Note: Descriptions are shown in the official language in which they were submitted.
2 1 98645
TE~E USl~ OF DlLLAPlOL AND llS ANALOGUES A~D DERTVATIVES
TO AEFECI ~1Ul~TIDRUG RESISTANT CE:LLs
1. ~IELD OF THE INVENTION
The present invention relates to a method of ~ t 1~ cells e~p~ g multidrug resistance (MnR~
activ~ty.
2. BAC~CGROUND Ol~ Tl~ I~VENTION
10 2.1 ~o:~
Multidrug Re~istance ~MDR) is the process whereby cells become r~;~tn~ll to structurally diverse
~h~ herapeutic age2Itg following exposure to a single drug ~Riordan and Ling, (lg85) Pharm-~L
77~er. 28:51-75). The MDR phenotype is characteli7ed by decrea~ed intr~-e~lular acalmulation of
dru~s or other chemical subst~nr~ and by cross r~C;ct~nee to other structurally- and filn~ti~n~lly-
15 unrelated drugs or sub~ . The hallm~k and clinically dcv..~ aspe~ of l~R-based
rP~i~anc E i9 that the r~ -e extends to a wide ranse of drugs.
hlI)R is observed in a wide range of settings, ranging ~om the clinical setting to pest control
management. In general, MDR can be observed whenever ~,he.,l ' s.ll,stnllrf.~ are ~Ised ro affect
20 cells or or~ ~:g~.~, and cells develop a re~ nce to the chemical agent. ~an~pl~: include such
dive~e chemical ~ ce~ as c.h~m-th~rapeutic agents, in.qectiri~e~ and nicotine. MI~R occurs in
pathologies such a~ Celtain cancers and diseases (eK. malaria), in ccrtain states of toler~n~e or
re~ist~nce (eg. to in.cectiri~l~), and in addictions (e~. to nicotine and opiates~.
A number of "-c~ have been shown to be involved in the MDR phenotype: a decteased
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up~ke of dru~; an increased ene~gy cle~ efflux of drugs from the cell; a lower affinity to intra-
cellul~ binding sites; and a slower conversion of drug~ to aLkali-labile materials as c r1F ~ ~ed to drug-
sfflsitive cells (Gottesman et al., In Caplan (ed.) Cell Bio~ogy undh~emhra7le Tn~ " Processes
(Ne.~v York: Academic Pre~, 1994) 41 3-15).
s
One n~c!~ by which cells can develop r~ t~nce to an array of ~t~ucturally-diverse drugs
involves an ATPbindmg cassene (ABC) superf~nily oftransport proteins (Higgins (l~ ) Ann ~?ev.
Cell~iol. 8;67, ~1yde et al., (1990) NalLr~ 34~:3~2). Me.~ e,~ ofthe ABC superfamily bmd ATP,
u~ing the free ener~y of ATP hydrolysis to drive particular biolo~ical ~ tinn~ These ~ranspolt
10 proteins are ~rnplmf~ of an active transport sy~tem that ~ the tr~nsport of ~olecules Elcross
the cytoplasmic l...,.l~r~e of cells Members of this ABC sup~ nily, which share c~nni~lf~rable
se~ence homology, include the followin~ the eukalyotic MDR P-~ycopl ul~ , the protein pf~)R
implicated in chloroquine resistance of the malarial parasite; the product o~the cy~ic fibrosis gene
CFI R, a ~n~ lna,~ cond~lct~ncs regulator that controls chloride ion fluxes; the product of the
15 White locus of Drosophila~ prokaryotic proteins ~Of ~ w~th membrane transport? cell divi~ion,
nodul~tion, and D~A repair; and the STE-6 gene product that mediates export of yeast a-factor
matin~ ph~rol.lone.
The MDR P-glyf"oprotein (P-~p) is a high mo~c~ r weight (1~0-170 kilodalton) e~karyotic
20 l~"~ ne protein. It is ~enned P-gp for its asF,ociation with the apparellt 1)~ '- 'iLy ba~rier
to dlugs that arcor-paries ~II)R. Most chemu~ ~u~ic drugF, are lipophilic and can enter cells
passively. Drug-resi~tant cells exh~it a large elevation of P-~Sp t~AIJi~7;on in concert with the
d~ of dmg re~ ~t~lc~. The ~-~p serves ~s an energy-depen~t puunp to efflux cytotoxic
drugs from the cell. This reduces the intracdl~lsr cQnr. ~ tion of the~,e d~ugs and ll~ cell
25 expo~ to the dnlg~ at a non-toxic level. At the ~ orhP~nical level, it ~ b~n fuund that th~ P-~p
pump is eno~nously catholic in iP, ability to bind and transport compounds. Recently, it has been
reportcd that the P-gp pump can ~ ,po, l a wide variety of lipidr~ (van ~elvoort et al., ( l 9~fi) Cell
87:507).
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2.1.1 MDR in the Trea~m~nt of r~
MDR cells have the ~bility to d~a~&sc intM~lula~ levels of Ghemotherapeutic drugs; the therapeutic
e~ècts of the-se drugs are then ~ ..n~ e~l Fw lhei ...ore, the oell~ become l..;.;sl~lL not only to the
5 drugs used in previous llb-~rn~nti but also .~n;rc~l resigtance to other dru~;s. MDR l~plesents a
major obs~le to ~e ~ l of such diseases as cancer, mala~ia, amoebic dysentery, and
l ~S~--~- ?~iS.
MDR is a cc~ ol~ ph. ~ .on in the treatment of cancer~ by ~ L..apy Tumors r~cponrl
1 () to a first ~ h~ap~ oi~01 fail to respond to a second t.~l~enl with the same dnug or w-th several
chemically-unrelated dru~s. l)rug-rcs;st~.~l tumor cell~ ov~ ss the P-gp pump and can thus
prevent cytotoxic cl o1~ ic agents, such ~/;.-~li;,ti.~e and taxol, from reachin~ the intracellular
levels needed to block cell divi~iun. This ph~nom~non has a negative impact on the success of cancer
chemotherapy and patient sur~val Resistance of malignant tumots to multiple chemotherapeutic
_gents is a myor cause of ~ t failure (Wittes el al., (19B6) C~cer Trea~. 12ep. 70 lO5~.
The M~R phenotype is also del- ;... ,~511 to malarial therapy. Ma]aria is caused by inf~ction by the
l"ulu~., P~an spp Tt is characterized by paru~y~..-s of chills, fever, and sweatin~,, nd by
anaemia, splenomegaly, . nd a chronic relapsir~ course The drug of choice for treating malaria is
20 ~hloroquine. Unfortunatcly, in some nlalariOUS areas, such as Afiica and South East Asia, malaria
is r~rnpant Drug-resi~tant malarial pls~m~liq are able to keep the cl~sic ~ntimsl~ri~l a~ent,
chloroquine, from reaching toxiç levels in the plq~nofl;ql cells' food vacuoles by a mechanism
involving the pf~R transport protein Control of re~ populations of malarial plasmodia in
individual patients ~vould benefit from syner~ists that in~el l~re w~th the MDR mechanisms.
ZS
Members ûf the ABC superfamily of transpûrt proteins have been imrlirqted in MDR in other
diaeaaes as well. En~soe~ hystoli~ is responsible for amoebic dysentery R~ ,e ofEntamoeba fystolitica to the clinically-used d~ug emetine is thought to imrolve a member of the
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~BC ~up~mily a S~ n etal., (l 990)Molec. BloL Purisitolof~ 38:281). Leis~)~ania spp
is the causitive a~ent of the tropical disea~e L~ "~ ie Clone~ of ~,ei.schmania ~pp. that show
cros~resistance to seve~l drug therapies displa~ amplification of the genes for transport proteins of
the ABC superfa~r~ly, ~I.~Ieas drug ~ensitive clones do not ~ ddlbOIl e~ al., (1992)Mc:le~. C'ell
~ioL l 2 2855).
2 l.2 MT~R ~ntl th~ Rlood Rra~n 13atrier
It has been d~ us the P-gp also flln~inn~ a~ Ihe mammalian bluod-brain barrier to protect
the brain a~ainst ~ g d~ugs (Schinkel et al., (1gg4) f~ell 77:491-502). In thi~ work, a
10 knockout mouse wa~ created ill which the ~ene coding for P-gp, mdrla, was inactivated. The
absence of Y-gp rendered tl~e mouse fa~ally ~lcc~rtil~le to a topically applied in~ctir;~ lecLisl~
a P-gp 6ubstrate: without the blood-brain barrier P-gp, brain levels of iv~ e~;~ and sen~itivity ~o
ivermectin ~ a neurotoxin both i,.~l~d by about two orders of m~gnihl(le P~ also a
component of the inse~t blood-b~ain barrier (Murray et al., (1994) J. Neurobiolog)~ ~25;23-34).
1~
N~ro&.,li.~e dru~s, whether thera~tic, r~,~ional, or inse~ ;d3l~ includin~ caloids and synthetic
nea~,~tuAil~ such as morphine, codeine, nicotine, and ivelmectin, mu~t cross tlle blood-brain barrier
to act on eentral nervous system (CNS) targets. Many of these dmgs are known or suspected
substrates of P-gp. For example, attempts to ~et chemotherapeutic drugs aGross the blood-brain
20 b~er can be hall"~eled by r-g~cu~r~ action at the barrier (Tamai and Tsuji (1996) A~v~nced
Drug J)eliveryReviews 19:401-424).
It i~ almost ce~tain that ~JS levels of neuroactive addictive ~ompounds are redu~ed to some extent
by P~ t the blood-brun banier. Inso~r as P-gp at the ~lood-brain barrier acts to limit access of
25 any of the~e compounds, it contributes to the ph~ llol~ r,n ordrug Lolerance. ~ur ~,,. . r !e, tv the
extent that P-~p at the blood-brain barrier pumps brain nicotine out of the brain and back to the
blood, hi~her penpheral nicotine is required by the addict brain to achieve the "desired'~ level.
Research har. shown that in specially adapted insects, an mse~:t P-~p serves to excrete nicotine such
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thal a hi~hly effective 'ol~od-br~ ba~ner can be mount~ against nicotine (Murray (19g6) "A P-
~,ly-iDp~tei.~-lilce l~lceh~ism in the Nico~ ~Res~ nt Insect, Ma~uca .sex~' Phl:) ~hesis
(U~ver~i~ of Oth~, ON, C~sda); Murray et al. (1994) .l. ~urcJbiolo~ 25:2,3-34) There is also
binf~e- ~' e~;~.,. ce that nicotine is a substrate fnr -: ~fia~l P-gp ÇHeather McI:~iarrnid Honour~
5 Thesis, :~ Sharom's lab ~Lll,n..lled to the Department of Ch~ and ~ioch ~ ry, University of
Guelph, Sept 1~95)
2.1 3 MDJ~ ar~ Pe~ CQ~trol
Peg~ control agento, have long been used to co1ttrol the undesired prolifera~ion of pests, such a~, weeds,
lû insect~, and rodents. Unfortunately, apprlla ~es using these agents may fail due Lo MDR. For
e~ le, over 500 cases of ~t r~eict~nce and/or muiti-realE~at~ce to insecticides have been
reported. Multi-resistance to insectiGides is thought to anse pr~a~ily from the in~ cti- n of
cytochrome P~50 mecli~ted polysubstrate monoux~ s (PSMOs). The PSMOs result in an
increased biotran~fonnation of ~e i.~.licirle to a more polar and excretable product; con~equ~ntly~
l 5 there is reduced e~cposure of cellular targets in insects to toxic levels of the in~ctiri~le. Resistant
~ns~s can become re~i~tant not only to the ;,-se~l;r,ilde that ~ red the enz~ne in~ ctinll but also
to a number of cbemically-~ ed in~ecti~d~ icide syner~ists t~at can prevent or inhibit
sp~ific deto~cation .,.~y-J~es are u~eful tool~ in the m~nag~m~nt of 5uch ~ases (l~a~a and Priester
(1989) J. Agric. ~ntomol. 2 2745).
