng:the ~t~t ~ or efflcacy of ~ a~ ~ide~ var~iety~` of ~ apoptosl` -lndùcing antineopIastic a~gent~ without a~corre~pon~ing ln~ré-6e~in ho~t t ~ic~ty.;~ Slnce~ap~pto~is i 8 it~el:~an energy-reguiring proc-~s`~ otter~et:~al~ Anti~ancer~Re~., 10:1153-11S9 990~ ;thi8~ re~ult~s~un~xpected~

The~; triplBi drug csmbination, PAW~-MNPR-6-AN, as well a~ ~all ~apo~to; i~-inducing ant1-cancer~ a~i~nts, have ATP
deplet~lon a 8 ~a~ba~ biochemical l~esio~, and comp1eme~t each~:other on thi~E ;baEi~. In additlon, the PA~A-NMPR-6-AN
combination produce:s a level of wide biochemica1 da~age in cancer~ells:that complements,~and `i~6~ complemented by, the apoptosls-lnducing;~lochemical eff~ect~3 of DNA-damaging a~ents,~and thereby re~ult in enxaDced ki~11 of tumor cell~. The ~EL~ addition sf:~-f1uor~urac~1, adrlamycin, taxol,: radlotherapy, mltomyci~:C, SUBSlllUrE~ SHEE~
~ ~` ` , , Wo93/23014 2 1 3 ~ ~ ~ 1 PCT/US93/0477~

cis-plati~um, cytoxa~, phenylalanine mustard, ~nd ~toposid~ with ~he PALA-NMPR-6-AN tr~ple combination demon~trate gr~ater anticancer sctivity than that ob~erved with the individual d~ug~ alone at maximum t~lerated dose tMTD~ or with the PALA-MMPR-6-AN combination alone.

Cellular Enerov DeP_etion Co~Pound~

IntracelluIar ATP has~been negelected as a primary :
target for cancer chemotherapy because ATP h~E long been considered too~important a ~ource of ~ner~y for all cell~
to warrant the expectation that primarily directed anti-ATP chemotherapy could~e selective for cancer cell~
and sae ~or ho t ~tiB~Ues.

The ~ub~ect~ppl~cat1on~document~ novel compo~ition6 and me *od6;~0r ~mpairing tumor cell energy ~etaboli~m, w1th~triXing therap~ut1c activity, a safe th~rapoutic index, and~an associated depletion of ATP l~el~ in the in yl~ treated ~umor~ :~ en~admini6tered in con~unction :with~agent~:which~trlgger apopto~i6 (programmçd celi :d~ath)~n tumors, the~nergy-depletin~ compositions o~ the m~nti~on~produce~therapeutie activity aga$n~t ~p~ntaneous tumor~ ~n~ y~y~ sub~tant~ially gr~ater than c~n ~e obtained by~either:~:-nergy~depletion comp~6i;tlon~ or apoptosi~-lnduc~ing~-gent~alone.
~: .
Althou~h glycoly~i ~pro~ide~ ~ome of ~e ATP needed by the~ell, the~major generation of ATP occur~ during :: :oxidative pho~phorylati~n in the mitochondria of mammalian c~ as electro~B ~ are tran ferred from reduced NAD (NA~) to 02 by a 6eri:eB of electron carrler~
reconverted to NAD with~concomitant conver~ion of ADP to ATP~: In addition to their central role in energy meta~olism, NAD and NADP (nicotinamide adenlne dinucleotide pho ph~te), are oxidizin~ agents wh~ch ~unction a~ coenzymes in criti~al bi~chemical r~a~tions.
: .
SlJBSTlTl ITF .C~l~FFT

WO93t23014 PCT/US93/0477~ ~
~36~ ~J~ ~2 Disruption6 in NAD 6ynthc~iQ and ~etaboli~m ha~ profoundly ad~rse effect~ on cellular inte~ri~y becau~e of ~he central role played by the~e coenzymes in i~termediary metaboli~m including the generation of ATP from ADP. A
limitati~n of adenin~ or NAD, or both, are key to ~TP
depletion. In the ~ubje~t invention, theoe metabolites are target6 ~or chemotherapy designed to result in depletion of cellular le~els of ATP.

: The cellular ener~y depletion compound~ of the vention are typically used in combination and typically include:

an inhibitor of purine nucleotide biosynthesls, 2) a ni~otlnamide antagonist, and optionally 3) an 1nhib1tor of pyrim~dine nucleot~de biosynthesis.

HPLC and~NMR moasurement~ of biochemical chan~e~
re-ulting~from treatment with PALA (an:inhibitor o~
pyrimidine biosyn~esi~s) + M~R (an: is~ibitor of purine b~io~ynthesi~) ~ 6-AN (a nicotinamide a~tagoni~t) in~icate a~evere depletion:of cellulsr energ~ levels in the treated tumor~.~ The ~ALA-MM2R-6-AN-induced reduction in ;all four individual ribonucleoside pools (Martin, Metaboli~m and Action of A~ti-cancer dru~. Ed. by ~arth Powis and Ru~sell A. Prough (London: Taylor ~ Franci~), pp. 91-140 (1987)), which generally correlate~ wlth a reduction in the corre ponding~deoxyribonucleo~de triphosphate pools (Hunting et al.~, Can. J. Biochem.
::
59:B21-829 (1981~)),:appears to not only deplete cellular energy ~ource~ and to inhi~it DNA synthesi~, but to inh$bit the potential for DNA repair ~ well. Because the DNA damage produced by many chemotherapeut1c drugE i5 ~ubject to repair, the cytotoxic activity of these DNA-damaging drugs is increased when the ~NA r~pair Dotential of the ~ell i8 decreased. A 3-drug combination, SUBSrlTl~E SHEElr ~ W~93/~3014 PCT/US93/0477~
21~6091 PALA-MMPR-6-~N, augments the antitumor activity of DNA-damaging drugs by ~irtue of its ability to deple~e ~oth the d~oxyr~bo~cleotide , and the energy source r~guired or the DNA repair processsfi.

InhibitQr~ of ~ ri~e Nucleotide Bio~ynthe~

Inhibitor~ of purine nucleotide biosy~the~i~ of the i~ention include ~he followin~:

a) Direct Inhibitorfi 6-methylmercaptopurine ribosidç (MMPR) 6-mercaptopurine Thioguanine Thiamipri~e Tiazofurin A2a~erine 6-diazo-5-oxo-L-norleucine Aci~cin '~
b~ Fola~e Anta~oni~ts Methot~exate Trim~trexate ~ ::
Pterspter~
Denopterin Didiazotetrahydrofolate (DD~ ~ F) Ad~antageous inhibitors of puri~e ~ucleot~de biosynthe~i$ are MNPR and folate antagonist~ which ar~
~elati~ly ~elective i~hibitor~ of ~he e~xyme glyci~amide ;; ribonucleotide tran~formyla~e, e.g. DD~THF or DAC~F.

~ieotinamide Antaaoni~ts Nicotinamide antagoni~ts of the in~ention ~n~lude the followin~:

6- aminoni cotinamide (6-AN) WO 93/2301~ PCr/US93/0477~

2~,3609~ ..

l~ionicotinam~ de o ~1, 3, 4-thladlazole 2-~thylamino-1, 3, 4-t}liadiazole 6-aminonicotini~: acid 5-methylnil ot~namid~
3-acetylpyr~ d~r~e itor~_~f Pyrimidirie BioEsynthe~i~

Inhi}:itors of pyrimidine nucleotide ~iosy~thesi~ o the inv2ntion inclucle! the following:

N- (pho~phonoacetyl ) -L-~partic acid ~PALA) 6~azauridine Triacetyl6-azsurOdi~e : Pyr~zo~uran Rre~uinar ~:; Ac ~eln * * *
: :
Whi le raot wi ~hi~g to be bound ~y theory, ~;he EGllOWln9 :~L8 offer~d as an explanat~on of th~ mec~a~ism of ~e combinatio~s ~f :~e in~r~tion. The triple con~i~ation o an~ inhibitor o pux ne bio~ynt1h~s, a nicotinamide a~ag~n~t and an i~ib~tur o~ pyrimid~ blo yrathe~i~
(~.g., PAL.A ~ M~R ~ 6-AN) wa~ designed to produ~e a mutually reinforcing blockade on pyrimldin~ arld purine de ~ovo ~io~ynthe~i~, a~ well a~ a ~F~ec~ attack on ~iAD
m-taboli sm, that in toto result primar~ ly ~n a damaging depletion of h~gh ~nergy nucleoside triphosphate~, parti~ularly .ATP .

PAhA i~ an inhi3: itor of de novo pyrimidine bio~ynthe~is (Collin~ et al., 3. B~ol. Chem. 246:6599-660 (1971~, John,son et al., C:ancer ~es. 36:2720-2725 (1976) ) .
PALA ha~ been found to be non-toxi~ to the hemopoietic S~91BSI ITIJTE SHE~

~ .~ WO 93J23014 PCI IUS93/0477~
21~iOgl ~y~tems in mice ~30~80n et al., Cancer Re~. 36:2720-2725 ( 1976 ) , ~larri ~Lon et al ., Cancer Chemvther . Pharmacol .
2:183-187 (1978)), and when administered at a low do~e ~ lOC) mg/k~) exer~ s~lectlve action in C138Fl brea~t tumor tissue, but not in th~ intest~ nal ~ ue of the ho~t ~Martirl ~t al., C ancer Re6. 43:2317-2321 (1983~ ) .

~ e~ tmtitumor tox~city of high do~e I~R as a ~ingle aS~2n~ is 3cnown to be a~E~ociated witll a general depletion of purisle nucleot~d~i that re~ults in the ~ ition of macromol~c:ular syr~the~is and tumor growth (Nelson et al., Cancer Re~ . 32: 2034-2041 ( 1972 ), Scholar et al ., Canc~r Re~ ~ 32: 259-269 ( 1972 ), Wara~ick et al ., Caneer ~e6 .
33:1711-1715 (1~73), and Woods et al, Eur. J~ Can~er 14:765-770 (1978)). Thi~ ~hiopurine-induced decre~e in tumo~ ATP le~ls ha~ been implicated previously ~ ~he cau~al mechani~m in the therap~uti~ ~ fect produ~ed in tumors (Atki~o~, ~egulatlon of energy m~tabol~s~:
Explolt~ble molecular mecha~lsms and noQplasia. The Unl~7er~ity of Texa~ M.D. Ander~on ~08pital ~nd Tumor In~titute at Housto~, 22nd Annual Symp~ium o~ Fu~dament~l Cancer Re~earch, 1968. (Baltimore: Willi2ms and WilXi~s~, 397-413 ~19~9)).

The earlie~t report~d ~tudie~ empha~z~ng AT~
depl~tion aE a chemotherapeuti~ approach ~a_Y~y~ employed as a ~inglo agent to iower ATP le~el~ i~ ~he tumor a~d ~hereb~ produeed effectiye anti~mor aetlYlty IDietr~ch et al., ~ancer R~s. 18:1272-1280 (19S8) and Martin et al., Cancer ~. 17:600-604 ~1957)). In more recently co~ducted in vitro ~tudies, 6-AN ~nhibiti~n of tumor cell grow~h wa~ accompanied by a depletio~ of purine and pyrimidine nucleotide~, and N~D, a~ well ~ ~y a reduction in the ATP to ADP ratio (Hunti~g ~t al., Biochem. ~harmacol. 34:3999-4003 (~98S)~.

The triple combination i~ le~s toxic when the interval between cour~e~ of PALA ~ MMPR ~ 6-A~ treatment S~SrlTtJTE ~HF~r WO 93/23014 PCI`/US93/0477 ~,13609~ l&

i~ ext~nd~d from 7 dayli to lO or ll dayE;, and thi~ change in the E~cb~dlule o~ aa~ninistration reduce~ toxicity suf~Eic~entls~ to penait the E;afe additioIl of o'cher drlsgs ~ e . g ., an apoptoE~ ducing agent ~uch as ElJra 3 every lO
or ll day~ to~¢ther with` the three drug ~:ombination.
.
The PAI~A-M~R-6~ indu~ed reduGtion o. ~ vels hafi been demon~trat~d by both H~LC and the non-inva~ive t~cbn~gue of ~ ~pectros~opy (Fig. 1 ) . Althou~h ~e degree of suppres~ion of ATP wa8 ~ nificant at early time points af t~r 't:he triple chemotherapy, by 72 hour~ the ~TP
level~ had returned to normal. In ~ome normal c:ell~
~ e . g., hepatocyte~ ~, maintenance of ~verely depreRsed level6 of ATP (20% of contr~l value~) for as long a~ 36-~8 ~our~ doe~ n~t ne;:e ~arily comprom~se viabllity as long a~
the adenine slucleotide concentration~ then rRturn to contr~l valtleE; ~ Far~r, Fed . ~roc . 32: 1~34-1~39 ( 1973 ) ~ .
8ust~ d or perman~nt 15~fiS of ATP is ~ncompatible with c~ urvi~al ( Schrauf ~tatter et al ., J . C:lin . Imre~t .
77:13~2-1320 (1986) ) . However, a tamporary ~48 ho-lr~
d~pl~t~on of ATP is n~v~r the 801e determina~t ~e~mpromi~i~g c~ll viability. Ar~y gignificant drop in ATP
concentratiorl ha~ many metabolic ~on~equeneeQ~ asld other induced p~r~urbati~ns to cellular biochemi~try (e.g.~, kho~e ~du~d by:PA~A and 6-AN) act in co~c~rt wlth ATP
lo~ to cau~ ~ell death (Hy~l~p et al., J. Biol. chom.
263sl665-1675 (1988)). For example, ~he pento e phosphate shunt provides reduci~g egui~alent~ in th~ form o NADP~
for certain anabolic reaction and a~so for the maintenance of reduced glutath~one, GSH. GSH i~ a major cellular reductant (Mei~ter et al., Pharmac. Ther.
49:125-132 (1991), Morrow et al., Cancer Cells 2:15-22 (1990), Doroshow et al., PharmO Ther. 47:359-370 ~1990), Keizer et al., Pharm. Ther. 47:219 231 (1990~, aDd in the process of detoxification of radical spe~ie~ becom~s oxidized glutathione, GSSG. NADP~ i~ re~uired for ~he conver~on of GSSG back to GS~ by the e~zyme, glutathione reductaRe. Thu5, '6-AN' ~ inhibition of the pento~e SUB5TI~JTE SHEET

. , WO 93/23014 PCr/US93/0477:-~136~91 pho~phate s~unt can lead to a lowering of NADPH levelswhlch in t~rn c~n p~event adequate GSB resynthe~is from GSS~. And, slnc~ ATP ~E3 roquired for the in~ ti~l synthe~is of GSH rom it~ con~tltuent amino ac~d~ (Mei~ter et al, Pharmac. l~er. 49:12~-132, (1991) ), ths Al~
depletiorl induced by the triple combinatiorl can limit suppl~e~ of G8~. l~us, the two effects of ~TP and ~DPH
depletion complement each other in thi~ r¢garc3, and when ~he level of C;S~ fall~ ~u~tantially after chemical injury, cell dea~ usual~y en~ue~ (Boobi~ et al., TIPS
10: 27~-~80 ~ 1989 ) ) .

