CA2190636A1

CA2190636A1 - Methods and compositions for reducing multidrug resistance

Info

Publication number: CA2190636A1
Application number: CA002190636A
Authority: CA
Inventors: John S. Coon; Mannarsamy Balasubramanian; R. Martin Emanuele; Himanshu Shah
Original assignee: Individual
Current assignee: Rush Presbyterian St Lukes Medical Center
Priority date: 1994-05-19
Filing date: 1995-05-19
Publication date: 1995-11-30
Also published as: WO1995031981A2; AU2689495A; EP0759753A1; WO1995031981A3

Abstract

The present invention comprises methods and compositions for reducing or eliminating multidrug resistance in cancers or certain infections by drug resistant microorganisms in patients. According to the method and composition of the present invention, a non-ionic amphipathic diester of fatty acids or a reverse poloxmer is administered to a patient in which a cancer or microorganism 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 90636 ~
W095/~19`1 1 ~ '167 METHODS AND COMPOS[TIONS FOR
REDUCING MULTIDRUG RESISTANCE

Technical Field The present invention relates to the use of resistance mrltlifir~ion agents in vivo to reverse multidrug resistance in human or anirnal cells and in drug resistant microo~ganisms.
More particularly, the present invention c~mrn~Ps pol~ yl~ c glycol polymers with either fatty acids or block polypropylene glycol on both ends of the pol~ llyl~,~e glycol polymer. These cb.ll~o~ilions are capable of reducing multidrug resistance in vivo.
Background of the Invention One of the major problems of cancer chemotherapy is the existence of drug resistance in tumors resulting in reduced resp~,n~ lcss to chemotherapy. Some human cancers, e.g.
kidney and colon Cal~ 0lll~, are drug resistant before treatment ~ 21 90636 ,, ~ !~
W0 95131g81 ~ '167 begins, while in others drug resistance develops over ~u,,c~;,;~, rounds of chemotherapy. One type of drug resistanee, ealled multidrug rociC~nr~, is ~1"~.,,( 1. . ;,. .1 by eross .~,.,i~..., to funetionally and strueturally unrelated dntgs. Typieal drugs that are affeeted by the multidrug resistanee are do~orubiein, vinrTic~in~ vinblastine, eolehicine and actinomycin D, and others.
At least some multidrug ~ Ldl.ce is a complex ph.,.lu~y~ whieh has been linked to a high expression of a eell Ill~ bldL~C drug efflu~ transporter ealled Mdrl protein, also known as P-glycoprotein. This mt~mh~n~ "pump" has broad specificity and acts to remove from the cell a wide variety of chemically unrelated toxins. (See Endicott, J.A., et al. "The Biochemistry of P-Glycoprotein-Mediated Multidrug Resistance", Ann. Rev.
Biochem. Vol. 58, pgs. 127-71, 1989.) Recently, a similar m~rh~nicrn of a broad spectrum drug resistance has been reported for certain microorganis_s.
These results indicate the existence of bacterial efflux systernc of extremely broad substrate specificity that is similar to the multidrug resistance pump of m~mm~ n cells. (See Nikaido, N., "Prevention of Drug Access to Bacterial Targets: Permeability Barriers and Active Efflux", Science, Vol. 264, pgs. 382-388, 1994; and Gottesman, M.M., et al. Annual Rev. Biochem.. Vol 62, 385 1993) S~hs~nrrc which reverse multidrug resistance are known as resistance mn~ifir~inn agents (RMAs), and are of importance in potentiating the cytoto~icity of rhrm..ll,..,.~ ;c agents to which a human cancer has become resistant. Although many agents have been identified as RMAs in vi~ro, a large proportion have little or no ~ P~c potential because of high toxicity in vivo at the doses required to reverse multidrug r~siC~n~ For example, metabolic poisons, such as azide, reverse multidrug resistance in vitro but have no usefulness in vivo. Most other highly effective RMAs, such as PSC833, appear to work as competitive antagonists of a drug binding site on the Mdrl protein. Mar~y of these ~gents also have toxicity which limits 2 ~ 9~636 WO 95/31981 1 ~ Jll '167 their usefulness in vivo. Col~ , there is a need to deYelop alternate pharmacological strategies for reYersirlg multidrug resistance to proYide RMAs with improYed aCtiYit~Y and lower oYerall toxicity.
Decreased intr~r~ r drug ~rcllmlll~ti()n through o~ ion of the drug efflux Mdrl protein is i~ Ol~t to, but apparently not the only factor, in the multidrug resistance phenotype. Altered intracellular drug distribution and binding, among other possibilities, also seem to play a role. For e~ample, the m~rh~nicm of reversing do7corubicin resistance using verapamil appears to be more related to altered intracellular distribution of doxorubicin than increased ~t~cllmlll~ion in the cell, as detailed in Schllllrh-lic, G.J., et al., "Quantitative determination of factors contributing to doxorubicin ~ ,e in multidrug resistant cells," J. NatL Cancer Insf., 81:1887-1892, 1989. In that report, it is shown that do~orubicin is c~
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 the nucleus in drug resistant cells. Thus, high affinity binding of drugs to Mdrl does not appear to be sufficient for optimal efflux, s~ ctin~ the existence of additional, rate limiting steps which may be susceptible to pharmacological intervention.
Certain non-ionic ~."~ 1,;r. s~ t~nrc. such as Tween 80 and CREMOPHOR~9 EL, have evidenced RMA activity.
(See Riehm H., et al. "Potentiation of drug effect by Tween 80 in Chinese harnster 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 phenotype with C~E~IOPHOR~ EL, a common vehicle for water-insoluble vitamins and drugs" Cancer Res. Vol. 50, pgs. 4199-4203, 1990.) However, Tween 80 potentiates drug toxicity in both parental and multidrug resistant cells, calling into question the specif1city of the Tween 80 effect on multidrug resistance. An effect on drug 35 efflux has not been demonstrated. CR~MoPHoR(~ EL is a ~ 2 1 ~0636 ~
W09~/31981 ~ 5 '17 complicated mixture of polyo,~ yl~_d esters of triglycerides of mainly ricinoleic acid (castor oil), the composition and RMA
active C~ t of which have not been i~lPntifi~d Use of CREMOPHOR~ EL in vivo is cnmrL/ ' by adverse 1~ A~ -F
release in some patients. There have also been reports of neuroto~icity associated with the administration of CREMOPHOR~
EL.
Cancer rh~moth~r~ry with. cytoto~ic agents can be successful only if the tumor cel~s are more sensitive than normal cells whose destruction is incompatible with survival of the host.
Success, defined either as cure or clinically ci~nific~nt remission, is not readily explained by the still popular idea that tumor cells are more susceptible to cytotoxic agents because they are dividing more rapidly than vital normal cells, e.g. hematopoietic ~ r cells. That rapid proliferation does not wholly account for the selective drug s~,nsiliviLy of tumors is d~ o~aled by the common observations that some drug-sensitive cancers are not rapidly dividing, and that many rapidly proliferating tumors exhibit resistance. To say that the m~ch~ni~ms accounting for the success or failure of ~hrmothPrapy for most human tumors is incompletely understood today is undoubtedly an lln~
However, recent evidence suggests that the ~el~,livily Of rh~mo~hr~apy for the relatively few tumors ever cured by drugs depends, to a large e~tent, upon their easy sllcc~rtihility to undergo apoptosis, i.e. to kill themselves. Many cytoto~ic drugs that kill cells by crippling cellular metabolism at high c..l~c~.ltl~tion can trigger apoptosis in susceptible cells at much lower c~llc~ tion. This appears to account for the unusual rhl~ml-S~ncitivity of many Iymphoid tumors, since many normal Iymphocytes are "primed" to undergo self destruction as an essential part of the m~rh~nicm for generating and controlling diversity of the irnmune response. Increased susceptibility to apoptosis rnay also be acquired by tumor cells as a byproduct of the genetic changes responsible for m~ n lnt transformation.
For example. tumor cells with constitutive c-mvc expression may .