Lignans and their derivative~ have been observed to enhance the to~ncity of in3ecL;~ by inhibitin~
the acti~nty of the PSMOs; for example, in an insect miero~omal prle~: alion~ dillapiol and
bios~.~Lti~:ally-related lignan~ were shown to be effective inhibitors of PSM-based aldrin epoxidase
activity (~er~ d et al., (1~8~) P~toc~e~ 28; 1373).
MDR to pest control a~ents may al~o involve the ABC s~ .ily of tran~port prntein~. P-~,p is
expressed t{~ varying level~ in many insect tissues, and its tA~,rei,sion varies devel-.pi~e~4~l1y; for
exarnple, to~acco-feeding caterpillars have more blood-brain barrier P-~p than the no~ n~, adult
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fo~n of the same in~ . Pressure ~om applied pe~ic;~ could cause Darwinian selec~inn of
pesticid~res;~ ;~i....S that O~ A~)Ie~9 P-gp in any tis~ue and in any d~iv~lop~ tal stage7
leading to MDR in those organisms. Insect M~lpighian tubules have P-gp: they ~u~ively excr~te a
wide range of allcaloids, in~ riing nicotine and vinca alkaloids, and the P-gp inhibitor verapamil
inhibits this excretion (~lurray (l 996) PhD Thesis, ~Jniversity of Oth~a, O~, Canada).
2.2 ~Peie~ to ~vP~e MDR
Sllal~gies d~ ne~d to block ~ ,r~;on of the ~DR ph~notype and to circumYent dmg res~ nre
are being ~ou~ht. Rever~al of the ~)R phenotype c~n be accompli~hed by using compounds that
are capabl e of blocking the filn~tion of ~ ,ml~er~ o~ the ABC superfarnily of transport proteins,
including P-gp and p~R These compounds are called ~h~-mr~ ti7çrs~ MDR inhibitors, or
reversmg agents. Particularly desirable are cl~e~l~os~ .~";ti7pn th~t ac~ at conre~ alions with little or
no cytotoxic ei~ect.
The ~ fi~ng;tizers descrl~ed to date may be grouped into $iX broad ~ B~ ~s: CalciuM chatmel
blockers, calmodulin a~ntli~ts; non-cytotoxic ~ntracycline and Vinca alkaloid analogues; steroids
a~ homlonal analogues, mi~cp1l~sne~us hydrophobic compounds; and cyc1Osponnes. Most ofthe~
compounds a~ c~ el~ lipophilic, and those in the fi~t five ~roups are all heterocyclic, amphipathic
sub~ ccs ~Ford el al., (1990) Phc~macol. ~elJ. 42~ 19~).
Verapamil, a calcium channel blocker, was the first d~ il~ MDR mo-~ifi~r. Nume~ous
invegtigators have des~.il,ed il~CI~aSeS in a~ç~ ti~rl of and d~ os in ~ .nr~ to natural
product ~4 ~ h~peutic dn~gs in a number of di1~erent MDR cells treated with v~l~p~llil and other
calGium channel blocker~. Vera~amil has been used w~th ~ome success in multiple myeloma pfltients
who no longer re~pond to sal~ge çh~n~tl~rapy ~Dalton e~ al.J (1989~ .I. C'lin. Oncol. 7:41~).
The mechanism by which verapalr~l, nther calcium chanrlel blockers, and calmo~ulin ~ntagonists are
thought to i~ drug flccl ~m~ tinn is by colllp~llng with at~ti~ncer drugs for binding to P-gp,
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thereby inhibitin~ ef~ux of drug (Con~well el al., (1986) .J. 13iol. C~ m. 261:7921-7928; Cornwell
et a~., (1986) J. Biol. C'hem. 262:2166-2170; Safa et aL, (1987) J. Biol. ~'hem. 7884-7888; and
A~iyama et al., (1988) Mol. Pha~macol. 33 14~147) The major problem a~soci~ted with usin~
vaapam~ to reverse MDR in patients is that it has dose-limiting cardiac toxicity due to blocking of
S the atrioventricular node. This toxicity ~ its use at cnn~ tions r~.lu,red to reversc MDR
in vr~o.
A number of calmodulin ~ulL~,u~ ~ have been found to bc g,ood ~1DR inh~ibitors in ~ra
Tri~luop.,.~, a ph~ hiazine a~ b~ihotic drug, has been noted to increa~e drug ~ce~mul~tir n
1 () and d~ Gase .~ s ~ts.nce in M~R cell lines (Tsuruo et al., ( I 983) ~'~ncer Res. 43 :2905-2910; Ford
et al., (1 989) ~ol. P~ ~ n~Ql. 3~: 105~ ). Neurotoxicity of this compound, however, is dose-
lirr~t1n~
Cyclosporins are imm~ os~ppressi~e agents which can also pol~.~lidle toxicity of anticancer agents
15 at clinically achievable conc~tldtions~ ln some ceil lines, they appear to ~nh~ toxicity and
increase drug a,r, lmula~ion in sens~tive and r~ t~ ells ~Chambers et al. (l9~Y) Ccme~ ~es.
4g:627S~279) Cy~,lo~o~ A c~ll.pet;li~ely inhi~it~ s guCh aB ~ GnjjlLIe and v~nblastine ~om
binding to the P~ arr~i etal., (1~90) ~ Biol. ~hern. 26~ 509-16~13) The u~e o~cyclosporin
A to mo~ te drug re~ist~nce may be ha~ )er~d by i~reversible n~ph~ o~i~ity and
2n i~ """~ ;on in patien~ already coll.pr(j",.~ed by myelosluppressive c~emo~herapy The use
of non-imm~1nos~lwre~;.,re cyc~osporin an~logues may be less toxic.
Horrnone analo~ues, such as the antiestrogens ta-m-oxifen and tole-~;rene~ are 1~ p~oyed in the
chemotherapy of breast canoers. These compounds can also mod~ e r~e~e~n~e of estrogen
r~l~lo, -negative MDR cell lines ~ia estrogen . ~e~ e~ ~ amu ef 41, ( 1 ~84)
CancerRes. 44:43~24395; and Berm~n et aL, (1991) Bload77:818~82~)
Som~ compound~ act a~ by interfering with the ener~y required to pump cytotoxic
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dlugs out ofthe cell. These inchJde 2,4 di~ ,ophwlol, 2,4d~ucl~3Ol, and hydrogen cyanide Mo~t
pO~ . that i~lLt7rl~ e with the energy required to pump cytoto~c drugs out of cells are toxic t~
no~nal cell8; thu5, they are not ideal fo~ clinical use.
5 Ch~ s ~ ~;ti~rs have al~o be~n used in ~e treatment of MDR in malaria. lt has been shown that
the treatment of monkeys infected with chloroquine~ .;s~ laria p~iLes iS improved in the
l~rcA~.,ce of a rh~mos~nsitizer (Juranlca el aL, ~1~89) FAS'EB ). 3 :2~'83).
The identification of sev~al phafmacologic agents that can re~erse the M~R phenotype m vilrr has
I n not, to date, identified MI~R inhibitors with good elini~al efficacy. This is primarily due to the ~àct
that in ~vo co~ l;o~ ~y to reverse MDR cannot be achieved without sub toxicity
to pa~ients. For this reason, there rem~ins a nee~ for an effective clinical nlethod of treating MDR
2.3 Collater~l Sen~it;yity
l 5 An a~ternative strale~y invalves deYeloping rl~ ~ln~l~s~ el~ that are 5~ 'lcal.1y ~uluiuC to MDR
cells, lea~ing no mal cells u..~ceL~d. The d~clopn~ of ~l)R is o~en ~ nied by an
irlc~d sensitivity to membran~aetive agents; this ph~n-lm~n~n i~ known as collateral sensitivity.
lt has been reported that collateral sensitivity occurs in MDR cells with the Use of membrane active
agents including d~t~ hf t;cs? and steroids ~Bech-~ansen et al., ~ 1976~ J. Cell. I'hys~ol.,
88:23-32, Riordan and ~ing (1985)P)~armol. Ther. 28:51-75; Loe and Sharom (19g3) ~r. J. ('arlcer
68:342-351). As well, collateral sen~itivity to dru~ has been re}lnrted with tr~nsfe~t~ntc ~Juranka
e~ al., (1989) FASEI~ J. 3:2583-2592). Unfortunately, many detergents are poor c~nt~ tes t'or
therapeutic use because of their unde~irable non-tAr~et effects, especiatly on membranes.
S-~me lignans luve be~ reported to enhance cytotoxicity with cultured MDR cells (Pe~utto (19$13)
NaL Pro~ 56. 233-239). For eY~mplc phyllanthin, an oxodiarylbu~ane, ha~ recently been found
to be more toxic to an MDR cell line than to its dru~-~itive parental cell line (s~ n~ba~h~ t
al., (1993) J. N~l. Pro~" 56(2):233-239).
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Several ch~mos~ , including calmodulin inhibitors (eg. trifluop~ c), calcium channel
blockcr~ (eg. verapamil, bepndil), and other classes of non-related compound~, have also been
repo~ted t~ be more to~uc to MDR ceJI~ line~ t}îan to their parental cell Unes (Schuurhuis et uL, (1990~
Int. ~ ancer 46:330-336) Despite these fin~lin~, the clinical llse of these c.h~rnr.~ is
que~tir '10 due to their dose-limiting toxicity
This revieur of chemos~ ;ef~ currently available indic~ o~ that ~?th~u~h these comrolm.lc are
effective in ~ the MDR IJL~uly~e in vitro, ~he cnn~ ;nn~ n~c ~ - ~ y to produce thi~ result
limit their use in vivo, thus, a need remain~ for a clin~cally a~ c method oftreatirl~ ~IDR
2,4 Dillapiol Compounds
Dillapiol is a mono~ol (phenylp~opanoid cleriY~hv~ t~at is produced biosy~th~tic~lly via Lhe
Shi~c acid pattrway. It has been It;pO~ led that dillapiol is sy~ A3~ in only a few species of hi~her
plar~ and i8 not currently ava~lable co.,.~ ,~;.dlly. The stlucture of dillapiol is ~epi~ed in T~onnula
1.
~ OC113
<o~
Fonnula I
Tn addition to the core stru~ure of d;ll~T)iol~ a number of analo~ues and derivatives exist (hereindl~er
referred to as dillapiol co~o~ ,se.lIed, for e~cample by the follo~,vin~ Formulae (JI) and (TTT)
-
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S o ~ R2
<o~~
R4 R3
Fo~nula
where~4
Rl is selected ~om H and CNCH3; and
1 ~ R~ i~ sel~ed ~om CH3;
R.