The t~r~n "bioch~mical modulation" ref~rs to the pharmacologic manipulatic~n of meta~olic pathwayE by an agent (the modula~in5~ agent) to produ~e the ~ele~tiv~
enhancement of the antitumor eff~ct of a ~econd agent (the "e~EI3ctor" Elgent) (Mart~n, Biochemi~al modulatie~n --Pe~rspectlYes and C)b~cti-~s. In: New~
. Ed . ~y ~es~
~arrap, I.c~dosl, Engl~nd ( 1987 ) ) . In thi8 ~:on~x~
triple combination, P~A ~ ~R ~ 6-~, Rla~ be ~viewod as biochomieal modulation employ~d to e~tabllsh ~n tumo2 cell~ a wide array of biochemical chan~s ~ ., a primary d~mi2~ution o~ ATP lowe~rin~ of all of the rlucl~ol3~de tripho phate~s and NAD, i~ibitlon of m~crocmolecular Isyn~hesis, and ~upre~sion of the~ f~ux thrc~u5~h the p~nto~e phosph~te shunt sncl glycolytlr:
pathway~ -- thereby establi ~hing a level of di ~rupted meta~oli~m in can~er ~ell~ that compleme~t~, and i~
complemented by th~ cascad~ of similar biochemical dexangem~r~t~ induced by tbe apopto&i~ effe!ct2 of DNA-dama~inq antit:ancer agents (e.. g., FUra, ci~platt~um, BC~U3 (Barry et al., Bioc~em. Pharmacol. 40:2353-236~
1990) , Berger et al ., Antî Cancer r)rug De~i~n, 2 : 233-Z10 (19B7~ ) . This modulation and complementat~nn r~ult~ in enhanted cancer cell deaths which r~f 12ct i21 improved tumor regre~sion rate~.

SUE3S~ITU~E SHEET

W093~23014 PCTIUS93/0477~ .
~36~9~ 18 `

I~tracellular ATP ha been negle~ted a~ a primary target for cancer chemotherapy becau~e ATP haF long been considered too important a ~ource of energy for all cells to warrant the expectat~on that primarily dirocted anti-~TP chemotherapy could be ~ele~tive ~or c~ncer cells and safe for host tissue~. However, the ~ub~ect ~pplication tocum~nts trlking therapeutic acti~ity, a safe therapeutic index, and a~ a~sociated depletion of ATP
leYels tn the in-vivo treated tumor~.

The relati~e selecti~ity of the tripl.~ therapy for the tumor over ho6t ti~ue~ i8 explained by the finding that many of the enzyme~ affected by the antimetabolites are in lower conce~tration in neopla~tic than in normal t~ ~uefi (e.q., NAD-dependent enzy~es; Dietri~h et al., Cancer Re~. 18:1272-1280 ~1958), Martin et al., Cancer Re~. 17:600-604 (19S7), Glock et al., Biochem. J.
65:413-416 (19~7), Jedkien et al., J. B~ol. Chem.
213:27:1-280 ~1955) and Morton, Nature lB:540-S42 (1958)).
It Is~po~sibl- to ~nhiblt more ~eleeti~ely enzyme~ present ln cancer ti~ue~in ~mall amounts, while ~roducing muc~
:less inactivation of the 6am~ enzyme in nor~al ti~sues containing larger amountB (Ackerman et al., Proc. SGC.
Biol. Med. 72:1-9 (1949)).

:The therapeutic actl~ity of a widely diver~e group of antica~eer agents has be0n markedly e~ha~ced ~y this iochemical modulation approach u~ing PALA + MMPR + 6-AN
in the CD8Fl:murine ~reast tumor model. The addition of 5-fluorouracil, adriamycin, taxol, radiotherapy, mitomycin C, ~ plat$num, cytoxan, phenylalanine mustard, a~d etopo~ide with the PA$A-MMæR-6-AN triple combination have demonstrated greater anticancer act~Y~ty that th~t ob~erved w~th the indi~idual drug~ alon~ at MTD, or wlth the PALA-MMPR-6-AN combination alone.

SU13SrlllJTE SHEET

. WO93/23014 2 1 3 ~ ~ g 1 PCT/US93/0477 ellular Ener~ Depletion Com~ und~ Plus ~Ura In on~ embodiment of the in~ention cellular e~ergy depletion compounds are adminis~ered with 5-fluoroaracil as ~he apopto~is inducing agen~.

In a~ adY~nta~eou~ embodiment, the ~ubject in~ention rela~es to a hi~hly ectiYe chemotherapeutic drug ~ombination compri~ing:

N-(phospho~acetyl)-L-a~partate tPALA~, 6-m~thyImercaptopurine ri~side (MMPR), 6-aminonicotinamide (6~AN), and 5-fluorouracil (FUra).

~ A guadruple drug combination of PALA ~ MM~ ~ 6-~N
:: FUra, admini~ter~d in a lO-ll day ~chedule, produ~ an ~:~ i~pre~siv~ part~aI tumor regre~ion rate.
.

~ h~ underlyi~g moleeular mechani~m of fluo,opyrimidine-induced ~thyminele~ death~ h~ been how~ ~o be du~ to nprQgr~mmed cell death" t~poptosi~
acti~at~d by DNA ~trand ~reakage (Kyprianou et al., B~ochem. Biophys. R~s. ~ommunications 165:73-Bl, (1989~9. Measurement of ~iochemical changes i~ ~D8Fl ~r~a~t tumors after ~a_YLY~ admi~i~tration of the PALA-MMPR-6-~N drug ~ombina~ion re~al severe ATP 1086~
inhi~ition of macromoleculàr 8ynthe~i6, i~hibition of the pento~e phosphate ~hunt, N~D deplet~on, r~du~tlon of ri~o~ucleo id~ triphosphates and inhibition 9f protein synt~ , a patt~rn of findi~y~ which overlap with those reported in ~thy~inele~ de~th" and apopto~

In the subject invention, ~he additi~n of a~
apopto~is-inducing anticancer agent such as FUra (Kyprianou et al., Biochem. Biophys. Refi. Communicatio~
16~:73-Bl, (1989), Barry et al., B~ochem. Pharmacol.

SUE~STITUTE SHEET

., . , . , .... , . ... . .. .. ~, .. ..... ~ .. . , . . I .. .. .... . .. . . ....

WO 93/23014 PCI/U~i93/0477:~ .
~,t3609~ 20 4û:2353-2362 (199C~) ), to the triple combination re~ults in compl~mentary th~rapeutic activity.

PAI.A-M~R-6-1~3 indu~es elevatiorl o$ P~P l~vels and reduction of UTP pool6. Th~ triple drug com~ination al~
incr~as@~ the ther~peutic acti~7ity of EUra by increasing the anabc~ m of FUra to its nucl~ot~de~ well ag by avoring the competit~on of the analo~ ver the r~atllral pyrimidi~ interm~diate~ wh~e l~vel~ have been re~:lu~ed by PALA .

qhe el¢vation of PR~P level~ and the lowering of tlTP
le-7elg by the triple combination w~re expes:ted to f acilitate the cor~ver~ion of EUra into i t~ ~etiv~
nucleotide~ for ef~ective blockade of Xey Ellra-sen~itive enz~e~, and this w~s indeed ac~:omplished tTable 5).
Thu~ ddit~on of EUra, which at 75 mg~kg a~ a single agerlt cauEs~E~ few ( <5%~ regre~ionE; aa~d only inhi}slt~ t~e g~ow~h of a~on~anQou~ t:D8Fl ~rea~t tumorEs, markedly ~crea~d th~ tusnor regre~6ion rate of the triple eombinat~çln from 3B% to 1;7% (Table 2).

Thr~e of the2~e druçls (PAr.A, M~R, and ElJra~ c:urrently are u~ed c:linically a6 component of ~,rariou~ dxuçl t~o~. A: combinatior~ of PAI~ plu~ ~Ura has prov~n to be ~ignific~rltly more active than EVra Ellone i~ th~
clir~cal trea'cment of coloFectal c:ance!r (Ardala~a et al., J. CIlI~. Oncol. 6:~053-1058 ~1988), O'Dwyer ~t al., J.
Clisa. Ont:ol. 8:1497-1503 (1990)). M~?R can r~;ult in the elevation of ~RPP le~ls in human tumors suc:h a~ colon, ovary and brea~t (Pet~r~ et al., Cans:er Ch~moth~r.
Pharmacol . 13 :136-138 ( 1984), O'Dwyer et al ., J. Natl .
Can~er Irl~;t~ 83 :123~-1240 ( 1991 ), Wiem~nn et al ., M~d.
Oncol. & ~harmacother. ~(2) :113-116 (19B8~) . The addition of MMPR augments the metabc~lic aetivation of ErUra in human tumor~ .

SUBSI~ITUTE SHE~

,.. w093/~30l4 21 3 6 Q ~ 1 PCT/USg3/0477~

C~llular En~rqy DePletion Co~Pou~ds Plu~ Adri~mYcin In th~ ~mbodi ment of ~he in~ention, cellular ~nergy deplet~on compounds are admi~ister~d with adriumycin ~6 the apopto~i~ i~ducing agent.

Ano~her advantageou~ embodiment of the invention ompri~s:

N-tpho~phonacetyl)-L-a~partate (PALA~, 6-methylmercaptopurine ri~oside (MMPR), 6-aminonicotinamid~ (6-AN~, and Adriamycin (Adr~a).

Thi com~nation yiolds signi icantly enhanced anti-cancer activity o~er that produced by e~ther Adria alon~ at max~mum tolerated dose (MTD~, ~r by ~he tr~ple drug combi~t~on, ag~qn~t large, ~pontan~ou~, autoch~honou- muri~e br~ast tumors. The augmented therap~ut~ r~ult~ were obtained w~th ~pproximat~ly one-half th~ ~ o~ Ad~ia a~ a ~i~qle agent, and pro~ides the:cl1~ical ~nefit of lo~ger and moxe e~ecti~e tr~atment with incr~a~ed ~afety. ~he combi~at~on of an ATP-d~ple~i~g dru~co~bi~at~on administ~red prior tQ Adria r~sult~d i~ a 1~ umor regre .io~ rate (12~ CR; 8BX PR) population of treated ~pontaneou~ tumor~ indicat~
that the~enorgy-depletin~ combination o~ercome~ re~i~t.nce me~ani~m~ to adriamycin.

, ~ , Adriamy~in ha~ ~een ~h~wn to induce death by apoptosi~ in cancer cell~ i~ ~itro (MarXs et ~1., Biochem.
:~ Pharmacology 42:1859-18~7 (1990~ be addition of Adria : to the triple d~ug combinati9n ~ignificantly ~nh~es ~ ant~-cancer actl~ity over that produced by ei~her Adria : alon~ at MTD, or by ~he triple drug combinat~on, ~ain~t advanced 6pont~aneou~, autochthonou~ murine bre~t tumors.
The augmented therapeutic results are o~tained with SUE15rlTl.llE SHEET

WO 93/23014 PCr/US93/0477~
4~36~ 22 approximately one-half the MTD of Adria as a 6irLgle agent ~

The therapeu~ic re~ult~ of t~e mo~t effective ~ingle agent against human brea~t canser, P.driamyein, are great~y er~hanced by the prior administration of ~e triple combi~tltion. These re~ult~ are obtained with e~entially hP~lf the do~e of the MTD of Adria a~ a ~ingle agent.
Adriamycin car~not be gi~en to br~alst cancer patle~nt~; for long periods becauEse ~ts cumul ti~e dosage rea~he~
lie-t~reatening heart damage; ~herefore, the u~e of much lower dol3e~ of Adria, offe~ the benefit of lonqer and more ~3ffec:tive treatmerlt with increased ~afety. Eu~ther, the ~liniclll developmerlt of Adriamycin re~istance i ~
f requ@ntly ~a~ed on energy-d~pendent mechani sm~ ( e . ~ ., multiple drug resi~tance Smdr) eacpre~sion: P-çslYcoProtein) i~ ~ car~c~r c:elIs ~G~rlach ~t al., C~ er Sur-vey~
5:26-46 (l986t and Ver~antvoort et ~l., C~cer ~o~.
~2 ~ 23 ( l992 ) ) . R~ult~ wit~ the triple drug co~ at~ o:~ are p~ rily ~a~d oal depletion o~ cancer c~ norgy level~ 8 Al~-deplet~ng tlpproach in combinat~ with Adr~a ~ever~es ATP-dependent m~ehanism~
r re!sistance to Adria n v~vo, a~ has been d~monstrated in Y~tro (Gerlach ~t .l., Caneer Sur~ey~ 5:26-46 619,86) and I)~no, Bloehim. Blophy~. Acta. 323:466 (1973)).

