CA2125279A1

CA2125279A1 - Methods and compositions for reducing multi-drug resistance

Info

Publication number: CA2125279A1
Application number: CA002125279A
Authority: CA
Inventors: John Sayler Coon
Original assignee: Individual
Current assignee: Rush Presbyterian St Lukes Medical Center
Priority date: 1991-12-10
Filing date: 1992-12-09
Publication date: 1993-06-24
Also published as: EP0616493A1; IL104063A0; AU3243393A; CN1076358A; EP0616493A4; WO1993011668A1; JPH07502274A; MX9207150A

Abstract

METHODS AND COMPOSITIONS FOR REDUCING MULTI-DRUG RESISTANCE

ABSTRACT

The present invention comprises methods and compositions for reducing or eliminating multidrug resistance in cancers in humans or animals. According to the method and composition of the present invention, a non-ionic amphipathic ester of a fatty acid is administered to a patient in which a human or animal cancer exhibits multidrug resistance to the chemotherapeutic agent. The method and composition of the present invention may be employed with particular efficacy where multidrug resistance to any chemotherapeutic agent has been conferred upon a cancer.

Description

2 1 2 5 2 7 9 PCr/~'S92/10563 METHODS AND COMPOSITIONS FOR
REDUCING MULTI-DRUG RESISTANCE
Technical Field The prescnt inven~ion relates to ~e use of resistance modi~lcation agents in vivo to reverse mul~idrug resistance in human or animal tumor cells. More particularly, the present invention relates to the use of certain non-ionic surfactants comprising cenain amphipathic es~ers of fatty acids as resistance modif~cation agents.
Background of the Invention OIle of thc major problems of cancer chemotherapy is the existence of drug resistance in twnors resul~ng in reduced responsivc~ess to chemotherapy. Some human cancers, e.g.
~idney and colon carcinoma, are drug resistant before trea~nent begins, whilc in others drug resistance dcvelops ovcr successive rounds of chemotherapy. One type of drug resistance, called multidrug resistance, is characterized by cross resistance to functionaDy and structuraDy unrelated drugs. Typical drugs that are effccted by the multidrug resistance are doxorubicin.
vincristine, vinblastine, colchicine and actinomycin D, and others.
At least some multidrug resistance is a complex pheno~ype which has been linked to a high e~pression of a ccll membrane drug efflux transporter called Mdrl protein, also known as P~
glycoprotein. This membrane "pump" }us broad specificity and acts to remove from the cell a wide variety of chemically unrelated toxins. (See Endicott. J.A., et al. "Ihe Biochemistry of P-Glycopro~ein-Mediated Multidrug Resistance", Ann. Rev.
Biochem. Yol. 58, pgs. 127-71, 1989.) Substances which reverse multidrug resistance are known as resistance modification agents (RMAs), and are of importance in potentiating the cytotoxicity of chemotherapeu~ic agents to which a human cancer has become resistant. Although many agents have been identified as RMAs in vitro, a large proportion have little or no therapeutic potential because of high toxicity in vivo at the doses required tQ reverse rnultidrug resistance. For exarnple, metabolic poisons, such as azide, reverse multidrug resistance in vitro but have no usefulness in vivo. Most other highly effective RMAs, such as velapan~il, appear to work as competitive antagonists of a drug binding site on the Mdrl protein. Many of these agents also have toxicity which limits their usefulness in vi~o. Consequently, therc is a need to develop alternate phannacological stratcgies for revcrsing multidrug resistancc to provide RMAs with improved activity and lower overall toxicity.
Decreased intracellular drug accumulation through overexpression of the drug efflux Mdrl protein is impcrtant to, but apparently not thc only factor, in the multidrug resistance phenotype. Altered intracellular drug distribution and binding, among other possibilities, also seem to play a role. For e~cample, the mechanism of reversing doxorubicin resistance using verapamil appears to be more related to altered intracellular distribution of doxorubicin than increased accumulation in the cell, as detailed in Schuurhuis, G.J., et al., "Quanti~ative determination of factors contribu~ing to doxorubicin resistance in multidrug resistant cells," J. Natl. Cancer Inst., 81:1887-1892, 1989. In ~at report, it is shown that doxorubicin is concentrated almost exclusively in the nucleus in drug sensitive cells, and mainly in the cytoplasm in drug resistant cells. With the addition of verapamil, doxorubicin is localized mainly in ~e nucleus in WO g3/1 ~668 2 1 2 ~ 2 7 9 PCI/IJS92/10563 drug resistant cells. Thus, high affu~ity binding of drugs to Mdrl does not appear to be sufficient for optimal efflux, suggest~ng the existence of additional, rate limiting steps which may be susceptible to phannacological intervention.
Certain non-ionic amphipathic surfactants, such as Tween 80 and Cremophor EL, have evidenced RMA activity.
(See Riehm H., et al. "Potentiation of drug effec~ by Tween 80 in Chinese hamster cells resistant to actinomycin D and Danomycin"
Cancer Res. Vol. 32, pgs. 1195-1200, 1972 and Woodcock, D. B., et al., "Reversal of the multidrug resistance phenoeype with Cremophore EL, a common vehicle for water-insoluble vitamins , j and drugs" C~ncer Res. Vol. 50, pgs. 4199-4203, 1990) However, Tween 80 potentiates drug to~cicity in both parental and multidrug resistant cells, callil~g into question the specificity of the Tween 80 effcct on multidrug resistance. An effecs on drug efflw~ has not been demonstra~cd. Cremophor EL is a complicatcd mixtwe of polyo~cycthylated cstcrs of triglycerides of mainly ricinoleic acid (castor oil), the composidon and active component of which have not been identified. Use of Cremophor EL in ~iw is complicatcd by advcrse histamine rclease in some patients.
Thus, wha~ is needed is a clearly identified class of compositions that reverse multidrug resistance in vivo. The composidon should have a low occurrencc of adverse sidc~ffects.
The composidons should inhibit drug efflu~t by a mechanism different fr~m antagonistic competition for a drug binding site on thc Mdrl protein, thereby broadening the pharmacological repcrtoire which may be employed to reverse multidrug resistancc.
Summary of tbe Invention The present invenion compnses ccrtain compositions that e~hibit substandal RMA activity in caDcers. One e~nple of such a composidon is a non-ionic amphipadlic surfactant, ~own by the trade name SOLUTOL~ HS 15 (BASF Corporation.