<o~ 2
Fonnula lII
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where~
Rl is selected ~om H~ OH, and OCH3,
~ is selected ~om ~ OCH3, ~ld C~C~CII3;
R3 i~ ~elected ~om ~ CKCH3, C~H~CEI=C~, CH=C~CH3, CH2c~C~3, and
CH~O¢H~CHkCP~H2C~OC4~; and
R~ i~ selected ~om H~ CH2C~ , and OCH3.
~Yhere R, is OC~H3, R~ iS OCH3, R3 is HL and F~ i~ CH2CH-~CH~ the compound m~y be called
pseudodil1apiol. ~VhRfe Rl is CPCF~, U~ i~ E~ ~ is CI~CH=~CH~, and R~ is C~C~, the cornpound may
bc callcd apiol. Whcre R~ is OCH3, R~ is OC H3, R3 is CH{~HCH3 and ~ i5 ~ the compound may
be called isodillapiol. Wllere Rl is OC~H3, R2 iS OCH~, R~ iS CH~CH~C~, and R4 is 4 the compound
may be called dihydrrulil'~-ic~l. Where R~ R2 is CEI~CH2CH3, R3 i~
CH20C~ltCH20CH2CH20C~, and R4 i~ ~, the compound may be called piperonyl butoxide.
Dillapiol i~ a myor c~n~tihlMlt (27%) of Indian dill (Ane1*um .sowa Roxb.) seed oil. ~ method of
C.AII' '~i~ dillapiol ~om CO~ ' dill seed oil ha~ been r~ romar e~ aL, (1979) J. Agr. Food
C~lem. 27:547). Dillapiol has also been i~olated ~om the dned leave~s of P~PL~r a~r~m ~,. (Oljala
e~al" (1993)P~taMec~ca 59:546-551) Methods for sy--tl~ 7it~g dillapiol are also known in the
art(BakerandSubrahmanyan(1934)J.Chem.S~c. 1681; Da~ Pr(1969)~-hem.B~ricl~e
102;2663-2668;Cannone~a~, (1980)J. Sci.5'~. Th~ilun~6;~9)
The nabun~ i~,olate ps~ldo~ riol has been extracted from the Jarn~can Pipe~ ,. and Piper
h~p~o~n ~3urke and ~ar (1~S6)P~y1ochemi.~lry 25: 14~7-1430). Methods of ~ ing dillapi~l
compounds have ~so been l~vll~d ~ the ~terature ~U.S. Patent Nos.4,~,76,277 and 4,803,290;
Mu~e ~ - - and Burke (199O) ~ A~ d C~m.38; 10g3- 1096, T~war ~ al., (1966) InG~N~erJ~mer 10:43; Saxena et al., (1977) ~relhl~on Posl 14:41; Tomar e~ aL, (1979) J. Agr. rood
(~he~. 27:547; Di~cian and Sliber (1896) BeriehJe 29.1799, Tomar et a~" (1979) A~r~e. Riol.
C'hem. 43:1479-1483, Da~acker (196~) ~hem. Benc~te 102:2663-Z668)
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There ~h ve be~ reporls ~n the use of dillapiol as a synerei~t with in3~,lir ~ , dillapiol compounds
have been used for the m~nagement of horticultural, silvicultural, and agricultural pests. These
compounds, il~c~ piperonyl buto~ide, have been sho~vn to have ~ynergi~ic activity with
pyrethnlm inAecticides (Nair el al., (1985) Agric. Biol. (~2em. 50:30~-3058; Devah~nar el al.,
~198~)A~iG ~iol. Chem. 49:725-728; Tomaret~l., (lg79)A~ic. ~ioL C,'hem. 43;1479-1483;
Indian Patent No. 128,129 (1969)). Dillapiol h~s also been shown to have synergistic activity fior ~1-
mcthyl C~~ dlCo {~ .'ht~lAt~ et al., (1974~ J. Agr. Food ~ m. 22:658, Tomar e~ Cl~., (lg78)
~ 40: l l 3). In this regard, dillapiol has be~ shown to fimction by inh;~itin~ polysubsh~te
~nono-u~e,~ s (PSMOs) (13emard etal., (1989) P~ytochem. 28:1373).
Monon~s of Ggnans, such a~ apiol, dillapiol, and safiol, all contain a n~eIh~ ~i.,xy~,h.,.-yl (h II)P)
ring (Ca~ida e~ al" (Ig70) J A~r. ~cJud Chem. 12 24-25). This M~P nne is thought t~ be
responsible in part for the abilil;y of these ~ "~. .. s to act as potent syner~ists of several classes of
inser~t~ inrll-tling pyrethrins, carbama~es and organoFh- p~-~es (~ irl~le~ e~ aL, (1~7~) J.
. ~ood C'hem. 22:~5~ 664; Mukel3ee et a~ 79) J. Agr. Food ('hem, 27(6): 1209-1211). The
~e-type synerg~ists and their synthetic derivatives act by i.~ Çe g with the rate of
(let~xification ofthe i.. ~ I;ri~e (Will~on and Hic1ces (l969~r, A~. Food Chem. 17:82~-836). This
interference, due in part to the presellce of the MDP ~ 3, is çaused by p.~,f.,.~..Lial binding of the
compounds to the active 9ite~ of the main detoxificat~on er~ymes in insects, the cytochrome P-450
dependent PSMOs (Ahmad el al. ~ .B. ~lat~ el~ and S. Ahmed (eds), Molecul~ As~ects ~)f
In,~ec~-PJfmf A.~iations ( New York; Plenum Press, 1986) 73-l 27).
Dillapiol compounds ha~e also beell shown to be usefill as a~t ~ obi~l agent~ and, more s~e~ifi~ -'ly,
ao, antifimgal and antibacterial agents (U.S. Patent Nos 4,876,~77 and 4,~0~"2~0, Nair and Bur~
(1990)~ Agnc. ~ooclCf~m. 38:1093-1096).
This ba~uJnd i~ iOi~ inrlir~ s tha~ there remains a need for an ei~èctive method oftreating
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MDR in ",~ and in pest control tnanagement
This b~c~ulmd ..&. ..~dlion is provided for the purpose of malcing known i,.rol",alion beCeved by
the a~plicant to be of relevance to the presen~t invention No ~ ;on is ~ ily in~nrlPrl, nor
should be construed, that any ofthe prcGel~lg i.lfD~ion c~ x~ ~ prior art a~ainst the present
invention. Public~ions refenred to Ll~,ughollt the ~re~ifi~2tion are hereby incorporated by reference
in their enPretie~ mto this appl:calion.
1~ 3. SUM~1ARY OF T~ INYENT~ON
lt is l]~e,~rul~: an ob~ect nfthe present ~nvention to provide a method of reducing ~)R activity andk~r
killin~ cells ~"~pr~ an MDR phe,loLype.
15 The pre~ent inv~ntion d~scri~s a method of using dillapivt ()r i~ ~ o~ and derivstives to a~ect
cells that express MDR acttvity. The ~tmcture of dillapiol is depicted in Formula I
OCH3
O_~oCH3
< I 11
o ~
Formula I
In addition to the core st~ucbJre of dillapiol, a class of dillapiol compound~ is jr~l lded as the method
ofttlis invention co.,.p,ising ~ ogues and derivatives"ep~e~e-lLed~ for ~ p'e by the following
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Fo~nulae (~ and Ll)
o~R2
S <c~ ~
R~ R3
Fonnula
l 5 vvherein
R~ is sele$ted ~om H and OR, whe~n R is selected from C~ a~kyl, alkenyl, and aLkynyl
groups; and
~ is selected ~om C~3 and OE~r; and
Rl
< ~ R2
Ponnula Ll
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21 98645
wherein R" R~, R3, and R4 may be the same or d~erent and are ~elected ~om the following
R1 ~ R3 R~
H H H
S R5 Rk R~ R~
OH OH OH OR5
OF~ OR5 OP~
R~OH' R~Oli
R50P~ R50~5
C~10 CH(OR~R~
OC~10 CH2OCH2C~OCH~CH20CT~C~I~CH3
Cll(OR6)CH2C',H3 CH(OI~H2CH3
~C~OR~CF13 C~CH(OR~GH3
C~c~2cH2oR6 CH2CH2CH20~6
15CH[OC(O)R6]CH~CH3 CH[OC(O)}~6]CH~CH~
CH2cH[oc(o)R63cH3 CH2CH[OC~O)R~]C~3
CH2C~2C~20C(O)R6 C~2CH2CIl20C(03R~
C(O)R6 CHCHCH2C(OH~PhPh
CH-~HCOOCHs
CEI2COCH2X
C~12OCHzC~O~OCEl~CH3
where~ ~
~ s~lected ~om tl and Cl-C, ~kyl~ alkenyl, ~nd ~ynyl groups;
R~ is selected ~om R~, phenyl ~roups, and C~H~(OCH3)~ where n=1,2~ or 3;
X is a ~ en
It i~ to be ~ndw~lood th~ new~ dc~loped ~s~n~ or isolated) m~ b~ of ~he cla~ o~
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2 1 98645
~npo~-n-1~ of which dillapiol is a .~pr~3~nlatiw mernber are considered to be within the ~cope of this
inventio~ Moreover, derivatives such as hvologe~ ed or oll.. l~;se o ~hst~ ted derivatives based on
the dilla~iol class of compounds are also considered to be within the s~ope of this invcntion. It is also
to be u..dc;.btood that those ll.c~ whicb demonstrate biolo~ical activity in terrns of their ability
5 to exert a negative effect on MDR are the compound~ fnr use in the method of this in~ention
In one embodiment, the present invention i8 ~ed to treat po~ .cg;~s and phy~iolo~ies in which M~R
plays a role. ln a specific embodimcnt, the present invention is used alone to adversely afFect MOR
cells involved in patholog~es and physiologies such as cancer and malaria.
ln another specific embodiment, the present invention is used in conjunction ~nth other chemical
agents to treat pa~holog~es and phy,s;c'~~ in which~)R plays arole. ~illapiol or its analogues and
d~,~ivalives i..~.rea~e the net accumulation of these agents in MDR eell~ an~l thu~, inerea~e their
efficacy. In a particlllar embodiment, this method is applied to ~educe ~R astivity in can~erous cell~
1 ~ in order to restore the efficacy of chemotherapy. In another particular embodiment, this method is
applied to cells, microbe~, and pathogens th~t have become Ml~, such as the malaria-causing
parasite P~noct~tlm spp ~ Leischffl~nia spp. r~sponsible for Leschrnaniasis, and the causitive agent
o~amoebic dysentery En~amoeb~ spp. In yet another particular embodiment, this method is used ~o
i~bit the Ll~o.l of dn~, across the blood-brain bamer in order to assist in tberapeutic treatments.
In yet another embodiment, the present invetltion relates to a method to a ~ nt addiction and
tolerance therapies, COmpri5111g adn~iniste~ing dillapiol or its analogues and derivatives in Gorljunction
~vhh other chan~ical a~ents. ln a particular embodiment, this method is used in a smokin$ eessation
therapy to md~ ;4~; the arnount of nicotine that remains within the Ci
2~
ln a filrther ~ oL..~.4 the present invention relates to a method of reducin~ M~R to pest control
agents, co~ d ~ist~ g di~lapiol or its analogue~ and deriv~tives either alone or in
C; ~ Jn~lion Witll other d~nical a~ents. In particular, this method is applied to improve the cfficacy
16
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21 98645
of ~ çs.
ln another embodiment, the present inve~tion is used in vifro to identif~r chçln~ compoullds ~hat
increase in effectiveness in the p~ ce of dillapiol compounds.