The re~sult~ obtained with th~ 4-drug combination in Cl:?8Fl mice b~ari~ p3:~ntan~0u~, autoch~nous ~r~ast tumors represent a ~ignificant th~rapeutl~ breakthrough ~n two r~pect~ ( see Ex~mp}e 2 ) . Fir~ ly, th~ therapeutic efec~ oî treatment increa~ed from a t~lmor regr~ion rate of 66% after the fir~t course of treatm~nt to 93 and 100%
ater ~seeond and th~ rd course~ of tr~atmea~t, re~pectl~ely. In corltra~t, although the ~ Bt course of Adria alone at it~ MTD produced a re~pecta~le 43%
regre~ios~ rate, the therap~utic v~lue of this drug dimini~hed with ~ubse~uen~ cour~e~ c~f treatment ( i . e ., 2}%
regre~ions after the second course and 16% after the Sl)BSrlTlJlE SHEET

., WO93/~3014 2 P(~/US93/0477:`

t31irel c:our~e ) . This decrease in chemotherapeutic acti~rity on ~ucces~iive courE~es s:lf treatment ha~ been ob~erved in preclinieal snd clis~ical ~rial~ with Adria, and wit.h mo~t chelaotherapeutis: drug~. Thi~ phenomenon ha~ u~ually been a~3cribed to the ~selection by t~e drug of c~ll& that were innately rc~ tant to the pecific drug, and preferes~tial growth of thi~ sub-populat:ion ~fter the drug-~e~sitive cells have b~en kil~ed by exposure to the drug.
Sub~eqlent drug e~cposur~s are con~ronted with relat~vely more resi8tant cell~;, a~d the resulting therap~utic ob~servat:ion iE; drug resi tance. A~ showsl in Example ~, the appear~rlce of incr~ased resistance ts Adria alone was ob~ervabl~ a~i early a~ ~e ~econd course of treatment ( on day ~O). ~Iowever, no operational re l2;tance to the PAI,A -M~R - 6-AN co~irlation could be detected throughout the 3 cour~e~ of tr~al;ment segimen and 28 day observation period ( Eig . 2 ) . And, importantly, the admini &tratios~ of ~?AL.A
~R - 6~ n conju~ction wlth Adria Preventçd the manife~tat;lo~ of re ista~ce to Adria. In act, t:he r~gr~s~ n rate ob~erv~d wit}~ th~ 4-drug treatment ~ncrea~ed o lOOX ater the la~t cour~e of treat~slent (~ig.
2)-A~ en~ionod abo~r~, the fact that t~e 4-dru~ r~gimen re~ulted in a lOOX tumor regres ion r~te ~n ~sch o t~e three ucce~sive experiment~ ~ a total of 42 ~pc~ taneou~
tumor~ ) was unprec~dented in t~i spo~taneou~ mur~ ne brea~t tumor syst~m. Spontaneou~ tumor are unlik0 ~:olmnon tran~;plantable tumor models, whi ch have been repeat~dly transplanted ~or years, ànd where all of the ~umor~ in a particular experiment, although ~omew}~at h~terogeneou o~
the cellular level, are n~vertheless guite 6imilar frc~m one indiYidual ~o~;t to another. II1 contras, ~pontaneous tumor~ in these mice, like in humans, differ in drug ~er~sitivity rom one individual to another~ For example, although on average approximately ~O% of the population o~
spontaneou~ CD8Fl brea~t tumor~ respond wlth partial tumor regreGsion on exposure to opt1mal treatment wi~ EVra SUBSTllVTE SHEEr WO 93/23014 P~/US93/0477~ ..
?~36~9~ 24 alone (Stolfi et al., J. Natl. Cancer In~t. 80:52-55 ~198B) ), 80% of thi~ population of tumors doe~ not refipond to t}~i~ extent. Ial fact, if on~ meaE~ure~; ind~vidual tuJnor ~ur~ng and ater treatment with ~.q. EUra, one ~ee~
~rl array of re~po~e& in indi~idual tumor~ ~aryislg from p~rtial regr~ion to 6t~ to progres~os~. ~owever, despite thi~; het~roqer~eity in drug ~u~cepti~ ty among indi~idual tumor6, the 4-drug treatment produs:ed regre~sion ln all of the t~mor~ in t~he population.
P.lthough thi ~ treatment doe~ not s: vercome hetero~eneity in l3usceptibll~ty compl~tely, aB eVidQnC:ed by t~e ~a~lure to achi~ve 100% cure of ~hese tumor~, thi5 i~ a ~ew level of therapeutic acti~ity. Further, the ~pontaneoug, autochthonu~ CD8Fl breafit tumor model has demonstrated a remarkable 100% therapeutic correlation wi~h human brea~t cancer in term~of ~th po~itive and negativ~ sensitivity to i~di~idual chemo~h~rapeutic drugs u~ing tu~or r~re~lo~ a~ the ~riterio~ ~or evaluat~on (Stolfi et al., J. Natl. Cancer In~t. 80:S2-~5 (19B8).
~:

: ~ellular Ener~ay DePletion Com~ound~ Plu~ Ta~ol In thi~ embodiment of the invention c~llular on~rgy depletlon comp~unds ~re a~mini~tered with taxol a~ the a~opto~i inducing ag~nt.
An ad~antageou~ embodiment of the i~entlon c~pri~e~:

N-~phosphonac~tyl)-L-aspartate (PALA), 6-methylmercaptopurine r~oside (~MPR), ~6-ami~onicotinamide (6-AN~, ~nd lt~XC3l ~

The abo~e combination yields ~ignifiea~tly enhanced anti-cancer activity over that produc~d hy either taxol alone at MID, or by ~he triple dru~ combination, against ad~anced, first pasEage ~pontaneous murine brea~t tumor~.
~he augmented therapeutic re~ult~ are o~tained with SOBSrlTUTE SHEEr W~ 93~2301~ PCr/US93/0477:.
25 21360~1 approximately one third the MT~ of taxol as a single agent. The latter i8 an important conEsiderativn with taxol b~cau~ of the~ ~ev~re supply pro~lem~ Furt~er, taxol re~ ~tance ls fr~quently ba~ed on energy-dependsnt mecha~i sm~, ~nd theref ore the combinat~ on of an ~TP-depletis~ç~ drug combination administered ~n con~unction with taxol reQults in the inhibition of energy-dependerlt re~i ~tance mechani ~ms to t x~

The new ~ntimicrotubule agent, taxol, an antimltotic s~eslt and th~ fir~t ompound with a taxane ring ha~
demonstxated signif~c~nt antineopla~tic actl~lty ~ n patients with refractory ovarian cancer, refractory ~rea~t cancer, non-~mall cell l-mg ca~er, and ot}~er can~ers IRowin~ky et al., Pharmac. Ther. ~2:35-~4 (l99l) ) . Its antitumor activity, noYel mechanism of act~on~ and u~ique structure haYe gerlerated excitementA Taxol 18 a pla2~t p~oduGt (ol~ta~n~:l from tho bark of th~ Pac~ic Y~w tree, Taxol bre~r$ oli a ), ~nd ~ecaus~ it i ~ obtained :Er~m a limit~d xe~ource th~re is a supply pro~l~m cr~ tlcal to it~
wide pread cl~ l u~e.

Un~ike Adria, taxol is not consid~red a DNA-da~na~ g agent. Taxol i~ an a~timitotic agent that bind~
p~fer~ntially to microtu~ule~ in ~he ab~e~ce of the c:oa~kor~ tubulin and C:TP, a mechani~m unlike t~aat of other anti7nicrotuble agent~ in cancer chemoth~rapeutics (e. 5~., vincr~ stine, and c:oichiciIle) . It block~ c:ell~ in the mitotic pha~ of the Gell cycle ~o that ~h~e Gells #Ir~ unable to repllcate norma~ ly, arld cell ~ea~ esl~ue~
(Rc~winsky ~t al., ~?harmac. Ther, S2:3~-84 (1991) ) .
Importantly, however, the taxol-induced di~ruption o the microtubular n~twork of cancer cell6 induc:eas dea~ of cancer cells by apopto~is SMartin et al., Cell Tissue Kinet . 23 : 545-559 ( 1991 ) , and Le~non et al ., Cell l?rolif .
24: 203-214 ( 1991 ) ), a3 Adr~a does (Mark~ et al ., Biochem.
Pharmacol. 42:1859-1867 (1990) ~ .

SVBSTllUrE SHEET

~23014 PCT/~S93/~4~7~

~ ~3 6~g ~ 26 ~ `

Ano~her ~imî~arity between taxol and Adria ~8 that cells re~i~t~at ~o both agents usually di~play the multl-drug resi~tant (MDR) phenotype, p-glycoprote~n, an energy-dependent drug efflux pump that mai~tain~ each of the~e agents below an i~tracellular cytotoxic level (Gerlach et al., Gancer Surveys 5:26-46 (1986), and Endicott et al., Annu. Rev. Biochem. ~B:137-171 (1989)~.
The~e MDR cellF are cross-re~istant to both Adria and Taxol (Rowin~ky et al., Pharmac. Ther. 52:35-B4 ~1991)) and Gerlach et al., Cancer Survey~ 5:26-46 (1986)), and, since inh~bitors of energy production (e.g., azide) when added to ~uch resi6tant cell~ in vitro increa6e the net accumulation of drug in MDR-cell6 (Gerlach et al., Cancer Survey~ 5:26-46 (~986) and Dano, Biochim. Biophy6. Acta.
323:466-483 ~1973)), the ATP-depleting effect~ of the PALA-MMPR-6-AN triple combination rever~s Adria-and taxol-re~i~tant cell~ to chemo~en~iti~ity.

Stlll another similarity i~ that ~oth Adr~a,. and t ~ ol (~olme~ et al., Proc. Am. Soc. Clin. Oncol. 10:113 (l991)~a~d ~olme~ et al., J. Natl. Cancer I~t.
83:1797-1807 (1991)):, are effective against human breast cancer. Since the:CD8Fl murine brea~t tumor model ha~
demonstrated a~remarkable 100% therapeutic correlation with human breast cancer in term~ of both p~iti~e and negatlve BenBiti~ity to individual chemotherapeutic dru~s using tumor regre~sion as the criterion for evaluation (Stolfi et al., J. Natl. Cancer Inst. 80:52-55 (1988)), it was deemed likely that taxol, like Adria, al~o i8 effecti~e a a single agent again~t the CD8Fl murine breast tumor. Taxol s~ ~imilarly enhanced ~n ~herapeutic a~tivity by th:e prior admini~tration of the PALA-MMPR-6-AN
triple combination. A markedly lower do~e of taxol is nece~sary in the ~uadruple combination. Although taxol had been pre~iously reported to be ineffective again~t the CD8F1 murine mammary carcinoma (Rowinsky et al., Pharmac.
Ther. ~2:3~-84 (1991)), it wa~ considered ~hat the high degree of chemotherapeutic correlation that had been SUBSrl~UTE SHEEr ,~.. WO 93/230~4 PCl`/l~S93/0~77~
27 21~6~91 obser~red betwee~ thi~3 model and the human disea~e warranted re- as~esE~m~nt i~ the ~ame t:D8Fl ~;y~tem.

Example 3 ~how~ ~hat taxol alone i E~ actlve agains~
the CD8Fl t~aor, tha~ the admini stration of t}ae PAI~-l~R-6-AN triple drug combination prior ~o taxol ~ignificantly ~nhanced antitumor acti~ity over ~at p2~0duced by e~t,her taxol alorle at MTD, or 1:he triple drug nation alg~n0, and that only one-third the dose of taxol (i2, t:om~nation) 18 re~uired for t~l8 greater antitllmor acti~rity.

I~npcrtantIy, the ant~tumor effec:t of taxol were ignificantly enhanced ~y the prior admin~ ~tratioIl of the txiple com~n~tion, PA~ ~ M~R ~ 6-~, and the6e therapeut~c re~ults w~r~ obtained wit~ one~ d the MTD
of taxo} a~ a l3ingle Elgent (Table 2 arid 3 ~ . l~e potential beneflt, i~ additiorl l~o enhanced clialical re~pon~ea wlth taxol i~ hu~a~ breas c~ncer, i~ that the a}:~lity to employ lower effect~vo~ do~e~ of $~zo} ~hould all~viate the taxol ~upply pro~lem. An additional be:~e~t of thl~
part~cular dru~ combinatlDn is that, ~inC~ acq~ ed taxol re~ tan~e i~l~vol~re~ :the mdr phenotype with ~ lyc:oprot~in aY3 an en~rgy-dependent drug effux ~ump (Rowin~ky et ~1., P~armac. m~x. S2:3~-84 (1991)), thi8 ATP-depl~ti~g approach in coml~nation with taxol oYercome~ ATP-dependent mechani ~m8 o resi sta~ce to taxol .

ellula~l5nFray_~

In thi ~ embodiment o tl~e invention, cellular energy depl¢t~on compounds are admini~ter~d with radiation a~ the apoptosi2~ inducing agent.

The PALA-MMPR-6-A~a combination ~en~itlzes tumor to ionizing radiation therapy ( s~e Example IV~ .

Sl)E~ U~ SHEI~

WO 93~23014 PCI'/US93/0477~

~36o~ 28 ~E~i~ U~ of ~e Co~PO~ition~ of the_ Inyentior~

l~e drll~ ce~mbinations of l:he sub~ect inventiorl are u~eful i2~ tr~atlslg a wi~de variety of neoplastlc di~sea~fi.
Eor a partieular type of canc:er, the apopto8is inducis~g ager~t o~ th~ co~inatlon of the invention (whicb comprise~;
the c~llular ener~y depletiGn compounds plu~ an apopto~i~
induc~ng agellt), i~ ~elected on th~ basi~ of demonstrated antitumor ~cti~ity w~n it il; used without th~ cellular enerç~y deplet~ng ~ombination. T~e cellular ~ner~y depletion compound~ ( e . g . ~A~-MMPR-6 AN) are primarily intended to ~ensitlze tumor~ to apopto~ du~inç~ agent~
t~ which the tumor~ are already ~usceptibl~ to some degree .

For example, f luorouraci 1 ( ar~d th~ref ore, the co~i~ation of the ~nventios~ comprisialg flus~rouracll and the c~llular energy ~pletion sumpound~, ~.g.
PAI~-~?R-6-AN3 $~ u~ ul for tr~at~ xlg tumor3 of the colon, st~3nac~, ~rea~t, ~ead-and-neck, and ~ancr~a~.
Taxs:~l (snd therefore the combinat~on of~ taxol w~th tb~
cellular energy depletion compound~ u~oful for treat~ng can~ers o the ovary and brea~t. Adriamycin (and therefor~ ~e combialat~on of adriamycirl w~ the energy deple~ion compound~3 o$ the ~ubject ~n~ntton) i8 u~eful in a wide variety of t:umors: acute leukem~as, malignant lymphom~3, c~nc~r~ o the ovary, br~a~t, lun~, bladder~
thyrG~d, er~ metriu3n, t~ste~, pro~tate, c~rvix, h~ad-a~d-n~ k, and in osteogenic and ~oft tis~ue 6arcomas (The Pharmacoloç~ical Ba~i~ sf Therapeutic~, Sevemth Edition (19B5), ed~A.G. Gilman, L.S. Goodman, T.W. Rall, and F. Mur~d, Macmillasl Publishi~ag Company, New York, Ny, pp. l283-l285 ~ . Ionizing radiation ~ and therefore the combinatioa~ of ionizing radiatios~ with the cellular energy depl~ion cnmpound~ of the invention) iB u23eul or treati~çl a variety of tum~r type~, includi~g~ lymphoma, and car~cers of the breast, pelvls, and lung.

SUBSTITIJTE SHEET

. 1~ WO 93/2301 ~ pcr/lJs93/o477:~
~1~6091 Eurthermore ~ 13i~ce the energy depleting con~inations of th~ ~;ubject irl~ntion render tumor~ more ~en~itive to each etpopto~i 8 ~n~luci~a~3 agea~t, the range of tu~or~
~usceptible to tr~Atment with a ~iven apopto~i inducing ug~nt i~ combination w~th the cellular ener~y depl~ting combinations of the i~aventioIl is broader than the range of tumor typ~ treatabls~ wlth the apopto~is inducing agent alone .

A combination of two or more apopto~is inducing agent~ optionally administered in c:on~unctloa~ with an ener~y depl~ting composition of the invention.