WO 93/1 1668 PC~/l,'S92/tO563 212~279 Parsippany, New Jersey). This composition increases the cytotoxicity of chemotherapeutic drugs in multidrug resistant cell lines, but not in drug sensitive cell lines, indicating that the po~entiating effect is not due to the additive toxicity of the agent itself. The agent also prornotes chemotherapeu~ic agent accumulation in multidrug resistant cells thereby potentiating the effect of the chemo~erapeutic agent.
The present invention also comprises a method for reversing multidrug resistance in human or animal cancer cells and a composition for eliminating multidrug resistant human or animal cancer cells. One composition ~at is an aspect of the preseslt invention is a particular fraction of SOLUTOL~ HS 15 collected by reverse phase liquid chromatography. It has been found that the RMA activity in the SOLUTOLæ HS 15 resides in a narrow fraction from the rcvcrse phase liquid chromatography.
It has b~n fur~er determined lhat the toxicity to cells which is inhercnt in SOLUTOL resides in a fraction di~ferent from the fraction containing the RMA activity.
l'he present invention also includes a class of compounds which are ethoxylated fatty acids which cxhibi~ strong RMA acti~ity. These compounds haYe been found to be a fatty acid with bctween approximately 8 and 60 carbon atoms and betwcen appro~cimately 4 to 100 etho~cy units. The fatty acid component of the present invention can be unsatu~ated and can have one or more hydro~yl group. In general, the fatty acids wi~out the etho~y units have litt~e or no RMA acthnty.
The prescnt invention also includes compositions and methods for reducing thc resistance of certain microorganisms to chemothcrapeutic agents. It has been determined tha~ certain microorganisms contain p-glycoprotein-like pumping mechanisms that are similar to those found in mammalian cells and it is believcd that these mechanisms may be important in resistance to an~icrobial agen~s.

WO 93/11668 PCI/I,'S92/10563 Another embodiment of the present invention are the polyoxyethylene/polyoxypropylene copolymers with the following general formula:

HO(C2H4 ) b(C3H 6)- (C2H4O)b wherein a is an integer such that the hydrophobe represented by (C3H60) has a molecular weight of about 1200 to 9000, preferably 17S0 tO 4000, and b is an integcr such that the hydrophile portion represented by (C2H40) consti~utes ' approximately 10% to 50% by wcight of the compound.
Another cmbodiment of the prescnt invention are the polyo~cyethylene/polyo~cypropylene copolymers with the ' :
following general formula~
(C3H60)b(C2H40~a\ /(C2H40)a(c3H6o)b (C3H60)b~C2H40)a \(C2H40)a~C3H60)b wherein:
the mean aggrcgate molecular weight of ~he portion of the octablock copolymer rcprcsented by the polyo~ypropylene is between approximately 4500 and 7000 daltons;
a is a number such that the portion representcd by polyoxyethylene constitutes betwoen approxir,nately 10% to 20%
of the compound by weight, and;
b is a number such tbat the polyoxypropylene por~ion of the total molecular weight of the octablock copolymcr constitutes between approximately 80% and 90% of the compound by weight.
Yet another embodiment of thc present inYention are the polyo~cyethylene/polyo~ypropylene copolymers with the following gencral formula:

wo 93/1166821 2 5 2 7 9 PC~/~)S92/10563 (C2H40)a~c3H6o)b~ / (C3H6)b(C2H4)a (C2H40)a(c3H6o)b \ (C3H60)b(C2H40)a wherein:
the mean aggregate molecular weight of the portion of the octablock copolymer represented by the polyo~ypropylene is between approximately 4500 and 7000 daltons; ;
a is a number such that the portion represented by polyoxyethylene constituus between appro~umately 10% to 40%
of the compound by weight, and;
b is a number such that ~he polyo~ypropylene portion of the total molecular weight of the octablock copolymer constitutes bctween appro~imately 60% and 90% of the compound by weight.
Accordingly, it is an objeet of ~e present invention to provide a composi~on and method for reducing or eliminating multidrug resistance in human or animal cancer cells.
It is further an object of the present invention to provide a composition and method for treuing a human or animal with multidnlg resistant canccr.
It is fur~cr an object of thc present invention to providc a composition and medlod for reducing multidrug resistance which will not producc advenie side-effects.
2S It is further an object of the present invention to provide a cornposition and method ~at can be used to reduce the blood brain barrier thereby allowing certain ~crapeutic agents to cross the barrier from the blood hto thc brain.
It is ye~ another object of the present invention to providc a composition and mcthod that can be used to reverse mulddmg resistance to VP-16 and VM-26 in cancer cells.

wo 93/1 ~668 P~/US92/ 10563 212~279 It is yet another object of the present invention to provide a composition and method for reducing the resistance of microorganisms to cenain drugs.
These and other objects, features and advantages of S the present ~nvention will become apparent after a review of the following detailed description of the disclosed embodiment and the appended claims.
Brief Description of the Figures Figurc 1 shows frac~ionation of SOLUTOL~ HS 15 using reverse phase liquid chromatography.
Detailed Description The present invention comprises methods and compositions for reducing or eliminating multidrug resistance in cancers in humans or animals. According to the method and composition of the prescn~ invention, a non-ionic amphipathic ester of a fatty acid is administered to a patient in which a human or animal cancer c~hibits multidrug resistance to the chemotherapeudc agent. The method and composition of the present invention may be employed with particular efficacy where multidrug resistance ~o any chemotherapeutic agent has been confened upon a cancer.
As used berein, the tenn multidrug resistance means resistance or acquired or natural resistance of hlmOF or other cells to chemotherapeutic agents. The mulddrug resistance can be mediated by P-glycoprotein or can be mediated by other mechanisms.
'rhe present invention includes a method of treating a human or animal with a cancer that exhibits multidrug resistance to reduce or elirninate the multidrug resistancc which includes adrninistering to thc human or animal an effective amount of a non-ionic amphipathic ester of a fatty acid. A prcparation that e~hibits the desired biologic activity is SOLUTOL~ HS 15. This : : ~.