S
Tn another embodn-~ent, the present invention is u~ed ~n vi~o tc~ tailor therapies to individual patients.
~n yet another enbodiment, the present invention is used in con3unction with other chemo~
to enhance their effect.
Various ~ther obje~ and advantages ofthe present in~nlioll will become apparent from the detailed
de~ tion of the invention.
1~ 4. BRIEF DESCR:D~lON O~ l~E DRAW:INGS
While the ~ ifira~ion con~ d~e with claims particularly pointing out and di~in~y r.l~;ming the
subject matter reg~rded a~ fo~ng the present invention, it is believed that the invention will be better
under~tood from the ~ ,g preferred embodiments ofthe invention taken in cnnneetion with the
20 neco..~ ~J;.IE; drawings in wt~ich:
Fi~re 1 depic;ts ~rf a' uptalce of ~H ~ by drug s~,...,;lh~e and MDR cells. In this fi~ure,
panel A shows the results obtained w~th hamster CGIIS~ wherein the abbreviations are as follt)ws
AUXB1 in~icate~ parental drug se.l ,iti~,~e cells; C5 in.iic~tes M[)R cells, and B30 indi~ates Ml)R
~S Gells. Panel B shows murine cells, WI~G~ l shows the results of parental drug-sensitive cells and
G185 depicts the results of ~IDR cells Dat~ r~3e,l~ the mean of two (C5) or four (all other cell
types) GAIJGI ;~
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Pigure 2 de~o~ . di~t;r~ntial uptake of ~ vinblastine by the Chinese hamster ovary MDR-
selected Gells B30 and their dnlg ~ -;tive parental cell~ AUX~1 ~n the presenGe ot the P-
glycoprotein in~bhor ~e~ il (160 ,UM)J or various ço~ alions of dillapiol.
5 Figure 3 shows .filF~ e~l;al uptake of 3H-vinblastine by the murine fibrobl~l cells tran;.f~.;Led with a
human MDR gene G185 and ~eir drug-se..s;Li~e parental cells NIH a~er 60 minutes of incubation
with dillapiol and selected dillapiol derivatives. The abbreviations ~re as following. D~L is dillapiol~
65 rM; dc~l is denvative l, lS0 nM; der2 is delivat;v~ 2, 1 ~0 nM~ der3 is derivative 3, 150 nl~l; der4
is derivative 4, 150 r~I; derS is deriv&tive ~, 150 nM, and PBO is piperonyl butoxide, 65 nM. Uptake
in contro3 w~s 53,000 ~ 2,000 tlpm and 19,000 ~ 1,S00 dpmJlO6 cclls for ~ and Gl85,
respectively.
Figure 4 shows ~ ti~l uptalce of 3H v;-.h'9 ~ G by the Chinese hamster ova~y Mr)R selected c~lls
E~30 and their parental cell~ AUXB I, and by the murine ~l obla~t çells l~ le~ with a human
l 5 MDR ~e~e G185 and their parental cells ~I, a~er 60 minutes of incubation with 65 nM of dillapiol
('DlL)t 160 IlL of verapamil (VER), and 65 nM of piperonyl butoxide (~'BO)
Figure 5 shows the e~ects of dillapiol on the ~r . llmlllatinn of a-terthienyl in the tissue~ of 4~ instar
mosquito larvae, Aedes ~t~ vfJal~us (I)iptera: Culicidae). Mo~quito larvae were incu~ dt ~ ~ in water
20 with a-te. lb;~.lyl (S0 ppb) and dillapiol for 24 hour~ in darkness.
~, DETAILEI) DESCRIPTION OF T~E INVE~TION
25 5.1 Def
The following comm~n abbreviations are u~ed thro.l~hout the specifica~ion and in the claims:
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The terrn "an~logl~e" means a chernic~l compound hav~ng a stnucture ~imilsr to that o~ another
compound but di~ling ~om it in respect to a ~enain c~..po~ thus, a dillapiol analogue is a
chemical compound with a structure similar to that of diliapiol.
S The term '~çhf~rncse~sitizer~ means a compound t~at ~llows an i~cleasc in the net accllm~ tion of
toxic ~;ul~ ld3 in MDR cells.
The ph~se "concurrently administe~ 't means that a c~ ' agent and a diLlapiol compo~md are
adminiQt~ed either (a) ~m~llt~np~usly~ or ~b) at dilrer,.,t times durin~g the cour~e of a common
10 tre~nt ~e :ln' e In the latter case, the two ~rnr~ound~ are admini~tered At time~ ffi~iently close
for the diUapiol compo~ln.l to enhance the action of the chemical agent. Thi~ may be within one
month, one week, one day, one hour, or one minute.
The terrn uderivative" means any c.:e --1 compound derived from, or re~arded as bei~g derived
15 fi~m, another co."poulld either directly or by modification or partial suh~ctitlltiorl~ tllus, a dillapiol
derivati~e i~ a chemical cnm~o~nd ~t eitber ~as, is, or can be regarded a~ havin~ ~een derived from
dillapiol. For a~ample, ~ olln~le such as halngen~ted compound~ thst can be considered as derived
from a ~r ofthe di~apiol clas~ of compounds are ~ol.s;deled within the scope of this invention.
20 Thete~m "dillapiol compound" includcs dillapiol and its iln-lo~ es and derivatives. The nle.nbdl~ of
the dillapiol cla~s of col~.l)ou~.ds that are cons;d~,,d to be pa~t of the method of thi~ invention a~ e
members of this cla~s that ~e ~elected on the basis of their ability to exert a neg~tive effcc~ on M~R
The t~n ~'net ~cc~lmulation" dd~cl ibe~ the net co~equpnce of influx and çf~lux of dnlgs, ~o that at
25 a l~ ic Ievel in~ ,~ net ~Gcl.mnl~tion can oc~;u~ from enl~3i~ce~ influx, ~o~ inhibitin~
efllux, or from both.
The term "1\~1~" is an abbreviation for multidrug 1~ ~ance Multidrug resistance is the s~ate in
19
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21 98645
which cells are resislant to a variety of chemical agents. This reqi~t~nr~ i5 charact~ixed i.n part by
dc.,l~ascd intrrcP~ a~r chemical retention of the agents a~ a result of their increa3ed efflux. This
illcr~d effl~ Tnediat~d by the o~el~A~ ion of ~ ,llJbal~ of the ATP-binding cassette
supcr~ni~ of l~ OIl proteins As used here~n, multidrug resistance incl~1des multi-re~:6t~.,e to
5 pest control agen~.
The term "MDR cells" as used herein is used to denote cells G,.~,ressin~ an Ml)R phenotype or
1 n The t~ "pest control a~ent" indicates a substance ~at serves to repel a pest from a living organism~
or de~e~e or inhibit the growth, development, or destructiYe activity of a pest. A pest can be a
plant, an aI~imal~ or a microorQ.ani~,". FYr-llT~ pests include iILsectsl spiders, n~ e~, ~ngi~
weeds, bac~ and other microorganisms; thus, pe~7t wntrol a~ents include in~seGtiçi~leR~ pestic~
ffingi~de~, herbicides, n~m~tic;de~ acaricide, m~ lluscic;de~ a~ ic a~ents, antibiotics, and
l 5 anti-m~crobials A pest co~trol agent can also be a mixture of two or more agents. Pest control
agents ~re ~IIlll~-,;dlly a~able. An exemplary list of such ~ubstance8 can be found in U.S. Patent
No 4,911,9~2, the di~C~ re ot'~hich i5 incol~,~ted herein by rcference.
~ .2 ~n~ript;vn ~f ~on
The present invention resdes in the discovery that the natural n~n~nli~nol dillapiol and its anatogues
and daivatives affect cells eAI)r~."g M~R acti~ity. The method of this invention entails the u~e of
dillapiol compound~ to affiect cells e.~l)n,~ 7 MDR acti~ity. In one embodiment7 dill~piol
compounds can be used alone in order to exert a direct toxic effect on ~ells ~ ;n~ an ~)R
pl~.. vty~ LTI another ~ 7 dillapiol ~mr~nd~, can be used a~ chemo~ b~ to ~ C Zl~
~ents7 including dnugo7 such as ~ nthe~ ap~ ltic a~ents, il~ ;r;des7 and addictive s~hs1h~cF ~ such
as nnorpbine and nicotine, in order to increa~e the net ~ ml llntion of these agen~ in MDR cells ll~is
method i~ useful for r~e~u-,l., for determ~ning eir~,vLi~ Ihclapeulic combina~ions, for treating
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pathologies and physiolo~es in which MDR plays a role such as ~ertain cancers and diseases (eg.
malar~a), for treating states of addiction and tolerance (eg nicotine and opia~e ~ddiction~, for pesl:
curltrol mau t~ r~, and for e~ ancin~ the effects of other chelnt S~....:l;,-. ~.
5 5.3 U~e of Inyentj~ to Tre~lt I "ll rlr.E~ Ph,~r ~ ics whicb MnR Pl~ Rvlr
5.3.1 Use nf rnven~ion Alone
In one ar~di~t, ~e metl~od ofthe pr~sent inve~ion is used alone to adversely af~ect MI)R cells.
Becau~e dillapiol ~ol~q~ & are selectively toxic to MDK cells, they can be used to kill MDR cells
10 at conc~ iol1~ that are not toxic to other cells. This ~mbodiment pro~ides a method of treating
a subject compr1sing ~ L.,t.,lillg to the sllbject a lh~ eLlti~lly e~ective amount of A compound of
formula (I), (II~, or (1ll) above.
Subjects to be treated by the rnethod ofthe present i~vention include animals and h~m~n~, preferably
l 5 subjects are, amm~ n
ln a speci~c embodiment, dillapiol compounds can be 6!,l.,,;n.~r. ~d to cancer patients a~ a means of
treating cancer cells which have become MDR in the course of therapy.
20 In another specific embodimentl the method is used to treat malaria.
5.3.2 Use of lr~ iol~ in Colu--n~ n with of h~r ~h~ l A~ nt~
In another embodirnent, the method of the present invention is used in conjunction with therapeutic
agents to af~ h~rl)R cells. R~lue of the general efficacy of dillapiol compounds in increa~ing the
25 n~ m~lation of therapeutic a~ents ~n M~R cells, these compound~ ~an be used to increase the
effieacy and/or reduce the total administered dose of all types of therapeutic agents used in human
c~r veterinaIy mP~i~ne.
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This embodiment provides a ~nethod of treating a subject compri~ing admin~stering to $he subject ~n
effecti~e ~ount of a çonlpound of forrnula (I), (I~), or (TIT) above isl combination with nnc or mor~
therapel~tic agents. The pllalm~;eUti~ compositions and adn~inistration can be as described below.
The thel~p.,~tic agents t~ be employed in combination with the dillapiol compounds can be used in
therapeutic amounts as in~ic~ted in the current vers;on ofthe Physicians' Desk Reference~ or ~uch
therapeut~cally use;rul amounts a~ would be known to one of ordinary skill in tlle art
When administered as a combination, the therapeutic agents can be fonnula~ed as separate
compositions, which are ~iven either at the same tinle or at ~ times7 or as a single c~mposition.
