CA2198645A1

CA2198645A1 - Use of dillapiol and its analogues and derivatives to affect multidrug resistant cells

Info

Publication number: CA2198645A1
Application number: CA002198645A
Authority: CA
Inventors: Peter Juranka; Cathy Morris; Tony Durst; John T. Arnason; Claude B. Bernard
Original assignee: Individual
Current assignee: Individual
Priority date: 1997-02-27
Filing date: 1997-02-27
Publication date: 1998-08-27

Abstract

The method of this invention entails the use of dillapiol or its analogues and derivatives to affect cells expressing multidrug resistance (MDR) activity. In one aspect, dillapiol or its analogues and derivatives are used alone as agents that exert a direct toxic effect on cells presenting an MDR
phenotype. In another aspect, dillapiol or its analogues and derivatives are used as chemosensitizers to chemical agents, such as chemotherapeutic agents, insecticides, and nicotine, in order to increase the efficacy of these agents. This method is useful for treating pathologies and physiologies in which MDR plays a role, such as certain cancers and diseases (eg. malaria), different states of tolerance or resistance (eg to insecticides), and addiction (eg. to nicotine and opiates).

Description

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 SENT BY: ; 2-27-97; 5:54PM; 6135637671~ 16û46694351#---9876;# 5 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).

SENT BY: ; 2-27-97; 5:54PM; 6135637671~ 16046694351#---9876;# 6 2 1 9~645 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 SENT BY: ; 2-27-97; 5:54PM; 6135637671~ 16046694351#---9876;# 7 2 1 9~645 ~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 SENT BY: ; 2-27-97; 5:55PM; 6135637671~ 16046694351#---9876;# 8 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 S~T BY: ; 2-27-97; 5:55PM; 6135637671~ 16046694351#---9876;# 9 2 ! 98645 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, SENT BY: ; 2-27-97; 5:56PM; 6135637671~ 16046694351#---g876;#10 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 SENT BY : ; 2-27-97; 5 : 56PM . 6135637671~ 16046694351#---9876 ; #11 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).

SENT BY: ; 2-27-97; 5:56PM; 6135637671~ 16046694351#---9876;#12 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) -SENT BY: ; 2-27-97; 5:57PM;6135637671~ 16046694351#---9876;#13 ~ 21 q~645 S o ~ R2 <o~~

Fo~nula where~4 Rl is selected ~om H and CNCH3; and 1 ~ R~ i~ sel~ed ~om CH3;

R.

<o~ 2 Fonnula lII

SENT BY: ; 2-27-97; 5:57P~; 6135637671~ 16~46694351#---9876;#14 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) S~IT BY: ; 2-27-97; 5:57PM; 61356376711 16046694351#---9876;#15 21 q~3645 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 S~IT BY: ; 2-27-97; 5:58PN; 6135637671~ 16046694351#---9876;#16 21 9~645 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

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 SENT BY: ; 2-27-97; 5:58PM; 6135637671~ 16046694351#---9876;#17 21 9~645 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 SENT BY: ; 2-27-97; 5:58PM; 61356~7671~ 16046694351#---9876;#18 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~

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 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~

SENT BY: ; 2-27-97; 5:58PM; 6135637671~ 16046694351#---9876;#19 ~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 SENT BY: ; 2-27-97; 5:59PM; 6135637671~ 16046694351#---9876;#20 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 ;~

SEI~T BY: ; 2-27-97; 5:59PM; 6135637671~ 16046694351#---9876;#21 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:

SENT BY: ; 2-27-97; 5:59PN; 6135637671~ 16046694351#---9876;#22 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 SENT BY: ; 2-27-97; 6:00PM; 6135637671~ 16046694351#---9~76;#23 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 SENT BY: ; 2-27-97; 6:00PM; 6135637671~ 16046694351#---9876;#24 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.

SE~T BY: ; ?-27-97: 6:21P~1: 6135637671- ~3046~557'?:# ~1 21 9~645 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 SE~T BY: : 2-27-97 ; ~ 22P~l : 613~637671~ 60~6895~72: # ~
21 ~8645 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.

SE~T BY: ; 2-27-97 ; 6;~2P~ ; 6135637671- 60~6~95-~72:~ 5 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 -SE\IT BY: : 2-27-97; 6:23P~ 135637~71- ~30~ 9557?:# ~

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 SE~T BY~ ?7-97; ~ P3~: 6135637~71-~ 60~895.7':rr 7 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.

SE~T B~ 2-27-97 ; 6:~PM : 6135~37671- 60~6895~7~:X 8 , 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 SE~T BY~ 27-97 : 6:'';PM : 6135637~71- 6~ 95-~72'~ 9 21 9~645 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 SE?~T BY: --27-97: 6:25P~ 135637fi71- oO~895~7'~:#10 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 SE~IT BY~ 27-97; ~:26P~1: 6135637~71- 60~689557~

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 .
~E~lT BY: ; 2-27-97; 6:2~P~I; e1.~5~7671- ~0~689~ 7~2:X1~' 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 SE~'T BY~ 27-97 ; 6:27PM : ~135637fi71- 60~6~9~7~.~13 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

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~

SE~'T EY: ; 2-~7-~7; 6:28P~l: 61~5~37671- ~0i6895~7'~
21 9~645 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.

SENT BY: : ~ 27-97 ; 6:29P~ : 61~5637671- 6~ 95-~7'~
21 9Q~645 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.

SE~T BY~ 27-97; 6:~9P~ 135t~7671- 60~893a7':~l7 21 9û645 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.

SE~T BY : ~-27-97: 6:30P'i~l: 6135~37~71- ~0~6~9557':~18 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.

SE~T BY: ; 2-27-97 : 6 ~0P~ 135637~71- 60'~895-~7~:X19 21 9~3645 ¦ 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~

SE~T RY~ 7-97; 6:31P~ l5~7~71~ 9~572~ 0 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.

SE~iT BY: ; 2-~7-97; ~:31P~I; t3135637671- 60~895~7':~21 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 SENT BY: : 2-27-97; 6:3~PM: 6135~7~71- 60~9557-':#2' 2! 98645 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 SE~T BY~ 27-97 : ~:3''P~l ; 6135637~71- ~0~689557~ 2~!

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

Claims

1. A method for inhibiting or controlling MDR, said method comprising administering to a patient in need of inhibition or control of MDR an effective amount of one or more compounds selected from a class of dillapiol compounds comprising dillapiol, its derivatives and its analogues, wherein said compounds are selected on the basis of their ability to exert a negative effect on MDR.

2. The method according to claim 1, wherein said compound is administered as a pharmaceutical composition, said pharmaceutical composition comprising one or more compounds of claim x and an acceptable pharmaceutical carrier.

3. The method according to claim 1, wherein said compound is dillapiol.

4. The method according to claim 1, wherein said compound is piperonyl butoxide.

5. A method of adversely affecting MDR cells in pests, comprising administering to the pest an effective amount of one or more dillapiol compounds, wherein the dillapiol compound is selected from the group consisting of dillapiol and analogs, salts, or derivatives thereof, and combinations thereof, wherein said compounds are selected on the basis of their ability to exert a negative affect on said MDR cells in pests.

6. A method of sensitizing MDR cells in a subject, comprising coadministering to the subject an effective amount of one or more dillapiol compounds and an effective amount of a therapeutic agent, wherein the dillapiol compound is selected from the consisting of dillapiol and analogs, salts, or derivatives thereof, and combinations thereof, wherein said compounds are selected on the basis of their ability to exert a negative affect on said MDR cells in mammals.