WO 93/11668 PCr/lJS92/10563 2125279 ~

preparation is a mixture of vanous compounds with surfactant activities.
By fractionating the SOLUTOL~ HS 15 preparation using reverse phase liquid chromatography and then assaying the various fractions for RMA activity, it has been dete~nined that the RMA activity resides in a small fraction which contains fatty acid esters containing ethoxide units. This fraction has a much higher speci~lc activity than the unfractionated SoLuroL~ HS 15.
By synthesizing several fatty acid esters with varying etho~ide units, it has been found that compounds which are ethoxylated fatty acids exhibit strong RMA activity. These compounds have been found to be fatty acids or polymers of fatty acids with between approximately 8 and 60 carbon a~oms and between approximately 4 to 100 ethoxy units. The fatty acid component ~5 of the present invention can be unsaturated and can be hydroxylated and still e~hibit activity. In addition, the fat~ acid can be branched. The preferred fatty acids are straight chained.
In general, thc fatty acids without the etho~y units have little or no RMA activity.
The preferred compounds are fatty acids which have ethoxy units esterificd on the carbo~y group. The fatty acids have bctween 8 and 60 carbon atoms and betwcen approxisnately 4 to 100 cthoxy units. If ~e fatty acid is hydroxylated the etho~y units may bc esterified at thc hydroxyl group. The ethoxy units can be attachcd to the carbo~yl group andlor the hydro~yl group if a hydroxyl group is prese~t. The more preferred compounds havc a fatty acid with between 12 and 50 carbons with the most prefe~ed compounds with between 15 and 25 carbon atoms and betwecn approximately 15 and 60 cthoxy units with the most preferred compounds having between approximately 15 and 20 carbon atoms. The preferrcd compounds have between approximately 4 and 100 ethoxy ur~its, with the more preferred compounds having between 15 and 60 e2ho~cy units and the most preferred compounds having between 25 and 50 etho~sy units.
Preferred fatty acids are selected from ~e group consisting of WO 93/1 1668 PCI /US92/10~63 212~27~

steanc acid, 12-hydroxysteanc acid, oleic acid, palrnitic acid, and ricinoleic acid. The preferred number of etho~y units are between approximately S and 50 units.
While not wanting to be bound by the following theory, it is believed that cellular membrane transport proteins must form polymers, usually dimers or tetramers, to effectively carry out their transport functions. Thus it iS likely that ~he Mdrl protein can achieve its function of removing from the cell a wide variety of chemically unrelated toxins only after forrning polymers in ~he membrane. Non-ionic amphipathic surfactants exhibit membrane surface activity and are characterized by having a hydrophilic head and hydrophobic tail. In particular, non-ionic arnphipa~ic esters of fatty acids, inhibit the formation of such pro~ein polymers, and thercby inhibit drug cffl~
The estcr of the present invention has a hydrophilic head, which comprises polycthylenc glycol, and a hydrophobic tail comprising a fatey acid. Such a moleculc is amphipathic: The molecule is largc enough that each end displays its own solubility behavior.
The fatty acid component of the ester of the composition of the present invention can be selected from a wide range of fatty acids. It may advantageously possess at least one hydro~yl group oueside of thc carbo~cyl group. Such fatty acids can casily bc esterified with themselves, as is well known in the art, to produce polymcrs of the fatty acid. Por purposes of the present invention, the RMA can be formed not just from csters of a fatty acid monomer with polyethylene glycol, but such polymers of hydro~cylated fatty acids also can be esterificd with polyethylene glycol to form the RMA.
In a preferred embodirnent of the prcsent invention, the non-ionic amphipathic ester comprises polyethylene glycol ester of 12-hydro~ystearic acid. SuGh a formulation is a component of a commercialiy available preparation from BASF
Corporation (Parsippany, New Jcrscy) under ~e trade name SOLUTOL~9 HS lS.

.:

WO 93~1 1668 2 1 2 ~ 2 7 9 PCl/US92/10563 The ester may be adsninistered tO a patient either alone or in combination with a treatment program of at least one chemotherapeutic agent to which the human cancer is resistant.
Such a chemotherapeutic agent typically includes, but is not limited to, doxorubicin, vincristine, vinblastine, Taxol, colchicine, VP-16 and actinomycin D. However, there are many other chemicals used in chemotherapy to which multidrug resistance may appear during treatment, and the psesent invention may be employed equally well in such cases. In addition7 the present invention is useful for reducing resistance to platinum compounds by promoting accumulation of these compounds.
In gencral, at least one effective dose of the RMA of the present invention is administered for evcry dose of chemotherapeutic agent that is administered in treatment.
Preferably, an effective dose of the RMA may be administered at least daily tl~oughout the period between administration of successive doses of chemotherapeutic agent. The treatrnent period typically lasts about four weeks, depending upon the cancer being treated and thc chemotherapeudc agents being used. Altematively, the RMA may be continuously infused throughout said penod.
The administration of the RMA may also cornsnence prior to a session of chcmothcrapy, and contillue throughout and after the chemotherapy session. The amount of the RMA per dose will depend on which particuL~r non-ionic amphipathic fatty acid ester is employed according to thc present invention. However it is prcferable that tbe ma~imum dosage that may be tolerated vith negligible to~ic symptoms in vivo be used. At least some non-ionic amphipathic esters of fatty acids, such as SOLI~TOL~!D HS 15, are tolerated extrcmely wcll in vivo, and may be employed with no acutc toxicity at dosages which achieve equivalent or superior reversal of multidrug resistance to common chemotherapeutic agents as compared to dosages of the pEoto~pical RMA verapamil which produce marked toxicity.
The RMA of the present invention can be 3S administered ei~her intravenously or orally. It may be WO 93/1 1668 PCI/US92/10~63 administered separately from the chemotherapeutic agent, as may be dictated by the chemotherapy, in which case the amount of time between commencing administration of the RMA and administration of the chemotherapeutic agent should not be S substantial, e.g. typically within 24 hours, or as the chemotherapy permits. An exemplary treatment regimen comprises oral or intravenous administration of the chemothcrapcutic agent, followed by continuous adsninistration of the RMA throughout the period until the next session of chemotherapy, either by continuous infusion or oral time release capsules. A typical dose for a human of the SOLUTOL~ HS 15 is between appro~cimately 1 mgJkg and 250 mg/kg. A morc preferred dose of SOLU roL~ HS
15 is between approximately 5 mg/kg and 100 mg/kg. If a puri~led esterified fatty acid is used to trcat a human with multidrug resistant cancer, the prefcrred dosc is between appro~imately 1 mg/kg and 200 mg/kg with ~e more preferred dose between appro~dmatcly 15 mg/~g and 60 mg/kg.
Altematively, the RMA of the present invention may be administered in combination with the chemotherapeutic agent, compnsing continuous infusion or daily oral consumption of ~me relcase capsulcs of thc RMA commencing prior to the chemotherapy scssion, and continuing throughout and after the session, by way of e~ample. Thc RMA may be infused together -~
through the same needle with thc chemotherapeutic agent, or combined in a single oral capsule, as the chcmotherapcutic agent pennits, in which cases the RMA of d~e present invention may be used as an emulsifier of the agent, since non-ionic amphipathic esters of fatty acids commonly possess emulsifying characteristics.
Preparation of an emulsion of thç chemotherapeutic agent with the RMA will depcnd on the particular agents used.
Typically, the RMA and the chemotherapeudc agent are combined a~d heated abovc room tempcrature to a range h which bodl the RMA and thc chemotherapeutic agent are sdll stable, but h which the RMA becomes fluid, abou~ 50 to 80 C. Sterilc water is heated to ~c samc temperature and thcn added with vigorous WO 93/1 ~668 2 1 2 ~ 2 7 9 P(~/~'S92/10563 agitation in a proper amount to achieve a viscosity appropriate for administration. Other components may be added to the emulsion as necessary to prepare it either for intravenous or oral administration, as is well known in the art.
S According to another embodiment of the present invention, the RMA of the present invention can be administered together with other RMAs, such as verapamil. The RMA of the present invention and a second RMA can be infused separately or concurrently, or combined into one time release capsule for oral consumption, in effec~ive doses typically administered in treatment using each RMA alone, as perrnitted by the toxici~y of the second RMA.
The method and composition of the prcsent invention provide an important new mealls of overcoming multidrug resistance in human canccrs. The method and composi~ion have an efficacy equal to or better thcn bcst resistance modification agents kno~,vn to the inventor. Purthermore the agent used in the method and composition of the prcsent invention has a lower toxicity than other RMAs and fewer side effects than other potential RMAs.
Moreover, it is believed that ~e agcnt operates by a different mechanism on the complex phe~otyp~e of multidrug resistance, and thus can be combincd wi~h other RMAs to provide a more potent means of reversing multidrug rcsistancc.
The structux of SOLUTOL~!9 HS lS is dissimilar to that of verapamil or other typical RMAs. l~e rna~edly grea~er potency of SOLUTOL~!D HS 15 than verapamil for reversing VP-16 or colchicine resistance relative to the ability of each to reverse vinblastinc or doxorubicin resistance supports the hypothesis that SOLUTOL~ HS 15 opcrates by a MDR-reversing mechanism different from competi~ion for the drug-binding site on Mdrl protein found in verapamil. Colchicine is known to interact wealcly w~th the identified dn~g-binding site on the Mdrl protein, sincc colchicine does not compete for vinblastine binding. The fact that MDR cclls are nevertheless highly resistant t3 colchicine indicates dla~ colchicine efflux is less dcpendent on interaction WO 93/1 1668 2 1 2 ~ 2 7 9 P~/IJS92/10563 with this drug-binding site than is vinblastine. Since SOLUTOL~
HS 15 is a highly potent RMA for both colchicine and vinblastine, it may inhibit a second event necessary for eMux after drug binding, namely acnlal transport through the membrane. It is likely that SOLUTOL~ HS 1~, as a surfactant, inhibits formation of Mdrl protein polymers which may be necessary tO achieYe drug efflux.
Another important advantage of the RMA of the present invention is the fact that the compounds which are contemplated as part of the present invention are highly effective against the multidrug resistance against the anticancer drug VP-16. The prior art R~s, such as verapamil, arc not effective against VP-16 multidrug resistance. (See Schested, M, et al.
"Relationship of VP-16 to the Classical Multidrug Resistance Phenotype", Cancer Research, Vol. 52, pgs. 2874-2879, 1992.) Thc RMAs of the present invention have been found to be effcctive in reducing multidrug resistance against a broad spectm n of anticancer drugs.
Is is well known ~at cer~ain microorganisms contain mem~rane protcins which are similar in stn cture and function to the P-glycoprotein that is e~pressed by the MDR1 gene in mammals. It is contemplated as part of the present invention that the methods and compositions that make up ~hc prcscnt invention can be used to make certain microorganisms more suscep~ible to therapeutic drugs. For example, it is likely that the present inwntion will reverse chloroquinc resistance in malana.
Another embodiment of the present invention relates to the blood brain barrier. It has been reported that the P-glycoprotein pump exists in brain capillary endothelium. (See Tasuta, T., et al., Functional Involvement of P-glycoprotein in Blood-Brain Barricr", J. Biol~ Chcm., Vol. 267, pgs. 20383-20391, 1992.) The brain is a phalmacologic sanctuary in that many drugs administered systemically have limited aceess to the dssue parenchyma. In the brain, endothelial cells forming the capillary tube are joined by continuous tight junctions that prevent many substances from entering the organ. Nutrients needed for brain cells are selectively transported from the blood through specific channels or transporters in the capillary endothelial cells. Thus, the brain is a rigorously isolated compartment that is protec~ed by a blood-brain barrier.
Hydrophobic antitumor agents, such as vinca aLkaloid and adriamycin (ADM), cannot enter the brain, although other hydrophobic molecules such as nicotine and ethanol readily pass through the blood-brain barrier. Therefore, some mechanisms of the barrier that selectively block the penetration of lipid-soluble antitumor agents into the brain could exist. The presence of P-glycoprotein in the capillary endothelium has been reponed in bod~ brain and testis but not in the other tissues. This suggests the functional involvemcnt of P-glycoprotein in the blood-brain barrier. It is contemplated as part of thc present hvention that the methods and compounds described herein can be used to reduce the blood-brain barrier thcreby allowing beneficial therapeutic agents to cross the barrier.
Another cmbodiment of the prcsent invention are compounds that are effecdve in reducing multidrug resistance in cancer cells that are polyo~yethylene/polyo~cypropylene copolymers with the followimg general formula:
HO(C2H,,O)b(C3H60)~(c2H4o)b wherein a is an integer such that the hydrophobe represen~ed by (C3H60) has a molecular weight of about 1200 to 9000, preferably 17S0 to 4000, and b is an intcger such that the hydrophile portion represcnted by (C2H40) constitutes appro~cimately 10% to 50~c by weight of the compound.
In another embodiment of the present invention, the block copolymer comprises a polymer of hydrophilic polyo~ycthylene (POE) built on an ethylene diamine ini~iator.
Polymers of hydrophobic polyoxypropyler~e (POP) are then built on the bloclc of hydrophilic polyethylene (POE). This results in an octablock copolymer with the following gencral formula:

wo 93/1 1668 ~CI /US92/10563 ~
2~25279 ~ :

(C3H60)b((;2H40)a\ /(c2H4o)a(c3H6o)b N H2C ~H2 N
(c3H6o)b(c2H4o)a \(C2H40)a(c3H6o)b wherein:
the mean aggregate molecular weight of the portion of the octablock copolymer represen~ed by the polyo~ypropylene ::
is between approximately 4500 and 7000 daltons; ~ :
a is a number such that the portion represented by polyo~yethylene constitutes between approximately 10% to 20%
of the compound by weight, and;
b is a nulslber such that ~e polyo~ypropylenc portion of thc tohl molecular wcight of the octablock copolymer constinltes between approasimately 80% alld 90% of the compound by weight. :
In one embodiment of the present invention, the block copolymer comprises a polymer of hydrophobic polyo~ypropylene (POP) built on an ethyleneLiamhe initiator.
Polymers of hydrophilic polyo~ye~ylcne (POE) are ~en ~uilt on the block of hydrophobic polyoa~ypropylcne (POP~. This results in an octablock copolymer with the follo~g general formula:

(C2H40)a(C3H60)b\ / (C3H60)b(c2H4o)a (C2H40)a(C3H60)b \ (C3H60)b(C2H40)a wherein:
the mean aggregate molecular weight of ~e portion of dle octablock copolymer rep~esented by thc polyo~ypropylene is benveen appro~imately 4500 and 7000 daltons;

W093/11668 2~ 2~279 PCr/US92/10563 a is a number such tha~ the portion represented by polyoxyethylene constitutes between approximately 10% to 40%
of the compound by weight, and;
b is 2 number such that fhe polyoxypropylene portion of the total molecular weight of the octablock copolymer constitutes between approximately 60% and 90% of the compound by weight.
The octablock copolymers comprising the biologically active copolymers of the present invention include, but are not limited to, the block copolymers Tetronic(E~ and reverse Tetronic~ manufactured by the BASF Corporation (BASF Corporation, Parsippany! NJ). l'he triblock copolymers are sold under the traderna~ PLURONIC~ and are available from BASF Corporation.
This invention is furthcr illustrated by the following e~amples, which are not to be construed in any way as imposing limitations upon the scope thereof. On the contrary, it is to be clearly understood that resort may bc had to various other embodiments, modifications, and equivalents ~cseof which, after reading the description herein, may suggest ~emselves to those skilled in the art widlout dcparti~g from the spirit of thc present invention andlor d~e scope of ~e appended claims.