The c:ellular energy depleting combination of the islvention is al~;o useful for overroming multiple drug re~i~tance ~n patients. "Multiple drug re~i~tance" ~r~D)R) i~ a condit~on i~ whic~ tumor cells become insensitive to a ~r~r$ety of cytotoa~ic aslt~neoplatic agerlt~. MDR i~
typical~y due to the pre~ence of an ener~y-resau~r~ng pump syÆt~m which remove~ cytotoxic a~ents i~elud~
adriamyc:~, taxol, a2~d Yin~a alkaloid~ frc~m c:~ll (or critlcal regio~s of cells, ~uch as the nucleus) (~:;erva~oni et al ., Cancer Research, ~1: 49~5-4963 ( 1991 ) ~ . In Example 2, in mic:e treated with adri amy~in alone, the re2apo~e to :adr~amycin decr~a~ over time during the three cour es o:E
drug treatment, indicating that the tumor~ are dsv*lopialg re~ *ance to t,h~ dru~. .In co~tra~t, ira mice tr~at¢d w~th adriamy in plu~ :a~ en0ryy depletirlg combination~ of the ~r~vention, the re~pon~e rate actually improves over time during the three courl3e~ of treatment, indicating that the tumors are not dev~loping resistance to adriamycisl, and ir fact, an unprecedented 100% partial regre~ on rate wa~
ob~erved in the~e an~ma~ ( Figure 2 ) .

Admini~3tration and Formulation of 'che_Composit~on~ of th*
In~ention The compound6 of the in~ention are admini~tered in Sl.1~3STlTUTE SHEET

WO 93/23014 PCr/US93/0477~ , 2336~9~ 30 therapeutically effective amOUSl't5. The term "th~rapeutlcally e~ ective amount" as u ed h~rein refers ~o that amount which pro-~ides therapeu~ic ~ffe~ets for a ~iven condition and admini ~tration regimen . q~e compounds and compoFitions of tbe invention are admi~istered orslly, by par~t~ral injectioT~, intravenoualy, or topically, depending on the condit~on being treated.

The timiag and ~e~uence o~ administration of the compound~ and compo~tions of the invention affect the effica~y of the tr~atment. Typically, arl i~hibitor of pyrintidine bio~ynthesi~ ( e . g. PALA) i~ admir~istered lO to 24 hour~ prior to adsnini ~tration of an inhi~itor of purine biosynthesi~; and a nicoti~amide antagoni~t. An inhi3~itor of purine nucleotide biosynthe~i~ (e. g. MMPR) and a nicot~amide aT~ta~oni~t (e.g. 6-AN) are typically adm~ tered at approx~ mately the ~ame tim~
apoptosi~ lnducinçl agerlt(. ) i~ admi2~istered aflter t~e c:~llular eri~sgy depletin~ combination, typlcally about 2 to 3 h~ur~!; aft~rward~ (although the timing may be modifi~d for par~eular drug~ accordiny to obser~ d elinical benef ~t ) .

Th~3 do~ of the particular ag~nt~ are det~rmi~ed a~cord~ to clinieal re~pon~e, alteration~ ioch~mical indice~ o efficacy, and ob~erved 8ign~; of tox~c~ ty.

PA~A i~ typ~cally administer~d in a do~e of 250 ~g/6quare meter; thi~ do~e h~ been found to be su~table in clinical studie~ in which PALA wa~ admini~tered a~ a modulator of fluorouracil. A typieal sin~le dose of 6-AN
is 10 to 50 mg/~uare meter. The optimum do~e ra~ge for 6-~N i~ determined by takin~ tissue blop~ie~ before ~nd after admini~trati~n ~f increa~ing do~es of 6-~N and determining actiYity of enzyme~ reguiring pyr~dine nucl~otideF, e.g pho~phogluc~nate dehydroge~a~e. Th~
minimum do~e required to adequately reduce enzyme a~tivity i B cho~en for clinical admini~tration in ~ubs~quent SUE~STITUTE SHEE~I~

W093~3014 PCI`/US93/0477 cour~e~2 of tre~tment. A typical E;ingle dose of ~R i8 200 to 400 mg/~s~uare meter; a do~e of 225 mg/kg has been ~af~ly admi~istered to ~u~an~ in co~ination with PAI.A.

T~ae compou~d~ and composition~ of the ~n~e~tion are :onnulated iT~ pharanaceutically acceptable carrter~. The dos~ of t~be component~ t~f the energy-deplet~ ng combinat~on~ are cho~en with the intention of providing ~igslificant ~en~itizat~ on of tumor~ to apopto~is-inducing agents~ I~ unclue toxici~y due t~ one or more of the enerç~y-depleting ~ompt~unds i8 encountered, ~ubsegu~nt do~ or the inter~al between courses o treatment are mc~diied. Alternatively, appropriat~ ant~ dotes are admi2listered: ~ac~n or rliacinamide ameliorate toxicity due to 6-AN ( or other nicotinamide antagoni ~ts; uridirle, p~od~g~3 of ~lridine, or other pyrimid~n~ nucl~otide precur~or~ reverlse the bioch~mical deficits prod~c:ed by PA~ ~r other inh~ bi~:or~ o~ pyrimidin~3 ~ucleotide bio~ tb2~i~; and cellular purine nucleotide pool~
aff~cked ~by MMPR (or o~ r ln~ib~tor~ ~f purir~e nucl~ot~de ~ 5~th~Bi8) ar~ replenished by admini~terlr~g appropriate purlne ~ucleotide preGursors , e . g., ino~ e ~ AICM, adsno~ine, or bypoxa~thine, with or w~thout an inhibitor of ~purin~ d-gradatioa~, ~u~h a~ allopurinol.

E~o~e~ o~ apoptc~is-inducin~ agent~ are cho~en to op~imize the therap~utic index. Since t~e en~s~qy-depleting combig~atls:~n ~ensitiz~ tumor~ tQ
ap~ptoEi. -inducing agent~, doses of apoptos~s-indu~i~g a~er~t~ are typically les~ than or eq-aal to th~ do~e~ that would ~e adsnini6tered isa the ab~e~e o ~e er~ergy~deplet~g ~ompc~ition. A~ i~ demo~trated ~n E:xample~l3 II arld I I I, pretreatment with PALA~ 6-AN
p~rmit~ substantial reductions in the do~e~ of adriamycin and taxol required to produce optimum bene~t. Thi~ i~
very important, since adriamycisl produce~ cu~ulative I-ardiotoxicity, limiting the total amount t~at a patient can safely receive. Supplies of taxol are currently SlJB5TlTlJTE SHEET

WO 93~23Q14 P~/U~;93/0477:~

~,~36~9~ 32 ~

l~mited t ~$n e ~t ~ s obtained from the bark of the Paeific Yow, a rel~tively uneommon tree ) . A ~ t~ntial reduetion in the CIOE~I of tB5~0'~ ded t3 produee~ ~n opti~um elinical ef~elt (a~ ~ d~mo~rate~d in Example III), p~rmits more patient~ to b0 e ~eetively tr~tod with ta~ol (or o~er ~seare~ but e~feetlve apopt~ nducing agoslt~).

A eol.ar~e of troatment (PALA-M~R-6-AN followed by an apopto~ inducing age~t) ~ ~ repe~ted a~out ~v~ry lO
days. P~t~ent~ typieally r~eeive three or more eon~eutive eourseR o~ tr~atment. l~e pr~ei~ nu~;~er of ~ourEIes l~f tre~tme~t and the interval b~tw~!en i 8 ad~ ted by the E;kill~d per~on a~cording to cllnica~ d~termir~ation of efficacy and toxicity.

For par~s~toral administration by inj~ctio~ or i~tr~er~ou~ ~nfu~io2~ a compound~ and compo~ io~2~ of t~ l~vo~ios~ t~r~ di~solv~d or ~u~p¢nd~d i~ aguoou~ m~dium ~uch ~ ~t~le phy~;iolo~ line. I~ 1:h~ case of poorly-oolu~l~ compounds (e.g. taxol), ~olub~ l~z~ng agent~
llk~ e~anol, propyle:~e glycol, or polyo~yothylatod ca~tor oi l ar~ u~od .
: ~:

* * *

ollowing exampl~ are o~red to more fully ~llustr~te the inYe~t~on, but ar~ not tc~ be cc~nstna~d a2s miting the ~cope t~ereof.

~E{E E~I.ES
IE~ample I. PAI~, M~PR, 6~ nd E~13ra Mur~e Breast Tumo~D: Spontan~ou~3, utoc~thonou~ MammarY Carcinom~ -~ Cl:)8Fl hybrid ~
bearing single ~pontaneou~, autochthonou~ breast tumor ari~ing during the preceding week were ~lected from a ct~lony which ha~ been de~c:r$bed pre~iou~ly (Stolfi et al., SU85TITUTE SHE~

.. ~ WO 93/23~14 PCr/US93/0477:-Cancer Chemother . Rep . 5S : 239-25} ( 1971 ), Mar~ et al ., Caslcer Cheznother ~ ~ep . O Part 2, S: 89- lO9 ( 1975 ) ) . All ~umors were mQa~ur~d wi~ ca}ip~r~, and the mice w~re di~tr~buted among ex~perimental groups 80 that mice carryin~ ~umorE; of spprox~nately oqual weight wsre repr~es~ted in ~acb tr~atm~t group. Indi~ridual t~nors raaaged in ~i~e from lO0 t~ ~00 mg, and the average tumor weight in all group~ was 260 mg at the beg~ ng of treat3nent .

Murine 13reast Tumor Sv~tem: Firs Pa~E~aqe CDEIFl MammarY Carcinoma -- For each escperiment, the spontaneously ari~inç~ CD8Fl breast t amor~ were trax~ plant~d into syngeneic t~ree-month old miee. A~ in all spo~ltaneous tumorE~, whe ther human or murine, e~ch in~iv~lual eaneer hn a heterogeneoufi r~eopla~tic cell populat~o~. The ~irzt gQne~at~on tra2~spla~i:s of CD8F1 br~a~t tumsr~ a~ obtai~ed ~ro~ a tumor e~11 brei m~de ~y poolls~çl 3-4 ~pontarleou61y-ari~ t~orE;. ~u~, the indl~r~duHl tr~splant~ eaeh exper~nt ~lov~lop ~rom a ingle b~ei thGt:, although eo3runosl to all the ~ee in that xp~rime~t, has a neoplastiG eell eompos~ tio~ that ~s kely sl~ghtly diff~r@nt ~rom that i~ a~o~a~r ~xperime~r~t. ~er~ ore, quantitat~re .mea~urem~nt~ o any i~di~tidu~l paramet~r ( ~ ., TSase actl-~rity, or av~r~ge tumor siæe) may 1~ som~what differ~nt from experim~r~t to ~xp~a~im~nt, but the finding~ w~ll b~ quantitati~ely rele~srant within indi~idual experiments, ~ will ~imilar trend~ amoa~g experiment~ . The C3)8F1 f i~st generatio~
br~a~3t tumor 1~ included in the murin2 tumo~ te~t~2lg panel of the National Cancer Dr~ag Screening Pro~ram (Goldin et al ., Eur . J . Cancer 17 :129-142 ~1981 ) ) .

In appro~cimately three to four weeks, whesl transpl~nted tumor~ were measurable, the tumo~-bearir~g m~ ce were di~tributed among experimerltal group~ ~o that mice carrying tumor~ o~ approximately equal w~ ht werf~

SUBSTlTlJTE SHFEr WO 93~2~014 PCltlUS93/0477:~

9~ 34 repr~ses~t~!d ir~ each treatment group. The a~erage tumor weight wa~ clo-se to 125 mg at the beginin~ OI treatment.

1~ Q~sYD~ - ~ Two axe~ of t}~e t~nor ~ the lor~ge~t a~i~, L, a~d the shorte~t axis, W~ wer~ measured wit~ th~ aid of a Vern~er calip~r. Tumor weight wa~
e~t~mated aec rdislg to the ~ormula: tumor weight (mg) = L
(mm) x (W(MM~ )/2.

Chemo~erap~ut~ Aclent~ -- M~R, 6-AN Rnd ElUra were obtain¢d rom S~gxna Chem~cal Co., St. Loui , MO. Each of the~e agents was dis olved irl O.E~5~ NaCl ~olution immediately before use. PAI,A wa~ o~7kained from the Department of ~ealth, Education, and Welfare, USP~S of the ~atio~al Cancer Institute, Bethe~da, MD. P~LA wa~
dis~olved 1~ 0.8~% ~aCl solution, a~d th~ p~ wa~ ad~ted to 7.2 to 7.5 wl ~ lN NaOH before ad~ustment to final volume~ All agent~ wer~ admini~tered i.p. 80 th~t the de.ired do8e wa~ con~ained in 0.1 ~1/10 g of mou~e body w~ig~t.

The~ drugs were admini~tered i~ a timed ~gu2nce, with P ~ admin~tered 17 ~ours ~efor~ the ~imultan~ous admini~tratlon o MMPR ~ 6-AN, and wi~h S FUra admini~terod ~ ~our~ after MMPR ~ 6-AN. m ro~ghout thi~
: application, ~he timing of bi~chem~cal m~asurement~ i8 given in relat~o~ to ~he in~ctio~ of the la8t ch~m~therapy (~Oe., MMPR ~ 6-AN) in the chemo~her~peutic ~eguen~e~

Determination of Chemotpera~-Induced ~L~r e~r~ssion Rate -- The inltial ~iæe of each tu~or ln each trea~me~t ~roup wa~ reeorded pr~or to the initiat~o~ of treatment. Tumor ~ize was recorded weekly duri~g treatment and again at 7-9 day~ after th~ la~t cour~ o treatment. ~or eaeh experime~t a ~i~gl~ o~erv~r mada all mea~urements in order to a~oid varsation in caliper measurement~ from individual to individual. By SUE~SrlTUTE SHEEl WO g3t23~14 PCI`/US93/0477:-35 213~D~I

co~ention, p~rtial tumor regre~;sion is defined a~ areduct~c~n in tu~nor Yolume o~ 50% or greater compared to ~e tume~r volume at the time of initiation o~ treatm~nt.
The partial r~res~ion rate obtained from a past:icular treatment iE~ expre~d a~ a percentage; ~.e., num~er of partial ~ e sion~ per group/total sltan~er of an~mal~ per group x 100.

Stat~. stical E~.raluation -- D~fference~ i~ t,he nun~er of part~al tumor reqr~sioa~ ~etween trea~nent group~ were compared for ~tati~tie:al ~igr~ificance ~y chi~ uare analy~i ~ . Stud~nts t t~st wa~ u~ed or evaluatlorl of mea~ured biochemical differen~es between treatment groupE;. Di eren~es between group~ with p ~ 0. 0~ were corl~idered to be siç~i~ ant.

l~cor~ion f Prec~Ls~nto RNA ~nd DNA --Racl~ol~ed precurlsors, 3 ~ and (3~a-L-~eucine were admis~ ered i.p.; th~ labelins~ period was 2 hr1s; At the end of t:he label~ng period, animals wer~ asr~ced by cerv~eal ~ locatior~., Tumor ti~ es w~re homo~o~zled in TNJ~ bu::f-r cont~ini~ag lX Tril;on-X 100 ~ 0.01 M
Tri~ Cl~ pH~ 7.6; 9.15M NaCl; 0.001 M EDTA). ~e homog~n~t;e w~,briefly sonicated, and t}~en diçlested with Prona~ :Eor 60 min. at 37C (0.2 mg~ml c~r 2 hs ~ at 37C) and ~ally~ materlal was extra~ted with chlorofor~ oamyl alcohol ~24~ ol/~ol). Sampl~ of extract~d materi~l were pre~ip~tated with trichloroacetic acid to determi~e total radioacti~rity. Other ~aa~ples are f~r~t treated with alkali (0.4M NaOH, for 90 mir~ at 37C:) to de~ermis~e alkali- stable, trlchloroa~et~G
acid preGipitable radit~a ti~rity. The difference between the total and alkali- table radioact vity wa~ a~ cl to repre~nt radioact~Yity in RNA. ~5- ~I)Fluorouracll (20 Ci/mmol~, t5-3~1)3 thy~TIidine and ~5~ deoxyurid~ne monop~a~sphate (20 Ci/nunol) were purcha~ed from Moravek.