2~ 90636 ~
W095t31981 1~ '. '167 s undergo apoptosis in response to DNA damage by ~ntir~nr~r agents, whereas norrnal cells are able to pause at cl-c~,h~oi-lLO in the cell cycle to repair the damage, or may not be cycling at all, rendering them highly resistant to apoptosis in this setting.
Although some tumors are c~ vs~1 of cells which are highly sensitive to apoptotic stimuli, and others have a genetic changes which, in isolation, ~ ~,lLO~ose to apoptosis, most tumors tend to acquire other genetic lesions which abrogate this increased Sc~la;LiViLy. Either at ~L~,sc,.ldLion or after ~ attempts, the tumor cells actually become less sensitive to apoptosis than vital normal dividing cells. Such tumors are generally not curable by available ~ o~ ;f approaches. Decreased apoptotic response has been associated with increased m~Ti~n~nt potential and has been shown to confer pleiotropic drug l~,oiSL~CC
byorlco~ ctransferfrhniq~lPc Therefore, althoughpreviously recognized " ,f ~ l".";," .~ of drug .~OiO~n~,e, such as decreased drug uptake, altered intr~rell~ r drug lor~ ti-~n, deto~ification and alteration of drug target conti~ue to be regarded as illl~OIldllL factors, pleiotropic resistance due to defective apoptotic response has recently emerged as a distinct and ci~nifit~nt category of drug l~,Oi~ ,e in cancer. Resistance due to failure to trigger apoptosis is s~,",- I;ll~f~ called ''duw-ls~l-,~l-'' drug r~_Oi~Ldllce to tiictin~lich it from the "classical" I l lf~ mentioned above.
Thus, what is needed is a clearly itlf ntifif d class of compositions that reverse multidrug resistance in vivo. The co...~oOiLion should have a low occurrence of adverse side-effects.
In addition, what is further needed is a composition and method for stimlll~tin~ apoptosis in cancer cells.
Summary of the Invention The present invention ~o.-lL risf s certain compositions that exhibit sl~bst~nti~l RMA activity in cancers. One example of such a co",L~o~l;on is a non-ionic ~ l;c s~rf~rt~nt known by the trade name SOLUTOL~ HS 15 (BASF Corporation.

-~ 2 1 90~36 '~
W095/31981 r~ C167 Pd,~ uy, New Jersey). This composition increases the cytotoxicity of ch~m~ ic drugs in multidrug resistant cell lines, but rlot in drug sensitiYe cell lines, in~irAtin~ that the p~t~ effect is not due to the additive to~icity of the agent S itself. The agent also promotes ~ ulll~ l;c agent ~f~cl-mlll~tion in multidrug resistant cells thereby potentiating the effect of the chemotl.~.a~ Lic agent.
The present invention also comprises a method for reversing multidrug resistance in human or animal cancer cells and a composition for elimin~ting multidrug resistant human or animal cancer cells. One composition that is an aspect of the present invention is a particular fraction of SOLIrrOL~ HS 15 collected by reverse phase liquid chromatography or super critical extraction. It has been found that the RMA activity in the SOLUTOL~ HS 15 resides in a narrow fraction from the reverse phase liquid chromatography. It has been further d~L~.IIilled that the toxicity to cells which is inherent in SOLUrOL~9 resides in a fraction different from the fraction cont~inin~ the RMA activity.
The present invention also includes a class of compounds that are ethoxylated fatty acids that exhibit strong RMA activity. These compounds have been found to be a polyethylene glycol with between approximately 4 to 100 ethylene oxide units with two fatty acids of between i.~yluALIlllstely 8 and 60 carbon atoms attached to the ends of the polymer in preferably an ester linkage. The fatty acid Cu~ ul~ t of the present invention can be unsaturated or can have substitutions in the carbon chain. The ethoxylated compound is preferrablyof the present invention has the following general structure:
Fatty acid Polyethylene glycol Fattyacid H3C(CH2)aXH2CCH20(H2CCHO)bH2CCH2X(CH2)aCH3 wherein the average of "a" is between appro~imately 8 and 30 and "b" is between 4 and 100 and X=CO-O (ester), CO-NH
(amide) or O (ether). In a the preferred molecule, "a" is between ~ = = = = = = = -- -- , = = = = = == = = . . . , = ., ., . = . , -~ 2 1 9'~636 wo 95131981 F~ I/LJ.. ,_I~ '167 15 and 25 and "b" is between 12 and 40. In the most preferred molecule '~a" is 16 and "b" is 18 and the molecule is a diester.
This molecule is ipci~n~t~d CRL 1095 and is available from CytRx Corporation, Atlanta, GA.
The present invention also includes comrocitions and methods for reducing the ~ u-ce of certain microorganisms to chemo~h~r~re~ agents. It has been ~l,otermin~d that certain microorganisms contain P-gly~u~,lvtc~ ke pumping m,orh~nicmc that are similar to those found in m~nnm~ n cells and it is believed that these ~ ",~ may be important in resistance to antimicrobial agents. It has been reported that tetracycline is particularly suscep ible to multidrug resistance in cells. Another embodiment of the present invention is the use of the compositions described herein to increase the crr~,.ti~,~,u~SS of tetracycline. According to the present invention, by administering tetracycline with the compositions described herein, the tissue conc~ntr~tion can be ei~nific~ntly increased. In addition. the duration of drug presence is also increased.
AQticancer drugs often kill tumor cells by inducing apoptosis, a form of cell death requiring actiYe participation of the target cells. Impaired ability of cells to execute an apoptotic response results in pleiotropic drug resistance, and modulation of apoptosis resistance might improve the t,rr~ ue~S of chemotherapy. Prominent involvement of the cell ll.c~ lane in the pathways leading to apoptosis suggests the o~UI~u~y to modify the cellular sensitivity or threshold for apoptosis with membrane inL~.d-,livc agents. It is cv..l. ,~I-l .t~ d as part of the present invention compositions that are novel sllrf~ft~n~c that reverse P-~lycuL~ut~l~-mediated multidrug resistance with negligible toxicity of their own. Some of these agents can also potentiate cytotoxic drug-induced apoptosis, irrespective of P-glycoprotein expression. It is further an aspect of the present invention to provide compositions and methods for inhibiting the expression of genes that code for P-~ly~v~L~t~;u or other proteins 35 that mediate the transport of chemotl,~ lic agents. It is to be , 21 ~a636 ~0 95/31981 ~ . '167 understood that the present invention includes the use of the disclosed compositions prophylactively in cancer therdpy before the use of ~ tll.,.d~y.
Another embo&ment of the present invention dre the S polyuA~ l,ylene/polyuAy~,-u~.yl.,~lc co~olylll.,.~ with the following general formula:
HO(C3H60)a(C2H40)b(C3H60)aH
wherein the average of "a" is bet~veen dpu.uAi.~-dlely 2û and 60 and "b" is between ay~luAilll~llely 10 and 30. A more preferable range is "a" of between d~io~ d~ely 30 and 50 and "b" of between approximately 15 and 25. It is to be understood that the propylene copolymer can be s~lbst;hlt~d with a butylene copolymer or even rnixhures of butylene and propylene units.
The preferred copolymer is a polyoAyethylene/poly~Ay~.uL,ylene copolymers with the following general formula:
Ho(c3H6o)a(c~H4o)b(c3H6o)aH
wherein the average of "a" is ~luAillldlely 40 and "b" is dy~lUAillldltly 20 and is lPCi~n~d CRL 1605.
Accordingly, it is an object of the present invention to provide a composition and method for reducing or -limin~in~
multidrug resistance in human or animal cancer cells.
It is further an object of the present invention to provide a composition and method for treating a human or animal with multidrug resistant cancer.
It is further an object of the present invention to provide a composition and method for reducing multidrug resistance which will not produce adverse side-effects.
It is another object of the present invention to provide a composition and method to block expression of the MDRI gene or related genes.