2~ 25279 Example I
Human epidennoid carcinoma cell lines KB 8-5 and KB 8-5-11, which exhibit multidrug resistance, and their parental cell line KB 3-1, which is drug sensitive, were treated in vitro with SOLUTOL~ HS 15 in combination with various chemotherapeutic agents, namely colchicine, vinblastine, and doxorubicin. The details of the trea~nent are described in Coon, J.S., et al, "SOLUrOL~ HS 15, nontoxic polyoxyethylene esters of 12-hydroxystearic acid, reverses multidrug resistance", Cancer 0 Research, S1, 897-902, 1991, which is incorporated by reference.
Briefly, cells from the three lines were plated as is well hlown in the art in 96-well plates, with increasing concentrations of cytotoxic drug along one axis of the plate and increasing concentrations of the RMA along the other axis of the plate.
After incubation for five days, the plates wcre washed and dyed according to methods known in the art, and a cell count was determined. The mean concentration of ~c cytotoxic drug that caused 50% inhibidon of cell grow~ compared to co~trols (IC50) was plotted at various concentrations of the RMA. Complete rcversal of ~e MDR phenotype in ~CB 8-5 and KB 8-5-11 cells was achieved by SOLUTOL~ HS 15, while thc RMA did not potentiatc drug toxicity in drug-sensiti~e KB 3-1 cells, indicating the potentiating cffect was not due to any toxicity of SOLUTOL~
HS 15 itsclf. At a concentration of 10% of its own ICS0, SOLUTOL~E9 HS 15 produced a 35-, 28-, and 42-fold reduction in the resistance of KB 8-5-11 cells to colchicine, vinblastine, and do~corubicin, respectively.
Identical platings were also performed for the prototypical RMA verapamil. Une~pectedly, the rclation between the effects that SOLUTOL@~ HS 15 had on thc three cytotoxins was different from the relation between the eff~cts that verapamil had on the three cytoto~cins, indicating SOLUTOLtl9 HS 15 and verapamil af~ect mul~idrug resistance by tiffere~t mechanisms.
SOLUTOL HS lS was relatively much more potent ~an verapamil ---. WO 93/ 1 1 66~ PCl / USg2/ 1 0563 for reversislg colchicine resistance, as compared to the ability of each RMA to reverse vinblastine resistance.
Example II
S Efflux of rhodarnine 123 from MDR cells was also examined to provide direct information about the action of the transport protein Mdr l. Briefly, prepared cells from the KB 8 5-11 line were washed and ineubated in 0.5 !lglml rhodamine 123 and 24 IlM verapamil for 3 hours at 37 C. The cells were washed in ice cold DMEM, split into 3 aliquots, and incubated in either complete medium alone or comple~e medium with 24 ~LM
verapamil or 70 ,uM SOLUTOL~9 HS 15 at 37C. ~e rhodamine 123 fluorescence of the cells was measured periodically by flow cytometric analysis as descri~ed in Coon et al. The rhodamine 123 studies showed that SOLUTOL~9 HS 15 promotes drug accumulation in MDR cells, and furthe~more that such accumulation is at least partly due to a pronounced decrease h the rate of dmg efflu~.
Example III
SOLUTOL~ HS 15 was fractionated using reverse phase liquid chrom~tography to determine wherc the activity resides in thc preparation. An approximately 50% solution of SOLUTOLt!9 HS 15 was pTepared in 100% acetonitrile (ACN) and water. One ml of the SOLUTOL~l9 HS 15 solution was injected onto a Phenomen~ Sil reversed phase column. The column has S ~un palticles, and is 4.6 mm internal diameter by 150 mm.
The flow rate was 2.0 mUmin. Thc mobilc pbase was as follows:
A=5-% ACN and B=100% ACN. The gradient was linear with 100% A to 100% B in 15 minutes, then was maintaincd at 100%
B. Fractions wcre collectcd at 30 second i~ten~als. The various fractions werc assaycd for RMA acdvi~y as described in E~ample II. The results of the fracdonation a~e shown in Figure 1. In addition the same fractions were assaycd for to~icity by measuring 50% inhibitory concentrations (ICso) as described in - ~:

wo 93~11668 PCI/US92/10563 212~279 Kessel D., "Exploring Multidrug Resistance using Rhodamine 123, Cancer Communications Vol 1, pgS. 145-149~ 1989.
As can be seen in Figure 1, the RMA activity is confined in a single peak which elutes at approximately 20 S minutes into the chromatographic run. The toxicity is confined to another peak that elutes before the activity peak and slightly overlaps the RMA peak. However, it is clear that most of the material that is responsible for the RMA activity is non-to~ic.
Example IV
Using nuclear magnetic resonance spectroscopy and mass spectroscopy, the material that eluted under the activity peak in Figure 1 was analyzcd, it is found that several species molecules are present. The molecules found under the activity peak in Figure 1 appcar to be esterified fatty acids. To identify the chemical compounds having RMA activi~, several fatty acid esters were synthetically preparcd and tested for RMA actiYity according to E~carnple II. Preparation of the etho~ified fatty acids is wcll known to thosc of ordinaly skill in ~e art. Synthesis of fatty acids and their deri~ratives with ethylene oxide are described in Bares, et al. Tcnsidc De~ergcnts, Vol. 12, p. 155 1975 and Wrigley, A.N. J'. Amcr. Oil Chcmfsts's Soc. Vol. 34, p.
39, 1957. All compowlds wcre administered to KB8-5-11 (multidrug resistant) cells in vitro at 100 llg/ml in tissue culture medilml at 37 C. Rhodamine 123 at 0.5 llgfml was also present.
The results of these measuremcnts are shown in Table I.

i.,.`' ' ' " ' ' ' . " '''~' :;''' ' .'' . . ":: :' "~ ''':':',,.,.''`"' .'. ' ''' :":, ': ' ."