SUBSrlllJlE SHEEI~

W093/23014 PCT/US93/0477; ~
~36~ 3 6 :?

Substrate_Accumulations -- C~llular levels ~f 6-pho~phogluco~ate, gluco~e-6-phosphate and ~ru~to~e-6-pho.pha~e were measured on perchlor~c acid extract~ by published method~ (6-pho~phogluco~ate (Haid, Methods of_Enz ~ (Bergmeyer, ~OU., ed). New York: Academic Pres~ pp. 1248-12~0 (1974~);
gluco~e-6-pbo~phate a~d ruc~o~e-6-pho~phate (Lang et al., M~thod~ o~ en~ymatic analy~is, Vol. 2 ~ed. ~.U.
Bergmeyer)).

14C-Qrotic Acid A sa~_for PRPP~-pho~phoribosyl pyropho~phate) -- The a~ay i8 ba~ed on the co~er~ion of ~4C-orotic acid to uridine monopho~phate with r~lease of 14C02 by orotidine-5-pho.phate- pyropho~phoryla~e ~
orotid~e-5-pho~phate decarb~xyla~e (Houghto~i et alO, Mol.
~h~rmacol. 22:771-778 tl982))- An aliquot of the homogenat~ was as~ayed for protei~ content ~y the method of ~owry et al~, J. Biol. Chem. 193:~6~-275 (1951).

Proce ~ina Df Sa~Ples for ~ ~opt~nt -- Froze~ tu~or : pecimen~ were homo~enized ~n ice-cold 1.2 N perchloric acid. The acid-in~olu~le fraction was removed ~y ce~triugatlon (7000 rpm for 15 min~. The acld-~oluble frn~tion a~ neutrallzed ~y e~trac.tion wlth a ~i~tur~ o~
freon and txi-N-oetylam~e ~2:1). æhe ~xtract Wa8 t~e~
~ilter~d through a 0.22 u ~illipore membrane filter prl~r to EPLC analy~i~. NSP eontents in tumor w~re normaliz~d to the protein cont~t o ~he ac~d-in~o~u~le fraGtio~.

Measurament~of_NT~P_Level~in~Tu~or - ~PLC analy~i~
in tumor wa~ perform~d on a Water~ 84~ ~PL~ Ry~tem with a WISP automatic ~ampler. NTP }ev~ls were analyzed ~y ion-~xchange grsdie~t chromatograp~y u8in~ a Water~ SAX
column startin~ wi~h 3 mM NH~H2P04, pH 3.~, pr9ceeding ~n two ~tep~ to 70% 0.5 M NH4H2P04, p~ 5.0, plu8 30~ start~ng buffer. The run time for each 100 ul ~f extracted sample wa 60 min. Tumor NTP levels are expre~s~d aB microgram8 nucleotide tripho~phate per milligram of pr~t~in.
SUBSTITIJTE SHEI~

~ ~ WOg3/23014 ~1 36 PCr/U~93/0477~

31P ~ ~Nuclear Ma~netic: ~e onance ~ SPectra -- 31P
NP~R spectra were o~tained u~ing techni~ueR de~cribed prevlou~ly (Koutcher et al ., Cancer Re~; . 5C): 7252-7256 (15~90) ) . ~rl~rly~, spectra were obtained on a Gen~ral ctr~c NT-300 wide ~ore spectrome~er op~r~ting at 121. S
NHz. Experime~tal parameter~ lncluded a ~pectral wid1:h of ,OOC) ~Iz, 60 t~p angle, recycle delay of 2 seeonds, 512-1024 averaged- free induction decays (FID'~), nd 1024 data pC~i;lt~. l~e E;pectra are partially fiaturat~d u~in e exp~rimental c:ondition~. Four turn ~olenoid coil~
w~th a Faraday ~3~ield ~Ng et al ., J . M~ . Rc~on. 49 : S26 (1982) ), po~sitioned between ~e bt~dy of the mou~e Emd the coil were u~ed to detect the NMR ~ nal. Control experiments ~erified that rlo sigrlal WE15 o}:taisled from non-ttamor bearins~ a2limal~ mounted in an identical manner.
Spectra were analyzed using 2S ~z expo~ential multiplication followed by Fouri er trans~ormation.
Speetr~l peak areas were estimated by fitting ~he sp~ctra to a ~ri~ o~ Lorentz~an peak, u~ing a progrun (GE~qC:AP) a~ailabl~ o~ the ~pectrometer, after *lttir;lg the Iba~eline to a *hird order polynomial (u~ing E;tandard ~n~rnl ctric softwar~). Si~lce other peak~3 ov~rlap ~e alpha-and ga~una-~uc:leotide triphosphate (NTP) peak~;, th~
b~ta~ wa~ u~ed for calculating NTP peak area ratios.

~ idine X~rla~e and T~idvlate_s~ e aæ~aYS --Ti~isueE~ w~re homogenzi~d (Potter-Elvehiem homogenizer) a~
a 20% ~wt/vol) olution in ~ris-Cl ~100 ~rM, p~ 7.6), ~-mercaptoethanol ~20 mM) and sodium fluoride (100 n~.
Homog~a~es were ~entrifuged at 4 (100,000 x g, 60 mi~. or 10,000 x g, 30 m~n) and th~ ~up~rnatant f~ct$o~ retained on ice. En~yme a~ays were per ormed either on i~dividual 6amples or on pooled ti~Rues from thr~e an~m~l~. Tumor~
wer~ between 300-500 ~g. Thymidine kina~a wa~ measured immediately after cyto~l preparat~on by mea~ of a DE81-fi~ter-binding a~ay (Ives et al., Anal. Biochem.
2a-192-205 (1969)). ~e as~ay mixture contai~ed Tris-Cl (100 mM, p~ 7.6), ATP (5 mM), MgC12 and ~-C3H3)thymidi~e SVBSTlTlJl~ SJ IE~T

WO 93/23014 ;~9~ 38 PCI/US93/û477 ~25 uM, 1. O Ci/mmol ) and ~:ytQE~c~lic prctein. Thymidylal;e ~yntha~e a~ ity was mea~sured ~y the rel~a~e of tratium from (~-3H)~ 10 uM, loO Ci/n~rtol), CH2H4Pt~l:;lu ~100 uM) arld c:yto~olic frac~ion (25 uL) (Robert. ~ ~iochemi~try 5:3546-354E3 (1966~). Reaction~ were termi~ated by the addition of perl:hloric acid (10 uL, 0.7M). Protein wa~
determined ~y the method of Lowry et al., J. Biol. chem.
193: 26~-~75 ~ ~9Sl ~ .

In~o~orat~on~la a into RNA -- I~corporation of FUra into RNA wa~ deten~ ed by i~olating tumor RNA by the acid-guanid~ne iso~iocyanate procedure (Chomczyn~ki et al., Anal. ~Sioc:hem. 162:156-159 (l9B7) ), a~ter treatment Wit}l (5-3~ElJra (2.0 mCi/mmol). Ti~sues were harve~ted 4-5 hr after tra~atme~t, i~unediately frozen in 1~ quid nitroç~en atld ~tored at -70 . Ti~sue~ w~re homogenlzed ( 80 ~g/ml ~ in the de~aturi~g solution ( cltrate a~rcoæyl lausate-2 mercaptoethanol ) ~nd ~tracted wlth one ~rolume of ph~nolschlc~roform:i~oamyl alcohol (50:4û:2~. .After extractlon the RNA wa~ precipitated with alcohol, wa~hed, redis~oltv@d and quantitated by u~, a~d tho~ pr~c~pitated with pé~Ghloric a ld. T~is flnal precipittl~:e wa~
collected Qn GF-G giiter~ and the radioacti~rity wa~
determl~d .

Che~motheraP~Effect~ on_ESrea~t Tumor Tra~lants In a ~er~s of 5 ~m~lar ~xper$men~, a group (Group 1) of C3)E3Fl m~ca bearing advanced, firE;t pas~age ~porltenaou~ CD8Fl breast ttasnor tran~pla~t~ were treated with the t~ipie co~l~ation of PAI~ ~ ~qeR ~ 6-AN. PALA
( 100 mg/Xg) waG administered 17 hour~ ~efor~ M~R ( 15C) m~/kg~ plu~ 6-A~3 (10 mg~k~) (Table 1). A ~ ond sroup ~Group 2~ recei~d the ~ame treatntent with ~?AI~ ~ M~?R
6-AN, foll~wed 2~ hour~ later by El~ra ~75 mç~/kg).
Treatment wa~; rep~ated at 10 or 11 day ~nter~r~ls for a total vf 3 courses, and o~ervations were ~ecorded 7 days after the last course of treatmerlt.

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Partial tumor regressions were ob~erved in 8 of the sur~i~g 48 mice ~17X)~ treated with PALA ~ MMæR ~ 6-AN
(Group 1). The range'of regre~ion rate in indi~idual experimentE varied ~rom 0 to 30X. The add$tlon of FVra to the same regimen of PALA ~ MMPR ~ 6-AN (Group 2) produced a sig~if~cant and meaningful ~ncrea~e in therapeutic activity. Thirty-seven of the 50 treated mice (i.e., 74X) experienced partial tumor regres~ions (with a range in the individual experiments of 60-90X). Thi~ level of therapeutic acti~lty was fo~nd to be significantly better than that achieved wlth the three-drug combination (without FVra,~Group~l, p < 0.001), and mo~t importantly, thi~ increase in antitumor acti~ity wa6 achieved without mortality. It 6hould~be noted that FUra a~one at 75 mg/kg did not prod~ce any~ tumor: regreBsions after 3 cour8eB of treatment ln nine separate experiment~ ~0/B8 mice, not 6hown).

; I~ 3 ~eparate:~::experimentg the therapeutic acti~ity of the PA~A: MMPR~ 6-AN ~ ~ ra combination was compared with that o ;the~combinati~n of:PALA ~ MMPR ~ FUra (i.e., wlthout:~6-AN)~at t~e~ame~doses ~nd~ admini~tration chedule. One~week after three cour~e- of treatment the four-drug com~inati~on yielded a 71% tumor regre~8ion rate (20 tumor regre~sion6~n 28 ~urviving mice) wherea~ ~he thre~-drug combinat~on without~6-AN yielded a regre~6ion rate~of only 41X~;(12 tumor regreosions in 29 6ur~iving mice). The diference in regression rate between the~
two treatments was found to be ~tatistically 6ignificant, p ~ 0.0~, indicating that~the low-do~e 6-AN W~8 contributing to:~t~e therapeutic activity in the four-drug eombination.~ ~
ChemotheraDeutic Effects on Spontaneou~, Autochthonous Breast Tumor~

The PALA ~ MMPR + 6-AN regimen, witb or without FUra, wa~ administered at 10-11 day int~rvals 1~ a &eries of ~ix SUB~I~UrE SHEEl ,~. W093/23014 2136~ ~1 P~T/US93/0 experiments to large nl~ber~ (a total of 66 or 67 mice in eac~ treatment arm) of CD8Fl mice bearing advanced, spontaneous, autochthonous breast tumors averagi~g 260 mg~. Re6ult~ were observed at 6 w~ek after the in~tiation o~ treatment (i.e., g day~ after the fourth course of treatment) (Table 2~.

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W~ 93/23014 P~r/U~93/0477~
2~350gl ~ e PAI~ ~ M~R + 6-AN regimerl ( group 1~ produced a partial t~nor rèS~re~sion rate of 38% in mice bearing spon~ag~eous, autoehthonous ~rea~t tum~rs ( 24 pas~ti al tumor regre~sion~ ~n 64 survi~in~ miee) with an ac~:epta~sle mortality rate (4%, 3 deaths in 67 treated mice). The addit~o~ of FUra to the three-drug r~gim~n (G~oup 2 ) re~ulted in 41 partial regre~ionE; in th~ 61 sur~iving mie0, or 675~ (wi~ a rarge of 50-90% in the indi~ridual expariments ) without an increase in the mortality rate 6 0~1Y 7%), and w~ only a 10% bocly we~ght 1088 . A8 in the prev~ou~ experiments in mice bearins~ flr~t pas~a~e t~amor~;; the addition of FUra to the ~ALA, MMPR, 6-AN
regimen re~;ulted in a E;igr~ificant increase ln tumor reçlre~o~ (p c 0.01) in mice bearin~ ~po~taneoust u~clc~ u~ r~a~t tumor6. Again, lt should be noted at EUra alone~ at 75 m~ produ~:ed ~ ~X r~gre~iorl~ of pontaneouE;, auto~hthor~ou~ CD8F1 brea~t 'cu~nors ( 1 r~gre~ssio~ ~n 24 treated tumor~ ), ~d spon~:a~eous reg~e~s~o~ of theBe :tumor~ ha~ not be~n ob~ ed.
:
BIOCHEMICAL FINDI~aS
MacromoIecula~ ~nthesi -- Adrn~nistration of R-6-~ ~ re~ulted ~ n signif~cant i~ tion o macromol~cular 6y~the~i~ in fir~t pa:ss~ge CD8Fl ~re~t ; tumor dete tabl~: a~ th~ earlie ti~e p :3int examined, 2 . 5 hours aftsr drug admini~tratiorl, and pro~re~ins~ to 80%
inh~bi~ion of DNA Byrlthe i~, 8~% i~ibition of ~P.
6yn~he~i~ and 70X ir~i~ition of protei2 ~ s at ~8 oùr~.

