Note: Descriptions are shown in the official language in which they were submitted.
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:
6--AN
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
WO93/23014 PCT/US93/0477
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
13
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
17
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~
21
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
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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) ~ .
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~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
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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.
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~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
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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
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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
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~,~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~
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2136091
33
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
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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
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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$men~, a group (Group
1) of C3)E3Fl m~ca bearing advanced, firE;t pas~age
~porltenaou~ CD8Fl breast ttasnor tran~pla~t~ were treated
with the t~ipie co~l~ation of PAI~ ~ ~qeR ~ 6-AN. PALA
( 100 mg/Xg) waG administered 17 hour~ ~efor~ M~R ( 15C)
m~/kg~ plu~ 6-A~3 (10 mg~k~) (Table 1). A ~ ond sroup
~Group 2~ recei~d the ~ame treatntent with ~?AI~ ~ M~?R
6-AN, foll~wed 2~ hour~ later by El~ra ~75 mç~/kg).
Treatment wa~; rep~ated at 10 or 11 day ~nter~r~ls for a
total vf 3 courses, and o~ervations were ~ecorded 7 days
after the last course of treatmerlt.
SUESTITUTE SHE~
~ WO 93/23~14 PCI`/US93/0'177:-
39 2136~91
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SlJBSrl~UTE SHEET
wos3/23ol4 PCT/US~3/0477~ ,~4~
"~. .;~
~,609~
Partial tumor regressions were ob~erved in 8 of the
sur~i~g 48 mice ~17X)~ treated with PALA ~ MMæR ~ 6-AN
(Group 1). The range'of regre~ion rate in indi~idual
experimentE varied ~rom 0 to 30X. The add$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
41
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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SUE~ UTE SHEET
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WO 93/23014 P~/VS93/0~77
o~f~ 42
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SWBSTITUTF SHEE~T
W~ 93/23014 P~r/U~93/0477~
2~350gl
43
~ 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 .
.
SUBSTlTl~E SHE~
-1 wo g3/23014 2 :~ ~ 6 0 9 1 Pcr/us~3/0477
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WO 93/23014 PCI'/OS~3/0477
46
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~
47
T~l~ 4
P~ oQ ~ ~P~50 ~ 6~ o~ $u~o~ A~
~B~l ~e~al~ ~c~ ~ug/~g p~ot~ln ~ S~
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:~ 0.32 :~: 0.32
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b~y w~h~; ~LD~
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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
49
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
PALA-MMPR- 6 -AN
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
WC3 93/23û14 PCr/U~i93/0477~
~6Q9~ 50
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~ ô o ~
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: ~ ~ `
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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~
.. .. . . . , ~
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~
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59
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.
,
:: :
:
: ~
.
:
SUE~grlTUTE SHE~T
r~wo 93/23014 ~ ~ 3 6 0 ~ 1pcr/us93/o477:~
61
~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~
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?,~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~
63
nt
;~ .
.7 ~~~o
a or~o ~o~
a ~as~ .7 ~ ~J0
6-A~ o ~ ~xol~ -~.5~
~xol~ ~ x 7 -2~ O~O ~0~ ~7~-
oo~ O -2~ orlo (0%~
a. ~ oo~l7 hr~ 0
o-2.~ hxol~0 -~ 2/lO ~oO
1. PJ~ "o-17 ~->~QP~o
o . -22 1~ ) 27
~as.a o~l7 ~ o
o ~~-5 ~ axoa~O -~ 0/~ ~0-~
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 ~.
:
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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.
a. ~~ ao ~o~ o~
. ~u~ol80 ~ 5 o/ao ( 089 s5~
aaoo~~ Nao ~~ aJao 1[~0%~ S5~'
~- ~a~o~~7~~WR
_~.5~o$u~~ 9r~0 t ~ ~~
s~ao ~S0~) ~,~a
2. ~ 0 21~0 g~OS~ ~.,~o~
. PaS~o-l7~-9~KpRl~o~ o ~5 1~ ~ ~ 3a2~-
,90-17hS-W~o~ o
.shro~ora~ -ao 0/10 ~ 0~) s~
A~anc-~: ~or ~/Q15~ht Vh~ a~9 ~,~Xp. 2S~ nd als ~ t~- as~ t ~e
og ~nltl~tio~ o~ ~rs~t.
051 601~5i~ o~ n~t~d ts-L~ o~ l V~ d~n~8t~ l/~
~-al ~y lrtest~al b~l~cvo~. cou~ . O~ t~oN YIU~ 0~ i
~t~ cou~ ot ~r-at~e.
Ot~p~ 5~2a c~ t b~ c~lly ~:o~pas~ O~
Sal~n0 eomt~olB, Y~2~ OT'lly ~ or~ nsl ~ou~o ~ 0% ~llt:y
o ~t~at~ or S~~-
p~O.054 s/h~n gsot~p ~ cosllpa~ A to ~ou~a~ 2 ~ 3 ~. 2~a, a~
~; SSOuP 2 ~s colop~ So ~~? a ~- as~
coOooa l,rh~n SF'~P ~ 1~ ec~lDp~ to g~ ~. 2 4
~n ~xp. 254~ oup ~ , 40'~ partl~,l t~ar ~r~ se$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:~
67
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~
~:::: :
- 5l)~3STlTUTE SHlEEr
r ~ ~ WO 93/23014 2 ~ 3 ~ ~ 9 ¦ PCr/~JS93/0477
69
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SUBSrllUTE SHFE~l