~, ~ 21 90636 ~
W0 9513~981 ~ ';C7 It is further an object of the present invention to provide a composition and method that can be used to alter the blood brain barrier thereby allowing certain ll,. .,.l.~.,lic agents to cross the barrier from the blood into the brain.
It is yet another object of the present inYention to provide a composition and mèthod for increasing the sc.l~iLivily of drug resistant micro~ al.i~,l.s to ~hfm~LI,f.a~uLic agents.
It is yet another object of the present invention to provide a c~ o~iLion and method for increasing the efficacy and bioavailability of certain antimicrobial drugs such as tetracycline.
It is another object of the present inYention to provide compositions and methods for altering the threshld for inducing apoptosis, especially in apoptosis-resistant cancers.
It is yet another object of the present invention to provide a col.. ~o~i~ion and method that can be used to reverse multidru~ resistance to VP-16 and VM-26 in cancer cells.
It is yet another object of the present invention to provide a composition and method for reducing the resistance of microorganisms to certain drugs.
These and other objects, features and advantages of the present invention will become apparent after a review of the following detailed description of the disclosed embodiment and the appended claims.
Brief Description of the Figures Figure I shows fractionation of sOLurOL(~ HS 15 using reverse phase liquid chromatography.
Figure 2 shows the effect of CRL-1095 on Rhodamine 123 (P-gpsubstrate)~cllm~ tioninMD~KB 8-5-11 cells (In Vitro).
Figure 3 shows the effect of CRL-1095 on modulation of colchicine resistance on MDR ICB 8-5 cells in vif ro.
Figure 4 shows the effect of CRL-1095 on modulation of VP-16 resistance on MDR KB 8-5-11 cells in vftro.

-~ 21 90636 wosa/3lssl r~"~ 167 Figure S shows the effect of CRL-1095 on modulation of doxorubicin resistance on MDR KB 8-5-11 cells in Yitro.
Figure 6 shows effect of CRL-lO9S on m~ tion of actinomycin l~,alaLdilce on MDR KB 8-S-l l cells in vitro.
Figure 7 shows the effect of CRL-1095 on morllll~ti~m of col ' ^ resistance on MDR KB 8-5-11 cells in vitro.
Figure 8 shows the effect of CRL-1095 on morllll~tiorl of Taxol l.,siala,lce on MDR KB 8-S-I I cells in vitro.
Figure 9 shows the effect of CRL-1095 on mo~ tion of daunorubicin l~a.alallce in MDR erythro-leukemic K562/III cells.
Figure 10 shows the cytotoxicity of CRL-1095 on KB

3-1, KB 8-5, KB 8-5-11, and K562mI cells.
Figure 11 shows the effect of CRL-1095 on vincristine ~cc~mlll~tion in KB 8-5 human xenograft tumor tissue in mice.
Figure 12 shows the effect of CRL-1605 on modulation of Taxol l.,a;alilucc on ~)R KB 8-5 cells.
Figure 13 shows the effect of CRL-1605 on modulation of daunorubicin resistance on MDR K5621III cells.
Figure 14 shows the effect of CRL-1605 on vincristine ~c~-m~ tion in KB 8-5 human xenograft tumor tissue in mice.
Figure 15 shows the mo~ tioD of tumor growth in KB 8-5 human xenograft model by CRL-1605.
Detailed Description As used herein, the terrn "multidrug resistance"
means resistance or acquired or natural resistance of tumor or rnicroorganiâms to ~h~mnth~r~rellti~ agents. The multidrug resistance can be mediated by P-gl)~ o~-uteill or can be mediaoed by other m~ch~ni~m~ The term "patient" means either a hurnan or animal with multidrug resistant cancer or a human or animal i~ 2 1 9~6~6 ~
WO 95/31981 F~,lll . 5. ~ ~167 infected with a multidrug-resistant microorganism or a c~mhin~tion of micluù~auis~
The present invention comprises methods and cu,..~o~iLions for reducing or ~1;.";,.~;"~ multidrug resistance in S cancers in patients. According to the method and c- mro~itinn of the present invention, a non-ionic ~",~ l,;c ester or diester of a fatty acid is ~rlminict~ored to a patient in which a cancer exhibits multidrug resistance to the cl'~..-otl.~ uuc 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.
Another embodiment of the present invention is directed to compositions and methods for increasing the tissue concentration of certain antibiotics such as tetracycline. For example, tetracycline is believed to be excluded from tissues via the multidrug resistance pump. By ~fiminict~rin~ the compositions described herein either with the ~LLa.,y~,lhlc or shortly before or after administration of the tetracycline, the tissue concentration of the tetracycline can be increased.
The present invention includes a method of treating a patient with a cancer that e~hibits multidrug ~ ~ ce to reduce or eliminate the multidrug resistance which includes a~miniC~.-ring to the patient an effective amount of a non-ionic ~-.~hipaLl ic diester of a fatty acid or a polyo~ypropylene/polyù~ ,ylene copolymer. A preparation that exhibits the desired biologic activity is SOLUTOL~ HS 15.
This preparation is a mixture of various compounds with s~ c-~nt activities.
By fractionating the SOLUrOL~ HS 15 preparation using reverse phase liquid chromatography and then assaying the various fractions for RMA activityl it has been determined that the Rl~IA activity resides in a small fraction which contains fatty acid esters and diesters containing ethylene oxide units. This fraction has a much higher specific activity than the unfractionated SOLUTOL~ HS 15. In addition, fractionation of ... ... . .. . .... ... _ . _ .. . ............... . .. _ . . _ . _ .

^-~ 2 1 9063~ ~
Wo95131981 ~ . q . '167 .
CREMOPHOR~ EL by either reverse phase liquid chromatography or by super critical Py~ tinn shows that the RMA activity resides in a fraction which contains fatty acid esters and diesters containing ethylene oxide units.
By testing several fatty acid esters and &esters with varying ethylene oxide units, it has been found that CUIIIIJUI~
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 d~loAillldl~ly 8 and 60 carbon atorns and between d~lo~ at~ly 4 to 100 ethylene oxide units. The fatty acid component of the present invention can be Ull ~ ' and/or substituted and still exhibit activity. The preferred fatty acids are straight chained. In general, the fatty acids without the ethylene oxide units have little or no RMA activity.
It has been determined that the most active fraction of SOLUTOL~ HS 15 and CREMOPHOR~ EL are &esters of polyethylene glycol. For example, when native CREMOPHOR~
EL was fractionated using supercritical ~x~ tion as shown in Example VIII, 35 percent of the total preparation of-CRE~IOPHOR6~ was extracted. The unextracted portion of the CREMOPHOR3 showed little or no activity. Approximately 14%
of the total CREMOPHOR@~ EL mixture was found to have 5i~nific~ntly higher (lOX) RMA activity compared to native CREMOPHOR~ EL. The active fractions of CREMOPHOR~ EL
are less toxic than the native CREMOPHOR~ EL. Infrared analysis of the active fraction shows that the fractions are polyethylene glycoVfatty acid diesters.
Thus, the present invention includes ethoxylated fatty acids The fatty acids of the present invention have between 8 and 60 carbon atoms and between approximately 4 to 100 ethylene oxide units. The polyethylene glycol polymers have fatty acids attached, preferably via an ester linkage to both ends of the polyethylene glycol polymer. The fatty acids that are components of the RMA molecule of the present invention have between approximately 4 and 60 carbon atoms. The preferred compounds 2 1 90636 f~
WO 95/31981 I~ J., '167 have a fatty acids with between 12 and 40 carbons with the more preferred compounds with between 8 and 30 carbon atoms with the most preferred compounds having between a~Lu~ dt~ly 15 and 20 carbon atoms. It is to be understood that the two fatty acids in the diester can be different fatty acids. Preferred fatty acids include, but are not limited to, stearic acid, linoleic, oleic acid, palmitic acid and linolenic acid.
The preferred polyethylene glycol polymers in the RMA compounds have between a~lu~illlat~ly 4 and 100 ethylene oxide units, with the more preferred compounds having between 8 and 30 ethylene oxide units and the most preferred cu".~uul..b having betweerl 15 and 25 ethylene oxide units.
While not wanting to be bound by the following theory, it is believed that cellular ~ 1llf transport proteins must form polymers, usually dimers or t~tr~m~orc~ to effectiYely carry out their transport functions. Thus it is likely that the Mdrl protein can achieve its function of removing from the cell a wide variety of ch.qmi~lly unrelated toxirls only after forming polymers in the membrane. Non-ionic Alll~ ;C sllrf~ct~3nt~
exhibit membrane surface activity and are characterized by -- having a hydrophilic head and hydrophobic tail. In particular, non-iorlic ,..,.L~ I.;c esters of fatty acids, irlhibit the formation of such protein polymers, and thereby inhibit drug efflux.
The ester of the present invention has a hydrophilic head, which comprises polyethylene glycol, and a hydrophobic tail comprising a fatty acid. Such a molecule is amphipathic. The molecule is large enough that each end displays its own solubility behavior. In another embodiment of the present invention, the polyethylene glycol has two hydrophobic fatty acid tails on either end of the polyethylene glycol polymer. The fatty acid tails are attached to the polyethylene glycol polymer preferably via an ester linkage, although the linkage can be via other bonds such as an ether linkage or an amide or similar linkage. The molecule has the following general structure:

~ 2 1 90$36 ~
~095/31981 r~ o.. , . '167 Fatty acid Poly~ yl~ glycol Fattyacid H3C(CH2)aXH2CCH20(H2CCHO)~H2CCH2X(CH2)aCH3 wherein the average of "a" is between d~o~ at~ly 8 and 30 and "b" is between 4 and 100 and X=CO-O (ester), CO-NH
(amide) or O (ether). In a the preferred molecule, "a" is between 15 and 25 and "b" is between 12 and 40. In the most preferred molecule "a" is 16 and "b" is 18 and the molecule is a diester.
This molecule is ~..ci~n~cl CRL 1095 and is available from CytRx Corporation, Atlanta, GA.
Another embodiment of the present invention are compounds that are effective in reducing multidrug l~ in cancer cells that are polyoxyethylene/polyo~ypropylene copolymers with the following general formula:
HO(C3H60)a(C2H40)b(C3H60)aH
wherein the average of "a" is between approximately 20 through 60 and "b" is between approximately 5 and 50. It is to be understood that the propylene copolymer can be ~ il"l~ d with a butylene copolymer or even mixtures of butylene and propylene units.
The preferred copolymer is a polyoxyethylene/polyu,~y~.u~ c copolymers with the following general formula-HO(C3H60)a(C2H40)b(C3H60)aH
wherein the average of "a" is ~IUAillldt~ly 40 and "b" is approximately 20 and is ~ cign~t~d CRL 1605.
The molecule may be ~minic~red to a patient either alone or in combination with a treatment program of at least one chemoLl.~ uc.lLic agent to which the human cancer is resistant.
Such a ~h~moll,. .~. Lic a~ent typically includes, but is not 2 1 91~636 ~
W095131981 r~ 167 limited to, doAorubicin, vinnrictin~ vinblastine, Taxol, colchicine, VP-16 and a~li.,or..y~;hl D. However, there are many other chPmir~lc used in ~ )y to which multidrug resistance may appear during treatment, and the present invention may be employed equally well in such cases. In addition, the present invention is useful for reducing ~ ,ia~ce to platinum compounds by promoting ~rc~m~ tion of these culll~uull~s.
In general, at least one effective dose of the RMA of the present invention is ~rlmini.ct.ored for every dose of chemoll,. ., ~ ;c agent that is ~mini.c~red in treatment.
Preferably, an effective dose of the RMA may be ~11lll;ll;~. ,~,d at least daily throughou~ the period between administration of successive doses of ~h~mo~ LiC agent. The treatment period typically lasts about four weeks, drp~on-iing upon the cancer being treated and the ~ ,.llic agents being used. Al~ ,ly, the RMA may be col.l;,~ùll~ly infused throughout said period.
The administration of the RMA may also c~...,.,..,~e prior to a session of ~ loLh~l~y, and continue throughout and after the chemotherapy session. The amount of the RMA per dose will depend on which particular non-ionic ~",~ I,;c fatty acid ester is employed according to the present invention. However it is preferable that the m~-~iml-m dosage that may be tolerated with negligible toAic ~ylll~ulull~ in vivo be used. At least some non-ionic ,.",I.h;~ l,;c esters of fatty acids, such as SOLUrOL~ HS 15, are tolerated eAll~ ely well in vivo, tmd may be employed with no acute toAicity at dosages which achieve equivalent or superior reversal of multidrug resistance to common ~ -II;C
agents as compared to dosages of the prototypical RMA verapamil which produce marked toAicity.
The RMA of the present invention can be a~minict~red either intraYenously or orally. It may be ~minictf-red separately from the rhPmn~h~rapeutic agent, as may be dictated by the chemotherapy, in which case the amount of time between comm~ n~in~ ~minictr~rion of the RMA and ~minictr~ion of the chemotherapeutic agent should not be ~ 2 1 90636 ~
WO 9S/31981 F~ .. . '167 sllhst~nti~l e.g. typically within 24 hours, or as the ~ Ot~
permits. An exemplary treatment regimen cnmrric~c oral or illtLd~C~IUU~ ;on of the RMA, followed by ~t~minictr~tion of the chemo~hPr~relltic agent throughout the S period until the next session of cl, ~ y~ either by contir~uous infusion or oral time release capsules. Although the optimal blood collc~ "~;on of the RMA of the present invention will vary according to the cl~f Illull~ ;c agent being used or the type of cancer being treated, a typically preferred plasma c-)"c~.",~-;on is between 0.1 to S mg/ml. A more preferred plasma con.,~,.ltl~.tion of RMA of the present invention is bet veen approximately 0.5 mg/ml and 2.5 mg/ml. If a polyethylene glycol fatty acid diester or a reverse poloxamer which has been synthesized or purified is used to treat a patient with multidrug lS resistant cancer or microorganism infection, the preferred plasma ~,OL.cc.ltl~ltion is between ~ylu.ullla~ely 0.01 mg/ml and 2.5 mg/ml with the more preferred plasma collc~ ,nion betveen approxirnately 0.1 mg/ml and 2 mg/ml.
AItc.llcLti~ly, the RMA of the present invention may be ~lminictpred in combination with the chemotherapeutic agent, comprising continuous infusion or daily oral ~,ollau~ tion of time release capsules of the RMA c~ f ". ;n~ prior to the chemotherapy session, and co~timlin~ throughout and after the session, by way of example. The RMA may be infused together through the same needle with the cl~.. llolll.. d~.ltic agent, or combined in a single oral capsule, as the c~. ,Ill lllrl,.rl~,~lic agent permits, in which cases the RMA of the present invention may be used as an Pmlllcifi~r of the agent, since non-ionic ,,,,,l,l.;l.,~lllic esters of fatty acids commonly possess emulsifying CII~IL~ tl ricti~s The RMA of the present invention preferably should be ~minictP~ed at least one hour prior to and during administration of a cancer chemotherapeutic agent. The RMA of the present invention should be given for at least 12 to 24 hours after adrnirlistration of the cancer chemotherapy. However, the _ _, . . . . . ... .. .

~ \ 2 9 ~ 6 3 ~ r~
. WO95/31g81 1~1/1 -167 ~t~minictr~tion protocol will be f~ upon the p~l" ".~.~okinetics of the .~ L1~ y and RMA.
P-~d aLion of an emulsion of the ~l- l)"L~
agent with the RMA will depend on the particular agents used.
S Typically, the RMA and the ~ agent are c-" "1~ f d and heated above room tf ~ to a range in which both the RMA and the Cl,.'."~)tl,. '''l" .-l;c agent are still stable, but in which the RMA becomes fluid, about 50 to 80 C. Sterile water is heated to the same tf ~ and then added with Yigorous agitation in a proper amount to achieve a viscosity appropriate for adrninistration. Other cvllll~ may be added to the emulsion as nec~3sd~ to prepare it either for i~,d~ vus or oral administrarion, as is well known in the art.
According to another embodiment of the present invention, the RMA of the present invention can be ~minictf~red together with other RMAs, such as verapamil. The RMA of the present invention and a second RMA can be infused sf*,~.tl,ly or concurrently, or combined into one time release capsule for oral consumption, in effective doses typically ~t~minictfred in treatment using each RMA alone, as permitted by the toxicity of the second RMA.
The formulations include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (inrlllflin~ s~b~ r~u~ ;..r"."...~ r, iuLLd~ nvus, in~ra~lrrm~ tldl.~a~ll.,dl, and epidural) ad,~ilialldtion. The formulations may conveniently be presented in unit dosage form and may be prepared by conventional pl~ l t~t~hniq~ c Such ~rhnir~ c include the step of bringing into association the RMA or the present invention and the ph,..".~.,l;r~l carrier(s) or e~riri~n~(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then. if necessary, shaping the product.
~ Forrn--l~tion~ of the present invention suitable for oral administration may be presented as discrete units such as .. .... ..... .... .........