Pentose Shunt and G1YC~1~tiC Intermed~ates ::~ :
6-A~ haR been reported to inhibit ~h¢ pento~e phosphate pathway ~y producing a met~olic ble~ on 6-pho phoç~luconate ( 6-PG~ dehydr~genas~ ~Herken et ~1 ., Biochem. Biophyo. Re~. Comm. 3~:93-1~)0 (~969) ) . l~e SUE~STI~ SHEEl WO 93/23014 PCl`/US93/0477~

~ 3~9~ 44 a~ccumulation of thi ~ strate re~ult~i in feed-back i~ibition of pho~phogluco~e i~merase and prevents the ormation o ructose-6-phosphate from glucose-6-phs phate (G 6-;1?~ (Racker, In: Mechani~;m~ in Bio~ne~etics.
Acade~mie ~ress, New York-London, p. 207 1~l965), ~lorecker, B.C. In: Car~oh~rate Metaboli~m_ancl It~ Disorders.
Vol. I. Agademic Pre~, New Yorlt-Londo~ (1968)). AR i~
evide:nt ira Table 3, administratlon of 6-AN did~ ln ~act, re~ult in ~ign~ fis~:ant elevation of 6-PG ( 167-~old increa~e above ~aline-treated coa~trol~, and G-6-P ~3-fold increa e a~ove control ) in CD8Fl breast tumor~ . Slmilar re~ult~
w~re obtained in t-lmors from mice treated with the 6-~-containing 3-drug combirlation (PALA-M~?R-6-AN). The 3-dr co~r~aination reduces NAD level~ in this tumor (Martin, Metabolism arad Action of Anti-c~nçer dru~. Ed.
by Garth Powi~ and Ru~Esell A. Prough (London: Taylor &
Franci~s3, pp. 91-14t) ~1987) 3 .

.

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WO 93/23014 PCI'/OS~3/0477 Egtimation~ of C~ellular EneraY Level6 by NMR ~See Eig.
pectra were o~tained frcsm fir~t pas~age CD8F1 }:~re~3t ~ orY~ prlLor to treatment, ~r~d at 2, 10, and 24 hour~ po~t treatment with PA~-M~R-6-AN. BaE~eline spectra were similar to tho~e vbtained in pre~ious ~tudie~
(1Coutcher ~t al., Mag~etic Resonsnce in Med~ ne 19:113 123 ~1991)). Po~t-treatm~nt, a deeroa~e in the B-NTP peEk relatlve to ~ norganic phosphllte W~13 noted. The result~ obta~ned from 7 tLunor~ aring animal~; treated in thi~ mlanner are Eummarized in Fig. 1 whlch 6how~ that both pho~phocr~atine~inorganiG (PCr/Pi) and NTP ~nucleos~de tr~pho~;phat~)/Pi ratios were decreased ater treatment wi~h thi~ drug comb~nation, whi~ indicat~ve of energy depletion. The changes i~ ~Cr/Pi and NTP/Pi at 10 hour~
were ou~d to be ~tati~tically significant.

Bios:hemiqal Mea~url~ment of ATP Levels ergy depletlon in dru~-tr~ated tumors also wa~
ma~fe~t ~n decrea~ed Al~ p~ol~ a~ shown ~ Table 4. At 6 arld Z4 hour~ af~er admir~il;tration of N~R alone (Group 2), 6-~ alo~e (Group 3 ~, or the 3-drug combi~at~o~, P~ R- 6-~N (Group 4), AT~ le~el~ ~n fir~;t pa~age C:D8~1 breast tumor~ wer~ ~gn~f~cantly depre~s~ed, ~ chi~g Q lerel of 32X of cont~ol ~n t~mor~ from m~ce treat~d with t~e 3-drug s;ombinatioIl at 24 hours po~t treatn~or~t.

SUE3Srl~JTE SHEE~

~ WO 93/23014 21 3 6 0 91 PC~/US93/0477~

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SUBSrlTUTE SHEET

WO93/23014 ~36~9 48 PCT~US93/0477 Biochemical Chanaes Favorin~ the Activation and Com~etiti~e Acti~itv ~f FUra -- A~ a con~eguence of the block of de novo pur~e ~ynthesi~, the admini~tration ~f MMPR has been sh~wn ~o result in the a~cumu~ation of PRPP
in CD8Fl tumor cell6, a~d when NMPR is adminlstered appropriately befo~e FUra, the incr~ased levels of PRP~
reæult in enhanced acti~ation of FUra (Martin et al., Nucleosides and Cancer Treatment (M.H.N. Tattersal and R.M. Fox, ed~.~, Academic Press, Australia, pp. 339-392 (1981)). Becau e of the pos~ibility of complcx tnteractionR among d ~ g~ in combination, lt was necee6ary ~; to veriy that the MMPR-containing 3-drug ~ombination al~o was capable of producing thi~ elevation of ~RPP in tumor cell~. Accordingly, PRPP levels were measured ln untreated fir~t pas~age CD8Fl tumor, and in CD8Fl t.umors at 3 and 24 hours after treatm~nt with PAhA-MMPR-6-~N.
Mea uremeDt~ ~rom 4 untreated tumors in each of 3 ~eparate experiments yiclded an aversge PRPP level of 28B pmol/~g with a standard error of 19 pMjmg. PRPP levels rose to 490 63 pmol/~g~(i.e., 2.2-fold increa~e, p c 0.01) an~
833 ~:153 pmol/mg (i.:e., 3.7-fold i~crease& p ~ 0.05) at 3 .
:and 24 h~urs, respectively, after adminifitration of PALA-MMPR-6-AN.

: : :
As a conseguence~o~ the inh~bition of aspartate transcarbamyla~e, the administrat~on of PALA alone ha~
been demonstrated t~ re~ult in depletion of VIP pool~ in CD8F1 tumors, and when administered appropriately before FUra, this re6ult~ ~n an augmentation of the actlvity of the competit~ve FUTP analog (Martin et al., ~anGer Re~.
43:2317-2321 (1983)). Again, UTP level~ were measured in first pas age CDBFl breast tumor~ at 24 hours after administratio~ of PALA-MMPR-6-AN and found ~tati~ti~ally ~ignlficant depress~on of UTP pools compared to saline-treated control tumor~ ~data not shown).

SVBSTITUTlE SHEET

WO 93/23014 ~ 1 3 6 0 g I ~Cl`/US93/047 l~sre~ore, two of the drugs (MP~R and PAL.A) . in the 3-drug combi~ation produce the l;ame biochemical alteratio~s ~n C:D8Fl brea~:t tumor cell~; when ~dmi~il;ter~d in t~e 3-drug comblnation as they did wh~n admini~tered as sin~le ag~nts, a2~d th~e alteratio~ ha~e be~n de~on~trated to re~ult in the augmentation of ~ equently admiTlistered FUra.
Biochemical MeasurementE of EVra Activity when Administered~ Two- and One-half Hours a ter Since the inhibition of RNA ~ynthe%i~ i one of the earliest mea~;urable es~oents after the admis~i~tration of the triple combinatic~n, the effect of the combination upon the incorporation of EUra into RNA is of inter~t. The amsunt o t~or (EU)RNA in the group which receiv~d PALA + MMPR
6~ EUra wa~ appreciable (355 :~ 127 epm/mg RNA). Thu~, althouçh RNA ~ynthesis wa~ significantly inh~bited ~llowis~o trea~ment - with PALA-MME'R-6~ the i~co.rporation o~ ~Ura ix~:o residual newly synthesized RNA wa~ ~o1:
it@!~O

Thr~e group~ of ~ CD8Fl mice b~aring e~ sized fir~t pas~age br~a t tumors were treated with saline, : ~ PAI~-MM3?R-6-~N, or PAI~-N~R-6-AN followed 2 ~ 5 hour~ later w~th E'Ura. Measurement~ o~ ~hymidin~ ki~a~e a~d thy~n~dylate oya~ e aetivity 24 hour~ after treatm~nt are ~hown in Table 5.

SlJBSTlTlJlE SHEET

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SUBSTITUTE SHFET

WO 93/23014 2 f ~0~ 1 PCl`/US93/~477~

Thymidylate ~yntha~e acti~7ity was d~creased by nearly 50%
~ter treatment with PALA-MMPR-6-AN, even without the add~tiora of FUra (Group 2, Table 5). l~i~ depre~ion of @rlzyme actlvlty 1~ likely due ~o the general inhi~ition of protein synthe~i~ followin~ treatment with the 3-drug co~ination de~cribed abo~e. However, th~ ~ddition of EVra 2 . 5 hour~ follow~ nq treat~slent with the 3-drug com~ination (Group 3, Table S~ resulted irl more than 76%
i~hibit~ on of thi ~ enzyme . l~i ~ increasd~d level of i~hibition i8 believed to be a re~;ult of Fd~lMP ~ nhibitlon of refiidual thymidylate E;yntha~e actitrity.

Nord et al ., Biochem . Pharmaco} . 42: 2369-2375 ( 1991 ), reported that thymidine kina~e a~tivi~y i~ lo~t over 24 h~ur in CD8Fl tumor~ from mice treat~d with the maxi~num tolerated do6e (100 n~g/kg) of ElJr~ alorle. The trlple combi~atio~ (Group 2, Table S) produced approx~mately 80%
~ibitlDr~ o$ TKa~e ( appar~ntly due to ar~hibition of RNA
~s~e~;i8~, while th~ additic~ of EVra (75 mg/kg) to the triple combinatio~ ( ~roup 3, Tabl~ 5 ) pro~Tided ur~er inhi~ition t93 . 7%) Of TKa~;e acti~ity .

~ rapeutic acti-Jity has been mea~;ur~d in the~e E~udiels u~inS~ ~;trinçlent clinical ~:riteria of tumor' regre~ion ( i . e ., 507~ or greater clru~-indu~:ed decr~a~e i~
tumor size ~, rather than the more coa~ventional a~mal model criteria of tumo~ growth inhi~ition. It shc~uld al~o be not;ed that the spontaneous, autochthonsu~ )8Fl br@a~t tumox model ha~ demonstrated a remarkabl~ correlatiorl with huma~ brea~t canc~ in t~rms of both poEitive and n~gatiYe sen~iti~ity 1tc~ indiYidual chemotherapeutic drug~ u~ing ~usnor regr~;sion a~ the criterion for ~aluatio%l (Stolfi et al., J. Natl~ C:a~cer In~t. 80:52-~5 (1988) ) .
Thi example fihows an impressive i~cr~ase in umor regresRiorl rate~ when E'Ura was admirlil3tered lr~ con~un~tion with P~LA, MMPR and 6-AN in the therapy of either advanced first pas~a~e, or spontanecus, murine br~ast tumor~, and SgJE~ UTE SHEET

WO 93/23014 PCI /US93/0477~

~ 5 52 E;~OW8 the result; o~ measureme~ts of biochemical parameters affected by`treatment.

E:x~ple 2: PAI,A, ~R, 6-AN and Adria~yc~n ~

Spontançou~, utochthonoll~ ManunarY Carci~oma CD8F1 hybrid mice bearing ~in~le ~pontaneou~, autochthonous brea~t tumors arisinq durin~ the preceeding week were 6elected from a colony which ha~ beerl described pre~riou~;ly (Stolfi et al., Cancer Chemother. Rep.
5~ ~ 239-251 ( 1971 ) a~d Martin et al ., Cancer Chemother.
Rep ., Part 2 , 5 : 8~-lC)9 ( 1975 ~ ) , and i ~ in~luded in the murine tumor t~;ting panel of the National Cancer Dnlg Screenir~ ro~ram (Goldin et al., Eur. ~. C~ncer :`
17:1~9-142 (1981)). All tumors were mea~ured wlth ~ , .
calip~rs, afld ~the mic~ were distributed unonq experimental :group~; E;D t: at mlce carrying tumor~ of a~proximately egual w~ h~ w~re sepre~nted ~n each treatmer~t ~roup.
In~ividual tumors raa~ged in size from 100 to 500 mg, a~d the a~rer-ge tumor wei~ in all group~ was 304 mg at the be~ ng of treatment. A~ in all ~pontaaeou~ tumors, whsther ~uman or murine, each indi-vidual c:anc~r ha~ a heters~gerleous~ cell~ population and therefore, u~like long-tra~3planted ~tu~Dor: lines, on~ ~pontaneous tumor may dif~er from another of t:he ~ame histiotype in ~u cept~ ~i li ty to a given drug treatment .

Tumor Measurement Two axe~ of th~ tumor ( the longel;t a~is, L and the shorte8t axi ~, W) w~re measured w~th the aid o~ a Vernier caliper. ~mor waight wa~ estimat~d according to the formul~: tumor wei~t (mg) = L ~ x (W(INn)2)/;~.

Chemoth_raPeuti c Aclents SUE3STlllJTE SHE~

. ~ WO 93/23014 2 1 ~ fi ~) 9 1 PCr/US93/0477~

M7~ and 6-AN w~re obtained from Sigma Chemical Co., St. Louis, MO. Adria wa~; oi:~tained from Adria I,a}:~oratorie~, Columbu~;, Ohio. Each of t~e~ agentg war-di~3EolY2d in 0.85% NaCl solution immediat~ly ~efore u~e.
PAL~ waEs obtained from the D~partment of ~l~alth, E:due:atiosl, and Welfare, US~HS of the National Cancer Institute, Bethe~3da, MD. I?~ wa~ di~sol~red in O . 8~% NaCl ~oluticn, and the pH wa~ adju~ed tc~ 7 . 2 to 7 . ~ wit~ 1~
~3aOH ~efore adjustment t~ f~nal ~olume. All agent~ were admini~tered ~o that the de~ired dose wa~ contained in 0.1 ml/10 g of mou~3e body weight. Adria waE; adsnini~tered i.~r.
and all other agentE; were administered i.p.

The e drug~ were a~ini stered in a timed ~equence, with PAI,A ~dminil;~ered 17 hour~ be~ore MMPR ~ 6-AN, and Adria ~d~aini~tered 2 1/2 }~ours after MMPR ~ 6-~3.

Determir~ation o Che~othera~v- Indu~ed Tu~nor Reares~ion Rate ~ i4ial locat~ on and ~ize of each tuallor in ~ach t~eatment group wa~3 recorded prior to ~e ~nitiation o~E
treatment. Tumor gize was recorded we~kly duri~g treatment a~d again at 7 days after the lllBt cour-;e of treatmetlt. For each experin~nt a ~ingle obs~rver m~e all m~al3uremer~ts in order ~o a~oid ~ariation i~ c:aliper meafiurements f rom individual to indi~idual . By convention, partial tumor regre sion i~ degir~ed as a reductic~n in tumor volume of 50% or 5~re3ater co~nparee~ to the tumvr volume at ~:he time of initiation of treatment.
The par~i~l regreesion ra~e obtained from a particular t~atmerl~ i8 expre~ed a6 a percentaS~e; i.e., Number of partial regressio~s per group~Total nun~er of an~mals per group x 100. Complete tumor regressie~n wa~ defined as the inability to detect tumor by palpation at the initial site of tumor appearan~:e.

Statistical Evaluation .

SUBSrlTUTE Sl IE~

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W O 93/23U14 '~ 609 sg PC~r/US93/0477~ ~

Di~ference~ in the nuu~ber of partial tuunor regre ~ion~ between treatment groups were compared for ~tatlsti~l Qisnnificance by chi-~ ~ are analy~
Dl~fere~ce~ between group~ wlth p = O.G~, or le-s, were considered 8i gnif~ant.