Subjects to be treated by the method of the present invemion include both humans and anin-als? and
are preferably nl~lnm~
5. 3. '. I ~t~ Trea~ (.'ancer
ln a speci~c embodiment, dillapiol compounds can be administered to cancer patients in conj-lnctio
~ith a c~ncer chemotha apeutic agent as a means of treating c~ncer cells tha~ have become l~R in
the course oftherapy l;)illapiol compoLInds increase the net accumul~tion of caneer chemotheral}y
agents in MDR cells. In this ~nanner~ dillapiol compounds can be used to restore, completely or
par~ally, the efficacy of cancer che~notherapy agents in M~R cells
Cancer chemotherapeutic agents c~ be administered ~ith the dillapiol compound~. Th~
coad- u~teralion is designed to increase net accumulation of the chemotherapeutic agent follo~ing
re~,ersal ofthe MDR phenotype, c~using the agent to ~cu~ te ul amouIlts effective fo~ cytotoxicit~
to cancer cells. Any cancer chemotherapeutic agent can be administered a~cording ~o the present
in~ention. F,xamples of cancer chemot~lerapy agents that may be used in combmation with the
~lillapiol compounds are Vinca alkaloids such as vincrist;ne, vinblastine, anllydrovinblast;ne, and
vin~qin~; epipodophyllotoxins such as etoposide and teniposide; antibiotics; an~hracycline antibiotics~
actinomycin D; p~romycin, ~gramicidin D; taxol; ta~otere; colchicine topoisomerase ~ ~nd Il
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inhibitors, cyto~h~l~cin E~; emetine; maytansine; and amsacnne.
In order to interfere with MDR in cancer cells, dillaplol compounds need be administered only in
amounts sufficient to inte~fere with MDR. Dillapiol compounds may be administercd fir~t; the
5 chemothel~pe~ltic dtug may then be administered Ul a dose lethal to the cancer cells. Alternatively.
the ~cancer chemotherapeutic drug rnay be a~ministered simlllt~rleously with the dillalliol compound.
Exemplary of cancer cell~ thal e~press P-gp and are intrinsically resistant are adenocarcinoma cells.
pancreatic tumor cellsl pheochromocytoma cells, carcinoid tNmor cells, chromc cyelogen~us leu1ce~
10 cells in blast crisis, renal cells7 hepatocellular tum~r cells, adrenal cancer cells, and co~on cancer cells
Exetnplary of cancer cells having the ability to become MDR followin~ chemotherapy are
neuroblastoma cells~ adult acllte Iyrnphocytic leukemia cell~ adult acute nonlympkocytic leukemia
cells, nodular poorly differentiated Iymphoma cells, breast cancer cells, and ovarian cancer ce]ls.
Other cancer cells may also l)e treated by the methods of this invention.
S. 3. ~.2 ~ ear Di.~eas~ C.'a~ ed by Parn~i~e.~ and ~a~lo~ns
Irl another particular embodiment, the method of the present invention is used to restore sen~itivity
to therapeuti~ a~ents in parasitic organisms and in disease-causing rnicrobial pathogens expressin~
the MDR phenotype. Accordin~ to tlle metllod of the present invention, therapeu~ic agents are
2n adrninistered in conjunction with the dillapiol comp~unds. The co-administration is desi~ned to
increase net aCcum~ tion ofthe therapeutic agent ~uch that the a~sent is present in amounts effecti~fe
for cytotox~city to the para~itic organisms or micr~bial pathogens Any ther~peutiG agent can lc
a~Tn~ stered acrordin~ to the present invention. For example, the method of the present invention
could be used in conJuction with avennectin for Anl~lo.s-~oma d~o~enc~is, wi~h vancomycin for
2~ bacterial infections, with micran~ole nitrate for fimgal infections including C'an~i~7 a~hicans~ with
Suramin for river ~1indness caused by (?nchocerca v ~ lu~, and v~ith Nifiurtinlox for Afirican and
American trypan~omiasis.
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Dillapiol compo~nds are particular]y usefill in malarial therapy, wbere they is~hibit the pf~DR pump
of Pl~ n~tium spp. In a specific embodinlent, the method of the present in~entiun comprises
adn1inistering dillapiol compounds in conjunction with antimalarial agents such as q~Jinine,
chloroquine, mefloquine, Md arten~sinin (arteslln~te). In one examt)le dillapiol would be cotllbined
S with the antimalarial a8ent in a weight r~tio of 5:1, but the ratio could v~ om 0.1 1 to 100: 1 .
In another specific embodiment! the method ofthe present inventton i~ used to re~tore druL~ sensitivily
in ~::ntclmo~ a spp. and Lel.~chmqn~a spp., comprising adminstenng dillapiol compounds in
conjunction witll therapeutic agents such as emetine or n~etronidasole for ~n~mneJa ~ist~ t~ca and
10 sodium ~tibo~luconate for r,eischm~7~7~asis spp.
S. 3. 2. 3 'lc~ Treat ('NS' Drt~g T~ler(~t~e
ln another specific embodiment, the method of the present invention is used to conlbat Ih~
phenolnenon of CNS d~g tolerance. P-~p-mP~i~ted extmsion o~ compounds from the braitl reduces
15 their effic~cy. Dillapiol compounds increase ~he net accumulation of dnl~s and c~emicals in the brai
through their ability to inhibit P-gp at the blood-brain barrier~ thu~, dillapiol compounds can be uscd
in conjunction with drug~ ~nd chemicals targeted to the CNS. Dillapiol compounds allow smaller
~uFInti~ies o~c~rculalin~ drugs to achieve the desired CI~S effect, tllus reducing the required do~e and
any potentia~ periplleral side effects ofthe administered dnlg. E~ample~ of CN~ dn~gs ~h~t could b~
20 used in conjunction with dillapiol compounds are headache dmgs such as Gp-blockers, sertonin
modulators, and ergots; antipsychotics such as haloperidol; antidepre~.~ants with anticholinergic side
effects such as a~ y~lilene and doxepin; and ~lti,~ hinsoluaII drugs such as !eva-dopa. As an~ther
exarnple, dillApiol compound~ can be usefill cliniGally in cornhattln~ blood-brain bflrrier ext~ ion o~
calcium channel blockers such as dihydropyridines used to alleviatc excitotoxici~y in ~he c~se of
25 .~trok~.
5.3.2.4 r~, Tre~A,~ t ~n ~n~ ~olerance
ln another embodiment, the method of the pre~ent invention can be used to treat addiction an~l
24
-
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tolerance. In ~ specific embodiment, dillapiol conlpolJnds can be taken concurrently with addictive
sl~bst~nrç~ during ~al~ t for addiction to these substances. For examp3e, dillapiol compounds can
be taken concul~c;nlly with nicotine products, such a~ nicotine-containin~ che~ing gums, nicotine
patches, or other slow-release devices, adrn~nistered in smoking cessation therapies. The dillapiol
5 compounds allou~ ~maller quantities o~ circulating nicotine to achieve tlle desired C'NS e~rèc~7 lhus
red~lci~g the required dose and any pot~r~lial side effects of the administered nicotine.
5.4 Use o~thP ~nYen~Qn ;n Pest Contrsl M~n~P~
10 Dillapiol cornpo~mds are also ~ble to cnhance the uptake of pest contro~ ~ents, such AS insecticides,
pe~ticides, tùngicides, llerbicides, nematicides, acaricides, molluscicides, anti~ar~sitic ~ent.~,
antibiotic~, and anti-microbial~ in targét cells. Dillapiol compounds can be used to counteract the
protectivc effiect of P-gp-mediated dru~ transport at any site~ including gut, blood-brain barrier. or
Malpighian tubule, in insécts or otller pests. Dillapiol~s collateral sensitivity action wouid kill or
15 gener~lly disable MDR cells in the o~ , and thus disable MDR pests as they arise in a populati~)n.
This is a different mode of action from the known ability of dillapiol to inhibi~ PSMOs, thus, this i!;
a novel method for maximizing the efficacy of pest control treatn ents
5.4.1 ~IseofInYention~lone
~0 rn one embodiment, the mcthod oftlle pre~ent invention is used alone to adversely a~ect hll)R cells
Because dillapiol compounds are selectively toxic to MDR cells, they can he used to kil] MDR cells
at concentrations that Are not toxic to other cells. This embodiment provides a method of treatin~,
a subject comprising ~J,~ uslerillg to the pest an ef~ective a~nount of a compound of formula (I~
or (I~l) above.
~s a specific example, the tobacco llornworm overexpresses the P-gp in cer~ain tissues such as th~
neuropile and l~lalpighi~n tubules; this allows it to avoid the accumulation and toxlc e~ècts of the
natural inse~ticide nicotine present in its d~et (Murray et a~., (1994) ~upra'~. Dillapiol eompounds
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could be administered to the tobacco homworm to inhibit the P-gp p~lmp. allowing nicotin~ to
acc~1m~ te until it reaches toxic levels.
5.4 2 Use of Tnv~ntion in Cor~ln~tinn with oth~r Ch~lni~ gents
5 ln another ~-"I)o~ ~, the me~hod ofthe present invention is used in conjunction with pest CQll~tOl
agents to af~ect MDR cells for pest control. For exarnple, dillapiol conlpounds could be u~ed ~u
increase the accumulation and efficacy of nat~lral and synthetic inseclicides by inhibiting the P-gp
pump in insects. Exarnples of insectides include pyethroids, carhanlates, organophosphates. plant-
derived antifcedants s~ch as azadirachtin from nee~n and its derivatives, alpha ~erthienyl aud its
10 derivatives, gedunin, and in~ectici~t extract~ of other species of the fnmily Melia( e~. Exan:lples ot'
insect pests that could be treated by the method of the present invention include .~7c~do~r~7
fru~iperd~, Drnsc~p~ , flnd the mite ~ rt~c~, ~ll of whicll expr~ss the P-gp pump.
5.~ Use of Ir~v~n~;nn ir l~ rc~
one embodiment, the method of lhe present invention relates to t~e use of dillapiol comp( unds i
v~lt~ to identify chemical compounds that increase in efflectivene~s in the presence of dill~piol
eompoullds. MDR cells in vitro cau~ be exp~sed to a poten~ial compound in the presencc of dil]~pi~l
compounds. The potential compound could be labeled, using such labels as radioisotopes? or it could
be cold.
l~illapiol compounds could be used in standard dru~ llptak~ experiments. Ml~R cells m vi~ could
be e:~posed to radioactive compounds ~f interest in coniunction with dillapiol compolunds to
det~nnine if the uptake of the test compound increases into a therapeutically usefi~l ranL~c in th~
presence of dillapiol compounds.
Dillapiol compounds could also be used in toxicity studie~. MDR cells in vJ?r-~ could be exposcd t~,
test compnunds in conjunction with dillapiol compound~ to determine ~hether the ~est compounds
increase in cytotox~city in the presence of dillapiol compounds.
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,
Pos~le test ~iompounds include ~ytoto~c dmgs~ ca;ncer chemoth~ es, therapeutic agents, ~nd pe.~t
control agents.