-2 1 ~0636 ~
~-- W0 95/31981 P~ . 6i67 capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a ~ .,ciOI~ in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil S emulsion and as a bolus, etc. Desired parenteral formulation of the polyo~ypropyl~ olyuA~ block copolymers or the etho~ylated fatty acids may optionally contain ssll~hili~s such as CHREMAPHORE or SOLUTOL.
A tablet may be made by cu-l-y.~sjlon or molding, optionally with one or more accessory ingredients. Cu-l-~-~sel tablets may be prepared by cu-,-~.~us~iug, in a suitable mac_ine, the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface active or dispersing agerlt. Molded tablets may be made by molding, in a suitable machine, a rni:cture of the powdered compound mnicren~d with an inert liquid diluent. The tablets may be optionally coated or scored and may be formulated so as to provide a slow or controlled release of the active incredient therein.
Formulations suitable for topical administration to the skin may be presented as nintm,ontc creams, gels and pastes comprising the ingredient to be ~.1,.,;";.~ .d in a phA.",~. r,,~icAl acceptable carrier. A preferred topical delivery system is a transdermal patch containing the ingredient to be A~minict~red.
Fortnl-lAtio~c for rectal ~ ion may be presented as a ~u~uo~i~u-y with a suitable base comprising, for example, cocoa butter or a salicylate.
ForrmllArionc suitable for nasal administration, wherein the carrier is a solid, include a coarse powder having a particle size, for example, in the range of 20 to 500 microns which is A~minictl-red in the manner in which snuff is A~minicn~red, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose.
Suitable formulations, wherein the carrier is a liquid. for WO 95/3198~ . '167 ~minictration, as for exarnple, a nasal spray or as nasal drops, include aqueous or oily solutions of the active i,~ ,Lc.-l.
Formulations suitable for vaginal ~ ;nn may be ~, ~ S~ ~t~ d as pessaries, tamports, creams, gels, pastes, foams or S spray fnrml~1ati()nc cont~ining in ad&tion to the active in~re such carriers as are known in the art to be ~ .o~ e.
Forrnulations suitable for parenteral ~lminictr~tion include aqueous and non-aqueous sterile itljection solutions which may contain anti-o~idants, buffers, b~,t~,lio~ and solutes which render the formulation à~-u~i~-~t~.ly isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile su~ io,-~ which may include s~lcp.on~in~ agents and thic~Pnin~
agents. The formulations may be presented in unit-dose or multi-dose crJnt~in~rC for example, sealed ampules and vials, and may be stored in a freeze-dried (Iyophilized) con-iitionC requiring only the ad&tion of the sterile liquid carrier, for e~ample, water for injectjnnc~ i"""r.l;,.t.~ly prior to use. F~l,."l.l),,.,.~,,.lc injection solutions and Sllcp~ncionC rnay be prepared from sterile powders, granules and tablets of the kind previously ~i~crrihe~
Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose, as herein above recited, or an appropriate fraction thereof, of the ~minict.ored ingredient.
It should be understood that in addition to the ingredients, particularly mentioned above, the formulations of the present invention may include other agents conventional in the art having regard to the type of formulation in question, for example, those suitable for oral ~minictr~tion may include flavoring agents.
The method and composition of the present invention provide an important new means of overcoming multidrug resistance in human cancers. The method and composition have an efficacy equal to or better than best resistance modification agents known to the inventor. Furthermore the agent used in the method and composition of the present invention has a lower toxicity than 2 1 90~6 . ~ ,~, WO 95~31981 I~ I67 other RMAs and fewer side effects than other potential RMAs.
Moreover, it is believed that the agent operates by a different "~ " on the complex pll~,l.Vl~ of multidrug l-,sisL~,e, and thus can be cull~ with other RMAs to provide a more potent means of reversing multidrug l~,.,isL~ e.
The structure of SOLUrOL~ HS 15 or other RMAs comr~ (i of ethoxylated faty acids is dissimilar to that of verapamil or other typical RMAs. The markedly greater potency of SOLUrOL~ HS 15 than verapamil for reversing VP-16 or colchicine resistance relative to the ability of each to reverse vinblastine or doxorubicin resistance supports the hypothesis that SOL[rrOL~ HS 15 operates by a MDR-reversing 111~
different from competition for the drug-binding site on Mdrl protein found in verapamil. Colchicine is known to interact weakly with the i~ ntified drug-binding site on the Mdrl protein, since co!chi~in~ does not compete for vinblastine binding. The fact that MDR cells are nc~ l.eless highly resistant to col~hirinlo indicates t~hat colchicine efflu~c is less ~ on int.or~rtion with this drug-binding site than is vinblastine. Since SOLUrOL69 HS 15 is a highly potent RMA for both colchicine and vinblastine, it may inhibit a second event necessary for efflu~ after drug birlding, namely actual transport through the ~ b~C. It is likely that SOLUTOL~ HS 15, as a sllr~ t~nt inhibits formation of Mdrl protein polymers which may be nc~ to achieve drug efflux.
Another illl~ulLdUL advantage of the RMA of the present invention is the fact that the compounds which are collt~rnrl~t-od as part of the present invention are highly effective against the multidrug l~sis~ against the anticancer drug VP-16. The prior art RMAs, such as verapamil, are not effective against VP-16 multidrug resistance. (See Schested, M, et al.
~'Relationship of VP-16 to the Classical Multidrug Resistance Phenotype", Carlcer Research, Vol. 52, pgs. 287~2879, 1992.) The RMAs of the present invention have been found to be ~ 2 ~ 90636 ~
W0 95/31981 ~ '5~ 67 effective in reducing multidrug resistance against a broad spectrum of anticancer drugs.
It is well known that certain microorganisms contain 111' .l1111~..~f proteins which are similar in structure and function to S the P-gly-,uylut~;ll that is expressed by the Mdrl gene in m~mm~lc It is contemplated as part of the present invention that the methods and comrocitions that make up the present invention can be used to make certain microorganisms more s~.creptihl~ to therapeutic drugs. For e~ample, it is likely that the present invention will reverse chloroquine resistance in malaria.
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 Barrier", J. Biol. Chem., Vol. 267, pgs. 20383-20391, 1992.) The brain is a pharmacological sanctuary in that many drugs ~.1",;l,;- ed a~t~.lfi,,~lly have limited access to the tissue parenchyma. In the brain, en~th~ cells forrning the capillary tube are joined by contin~lo~c tight junctions that prevent many s~bs~nr.oc from entering the organ. Nutrients needed for brain cells are selectively transported from the blood through specific channels or LL~I~OI~ in the capillary endothelial cells. Ihus, the brain is a rigorously isolated co...~u Llll.,.lt that is protected by a blood-brain barrier.
Hydrophûbic ~IlLilulllol agents, such as Vinca aLlcaloid and adriamycin (ADM), cannot enter the brain, although other hydrophobic mol.oclllps such as nicotine and ethanol readily pass through the blood-brain barrier. Therefore, some mrrh~nicmc 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 reported in both brain and testis but not in the other tissues. This suggests the functional involvement of P-gl~o~.oL~ in the blood-brain barrier. It is contemplated as part of the present invention that the methods and compounds described herein can be used to reduce ... . .. . .. . . ... . . . . . . _ . . .