wo 93/11668 Pcr/uss2/ 10563 212~279 ~ 20 -Table I
Rhodamine 123 accumulation in KB8 5-11 (MDR) cells treated with e thoxv!ated fa ttv acids Fatty Acid Number of %
_ EO un~ts AccumulationJ
Noneb 3.2 SOLUToL~ Mixture 99.6 Stearic acid O 3.3 12-hydroxy stearic acid 0 3.1 Oleic acid 0 8.4 Ricinoleic acid 0 6 5 Stearic acid 5 7 2 Stearic acid 15 72.1 Stearic acid 45 99.8 12-hydro~cy stearic acid 5 33.1 12-hydro~cy stearic acid 15 35.3 12-hydro~cy stearic acid 45 90.0 Oleic acid 5 3.6 Oleic acid 15 55 Oleic acid 45 99.9 Ricinolcic acid 5 25.9 Ricindcic acid 15 nd Ricinoleic acid 45 92.0 a. Per cent cclrs show~ng rho ~a~Dinc 123 fluo csccnce in thc rang : of sensidve cell (KB3-1) in the same e~
b. ForKB3-1 ~sa~tive)cells, 100.0%a cumulatedrhoda~ne c. no~done Example V
To~cicity studics indicate SOLUTOL~9 HS 15 is e~tremely well toleraQed in vil~o. Pure-bred beagle dogs received intravenous doses of S, 25, 50 or 100 milligrams of SOLUTOL~
HS 15 per kilog~n body weight, daily over a pcriod of 4 weeks.
No signs of to~icity were found in doses up to 25 mg/kg. At 50 mg/kg, sporadic and transient pruritus, erythema, and/or urticana were obser~ed. After doses of 100 mg/kg, ~e dogs showed diffcrent dcgrces of pruritus, erythema, or urticana, most pronounced 5 to 10 minutes after injection, and no longer detectable after 60 n~inutes. These studics indicate SOLUTOL~ HS
15 is better tolerated in vivo than Cremophor EL.

A ,~

Example VI
The PLURONIC~ and TEcTRoNlc~(g copolymers were tested for RMA activity in a manner sirnilar to that shown in Example IV. The results of these measurements are shown in S Table II.

~ wo 93/1 1668 2 1 2 ~ 2 7 9 PCr/ iS92/10563 Table II
Rhodamine 123 accumulation in KB8-5 11 (MDR) Cells Treated with Polyoxyethylene/polyoxypropylene Block Copolvmer Compound . _ _ (70 UM) Accumulationa N~ _ l.2 SOLUTOL~ 100 72.3 VeraPami1C 2411M 98.2 Benzyl alcohold 5405 4.6 (SOmM) PLURONIC(9 . ~ COPOL~S
F-38 329 0.1 F-77 462 1.1 L-8 1 192 97.4 L-101 270 21.0 F-108 1022 0.3 L-121 308 64.2 : :
F-127 8~0 5.7 L-141 336 78.2 L-190.5 6~7 100.0 Tkl~oNIC
COPOL~S
T1301 476 99.7 T1302 539 89.7 T1501 553 42.8 REVERSI~ TEI~oNIC
COPOL~S
T1 10R-1 360 100.0 T130R-1 476 97.3 T130R-2 542 50.2 TlSOR-1 SO 87. 1 S a. Per ccnt cells showing rho l~minc 123 flua rescence in the rang ~ of sensitive cdl (KB3-1) in the same expenment b. For KB~l (sensitive) cells, 99.3% accumulated rhoda~ne c. Verapamil is a revasing agent O d. Non-specificmembranefluidizcr As can be seen in Table II, sevcral of the polyoxyed ylenefpolyoY~yethylene block copolymers are effective V

WO 93/1 1 668 PCI /US92t10563 212~279 in reducing multidru,, resistance in cancer cells exhibiting the activity.
It should be understood, of course, that the foregoing relates only to a preferred embodiment of the present invention S and that numerous modifications or alterations may be made therein without departing from the spirit and the scope of the invention as set forth in ~he appended claims.

Claims

What Is Claimed Is:

1. A method of reversing multidrug resistance in a cancer in a human or animal with the cancer comprising the steps of administering to the human or animal an effective amount of a resistance modification agent comprising a non-ionic amphipathic ester of a fatty acid.

2. The method of Claim 1, wherein the ester comprises a hydrophilic head selected from the group consisting of polyethylene glycol and saccharides.

3. The method of Claim 2, wherein the polyethylene glycol has between approximately 4 and 100 ethylene oxide units.

4. The method of Claim 3, wherein the polyethylene glycol has between approximately 15 and 60 ethylene oxide units.

5. The method of Claim 4, wherein the polyethylene glycol has between approximately 25 and 50 ethylene oxide units.

6. The method of Claim 1, wherein the fatty acid is selected from the group consisting of saturated fatty acids, unsaturated fatty acids, hydroxylated fatty acids and hydroxylated unsaturated fatty acids.

7. The method of Claim 6, wherein the fatty acid is selected from the group consisting of stearic acid, 12-hydroxystearic acid, oleic acid, palmitic acid, and ricinoleic acid.

8. The method of Claim 6, wherein the fatty acid has between approximately 8 and 60 carbon atoms.

9. The method of Claim 8, wherein the fatty acid has between approximately 12 and 50 carbon atoms.

10. The method of Claim 9, wherein the fatty acid has between approximately 15 and 25 carbon atoms.

11. A method for potentiating the cytotoxicity of a chemotherapeutic agent in a human or animal with a cancer exhibiting multidrug resistance comprising the step of administering to the human or animal an effective amount of a resistance modification agent comprising a non-ionic amphipathic ester of a fatty acid in combination with a chemotherapeutic agent.

12. The method of Claim 11, wherein the ester comprises a hydrophilic head selected from the group consisting of polyethylene glycol and saccharides.

13. The method of Claim 12, wherein the polyethylene glycol has between approximately 4 and 100 ethylene oxide units.

14. The method of Claim 13, wherein the polyethylene glycol has between approximately 15 and 60 ethylene oxide units.

15. The method of Claim 14, wherein the polyethylene glycol has between approximately 25 and 50 ethylene oxide units.

16. The method of Claim 11, wherein the fatty acid is selected from the group consisting of saturated fatty acids, unsaturated fatty acids, hydroxylated fatty acids and hydroxylated unsaturated fatty acids.

17. The method of Claim 16, wherein the fatty acid is selected from the group consisting of stearic acid, 12-hydroxystearic acid, oleic acid, palmitic acid, and ricinoleic acid.