5,6 Use of ln~Vpntioll to ASSÇ~C l~ru~ Ther~ipc
5 Dillapiol compounds can also be u~ed m ~~itPo to tailor drug therapies to individual patients. A tis.sue
sample could be talcen from the patient and used in vi~ to asses~ the efficacy of a l alticul~r
therapeutic protocol in combination with dillapiol compounds. ~s an e~ca~nple, a biopsy could be
taken ~orn a tumor, the cells ~rown in culture, and va~ious combinations of dillapiol compound~ and
k~}own or potential chemotherapeutic agents tested for their ~bility to affect the ~DR n~ or cells
10 This re~imen allows for determin~ion of effective ec~mbinations for chemotherap~ ~y demonstratin~
whicl1 chemotherapeutic d~ugs can be e~ectively accumulated in MDR cells as a result of tl1e addition
of dillapiol compounds.
5.7 U~e of ln-/e~¢ion to gnh~nce ~he Ff~ect of other ChpmDs~ Y;~
1~ Dillapiol compounds can bc used to el~lance the effect of other che~oset1sitizers. In rhis
embodiment~ the dillapiol compounds are added to A regimen that already includes the use ol' ~
chemoser-~itizer being co-~dn~;l,iste, ed with ~ chemotherapelJli~; agent whose intracellul~r
aCCUm~ ltiOn in M~R cells is desired. Dillapiol compounds should be a~ inistered concurrently ~ith
another known chemosen~iti~r to enhance the cytotoxieity or reversing properties 1)l' the
2() chemosensitiz~r.
5.8 Compnr;ti~ns
5 8 1 Pharm~rlltir-~l CoT~positions
25 The present in~ention involves pharm~ceutical compositions containing dillapiol compounds in
c~ ;n~lion ~v~th one or more pharnl~ce~ltie~lly acceptable, inert or physiologically active7 diluetlL~
or adjuvants. The compounds of the inves~tion can be freeze dried ~nd, if desired? combined vvith
ot?ner pharrn~ceutic-~lly acceptable excipients to prepare formulations for admi--istratiotl. The~e
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compositions may be presented in any form appropriate for the admin~stration route envisaged.
r)illapiol compound~ may loe adnunistered ~rally, topically~ parenterally, by inhalation or spray, or
rectally in dosa~e unit fiormulations co~tainin~ con~rentional non-toxic pharm~ceutically acceptable
S carriers, adjuvants, and ~h -~ The term parenteral as u~ed herein ineludes ~subc~ltane~us in~ections,
intravenous, intr~ sc~ r, or intrasterna~ injections, or infilsion techniqlles.
A pharrn~eeutic~l composition m~y be prepared comprising dillapiol compounds and a
~)h~ A~)tic~lly a~eFt~'e carrier. One or more dillapiol compounds may be present in associatioll
1~ with one or more nc~n-toxic pharn~c~ltic~lly acceptable carrier~ and/or dilllents and/or adju~al1ts and
if des~red ~~ther active ingredients. The phaumaceutical compositions containin~ dillapiol compc~-lnds
may be in a form suitable for oral use, f'or example, as tablets, troches, lo~enges. ;1~3ueous or oi~y
suspensions, dispersible powder~ or granules~ emulsion hard or soPt capsules, syn~ps, or elixir~.
1~ Composition.s intended f~r oral use may be prepared ac~;~rding to any metllod known to the art for
tl-e m~nllf~ct ~re of ph~ lti~l compositions. Such compositions may contain one or m--re agents
selected from the group consisting of s~eetenin~ agents, flavou~ng a~ents, c~louring A~ents, and
preserving agents in crder to provide phaumaceutically elegant ~nd palatable preparations. Tablets
contain the active ingredient in an admixture with non-toxic pharmacel~tically ~cceptablc excipient~
20 suitable for the mPn~f~ct~re of tablets Tllese excipients include inert diluents, such as calciutn
carbonate, sod~um carbonate, lactose, calcium phosphate, or sodium phosphate; ~ranulating and
disintegrating agents, such a.s corn starch or alginic acid; binding agent~, such as starch~ gelatin, or
acacia;and lub~ inL~ a~ents, sllch as nlagnesium stearate, stearic acid, or talc The tablets may b~
uncoated or they may ~e coated by te~hniques known to delay di~integration and absorpti~n in ;h~
g~stlo~ s~ fllkac~thereby providing a sustained a~ion over a lon~er period. For example, a time
delay material suc~l as glyceIyl monostearate or glyceryl diste~rate may be emplnyed.
Compositions for oral use may also be presented as hard gelatin ca.psules wherein the acti~e
28
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21 9~3645
ingredient is mixed ~;th an inert solid diluent; for example, calcium carbonate, calc;um phosphate,
or kaolin may be used. ~ternatively, compositions for oral u~e may also ~e prcsented as so~ ~,el~tir
capsules ~h~r~ the active ingredient is mixed with water or an oil medium.
S A~ueous suspensions contain active matcrials in an admixture ~ith e?ccipients suitable t~r the
manufacture of aqueous suspensions. Such excipients are ~uspending agents, such as sodiutn
carboxymethylcellulose, methyl cellulose, hydropropylmetbylccllulose~ sodium alginale,
polyviny~p~rrolidone, gum tr~ th, and gum acacia. Dispe~sing or wetting agents may in.clude the
following: naturally-occu~ring phosphatides, such as lecithin; cnndensation products of an alkylcne
10 oxide with fatty acids, such as polyoxyethylene steara~e, condensation products of ethylene oxide with
long chain aliphatic aicohols, such as hepta-decaethyleneoxycetanol; condensation produ~;ts of
ethylene oxide with partial esters derived firom fatty acids and a hexitol, such as polyoxyethyl~nc
~orbitol monooleale; or c~ndPn~tion products of e~lylene oxide with partial esters deri~ed l'rom falt~
acids and hexitol anllydrides, such ~s polyethylene sorbitan monooleate. The aqueou3 susp~llsion~
15 may also contain one or rnore preservative~, ~uch as ethyl or n-propyl p-hydroxy-bel~zoat~, one or
more colouring a~ents, one or more ~ia~ouring a~ents, or one or more sweetening a~ents, .~uch as
sllcrose or sacchann.
Oily suspensions may be fo~ ted by suspendin~ the active in~redients in a ve~etable oil ~ucll as
20 arachis oil, olive oil, sesame oil, or c~conut oil? or in a mineral oil such as li~uid paraff'ln. Th~ oil~
suspensions may contain a thickening agent, such as beeswax, hard paraffin or cetyi alc~
Swee~ening agents, such as those set folth above~ and flavourin~ agent~ may be added to pro~ide
pal~t~ble orai ~ d~ions. l'hese compositions may be preserved by the addition of ~n anti-oxidant
such as ascorbic acid.
2~
Pl~ nl~c~lll;c~l composition~ of the invention may also be in the form ~f oil-in-water emulsion~. The
oils phase may be a vegetable oil, such as olive oil or arachis oil, or a mineral oil, such as li~uid
pal affin, or mixt~ res of these. Suitable emulsifying agents may be naturally-occurrin~ ~ums, such as
2g
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g~n acacia or gum trag~c~nth, naturally-occurnn~ phosphatides, such as soy bean, lecithin, and estel s
or partial esters deri~ed ~om fatty acids and hexit~l, anhydrides, such as sorbit~n monole~te, and
conden~ation products ofthc said partial esler~ with ethylene oxi~le, such as pclyoxyethylene sorbttan
monoleate. The en-Ul~;or~s may also contain swe~tenin~ and flavounng agents. Syrups and elixirs may
5 be fo~ te~ with sweet~in~ a~ents, such as glyccrol, propylene glycol, sorbit~l, or sucrose Such
con~posilions may also contain a demulcent~ a preser~ative, and flavouring and colouring agent~
The pharmaceutical compositions may be in the form of a sterile ;njec~able aqueous or olcaginous
suspension. This suspen8ion may be formulated according to kn~ wn art using those suitable
10 dispersing or wetting a~ent~ and suspending agents which h~ve ~een ~nentioned abo~/e. Tt-e sterile
illje~;table pr~ alalion may also be a solution or suspension in a non-toxic parentally- accep~ahle
diluent or solvent, for example as a ~c lution in 1,3-blltanediol Amon~ the acceptable ~ehicles and
solvents that may be employed are water, Rin~er's solution, and iSOtOlliC sodiunl chlonde solution
In addition, sterile, fixed oils are con~/entionally employed as a ~olvent or sl~spendin~ medium. For
15 this purpose, any bland fixed oil may be employed in~;luding synthetic mono- or di~lycerides. In
addition, fatty acids SUC]l as oleic acid find use in the preparation of injectables
Dillapiol compounds may also be administered in the fonn of suppositories for rectal administralio
o~the dlug~ These compositions can be prepared by mi~cing the dru~3 with a ~uitable non-irritatin~
20 excipient that is solid at ordinary temperatures but liquid ~t the rectal temperature; thus? i~ will melt
in the rectum to relea~e the dn~g. Such mater;als are cocoa but~er and polyethylene glycols
Dillapiol compvunds can also be administered in the fo~m of liposomes As is known in the ~
liposomes are generally derived from phospholipid~ or other lipid substances. Liposnmes ar~ folm~d
'~5 by mono- or multi-lamellar hydrated liquid ~;lystals that are dispersed in an a~ueous medium. Any
non-toxic, physiologically acceptable and metabolizable lipid capable of f~rm~ng liposomes can be
used The present GOmpo~itions in lipo~ome for~n can contain, in addi~iurl to a compound of the
present inven~ion, stabilizers, preservative~, excipients~ and the like. The preferred lipids are the
.
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pho~pholipids and phosphatidyl cllolines (lecithins), both natural and synthetic. Metho~is to form
liposomes are known in the art (See Prescott (ed.) Methocl in ~.~ell ~iolo~ olume XIV (Ne~
York, Academic Press, 197~) at 33).
5 Dillapiol compounds may be administered parenterally in sterile medium. The con~pound, depending
on the vehicle and concentration used, can either be suspended or dissolved in the vehicle.
~dvantageo~lsly, adjuvants such ~s local ~--e~th~tics, prcse~vatives, an~ bufferiin~ agents can be
disso1ved in the vehi~;le.
10 For the compounds ofthi~ invention, the dose to loe administered, whether a single, multiple~ o r daily
dose, will vary with the particular compound being used. Factors to consider when decidin~, upon
a dose regimen include potency of the cornpound, route of administratiolL, age~ body weight, and
genaaJ llealth ofthe recipient, and the severity ofthe particular condition undergoing therapy The
do3age to be adminisl:ered is not subject to dffined lin~its, but will usu~lly be an ef~ective amount
5.8 2 Pest Control C~ osit;onc
~hen pest contr()l is involved, subjects to be treated by the method ofthe present inYention are pest~,
includin~ plants, animals, and microorganisms.
20 The pest control compositions are prepared in manners known to one slcilled in the art. A pe5t
control composition will comrnonly contain an active ingredient or pe~t cvn~rol agent1 ~ wettin~
agent such as a surf~tant, and a solvent such as waler. Optionally, an oil such as conventional cr(lp
oil, can also be included a.s a solvent for emulsion conc~ cs. The dillapiol compounds may be
nlixed with pest control agents and conventional inert ,lgronomically or physiologically acceptable
25 (i.e. plant and rnamlnal compatible an~/or insecticidally inert) diluents or extenders usable in
conventional compositions as is well known in the art. If desired? adjuvants ~uch as surfactants,
stabilizers, antifoam agents, and antidri~ agents may also be added. Examples of pest control
compositions include aq~leous or other agronomically acceptable suspensions and disper.si~ s, oily
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21 98645
disper~ions, pastes7 dusting powders, wettable powders, emulsifiable concentrate~, flo~ables,
granules, baits, invert etnulsions, aerosol comp~lsitions, and ~nu~ating candles.