WO 9~;/319B1 l ~ C. ~167 the blood-brain barrier thereby allowing beneficial Lhc~a~u~ ic agents to cross the barrier.
This invention is further illllc~rAtrd by the following ~Amrl~s, which are not to be construed in any way as imposing limitAtionc upon the scope thereof. On the contrary, it is to be clearly understood that resort may be had to va~ious other embodiments, mo~1ificAtionc and equivalents thereof which, after reading the description herein, may suggest thernselves to those skilled in the art without departing from the spirit of the present invention and/or the scope of the appended claims.
Example I
Cell lines and culture cor~l;tions The drug sensitive human carcinoma cell line, KB 3-1, and the MDR variants, KB 8-5-11 and KB V-l used in this study were generously provided by Dr. M. Gottesman, NC~I, Bethesda, MD. The p~u,u~,lL}es of these cells and their culture conditions have been previously described in detail (Akiyama, et al., Isolation and genetic ch~c~ tion of human KB cell lines resistant to multiple drugs. Somat. Cell. Molec. Gene~., II, 117-126 (1985)). The KB 8-5-11 cell line was IllA;llIA;,,~ medium containing 100 ng/ml colchicine (Sigrna, St. Louis, MO) and the KB V-l cell line was m-AintAin~ in medium COIIIA;II;I~ I mglml vinblastine (donated by Eli Lilly and Co., Indianapolis, ~).
Human epidermoid CAI ~ U~IIa 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 SOL~rrOL6~ HS 15 in comhinAtion with various chemotherapeutic agents, namely colrhirin.o vinblastine, and do~orubicin. The details of the treatment are described in Coon, J.S., et al, ' SOLU roL~ HS 15, nontoxic polyo~yethylene esters of 12-hydroxystearic acid, reverses multidrug resistance", Cancer ~esçarch, 51, 897-902, 1991, which is incorporated by reference.
Briefly, cells from the three lines were plated as is well known in the art in 96-well plates, with increasing conren~ionc Of 2 1 9~63~
"~ ~ ,_, . ..
WO 95/31981 r~ J.,. '1~7 cytotoxic drug along one axis of the plate and inrr~in~
concentrations of the RMA along the other a~;is of the plate.
After in~n~bation for five days, the plates were washed and dyed according to methods known in the art, and a cell count was .1~ t. .",;,.. ~.~ The mean c~"~- .. I,,.tioll of the cytoto~ic drug that caused 50~7a inhibition of cell growth compared to controls (ICso) was plotted at various c~-lc.,l.tlations of the RMA. Complete reversal of the MDR phenotype in E~B 8-5 and E~B 8-5-11 cells was achieved by SOLUrOL~ HS 15, while the RMA did not put~ntic~le drug toxicity in drug-sensitive KB 3-1 cells, in-iir~tin~
the potentiating effect was not due to any toxicity of SOLUTOL~
HS 15 itself. At a c-lnc~ntr~tion of 10% of its own IC50, SOLUTOL~ HS 15 produced a marked reduction in the resistance of KB 8-5-11 cells to colchi~in~7~ vinblastine, and doxorubicin.
Identical platings were also p.,~ru~ c,d for the prototypical RMA verapamil. U~ lly, the relation between the effects that SOLUTOL(~ HS 15 had on the three ~lu~u~
was different from the relation between the effects that verapamil had on the three cytotoxins, in~ic~in~ SOLUTOL~ HS 15 and veraparnil affect multidrug resistance by different ,~
SOL~OL~ HS 15 was relatively much more potent than verapamil for reversing col~hirin~ resistance, as c~llly~.~ to the ability of each RMA to reverse vinblastine .~ ~.ce.
Example II
Human epit~rrnoi~l 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 SOL~OL~ HS 15 in ~ ;on with various chemo~ .d~ ic agents, namely colchicine, vinblastine, and doxorubicin. The details of the treatment are described in Coon, J.S., et al, ~SOLUTOL~ HS 15, nontoxic polyoxyethylene esters of 12-hydroxystearic acid, reverses multidrug resistance", Cancer Research, 51, 897-902, 1991, which is incorporated by reference.

~ 2~9~36 f~
, W0 9~/31981 r~ ,Sa. 'lC7 Briefly, cells from the three lines were plated as is well known in the art in 96-well plates, with inrrr~cin~ COI~f~ l;f~nc of eytoto ~ie drug along one a~is of the plate and inr.
cr~ ..t...l~onc of the RMA along the other a~is of the plate.
After inr~lb~tion for five days, the plates were washed and dyed aecording to methods known in the art, and a eell eount was flf-rf-nnin~fl The mean c~ f.l ..I"~I;nn of the eytoto~cie drug that eaused 50qo inhibition of eell growth eompared to eontrols (IC50) was plot~ed at Yarious conc~,.lLLdLions of the RMA. Cornplete reversal of the MDR phf.~uLy~c in KB 8-5 and KB 8-5-11 cells was aehieved by SOLUTOL~ HS 15, while the RMA did not potentiate drug toxieity in drug-sensitive KB 3-1 eells, in~lir~tin~
the potentiating effeet was not due to any toxicity of SOLUTOL~
HS 15 itself. At a concc.lL,~Lion of 10qo of its own IC50, SOLUTOL~ HS 15 produeed a marked reduction in the L~Llce of KB 8-5-11 cells to eolrhirinf~ vinhl~ctinf-~ and do~orubiein.
Identical platings were also ~,.r~Ll.lcd for the prototypical RMA verapamil. Unrrrectr~ly, the relation between the effects that SOLUTOL~9 HS 15 had on the three l ~lutu~h~s was different from the relation between the effects that verapamil had on the three eytotoxins, in-lir~rin~ SOLUTOL~ HS 15 and verapamil affect multidrug resistanee by different .1-~ . I.,,,,l~...c SOLOTOL~ HS 1~ was relatively much more potent than verapamil for reversing colchicine resistance, as compared to the ability of each RMA to reverse vinhl:lctin~ resistance.
Example III
Measurement of cellular rhodamine 123 accumulation by flow cytometry Efflux of rhodamine 123 from MDR cells was also ~min~d to provide direct information about the action of the transport protein Mdr 1. F.nh~nred ~ccllmlll~tion of the P-gp substrate, rhodamine 123, in multidrug resistant KB 8-5-11 c)clls was used to measure RMA activity. Flow cytometric analysis was . 21~3~
, .,~ ! ~
WO 9~1981 }'~ '167 performed as described (Coon et al., 1991; B~lcl~in~h~m et al.
1995). Briefly, multidrug resistant KB 8-5-11 cells (0.5 X 106 cells/ml) were inruh~t~d with 0.5 mg/ml rhod~min-~ 123 at 37C
in the presence or absence of the putatiYe RMA. After 60 rnin., mean intracellular steady state rhr,~l~minr 123 ~luo.. ;,.,.,.~ was measured on an Epics Profile Flow Cytometer (Coulter, Hialeah, FL). Increased rhodamine 123 ~ecllm~ tion in MDR cells has been shown to correlate ~Iu~Pit~ively with inhibition of MDR
activity (Kessel, 1989; Efferth et al., 1989; Neyfakh, 1988).

Example IV
Cell proliferation inhibition assay ICso (the drug concentration that reduces cell proliferation to 50qo of untreated controls) was assessed as previously described (18). The chromogenic substrate, MTT
(Sigma), was used to ~".. ,.. ~-- viable cells. Cy~lo~ A was a gift from Sandoz (Basel, Switzerland) and verapamil Hcl was from Knoll (Wluppany, NJ). For the Resistance l~o~ifir~tion Index (RMI, Eliason et al., 1990), cells were prepared in microtiter plates and incubated with the indicated drugs and RMA. RMI was r~lclll~trd as the ICso f drug alone/ICso of drug + RMA. The average RMI was c~lr~ d from at least three in~ ,l assays.
Example V
SOLU roL~3 HS 15 was fractionated using reverse phase liquid chromatography to ~,~t~nnin~ where the activity resides in the preparation. An approxirnately 50qo solution of SOLUrOL~ HS 15 was prepared in 100% acetonitrile (ACN) and water. One ml of the SOLUrOL63 HS 15 solution was injected onto a Phenomene~ IB-Sil reversed phase column. Ihe colurnn has 5 ~Lm particles. and is 4.6 mrn internal diarneter by 150 rnrn.
The flow rate was 2.0 ml/min. The mobile phase was as follows:
A=5~7o ACN and B=100% ACN. The gradient was linear with 10090 A to 100% B in 15 minutes, then was m~in~:~in~d at 100%
,. . . . .. . . . .