18. The method of Claim 17, wherein the fatty acid has between approximately 8 and 60 carbon atoms.

19. The method of Claim 18, wherein the fatty acid has between approximately 12 and 50 carbon atoms.

20. The method of Claim 19, wherein the fatty acid has between approximately 15 and 25 carbon atoms.

21. A composition for treating multidrug resistant human cancer cells in a human or animal comprising at least one non-ionic amphipathic ester of a fatty acid and at least one chemotherapeutic agent.

22. The composition of Claim 21, wherein the ester comprises a hydrophilic head selected from the group consisting of polyethylene glycol and saccharides.

23. The composition of Claim 22, wherein the polyethylene glycol has between approximately 4 and 100 ethylene oxide units.

24. The composition of Claim 23. wherein the polyethylene glycol has between approximately 15 and 60 ethylene oxide units.

25. The composition of Claim 24, wherein the polyethylene glycol has between approximately 25 and 50 ethylene oxide units.

26. The composition of Claim 21, wherein the fatty acid is selected from the group consisting of saturated fatty acids, unsaturated fatty acids, hydroxylated fatty acids and hydroxylated unsaturated fatty acids.

27. The composition of Claim 26, wherein the fatty acid is selected from the group consisting of stearic acid, 12-hydroxystearic acid, oleic acid, palmitic acid, and ricinoleic acid.

28. The composition of Claim 27, wherein the fatty acid has between approximately 8 and 60 carbon atoms.

29. The composition of Claim 28, wherein the fatty acid has between approximately 12 and 50 carbon atoms.

30. The composition of Claim 29, wherein the fatty acid has between approximately 15 and 25 carbon atoms.

31. The composition according to Claim 21 wherein the chemotherapeutic agent is selected from the group consisting of doxorubicin, daunomycin, vincristine, vinblastine, taxol, colchicine, VP-16, camptotechin and actinomycin.

32. A method of reversing multidrug resistance in a cancer in a human or animal with the cancer comprising the steps of administering to the human or animal an effective amount of a resistance modification agent comprising a polyoxyethylene/polyoxypropylene copolymer with the following general formula:

HO(C2H4O)b(C3H6O)a(C2H4O)b wherein a is an integer such that the hydrophobe represented by (C3H6O) has a molecular weight of about 1750 to 9000, and b is an integer such that the hydrophile portion represented by (C2H4O) constitutes approximately 10% to 50% by weight of the compound.

33. A composition for treating multidrug resistant human cancer cells in a human or animal comprising at least one chemotherapeutic agent and at least one polyoxyethylene/polyoxypropylene copolymer with the following general formula:

HO(C2H4O)b(C3H6O)a(C2H4O)b wherein a is an integer such that the hydrophobe represented by (C3H6O) has a molecular weight of about 1750 to 9000, and b is an integer such that the hydrophile portion represented by (C2H4O) constitutes approximately 10% to 50% by weight of the compound.

34. The composition according to Claim 33, wherein the chemotherapeutic agent is selected from the group consisting of doxorubicin, daunomycin, vincristine, vinblastine, taxol, colchicine, VP-16, camptotechin and actinomycin.

35. A method of reversing multidrug resistance in a cancer in a human or animal with the cancer comprising the steps of administering to the human or animal an effective amount of a resistance modification agent comprising a polyoxyethylene/polyoxypropylene copolymer with the following general formula:

wherein:
the mean aggregate molecular weight of the portion of the octablock copolymer represented by the polyoxypropylene is between approximately 4500 and 7000 daltons;
a is a number such that the portion represented by polyoxyethylene constitutes between approximately 10% to 20%
of the compound by weight, and;
b is a number such that the polyoxypropylene portion of the total molecular weight of the octablock copolymer constitutes between approximately 80% and 90% of the compound by weight.

36. A composition for treating multidrug resistant human cancer cells in a human or animal comprising at least one chemotherapeutic agent and at least one polyoxyethylene/polyoxypropylene copolymer with the following general formula:

wherein:
the mean aggregate molecular weight of the portion of the octablock copolymer represented by the polyoxypropylene is between approximately 4500 and 7000 daltons;
a is a number such that the portion represented by polyoxyethylene constitutes between approximately 10% to 20%
of the compound by weight, and;
b is a number such that the polyoxypropylene portion of the total molecular weight of the octablock copolymer constitutes between approximately 80% and 90% of the compound by weight.

37. The composition according to Claim 36, wherein the chemotherapeutic agent is selected from the group consisting of doxorubicin, daunomycin, vincristine, vinblastine, taxol, colchicine, VP-16, camptotechin and actinomycin.

38. A method of reversing multidrug resistance in a cancer in a human or animal with the cancer comprising the steps of administering to the human or animal an effective amount of a resistance modification agent comprising a polyoxyethylene/polyoxypropylene copolymer with the following general formula:

wherein:
the mean aggregate molecular weight of the portion of the octablock copolymer represented by the polyoxypropylene is between approximately 4500 and 7000 daltons;
a is a number such that the portion represented by polyoxyethylene constitutes between approximately 10% to 40%
of the compound by weight, and;
b is a number such that the polyoxypropylene portion of the total molecular weight of the octablock copolymer constitutes between approximately 60% and 90% of the compound by weight.

39. A composition for treating multidrug resistant human cancer cells in a human or animal comprising at least one chemotherapeutic agent and at least one polyoxyethylene/polyoxypropylene copolymer with the following general formula:

wherein:
the mean aggregate molecular weight of the portion of the octablock copolymer represented by the polyoxypropylene is between approximately 4500 and 7000 daltons;
a is a number such that the portion represented by polyoxyethylene constitutes between approximately 10% to 40%
of the compound by weight, and;
b is a number such that the polyoxypropylene portion of the total molecular weight of the octablock copolymer constitutes between approximately 60% and 90% of the compound by weight.

40. The composition according to Claim 39, wherein the chemotherapeutic agent is selected from the group consisting of doxorubicin, daunomycin, vincristine, vinblastine, taxol, colchicine, VP-16, camptotechin and actinomycin.