Adhesives, such as carboxymethylcellulose and natural and synthetic polymers in the fiorm of
5 powder~, granule~, or lattices, such as ~um arabic, polyvinyl alcohol~ polyvinyl cellulose, ~nd
polyvinyl acet~te, can be used in the pest control compositions to improve the adherence of the pest
control agent. Furthermorc, a lubricant such as c~lrium stearate or magnesium ste~rate m~y be
added to a wettable powder or to a mixture t() be ~granulated.
lO Pest control compositions suit~ble for ~se with dillApiol compounds of the present invention includc
both package and tank n~ix compositions.
The compo~lents ernployed in the pest control composition can be combined in any order ~or
example, the cc~mposition can be prepared by starting with a pest control agent and adding the vnriolls
1~ components in any order. Water can be employed, if desired. The amount of water empl- yed tG
prepare the concentrate or final application concentration, a~ in a spray. is adj~lsted as necessary. Tl-e
concentrate and/or final composition ~ay be a dry composition.
A dillapiol compound of the present invention i~ incorpor~ted into a pest control cornposition at a
~0 c~nc~nlr~lion th~t will deliver enough dillapiol cotnpound to increase the e~cacy of the pest control
agent. Tho~e of skill in the art will reco~nize that the rati~ of pest contrnl agent to di~lapiol
cotnpound w~ll depend a gr~at deal upon thc nature and ty,~e o~ the pest colltrol agent ~lich is
pre~ent in the composition. 'lypically~ however, the weight ratio of pest control a~ent to dillapinl
compound will be in the range ~om about O l I to 1:100 and prefierably is in thc ratio of l S
2S
In addition to the aforementioned components, the compositi~ns of the present invention may a~s(:)
~ontain other l~e~t control agent adjuvants commo~ly employed in the art. Examp~e~ of such
adjuvants include crop oil concentrates s~ch ~s AGRlDEXr~, spreaders such as OR'rHOTM X-77~
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drift control agents, such as LO-DRlFl'rM~ deÇc,~ g agents, such as r)-FOA~IERT~ compa~:ibility
agents, such as E-Z MIXrM, and other adjuvants well known in the pest control agent a~t. It is
understood by those skilled in the art that the ~mount of tllese adj~vants in the pest control agent
~omposition can vary widely, and that the amount needed can be readily determined by ro~ltine
S e~pe.;.,l~l~lalion.
For any given pestiride~ the skilled a~tisan will readily amve at a pest c()ntrol compositicn having thc
optimum ratio of un~redients by rou~ e experimentation.
1~ ~.9 A~lv~n~yes
The use of dillapiol compounds in the method of this invention presents an impr~vemenl over
previously known methods oftre~tin~ M~R for a number of reasons. Dillapiol and its analogues are
powerful chemos~l-si~;7~,s that inhibit the ABC supe~rd~ oftran.qpolt protein~; thus, they are
1~ promising chemos~n.~itizers for I~IDR management in the treatment of disease, pest contl-ol, and
addiction therapies. In addition to this~ dillapi~l compounds are selectively toxic to ~f~R cells. Thi.q
collateral sensitivity is valuable; dillapiol c~mpounds act on the ~lDR phenotype, nol by inhi~iti
~ physiological fiJnction f~und in both healthy and cancerous cell3. The fact that dillapio] cvn~poul-d
both exhibit collateral sensitivity to MDR cells and are chemose~ er~ makes them ideal c~ndidate~
20 for chemosensitizers in chemotherapy.
Since dillapiol compound~ are highly toxic to MDR çells, b~lt not to dru~-sensitive cells or to non-
cancerous cells, these compounds can be used at lower con~entrations than other chemosensitizer~
their effectiveness is au~nented by their specific toxicity to J~R cells. For example, thc selectiv~
25 e~ect of dillapiol on l~lDR ceUs was obtained ~Nith much lower concentrations than with the stanc~ard
P-gp inhibitor, verapamil, as illustrated in Pigure 2. The use of lower eoncentrations of
~hemo~ensitizers avoids unacceptable side effects~ a problem associated with many of Ihe
chem~sensitizers eurrently available
SE~'T BY~ 27-97 : ~:2~P~ ; 6135~37671- ~U~6~95}72;~1
Dillapiol compounds increase the efficacy o~ admini~tered dm~s. This results in the reduction in
concentrations of drugs in~olved in the treatment of discases ~nd, consequently, a reduction in side
ef~ct~ to dn~g treatrnPtlt~.
Dillapiol compo~lnds also enhance the activity of pe~t control agents, decreasing the concelltrations
of ~hese agents needed to be effective. Thi3 is e.~l eZllely advantageous considering that the u~e of
chemical pe~t control agents pre~,nts the disadvantages of polluting the environment, creatin~
potential ha~ ls to agricultural workers and consumers, and causin~ detrimental effect~ on non-
target species.
I()
A filrther advantage of dillapiol compounds is that they exhibit low toxicity to noT7nal, u~ e~i~tant
cells. The cytotoxicity o~other chernoscl,si~e,~ to healthy tissues limits the therap~utic use of m~y
nfthese wmpounds. Dillapiol and the closely-related phenylpropanes, however, have been identified
in severa~ vvidely consumed food substance. This indicate~ that they are wcll tolerated in the human
digestive system. For example, dill~piol is a major constituent of dill and celery seeds; e~tra.L~ole is
used as a flavour in foods and lic~ueurs and is found in Piper bet~e, which is chewed with betel nuts
on a daily ba~is in ~sian countries; myristicin is found in carrots, nutrneg, and celery; ele~icin i~ found
iTl carrot~; and eugenol is found in nutmeg, oregano, cinnamorl, and cassia; and safrole is found in
basil and nutmeg (~arbonle and ~axter ~ed.) A HcmdbooJ of Bi~ac~ive C'o~np~un~s frlJm ~'~CI~f.S'
(London ~ylor and Francis, 1993) at 472-488).
Dillapiol compounds also act morc rapidly on ~:DR cells than do other chen osensitizers.
ln additiun to these advantages? dillapiol is cheap to produc~ since it can be extracted and purified
~5 ea~ily firom widely-cultivated plants. It is amenable to practical agrobotanical production and
purifieation. Further, dillapiol compound~ can improve the e~icacy of low doses o~ chemothcrapy,
thereby reducin~ the cost of drug treatments with ~e~v expensive therapeutic agenls.
34
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6. E~CAMPLES
Tlle pre~~nt invent;on is described in fi~rther detail in the followin~ non~l~miting examples lt is to be
understood that the examples described belo~ are not meant to li~it the scope of the present
S invention ~t i~ expected that numer~us variants will be o~vious to the person ~killed in the aJt to
which the prese~t illvention pertains, ~Arithout any departure ~om the spirit of the present inventio1~.
The ~ppended claimo" properly construed, form the only limitation llpon the scope of the present
inventlon.
EXAMPLE I
Tosicity Studies
The results presented in this e~ullp~ demunn,trate the resistance level~ of two series of MDR cell lines
(cell lines selecte~ for M~R as well as cells transfected with the P-gp gene) to chemotherape-lti(;
l 5 drugs, a known chemosensitizer, ve"~pa.~lil, detergcnt~, and dillapiol compounds.
Cell I ines:
The following cell lines were used: NIH13T3 (~IIH), established frorn NT~ Swiss mou~,e e-nbr~o
20 culture~.~, and their MDE~ derivatives transfccted with a human gene coding for P-gp, NIH-~)R-Gl 8 5
(Gl85) ~3~dley ~t al., (lY~8) B~ocJ~em. Bif phys. ~ a 948 :87-128) Both cell lines were provided
by the National Cancer ~n.etit~te~ Bethe~da, MD. Three Chinese hamster ovarian ce!l lines w~re als~
u~ed: the parental CHO-AUX~I (AU~l) and its two MI?R lines successive1y selected ~ith
colchicine CHRC5 (CS) and CHE'B30 ~B30) f~ing e~al" (1983) C'anc~?r T~eal. Rep. 67 86~-g74)
2~ C~RTl0 (I10) revertant, drug-sens~tive cells wese selected ~m C~IRC5 These cell lines were kindly
supplied by Dr. V. Ling of the Ontario Cancer It~stitute~ Toronto~ ON, Canada. B~th resistan~ ~ell
lines have ~I~i~.t~d stable levels of crl~ss-resistance to anthracyelines~ etopo~ide (VP-I~), al~d Vinca
alkaloids ~or s~/eral year~, with highest le ,els of P-~p in B30 (I,ing e~ al., ( 19~3) C'ancer ~r~al. ~p.
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67:8~9)
Cells were gro~n in pla~tic flask~ (~alcon) in a h~ ified atmosphere at ~TC, 5~,'o CO2. The mouse
ceils, NIH and G185, were n,~~ ;"~ in Dulbecco's modif~ed Eagle medium (l}l!lEM, Gibco BRL)
5 COllt~ g glllt~minp The hamster cells, AUXBI, C~! and B30t werc n~int~;ned in alpha minimum
eS~nti~l medi~n (aM~f, Gibco BRL) containing nucleosides and glycine. All culture media were
~upplemented ~ith 10% fetal calf senlm (Wittaker Bioproducts), per~icillin (50 units/ml), and
streptomycin (~0 ,ugtml) (Gibco BRL). Re~ist~ce levels were maintained with the additio~ to the
cultures of ColGhicine at the following concentrations: 0.06 ,ug~ml in G185~ 15 I,lg/ml in C5 and ~0
g!ml in ~30.
Determination of Resistance Levels-
A SCreening o~the toxic dose~ of various test compounds to the cell lines waS pe~formed hdherent
cells ~vere dnn I-~d with a solution of trypsin (0 ~% in PBS with 0.2% EDTA), centrifùged ~t 1000
rpm for 15 tn~nutes, and resuspended in medium to a final conce~tration of ~00 cells/ml. One ml of
the cell suspension was distributed in each welt of a 24-well plate (Falc~n), already cont~inin~ on~
ml or~le te~t compound at the approp~iate concentration (at least six concentrations). Control wells
without test compound conta~ned cells and the s~ ent (0 1% H20:~tO~I or DMSO) ~lsed to stahilizc
20 the test compound. Cells ~ere incubated for eight day~ at 37~C, 0.~% CO2 The media wcre then
discarded, and cell colonies were stained with 0.25% methylene blue (in 50% EtOH) for 15 minutes
The concentration beyond which cell ~~rowth ceased was u~ed t'or thc assessment of the tuxicity of
the compounds For a more accurate calculation of the ~nini~ m inl~ibito~y concentrations (h ll(~)
of the te~t compounds, a similar experiment was set up u~ing ~ar~er 3 5 mn~ diameter sterile petri
~5 dishes at two dilrer~.,t concentrations o~' ~ell: 500 cells/3 ml and 500(~ cells/3 ml 1)mL~
conc~ nlionsclu~ered around the ki~ing dose pre~ously dcl~."~.edin ~he 24-well plates. Colonies
were Gnunted, and cell growth was expressed as percent growth of the respe~tive control~. The
results are presented in T~ble 1.