ChemotheraPeutic Effect~_of the Triple DrugL~on~bi~ation and Adria, Alone and in Ç~adruPle Con~bination SPAr~9~MPR~6-P~N~Adria) on SPontaneous, Auto hthonou~ C~D~F~
IL~ Tumor~

The PAI~ ~ N~IPR ~ 6-~iN regimen, wit~h (Group 2) or without (Group 1) Adria, wa~ ac~ninistered at 10-11 day intervals in a ~ries of t~hree separate ~xperiment~ to a total of 44 C~D8Fl mice bearing ~dvanced, ~pontanoous, au~oc~thon~u~ brea~t tlunor~ averaging 304 m~s in ~ach group. In ~ach of the~e experiment~, a c~mparE~31e ~roup of mice wa~ treated wit~h Adria alone at 11 mg/kg whlch had been deterlnined prev~ou~ly to be the M~D of Adria alone when adbnini~tered ~ t~hi~ 10-11 day treatment s~hedule.
Re~ults w~re ob~e ~ ed 7 day~ after t~he t~hird cour~ o~
tr~a~ment.

.

SUBSTITIJT SHE~

".~ WO 93/23014 21 ~ 6 û 9 ~ PCI /US93/0~77' ._ ~_ ~~ ~ ~

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SUE~STITUTE SHEET

Wo 93f23014 PCr/lJS93/0477~
~ 3c~9~ 56 T~e Pl~ ~ Mq~R ~ 6-AN regimen ( Group 1 ) produced a partial ~umor re~gre~s~ion rate Os~ 76% ln mice beari~s~
~pontaneous, auto~hthonouE3 })rea~t ~umor~s (32 partial tumor regr~fiion~ in 42 ~ur~ ng mice with a ra~ of 50-92% in the lndi~rid~aal experiments ) with no toxic deat~ ~n the treated mice. The addit~on of ~dria to the t:hree~-drug reg~men (Group 2 ) resulted in 42 partial tumor regres~ions ln the 42 survi~inçl Jnice, or 100% (i.e., wlth no range in the indi~idual exper~ments), without a E;ignificant increa~e in t~h~ mortality rate (only 5%), ~nd w~th only a 16% body weight 101s~. The add~tiorl of P.~ria to the PALA, MMPR, 60AN regimen waæ found to result ~n a ~atist~cally siq2lificant increa6~ in tumor regreE~ion~ (p < 0. Dl ) in mice bearing E;pontaneous, autochthonc~u~; breaE~t tumors.
Moreo~rer, 5 of ~e 42 tumor regreEsE;ion~ in Group 2 w~re complete as~ ~ppo~ed to parti al regressions, a~d lt s~.~uld e not~d that complete regressiosl~ were not ob~erved i~
he other two ~roupE3. It should be ~oted that Adria alone ~t 11 m~/k~ produeed only 16X regre~slo~l; in 42 treat~d tumors, l~d ~ontaneou reçlre~ion~s of 1:hese ~umor~ ha~
not been o~erved.

~ critically important feature o the rel;ult~ Qf the~e exp~rim~3rat~ in mic:e bearing ~pontan~ous, autochthonou~ broa~t tumor can be seen in Fisr~are 1 where the per~ent of tumor~ regres~ion in each o t:he treatment groups i23 plott~d at 7 day~ after ~ach of the 3 coux~e~; of treatme~lt. Not~ thç diminishin~ therapeutic e~fect in mice tre~ted with Adria alone at it' 8 PqTD of 11 mg/kg every 10-11 day~. After th~ fir~t cour~e, 43X of the tumors had r~gre~sed ~o 50% or le~s of their ~nitial ~ize. Howe~er, 7 day after the second cour~, only ~1%
were partially regres~ed, as~d 7 day~ after th~ third course, only 1~% were ~till in partial regres~ion. In c:ontra~t, in mice treated with PALA ~ M~R ~ 6-AN followed by Adria at 6 mg/kg, the regre~ion rate wa~ 66% after the ~lr~t cour~e, and then it increa~ed to 93% after the SUE~ JTE SHEET

W0 93~23014 21 ~ ~ O g 1 PC~/US93/0477 secorid cour~e and ~o lOQ% after the third cour~e.. Nste that non~ of the tumors in the 42 mic~ ç;ur~riving three eourses of treatmeT~t ~ss:aped from the re~res~ion-inducing ac~i~rlty vf the guadruple-drug regimez~ l 0 i B an u2~prec~d~t~d respon~e ~ate in thi 8 ~;pontan~ou. , and hl~hly heter~çTerleou~ tumor model, and because of the high degre2 of chomotherapeuti s:orrelatis~n that haR ~een ob~e2~ed betw~ ; msdel and the human diæeaQe we believe that thi~ 4-drllg regimen may be a thorapeutic l~rea3Ythrough .

E~cample 3: P~ IPR, 6-AN arld Ts~col Murine Br~a~t lumor_ SY~tem CD8Fl hybrid mice b~aring ~ingle ~spontaneou~, autoch~tho2~0u~ ~rea~t tun~ors ari ~ing ~luri~ pr eced~ ng woek `we~e ~e~lected from a colo~y ( 5tolfi et ~l ., Ca~er t:hemoth~r. ~e~p. ~ 239 251 ( l97l ) and Mart$n ~t al ., Csncer Chemother. R~p., Pa~t 2, 5:~9-109 (l975~ ) . For e~ch: exper~m~nt, a tumor cell ~rei, prepared ~y pooling 3-4 ~pontarleously ari~ing CD8Fl ~rea t tumor~, w~s tra~splanted ~rlto sy~geneic three-month old mice. In ' ~pprox~mat~ly three to fcaur we~ks, when tran~pl nted tumors: wore measurable, the tumor-be~arin~ mice were di~tr$buted a~ong experiment~l group~ ~o 'chat m~ c~
s:arry~g tu~or~ of approxlmately equal wel~t were repre~nted in ~ach treatment grotap. Therapy wa8 begun when the tu~nor~ were advan~ed and relatively lar~e; the av~rag~ :tumor wei~t was close to 130 mg at the beginning : : of treatment~

A~ i~ al 1 ~pontaneous tumors, whether human or murine, each indi-.ridual caslcer ha~ a h~teroge~e~u~ cell pop~ tlon~ The ~ r~t gerleration tran pla~t~ of CD8Fl brea~t tumor~ are obtained f rom a tumor cell bx~i made by p~oling 3-4 spontane~u~ly arising? tumor~ Au~, the individual transplant~ in each experimerl~ develop from a SUB5~1~1JTE SHEET

WO 93/23~14 PCI/US93/0477~ ?~

9~ 58 sis~gle brei that, alt:hough common to all t:he mice in that experiment13, ha~ B neoplastic cell compoE~itic~sl that i~3 likely ~l~ghtly d~fferent rom that isl ano'cher ~xperiment. There or~, quantitative me~sur~ment of any indiv~idual par~smeter ( e . g ., average tumc~r ~i~e ) may be ~omewhat diff~rent fro~ experiment to ~xperimerlt, but the findings wlll be ~uant~tati-vely rele~ant wit~in individual expeximer~t~, as will fiimilar trerlds among experiments.
The CDBFl fir~t gerlerat~ on breast tumor ls in~:luded in the murine tumor te~tin~ panel of the National Canrer Dru Screenislg Program ( Goldin et al ., Eur . J . Cancer 17:12g-142 (1~81) ) .

~or Mea urement Two axe~ o~ t}~e ~umor (the longest ax~ 8, L and the shortoEIt a;~iE;, W~ were measured with the aid of a Jernier callper. Tumor weig~t w~ estimated according to the fonnula: ~umor weight ~mg) -- L (mm) x ~W(wm)~)/2..

Chemotherapeu~ic ~a~nt~
MMPR ~nd 6 AN w~re obtai~ed from Sigma Chemical Co., St. Loui, ~0. Each of the~e a~ent~ was di~solved in 0-85X Na~l solution ~mmediately ~ef~re u~e. PALA and taxol were obtain~d from the ~apartment o~ ~alth, Educat~on, and Welfare, USP~S of the Natio~al Canc~r I~titute, Be~he~da, MD. PALA was di~slYed in 0.85% NaCl ~olution, and the p~ was adju~ted to 7.2 to 7.~ with lN
N~0~ before ad~u~tment to final vol~me. Taxol wa~
rareived already ~oluabilizxed in polyoxyethylated ca~tor oil and dehydrated alcohol. B~caus~ of the k~own to~icity of this d~luent, the Taxol ~tock was dilut~d, depending upon the do~e to be admini~tered, a mini~u~ of 6-fold ln saline before inject~on. For do~e~ ~elow 10 mg/kg, taxol was admi~istered i~ 0.1 ml/10 g of bodyweight. For do~e~
above 10 mgfkq, an appropriate additional volume wa~
admini~te~sd. All other agent~ were administer~d ~o ~hat the de~ired do~e was co~tained in 0.1 mlflO g of mou~e body weight, with the except~on of taxol~
SlJE35TlTlJTE SHEET

WO 93~23014 21 3 ~û g 1 PCl`/US93/0477:~

These drug~3 were admini~stered in a timed ~eguence, - with PALA admini:;tered 17 hours before NP~R ~ 6-AN, and taxol sdmirli~tered 2 1/2 hours a ter~ ~?R ~ 6-AN. In one experin~ent (~xp. 2S36, Table 8), Taxol wa~ nister~d in a ~ract~onated ~chedule a8 fQl10WS: Taa~ol (4 ~kg) ~imultarleously with ~R ~ 6-AN then, 1 1/2 hour~ lat~r, Taxol ( 4 mg/kg ~ q 3 hour~3 x 7 .

Determination of Chemotherapv- Induced Tumor ~eares~ion Rate The initial st ze of each tumor in each treatment ç~roup was record~d prior to the i~itiation of treatmen~.
Tumor E;i~;e w~s recorded wg~ekly during tr~atmeIlt and again at 7 day~s a ter the laEst course of treatment. For each oxperiment a singl~ o~ srver made all msa~urements in order to avoid variation in caliper mea~urements from indi-ridual to ia~di-~ldual. By convention, partial tumor rogroE~sio~ i8 defisled as a reduc:~ion in tumor vc~lume of 50% or greater c:ompared to the tumor Jolum~ at t~he tlm1s o initiation of treatm~rlt~ ~he partial reg~o~Los~ rate obtai~ed from ~ particular trsatme~t i8 expr~s~d ~a3 a p~rceal*age; i . e., Number of partial r~gr~si3ns per group~kotal nun~er of animals per group x 100.
.
Stat~tic:al_~Svaluation Dif~rerlces ln the ~;ize of tlamors betw~n tr~atment groups wer~ compared ~or ~;tatiç~tical ~ignificanee by the Student's t-te~t. Di f~renc:es between group~ with p ~
O. 05, or le s, wsre c:on~idered ~igni f icant.

~: Chemothera~eutic Effect of Taxol Alon~
In the Treatmen _ of First pa~R~;a~a_ CD8Fl Murine Advanced Brea~t Tumors Table 7 repc~rts a ~eries o four experimellt~ in CD8Fl ~nice bearing fir~t pas~age sp~ntaneous CD8Fl ad~anced breast tumor transplants. Each individual experiment compared a group of ~aline-treated controls with a ~econd group that received ~e maximal tslerated do~e (M~) of 5UE3STI~UTE SHE~

WO 93J23~1~ PCr/US93/(~477~ ff~"

?.~3~ ~ ~ 60 .

taxol alo~e (B0 mg/kg)..in a Sl 10 11 day admini~tration ~chedul~ for a total of 3 courl;e~, and ob~ervation~ were re~orded 6 day~ after tha la6t cour~;e of treatment.

, :: :

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:
SUE~grlTUTE SHE~T

r~wo 93/23014 ~ ~ 3 6 0 ~ 1pcr/us93/o477:~

~y ~~ ~.

~0 ~a~ 5 9~L0 ~90~)7"15~
a~ 5 /~ ~ aO~4*
~2 1~ 5~n~ ~6 ~110 It90~,ao~
2. ~xol~ 3 0/ 0 ~ 0%a ~5~

l. sa~B ~ s ~ (s~%) ~,~5a a. Taxo~0 ~ ~ 2010 t20~l,a~0 L2 ~ t90%~
xo~ 7 0~10 ( 0%~ 8~

A~ cæds Tus~or ~ig~t a~Je~ag~d 130 SExp~. 25~0) 0 ~0 ~c~. 2~4Z~ J
l~S t~CP- 25~4~ lS5 ~xp. 2SS~) ~ag. ~t ~ o~ t~t~n o~! t~at~nt~
b T~ cous~; o~ ~h~ d~e~te~ ~rea~nt (~p) w@r~ ~d~inl$ter~d 7nth a 10~ ay llltor~al lbet~ee~ cour~ . Ob~er~tloaD ~Ee ~cord~ 6 d~y~ sfe~r ~h~ thl~ cour-æ of t~eaæ~t. Subl~crlpt ~5~ do~ ~t 80mg/~, ~h~ olgr~od do-~ of ~ l o~ eh~ y ~che~

SUB~Jl~qTE S~tEE~

WO 93/23014 PCr/US93/~)477:~

?,~36~9~ 62 At ~e end of the ob~ervation period, the untreated ~eial~e-treat~dl contrc~l~ averaged an 80% mortality due to unr~R~r~c:ted tumor grow~ n contra~;t, ~ four taxol-treat~d g~oup . evidençed little ~oxiclty ( average weiq~t lo ~3 - 5%; average mortality = 5%), asld there was marked inhi~it~on of tuml~r ~rowth. Taxol alone clearly ha~ trorlg anti~tl~mor activity in the C:D8Fl breast tumor mod~l .

Chemotherapeuti~ Effect~; of the A~g~axol to the TriPle Combinatlon of PALA + MM~R ~ 6-AN +
Taxol In First Passasle CD8Fl Advan~ed Brea~t Tumors l~e trlple drug regimen, with ( Group 2 ) or without taxol (Grc~up 1 ), wa~ a~mini6tered at lO-ll day intesvals in a s3eries of three s~parate experiment~, ~nd observations were recorded 6 day~ after the third course of tr~atment.

;: ~

SUBSTITUTE SHEET

W093~23014 2~36Q91 P~/US93/0477~

nt ;~ .
.7 ~~~o a or~o ~o~
a ~as~ .7 ~ ~J0 6-A~ o ~ ~xol~ -~.5~
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d~nc~ or v~ç~ ~v~r-g~ o ~ . 2S~6, 25~7) ~ 7 ~. ~.
25~ t U~ tl~ o~ lr~tl~tlon o~ tr--~t.
~hra~ eou~ o~ n~ t~ nt ~ r~ inl~t~rd Yl~
lo~ y 1~-~ 1 b-tv-~n cou~a~. Cb~ lon- w~ co~ ~y n~t~r ~cha tt~lrd cours- Or tr~ nt.
p ~ O.OS 1~. 25~6) or ~ o.o~ ~xp. 25~7) ~r~r~ grou~ ~.
2~s v~ gsoup ~ xp. 25~g), ~ut group ~ d ~ 5~) p~rtla~ ~or s~ lon~ co~p~r-~co only 119 ~ ) ~ ~oup ~.
:

SUE~STITUTE SHEET

WO93/X3014 PCTIUS93/0477~
9~ 64 Pooling the toxicity data of the three group~
receiving only the triple drug regimen, there wa~ an average wei~ht lo~ of 22X a~d a mortality rate of only 3X In one of the experiment~ (Exp. 2536), Taxol wa~
added to the triple regimen in the indicated fractionated ~chedule (Group 2). The weight 108~ (25~) and ab~ent mortal~ty (0%) in the taxol-containing four drug combination, Group 2, were identical to its three-drug-treated control without taxol, Group 1, but the therapeut~c acti~ity was siynificantly better ~p < 0.05) than that achieved by the three-drug com~ination without Taxol.