~.1 = 2 1 9~36 ~
' WO 95131981 r~ il67 B. Fractions were collected at 30 second intervals. The various fractions were assayed for RMA activity as described in E~ample III. The results of the fr~tinn~tion are shown in Figure 1. In addition the same fractions were assayed for to~icity by S mP~c-lrin~ 50% inhibitory cor, ~.~I,,.I;on~ (IC~o) as described in Kessel D., "E~ploring Multidrug Resistance using F~ho~i~""" r 123, Cancer Co~nm~nicAtions 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 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-toxic.
Example VI
Sy,n~hesis offatty acid PEG-fatty acid diesters Fatty acid diesters were ~ i~d by tr~ncesrpnfic~tion of PEG and fatty acid methyl esters kPmllc, 1956). Stearic acid diester (CRL 1095) was synthesized from 2 moles of methyl stearate (Sigma) and I mole of PEG 900 in a reaction catalyzed by zinc acetate. The reaction t~ c.~ltl,c was kept at 160~C for S hrs. under high vacuum (S
rnicrons) with slight stirring. After 8 hrs. of reaction, the product was heated with m~nPcitlm silicate adsorbent at 120C
for 6 hrs. and filtered. The product was washed twice with deionized water to remove the residual zinc acetate, discarding the aqueous phase. The final diester product was analyzed as 99%
diester by HPLC. Other diesters were synthesized in a similar manner by s--bstit--rin~ appropriate PEG and fatty acid starting m~Pri~lc The resulting diesters were hydrophobic and were therefore dissolved in 95~o alcohol to make stock solutions.
S~nthesis of distear l ether Distearyl ether was synthesized by reacting stearyl bromide (Sigma) with PEG-900 in the presence of potassium 2 ~ 90636 ~
W095/31981 F~ SI~ I67 hydroxide. Stearyl bromide (4.168 g in 10 ml toluene) was slowly added with stirring to 4.64 g of PEG-900 in 10 ml of toluene at 45C. The mi~ture was then stirred at 60C for 30 rnin. and refluxed for 6 hrs. The final product was treated with charcoal, filtered and isolated from toluene.
Synthesis of distearyl amide Distearyl amide was ~yl~ iL.,d by reacting amine-modified PEG (IT,I~ Austin TX) with fatty acid chloride (Sigma). Stearyl chloride (0.61 g; Aldrich) was slowly added with stirring to 0.9 g of amine-modified PEG-900 and 2 moles of potassium hydroxide in a carbon tetrachloride/water mixture at room ~ ul~. The carbon tetrachloride layer was separated and evaporated to produce the white powder of the diamide product (yield 68%).
S~nthesis offatty acid-PEG monoesters To optimize the yield of l~lOIIOC.~ in the ethoxylation reaction, a ~l~c~dul~; different from that for making etho~ylated oleic acid (Exarnple VII) was used. Approximately 1 mole of stearic, oleic or capric acid (Sigma) was mixed with I
mole of PEG 600, PEG 900 or PEG 1500 (Aldrich, Milwaukee, WI) at a ratio of 1:1.1 fatty acid:PEG. The reactions were carried out as ~srnbed (Wrigley et aL, Synthetic detergents from animal fats. Ethenoxylation of fatty acids and alcohols. ~.
Amer. Oil Chem. Soc. 34, 39-43 (1957); for 8-12 hrs. in the presence of I qo para-toluenesulfonic acid catalyst (Sigma). After removal of the water byproduct and the toluene, the final product was dissolved in dichl~lv.~ c and washed with water. The dichloromethane solvent was then evaporated. The reaction products were analyzed by HPLC and found to be approximately 67 to 73qo monoester, the remaining material being diester, PEG
and traces of free fatty acid.
This presumably reflects the l~.lUil~ llt for proper hydrophile-lipophile balance (HLB) of the active molecule. A

-2 1 90~36 ., f~
wo ssalssl r~ 67 series of PEG domains of average MW 600, 900 or 1500 was synth~ci7~1 The results from rho~rnin~123 analyses of these Cu~ )uullds is shown in Table II. The optimal average MW of the PEG domain in diesters was also fouDd to be 900, c~ o~rling to about 20 ethylene o~ide units (ethylene o~ide ~44 MW).
~sLE II
COMPARISON OF RHODAMIN ,123 ACCU~fULATlON IN MDR ECB 8-5-11 CELLS lREATED WII~I ~IESTERS: EFFECT OF DEGREE OF
' 10 ElHOXYLArON AND FATrY ACID
Fa~ty Acid PEG ~IW mg/ml MFII _ 15(No treatment) Capric (C10:0) 600 1 2 _ .'.
Stearic (C18:0) 600 900 1 ' lo '( Oleic (C18:1) 600 1 ,~
,, soo --h ^,~
Linolenic (C18:3) 600 1 .~
lo 40 lo 14 ICi3 8-5-11 cdls were e~posed lo each agent ror 60 mm. at 37C in the presence of 0.5 mg/ml 17~ Accumulation of ,' ' 17~ was oetemuned as descri~ed in Materials a~
Melhods. IMean lluorescence irl ensiy r~sulun8 frr)m imracellular L ' 173; a~era8e Or three c:cpcriments.

-- 2~ 90636 ~
W0 95/31981 3'~ . '167 Example VII
Syn~hesis of Ethoxylated oleic acid Etllv~.yl~t~,d oleic acid was ~yllil~iL.,d as described using established methods (Wrigley et al., Synthetic d~,t~,.~.. lLs from animal fats. Ethenoxylation of fatty acids and alcohols. J.
Amer. Oil Chem. Soc. 34, 39-43 (1957); ~klomll~. Production of alkylene oxide deriYatives. J. Amer. O~l Chem. Soc 33, 571-574 (1956)). Ell!U~YI~ oleic acid was ~y~ ,i~d by reacting ethylene oxide with high purity oleic acid. About 1.38 grams of oleic acid (Sigman Chemical Co., St. Louis, MO) was allowed to react with 4.45 grams of ethylene oxide (Proxair, Inc. Knoxville, TN) in the presence of cesium hydroxide catalyst (Cabot Performance Materials, Revere, PA) at about 100C. The final product is heated with m~n~cillm silicate adsorbent at 120CC for six hours, cooled, and eluted with tetrahydrofuran. The product was isolated by evaporating the t~ dlu~.,l~ solution.
Ethoxylated oleic acid was fractionated by gradient reverse phase liquid chromatography on a Waters LC Module I
with gradient controller (Model 600), auto-sampler (Model 717), -- a UV detector (Model 486), and Millenium Software (Waters, Milford, MA). The sample solution was 25'o ethoxylated oleic acid produced above in dichlulu~lh~c. The sample was applied to a Lichrosorb 5~1 Diol, 250 mm x 4.0 mm column. The injection volume was 200~11 with a flow rate of 2.5 mIJmin. The mobile phases were A: lOO~o dichlolu~l.~.c and B: 1005'o reagent alcohol. The gradient was as follows:
%A %B Curve Initial 100 0 l.O min 100 0 6 (Linear) 5.0 min 95 5 6 (Linear) 8.0 min 100 0 11 (E~amp at 8.0 min) `1 2190636 ,~
,-- WO g5/31981 F~ . '167 Gradient descriptions From 0 to I minute, mobile phase is 100% A and 0% B.
From 1.0 minute to 5.0 minute, mobile phase changes linearly from 100% A and 0% B to 95% A and 5% B.
From 5.0 minute to 8 0 minute, mobile phase CO~ ~ion remains at 95% A and 5% B.
At 8.0 minute, mobile phase changes to lOO~'o A and O5'o B (ramp).
The total run time was 12 minutes and fractions were collected every 30 seconds.
. The various fractions obtained from the reverse phase liquid chromatography were analyzed to determine the RMA activity in each fraction The fractions were then analyzed by infrared spectrophotometry Briefly, in the IR spectrum of unfractionated Etho~ylated oleic acid, bands around 2800 cm-l - 3000 cm-l can be assigned to aliphatic C-H stretching, whereas, band around 1740 cm-l and 1100 cm-l can be assigned to carbonyl stretchirlg (C=O) and ether stretching (C-O-C) respectively. Relative intensity of these bands can be used to distinguish ~etween a monoester and diester type species. For example, diesters have a higher amount of C-H and C=O linkages as compared to C-O-C
linkages on a molar basis. Thus for diesters, one would expect to observe relatively higher inton~ for aliphatic stretching (C-H, 2800 cm-l - 3000 cm-l) and carbonyl stretching (C=O, 1740 cm-~) when compared to intensity for ether stretching (C-O-C, 1100 cm-'). Analysis of IR spectra of active fractions of Ethoxylated oleic acid suggests relatively higher (or equal) ill~r~ s for aliphatic stretching (C-H, 2800 cm-' - 3000 cm-l) and carborlyl stretching (C=O, 1740 cm-') as compared to intensity for ether stretching (C-O-C, 1100 cm-l).
Each sample was analyzed on a Magna IR
Spectrometer, Model 550 When an IR spectral library search was conduc~ed for above fractions, best match was obtained with the 2 ~ ~0~36 WO 95/31981 ~ 6167 specTIum from the IR spectral libraTies. Based on IR results and the, T~ v ~l IR librar,Y search, the actiYe fractions of ElLuAyldtel oleic acid are ~ diester-type species.
The results of the analysis of the fractions are shown in Table III