36
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The r~si~t~nce levels of the cell lines were calculated ~s the LDso ~f t~le test compound for th~ cell
line ot' interest divided by the LD~o of the test compound for the parental cell line. The letllal
col~ce~ tion of the drugs colchicine, actinomycin D, vincristine, and vinblastine were hi~her for tl~e
5 resistant cell lines C5 and B30 th~n for their p~ w,l~l drug-sensitive line AUXB 1. Sin~ rly, the lech~l
concentration ofthese druL~swas 10- to 1~)Q-fi:)ld higher for the tr~nsfected M~R cell line G1~5 th~n
for its parent NIH.
T~le chemosen~itizer ver~pan~il was equally toxic to both the parental and MDR cell lines
]O
The MDR cell lines showed collateral sensitivity to the deter~ents NP-40, Tritnn X100, and phenolic
DBP, but not to T~een 80. This phenomenon wa~ elimirlated in revertant, d~ g-~ensiti~ e I l 0 cells.
indicatin~ that both hypersensitivity and resistance are an intrinsic par~ of the Ml)R p}lenotype in
these cell series.
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¦ Table 1. E ffectof Dru~ and Other A~ents on the RecistAnce L~vct~of C~ncer Cell Lines
_. ..... _ .......... ... ..... ..
CEI~L LlNES
Murine Hamster
human MDRl treated with colchicine
(transfected) ~lected)
S Nl~l G185 AIJ~I C5 B30 TlO
sensitiYe Ml)R sensitive MDR ~DR revert~nt
1 96 6X~ 2~3
l ~4
l 0~ 1 75
l tO . 1 50
I~/IVR Group of
Drug~:
colchicine
1~ actinomycinD 1 1 3 0 9 1.2
vincristine l 0 ~ 0.4 l l
vinblastine I 0 2 0.06 1 1
Chemos~nsitizer 1 0.5 0 09 1 2
fo~ MDR:
Lignan~ and
Derivatives;4
pod~phyllotoxi~ I ~ 2 l 10G
~IP-16 1 10 1 l0
dillapiol 1 0.61 l 0.00l
p i p e r o n y I 1 0.1 1 o 01
hutoxide
~ec~ eo I,evel - LD~o rcsist~t cell lir~e I LDso ~cnsitive parcnlal cell line
P. luranka, pervcnal cotn~nrnir~tioTl
~ ~cter~enls sb~w~n~ er l~icity to MDR th~n sensilive cells- LQC ~nd Sharom ( 199~) Br. J. ~~'ancer G8 342-3 51 .
~130urret-E~ernard (1995~ Ph~ Ihesis, University o~Ollau~
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Both M~R ~ell types, selected and transfected, showed ~]lateral sensitivity to dillapiol ~nd one of
its synthetic analogues7 piperonyl butoxide (see ~igure 4). Both compound~ were at least 10 to 10()
timeg more to~ic tn MDE~ than to p~rental cell~ The very low concentrations of piperonyl butoxide
that were effective (V.S and S ng/ml ~vith B30 and G185 respectively) are far be]ow the
5 conccntrations found in currently collu-le.-;ialized insecticidaT mixture~. Similarly, the very low MIC
of dillapiol (0.~ ng/ml with B30) is t'ar beluw the concentration found in plants, in p~rticul~r the
~iperace~e.
EXAMPLE lI
Drug AcclJmul,stion Studies Using Vinblastine
The effects of dillapiol wmpounds on the net accumulation in cells of the dru~ vinbla~tine is shown.
Cell lines used were the same as described in Example I
1~ This exarnple was canied out at room temperahlre ~ollow~llg a method modified ~om Le~ontt ~l c~.
(198~) C'anc~r Re5. 48:6348). In short, t~ypsinized cells were washed and rcsuspended in PB~i-
glucose (1~ l), at a concentration of 1.5 X lO6 cell~/mJ. A I ml cell suspension wa~ mi~e~l with
2 ~l 3~-vinblastinc1ml (specific activity l I Cilrn~ with or without tlle potential r~sis~ancc m~)di~yiIl~
cornpounds At specific intervals (1, 15. 30, 60, and 9~) minutes3, 300 111 of the mixture wa~ layered
2(~onto 250 ~Il of a silicone oil:mineral oil mixture (4:1, v;v, Aldrich silicone oil, d~-l.05:Fi~her li~ht
mineral oil), and immediately centrifùged 10 seconds at 500x~ on a countertop centrifil~e The
aqueous phase w~ removed, the upper part ofthe tube rinsed with 500 ~11 PBS to eliminate risks of
~ontam~nation of the pellet witll the unincorpora¢ed radioa~;tivity? the oil remGved, and the pellet
digested overnight in S00 ,ul ~laOH (0.1N). Five ml of scintillation liquid was added to the pellet;
2 ~ countiIlg of tritium activity was done on a Beckman LS 1701 scintiliation counter.
The validity of the system was e~tablished by using verapamil (15 ~IM), a specific P-gp inhibit-lr as
a po~itive control in eacll experiment.
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Ac~ tion of 3H-vint lastine correlAted with the resistance level ofthe S cell lines studied (E;igure
1). A plate~u was reached aflcer 60 minutes in all cell types. l'here was 2.5-fold more vinblastine in
N~l cell~ than in the MDR cells G185, the hamster dnlg-sensitive AU~3 I cells had 2 5 -fnld more
5 vinblastine than C5 cells and 4.2-fold more than B30 cells. Thexe ~;ounts represent the net ~;ellular
loadin~ that result~ from the con~bination of the uptake of the dn~g by cells~ minu.s its acti~.~e efflu~
by the l)-elnb- ~ne bound P-gp pump. Counts were stable from one experiment to the next oncc the
proper protocol was established. The rrsults were comparable to results reported ~om other studies
using vinblastine and other drugs recogn~ed and transported by the P-gp pump
1~)
Veraparnil a specific inhibitor of P-gp, reverses the MDR phenotype (Lemmont et al., (1988) CC~7C ('7'
~es. 48.6348-6353; Loe and Shar~m (1993) ~t. .J. C~cmcer 68:34~-35l~. It restored the level of
e acc~ ted by B30 cells to that ofthe drug-sensi~ive A~l cells (~igurc 2) Murinece}l responses to verapamil paralleled that of hamster cells (data not shown)
Dillapiol had a lar~er e~ect on the net accunlulation of 3H-~nblastine in the MDR hamster cells (B30)
lhan on the dNg-~ensitive cells (Figure 2). ~ile the lower doses (9 nM and 18 nM) h~l~ little
m~dulating ef~ct on the net uptake in sensitive cells, they increased the net uptake in resistance cells
by five ti nes ~DPM in B30 w~thnut di]lapiol - l 800 ~ 4000; with dillapiol = l 02 000 -~ 9~)00) ~uch
2~ result~ are cor~istent w~th toxici~ studies showing a hypersensitivity of MDR cells to dillapi~ T~blc
1).
The ~DR-~elective mod~ tin~T ef~eets of dillapiol on the 3H-vinblastine ~lptake were more rapid and
o~tained with ~nuch lower concerltrations than with verapamil. Aflcel 3 15 minute incubation of B30
25 cells~ the ~et accumulation of vinblastine in dillapiol-treated cells was three times that of vinbla~tine
in verapamil-treated cells (Figure 2). ~one of the dose~ assayed were toxic to the drug-~en~itive cell
line AU7~l ~lC for dillapiol - 500 ng/ml = 2.2
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A variety of dillapiol derivati~fes were sho~vn to h~ve a similar effect on net accumulation of ~H--
vinblastine in the MDR hamster cells, B30 ~Pigure 3).
EXA~PLE m
Drl~g AccumulAtion Ct~Jdies Using a-Terthienyl
The e~ects of dillapiol compounds on the net accumulation in cells of the drug a-terthienyl w~.s
investigated.
1 0 Chemical~:
Dillapiol was purifed and kindly provided by Dr. Krishnamurty (Tndi~). Alph~-terthienyl ~as used
~um a stock that ~a~ sytlth~sized as described in Philo~ene er al., (198S) IComplete cite needed~
1~ Effects of Dill~piol ~n the Aceurnulation of a-terthienyl in Mosquito Lar~ae.
~osquito larvae ~e~es atro/~a~t~ iptera Culcidae) were incubated in the presence of a constant
~ub-lethal concentration of a-terthienyl, I ~g/rnl, and in the presence of variable concentrations of
dillapiol ranging from 0 to I ~g/mJ. The amount of a-terthien~l in the tissues was then determined.
2~
Fourth instar larvae (13-21 larvae per replicate, 4 replicate~ per treatment) ~vere incubated in 7-ml
glass vials containin~ 5 ml of di~tilled vvater to which a-terthienyl had been added at ~ concentration
of 1 ~ nl, throu~h 20 ,ul of ethanol solution. All treatments received the same amount of eth~nol.
I,arvae were then incubated for 24 hours in darknes~ to prevent tbe photosen.~iti~Ation proeeSse~
25 mcdiated by a-tcrthienyl when exposed to near-UV w~velengths. Under these specific conditions
no larval mortality was observed in any treatment.
At the end of the incubation period, the larvae were slightly dried out on a paper to~rel then
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transferred in viaJ~ conatining 2 rnl of hexane and incubated with agitation in d~rktles~ 3 ~;~ys.
Plel;",;~aly ~,.t)e~ ts showed that the yield ofthis extraction protocol of oc-telthienyl ~vas higher
than ~2% An aliquot of 1 ml of hexane was then ~ampled for HPI ~ analysis and the tarval ti~sues
were put in an oven at 55~C for 24 hours to determine the dry weight.
I~:PLC Analysis of o~-Telthienyl
Alpha-~e. ~ yl wa~ analyzed by HPLC using acetor~itrile:water (7 ~ at a flow rate of 1 ml~min with
a C-l 8 ~everse phase colurnn (2~0 mm ~ 4.15 mm, Ber~T~n, USA). An HPLC Beckman Gold Sys~em
incl~l~lin~ a ~olvent module (model 126)? a W-detectnr ~model 16~)? and an autosampler (model
502) was used Undet th~e c(?nditions the retention time of a-tertllienyl was 11.4 min The specific
peak areas on the chl-on)a~ograrns were used to detennine the concentration of ~-terthieny3 A
calibration curve w~s made with pure a-terthienyl diluted to dilrerellt eo~celltrations in hexane 0 2~
1.0, 5 0 10 0 and 25 ,ug/ml. The coefficient of dete~n~nation values (r~) according to the peak areas
1 S measured was 0.994. The concentration of o~-terthienyl in mosquito larvae was expressed in ~lg of
u-terthienyl per gram of dly wei~ht l~nral tissues.
From the foregoing description. one skil]ed in the art can e~sil~,~ ascertain tlle ess~nlial characterislic.~
oft~s ~nvention, and without depa~ting from the spi~it and scope thereof, can make vanous chan~es
20 and m~d;fications to the invention to adapt it to variou~ usa~es and conditions. Such chan~es and
modifications arc properl~,r, equitably, and intended to be witl~in the fùll range of equivalence of thc
t'ollowin~ claims