In two of the experiments (Exp . 2~37 and 253~ ), a single bolu~ do~e ~of taxol wa~ added to the three-drug c~mbination ~Group 2). The pooled toxici*y data in the~e two taxol-cont~in$ng group~ (Group 2) avcraged 22% weight lo~ a~d llX mortality,~es~entially no different than the ave~age:of~ their; two ~three-drug control group~ (-23X
wei~ht lo~; SX mortality). The therapeutic acti~ity of the taxol-conta~ning~co~ination (Group 2) in E~p. 2537 was:iignificantly b-tter (p ~ O.01) than that of its three-drug control group without tAXol, Group l. In Exp.
2539,: the averago~tumor woight (138 mg3 of the three-drug regimen ~:Taxol, Group 2, although much ~maller than the aver~ge~tumor~weight (271 mg) of lts 3-drug control without taxol,~Group 1, neverthele~ was not significantly different from that ~f it~ control (Group 1). But ~here were 5/9 partial tumor regre~ion~, or a 55% PR, compared to only l/9 (11-~PR) in lts three-drug control, Group 1.

The data of Table 8 indicate that the level of therapeutic act1vity achieved by the quadruple dru~
combination~containing taxol wa ~ignifica~tly better in : ~all three experiment~ than that in the triple drug combination without taxol, a~d that thi~ improveme~t in anti-tumor activity was achieved without incre~e in toxicity.
SUBSTITUTE SHEEr -~ W~93/23014 21 360gl PCT/US93/~77~

Chemothera~utic Effects of the Triple-Dru~
Com~ination and Taxol at M D, Alone and in OuadruPl~ CombinatlQn ~PALA ~_MMPR ~ 6-AN +~Taxol In Fir~t Pa sa~e CD8Fl Murine Adyanced Rrea~t Tumor6 Two experiment~ are presented in Table 9, each ~on~i~ting of four ~roup~ of ten tumor-bearing animal~
each, a~ follow~: Group l, ~aline-treated control~; Group 2, Taxol at 80 mg/kg; Group 3, PALA ~ MMPR ~ 6-AN; a~d Group 4, PA~ ~ MMPR ~ 6-~N ~ Ta~ol at 25 m~/kg.
Treatment wa~ repeated at lO or l~ day interval~ i~ all ~groups~for a total of 3 cour~es, and observations were recorded 6 day~ after the la~t course of treatment.

WO 93t23014 ' P~r/U~93/0477 ~6 ~ 3.

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coOooa l,rh~n SF'~P ~ 1~ ec~lDp~ to g~ ~. 2 4 ~n ~xp. 254~ oup ~ , 40'~ partl~,l t~ar ~r~ se$a~10r~ Zl~ ~W t2 no ~ eoo~ s~gr~.S.o~ ln ~ oth~ ~o~lp~

:

SVBSrlTUTE SHEEr -~ WO 93~23014 21 3:6Q~1 PCI/US93/(~477:~

xp. 2542, Group 1, ~al ine, had a 90% mortality due to ~he unre~tric:ted ~rowth of untr~ated ~umors.
There~ore, ~ aver~e tumor ~ize in Group 2, Taxol~
alone, and in Group 3, the triple combinatiora, c~not be c:om~ared ~tatil;tically to the single tumor-b~aring mouRe i~ Group l, 6alir~e. ~lowever, from the differe~c~ in tumc?r-induced mor~al~;y rate~ between ~he~e three groups, it i8 cl~ar ~at ~e tumors of groups 2 and 3 were markedly ~ bited by their re~pecti-ve treatments. Group 4, the ta~col-contai2ling four drug combination, had tumor~
that were significantly i~hibited compared to the tumors i~ 'che three dnag e:oml:iination without taxol, Group 3, and to taxol alo~e, ~roup 2, and thi~ was aehieved wit~ ttle toxicity (~3% weight lo~; 0% mortality). ït E~hould be noted t~at the sup~rior ~rati-tum~r acti~ity of Group 4 was achieved with a doE~e of taxol (25 mg/kg) that wa~; le~
~n one-thlrd that of taxol alo~e (80 mg~kg), Group 2.

In Exp. 2544, t~xol alone at 80 mg/kg (Group 2 ~, and the tr~ple drug c:on~i~ation (Group 3 ), s~ icantly inhi}:lited tumc~r growth over that of the ~alin~-treated tumor~; ~ Group 1 ) . The anti=tumQr acti~ity of the thres-drug e:ombinatlon wi~ taxol (Group 4) i8 clearly ~uperi~r to all other group~ becau~e a 40% PR rate was induced ( ~e~ d, leç~er~d~ Table ~ ), ~d th~re were ~o parti~l tum~r r~s~re~;s~o~ls produced in any of the other ~roups. Aga$n, it should be noted that the ~aperior a~ti-tun~or ac~i~rity by the taxol-containing quadruple combinatio~, Group 4, wa~3 achieved with a dose of taxol ~25 mg/~g) approxima1;ely one-third that of the Ml~ do~e ( 80 mg/kg ) of Tax~l alone, Group 2, and thi 8 ine:rea~e in anti-tum~r activity was achie~ed without mortality.

I~:Rample IY: PALA, MP~?R, 6-AN and Radiati on ~ e PALA-MMPR-6-AN com~ination al~o ~ensitizes tumor~
to ionizing radiation therapy.

SUlBSrlTlJ I E SHI~ET

WO 93~23014 P~/~93/0~77~
?,~36~ 68 Mice w~th advanced tran~planted CDBFl brea~t umor~
t initial tumor weiS~ht 150mg ) wer~ divid~d ~nto our treatm~nt 5~roup~:

1 ) 5aline C:ontrol 2 ) PAI,A-MMPR-6-AN
3 ) PAI~R-6~ radiation ( 15 Gy, localized~
4) Radiation ( l~ Gy, lo~:alized?

Three e:ourses of *hese treatmentC werB admini tered, with a lO-ll day inter~val between course~ . Re~u~ t~3 and det~ of treat~nent t~ming and drug dose~3 are indicated in Table lO.

While radiation thexapy alone ~group 4~ ~ignifi~:ar~tly improved ~ur~r~ val and retarded tumor growth, no tumor regr~ iQns w~re :ob~sn?ed. In ~o~trast, ~h~n mi~:e w~re treat~ for~ i~radi~tion with PAIA-~R-6~ out of lO~ ce ~ad regr~ions (tumor ~i~e le s th~n 50% of oriçlinal we!ig~ 3~,~ a~d 3 o tho~e regres~io~æ wer~ comple:te.
~ ~ *

While the ~pre~en~ ~nventios~ ha~ been d~crib~d in term~ o~ preferred~ -mbodimen~, it i~ und~rstood that ri~t1o~ ~ -nd mod~icat1on~ wlll oce:~ar to tho~ ~kill~d in the asl;. ~erefore, i t i8 intended that th~ ~pF~end~d claims ~cover ~ uch~ equivale~t var1a~io~l3 whll:h ce~me with~n the cope v~f: the~ invent~on as claim~cl.

: i . i l~e f~t~res dis~los~d in the foreç~oin~ d@~cription, 1n~the :followi8lg slaim~ and,for i~ the ac~oYnp~rlying drawing~ may, both separately and in~ any com~ at~on reof, b~ materlal ~or realizing the irlventio~ ~n diver. e form th~rec~

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- 5l)~3STlTUTE SHlEEr r ~ ~ WO 93/23014 2 ~ 3 ~ ~ 9 ¦ PCr/~JS93/0477 ~ o al s:
I ~ o I O Y~ I
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SUBSrllUTE SHFE~l

Claims (27)

WHAT IS CLAIMED IS:

1. A pharmaceutical composition comprising:
aa)inhibitor of purine nucleotide biosynthesis, and b) a nicotinamide antagonist.

2. A pharmaceutical composition as in claim 1 wherein said inhibitor of purine nucleotide biosynthesis is selected from the group consisting of MMPR, 6-mercaptopurine, thioguanine, thiamiprine, tiazofurin, azaserine, 6-diazo-5-oxo L-norleucine, methotrexate, trimetrexate, pteropterin, denopterin and DDATHF.

3. A pharmaceutical composition as in claim 1 wherein said nicotinamide antagonist is selected from the group consisting of 6-AN, thionicotinamide, 2-amino-1,3,4-thiadiazole, 2-ethylamino-1,3,4-thiadiazole, 6-aminonicotinic acid, 5-methylnicotinamide and 3-acetylpyridine.

4. A pharmaceutical composition as in claim 1 further comprising a pharmaceutically acceptable carrier.

5. A pharmaceutical composition as in claim 1 comprising:
a) MMPR, and b) 6-AN.

6. A kit comprising:
a) a vial containing an inhibitor or purine biosynthesis and a niacin antagonist, and b) a vial containing an apoptosis inducing agent.

7. A kit comprising:
a) a vial containing an inhibitor of purine biosynthesis, b) a vial containing a nicotinamide antagonist, and c) a vial containing an apoptosis inducing agent.

8. A kit as in claim 6 or 7 further comprising a vial containing an inhibitor of pyrimidine biosyntheisis.

9. A kit as in claim 6 or 7 wherein said inhibitor of purine biosynthesis is selected from the group consisting of MMPR, 6-mercaptopurine, thioguanine, thiamiprine, tiazofurin, azaserine, 6-diazo-5-oxo L-norleucine, methotrexate, trimetrexate, pteropterin, denopterin and DDATHF.

10. A kit in claim 6 or 7 wherein said nicotinamide antagonist is selected from the group consisting of 6-AN, thionicotinamide, 2-amino-1,3,4-thiadiazole, 2-ethylamino-1,3,4-thiadiazole, 6-aminonicotinic acid, 5-methylnicotinamide and 3-acetylpyridine.

11. A kit as in claim 6 or 7 wherein said apoptosis inducing agent is selected from the group consisting of methotrexate, 5-fluorodeoxyuridine, 5-fluorouracil, 1-B-D-arabinofuranosyl- cytosine, puromycin, trifluorothymidine, cisplatin, etoposide, camptothecin, cytoxan, adrimycin, teniposide, podophyllotoxin, aphidocolin, sodium azide, N-methyl-N'-nitro-N'nitrosoguanidine, nitrogen mustard, bleomycin, 1,3-bis(2-chloroethyl)-a-nitrosourea, methyl glyoxal-bis-(guanylhydrazone), colcemid, vincristine, taxol, taxotere, dexamethasone, retinoic acid, purinergic P2 receptor agonists, somatostatin analogs, luteinizing hormone releasing factor analogs, and antibodies capable of inducing apoptosis.

12. A kit as in claim 8 wherein said inhibitor of pyrimidine biosynthesis is selected from the group consisting of PALA, 6-azauridine, triacetyl-6-azauridine, pyrazofuran, brequinar and acivicin.

13. A method of treating antineoplastic disease in an animal comprising the steps of:
a) administering a therapeutically effective amount of an inhibitor of purine biosynthesis, b) administering an nicotinamide antagonist, and c) administering an apoptosis inducing agent.

14. A method as in claim 13 further comprising the step of d) administering a therapeutically effective amount of an inhibitor of pyrimidine biosynthesis.

15. A method as in claim 13 wherein said inhibitor of purine biosynthesis is selected from the group consisting of MMPR, 6-mercaptopurine, thioguanine, thiamiprine, tiazofurin, azasevine, 6-diazo-5-oxo L-norleucine, methotrexate, trimetrexate, pteropterin, denopterin and DDATHF.

16. A method as in claim 13 wherein said nicotinamide antagonist is selected from the group consisting of 6-AN, thionicotinamide, 2-amino-1,3,4-thiadiazole, 2-ethylamino-1,3,4-thiadiazole, 6-aminonicotinic acid, 5-methylnicotinamide and 3-acetylpyridine.

17. A method as in claim 13 wherein said apoptosis inducing agent is selected from the group consisting of methotrexate, 5-fluorodeoxyuridine, 5-fluorouracil, 1-B-D-arabinofuranosyl- cytosine, puromycin, trifluorothymidine, cisplatin, etoposide, camptothecin, cytoxan, adriamycin, teniposide, podophyllotoxin, aphidocolin, sodium azide, N-methyl-N'nitro-N'hitrosoguanidine, nitrogen mustard, bleomycin, 1,3-bis(2-chloroethyl)-a-nitrosourea, methyl glyoxal-bis(guanylhydrazone), colcemid, vincristine, taxol, taxotere, dexamethasone, retinoic acid, purinergic P2 receptor agonists, somatostatin analogs, luteinizing hormone releasing factor analogs, antibodies capable of inducing apoptosis, and cytotoxic T-cells.

18. A method as in claim 14 wherein said inhibitor of pyrimidine biosynthesis is selected from the group consisting of PALA, 6-azauridine, triacetyl-6-azauridine, pyrazofuran, brequinar and acivicin.

19. A method as in claim 13 wherein step c) occurs after steps a) and b).

20. A method as in claim 14 wherein said administering a therapeutically effective amount of an inhibitory of pyrimidine biosynthesis step occurs before steps a), b) and c).

21. A method as in claim 13 wherein said inhibitor of purine biosynthesis is MMPR, said nicotinamide antagonist is 6-AN, and said apoptosis inducing agent is FUra.

22. A method as in claim 13 wherein said inhibitor of purine biosynthesis is MMPR, said micotinamide antagonist is 6-AN, and said apoptosis inducing agent is FUra.

23. A method as in claim 13 wherein said inhibitor of purine biosynthesis is MMPR, said micotinamide antagonist is 6-AN, and said apoptosis inducing agent is taxol.

24. A method of treating antineoplastic disease in an animal comprising the steps of:
a) administering a therapeutically effective amount of an inhibitor of purine biosynthesis, b) administering a nicotinamide antagonist, and c) administering radiotherapy.

25. A method as in claim 24 further comprising the step of administering a therapeutically effective amount of an inhibitor of pyrimidine biosynthesis.

26. A method of treating multiple drug resistance in an animal comprising the steps of:
a) administering a therapeutically effective amount of an inhibitor of purine biosynthesis, b) administering a nicotinamide antagonist, and c) administering an apoptosis inducing agent.

27. A method as in claim 26 further comprising the step of administering a therapeutically effective amount of an inhibitor pyrimidine biosynthesis.