2~0~36 ~
~1V095/31981 r~l" 167 Table III
Fraction # % DCE % EtOH Amount Relati-~e Mp/# of mg RMA peaks Acti~!ty Corltrol - - - 0-9 100 0 >0.5 24.7 2100 0 >0.5 0.9 399.69 0313 >0.5 0.7 854/2 499.06 0.934 >0.5 10.3 1151/3 598.44 1.563 >0.5 95.3 1363/3 697.81 2.188 0.8 55.7 1488/3 797.19 2.813 0.8 88.9 1586,360/3 896.56 3.438 0.7 73.6 1627/4 995.94 4.063 0.5 84.3 1671/1 1095.31 4.688 0.5 89.4 1693/1 1 19S S 1 92.5 1834/3 1295 5 5.3 87.2 1346/Bb 1395 5 4.8 26.8 18101B
1495 5 l.S 2.2 9.78/l 159S 5 3.8 1.8 131412 1695 5 >0.5 1.3 1432/2 1795-100 5.0-0 >0.5 1 1098/B
18100 0 >0.5 1.4 1282/1 19100 0 >0.5 0.8 1098/B
20100 0 >0.5 1 35711 Col. Wash - - - - 864/1 PEG-Oleic - - - 62.3 1380/2 Acid Oleic Acid - - - 355/l a. Per cen~ cells KB~-S-I I (MDR) showing rhodamine 123 n". Ilr~ ~1 ll r S in the range of sensitive cell (KB3-1) in the same e~perunent .
b. Broad peak Infrared analysis of each fraction irldicated that the active fractiorls con~in~d at least one polyethylene glycol diester.

2 1 90636 ~
W095/31981 r~ ., 5 ~ '167 Example VIII
CREMOPHOR~ EL was fr~-~tion~t~d by super critical extraction. Ap~u~ ly 25 grams of CREMOPHOR~ EL was thoroughly mixed with 20 grams of llyd~UllldlliA (silicon dio~ide) m~nllf~rhlred by Varian - Harbor City, CA 40710 and loaded into a stainless steel high pressure extraction cell. The extraction cell was m~in~in.od at 40C. Supercritical carbon dio~ide was pumped into the extraction cell at a rate of ~iUAil~t~,ly 3 liters of gaseous carbon dioxide per minute (4 rnl of supercritical carbon dioxide per ;runute). The extractable material was dissolved under the extraction contiitinnc, and separated from the matri~. The supercritical carbon dioxide solution with dissolved component was then d~ s~u.i~d to evaporate the carbon dioxide gas, and to ~ iLdl~ the solute. Extraction was done at various pressures of carbon dioxide and fractions were collected at eacb pressure from 2000 psi to 7000 psi with 400 psi intervals.
Under these r"~ 1 conditions, 35 percent of the total CRE~OPHOR~9 preparation was extracted. About 14 percenl of the total CREMOPHOR~ EL preparation was found to have significantly higher RMA activity than the native CREMOPHOR~ EL. The portion of the CREMOPHOR~ that was not extracted had rninirnal RMA activity. The results of the RMA
activity ~ asu~ -Ls on the super critical fractions of CRE~OPHOR~ EL are shown in the following Table IV.

~ 2 1 ~0636 f~
,~ W0 95/31981 F~ 67 Table IV
Agent Relative RMA
Activitya - None 0.5 SOLUrOL~9 (10011g/ml) 73 CREMOPHOR~ EL (10 lg~ml) 3.9 CREMOPHOR~ EL (1O~ ImI) 29 CREMOPHOR~9 F~sc-ions 45B lO,~Lg 1.5 45B 100 llg 4.1 46A 10 ~lg 7.5 46A 100 ~lg 56 46B 10 ~Lg 10 46B ~00 ~lg 73 46C lO~lg 19 46C lO0 ~g 94 46D lO,ug 26 46D 100 ~lg 97 47A 10 ~lg 30 47A 100 llg 99 47B 10 llg 30 47B 100 ~lg 98 47C lOilg 20 47C 100 ~g 97 47D 10 ,ug 24 47D 100 llg 92 48A 10 ~lg 9.8 48A 100 ~lg 82 49A 10 ~lg 11 49A 100 ~lg 74 49B 10 ~lg 6.7 49B 100 ~1~ 66 Per cent ~CB &-5-11 (h~R) cells showing rhodarnLne 123 n ~ ~e L'l the range of sensitiYe cell (KB3-1) in the same ~Y~nml~nt The active fractions were found to be about half as to:cic than native CRE~IOPHOR(~ as shown by ICso mea~ulc~ ."~.

2t 9Q636 f~
W095/31981 P~ 5 ~'167 Example IX
The RMA activity of various fatty acids, PEG
mtmo~st~rs and PEG diesters was l~ l.;..Pd by the method described in E~ample III. The results of these r~l~ .;",~ are S shown in Table V.

--~ 2 1 9~63~ f~
WO95B1981 r~~ 167 Table V
~FI
(T/U)b Agent ~glmla Relati~e KB3-1 KB8-5-II
RMA
Actj~itya None 0.6 1.0 1.0 None + 10 111 EtOH 0.5 1.2 0.9 Stearic Acid 100 2.1 1.9 1.4 Oleic Acid 100 2.7 2.1 1.7 Linolenic Acid 10 1.3 1.2 0.9 100 9.2 1.7 1.7 PEG-6oo-mn~nost.o~r~t~ 10 0.4 1.3 1.1 100 0.6 1.1 1.2 PEG-600-rlict~r~tlo 10 8.6 1.1 2.5 100 31 0.9 5.5 PEG-400-riict.-~rr~A 10 5 o ~ 0 1.8 100 13 0.8 3.6 PEG-900-mon~linolPni~ 10 45 0.6 3.6 100 96 0.5 8.9 PEG-900 dilinolenic 10 98 0.5 11 100 99 0.6 14 PEG-900 dicapric 10 8.7 0.8 1.3 100 21 0.8 2.0 PEG-900 ~ 10 96 0.7 9.8 100 97 0.6 9.9 , Per cent IC3 8-5-11 (MDR) ceDs sbowing rhoda lline 123 fluorescence in the ran8e of IC13 3-1 (sensitive) cells in t~e same eaperimenL
b Mean Flwrescence Intensitr resulting from intracellular rbodamine 123 e~sed ;Is mean nwrescenoe of t~ed oeDslmean fluor scence of untAealed cells (168 for K~33- 1 ~d 6~ forK~38-5-11).
As shown in Table V, PEG 600 stearic acid diester is more actiYe (31%) than PEG 400 stearic acid diester (13~o). The PEG 900 diesters containing two linolenic acids or two stearates had greater activity then the corresponding PEG 900 mlln~s~,-r~
15 The free fatty acids and the Ill.)~.O. ~ only show minimal ~MA

2 1 9~636 ., ~ .
W095/31981 r~ I67 activity. It should also be noted that the PEG 900 dicapric acid (8 carbons) showed minimal activity. The ratio of hydrophil (EO) to l.yd.upllobe (fatty acid) in the G 900 dicapric acid is not optimal.

Example X
FY~mrles X through XX are directed to CRL-1095.
This molecule has the following structure:
H3C(CH2)aXH2CCH20(H2CCHO)bH2CCH2X(CH2)aCH3 wherein the average of "a" is 16 and "b" is 18 and X is an ester linkage This molecule is available from CytRx Corporation, Atlanta, GA.
This exarnple shows the effect of CRL-1095 on ~ho-l~min~ 123 (P-gp substrate) a~cllmlll~tinn in MDR KB 8-5-11 cells in vi~ro.
The cell ~"~"~ in DMEM (5xlO5/ml) is inrllh~rrd with 0.5 ~Lg/ml of Rhodamine 123 and indicated concentrations of CRL-1095. After incubation for 60 min. at 37C, the cell suspension was pelleted and washed in cold D~EM
for ;"""~ flow cytometric analysis (See Example m). The term "MFI" means "mean fl~ x~G~Ice intensity" resulting from intr~r~ r Rho~l~minr-123. Figure 2 shows that CRL-1095 enhanoes the uptake and ~rcllm~ on of E~h~ f 123 within KB 8-5-1 l(resistant) epidermal carcinoma cells in a dose clrp~n~l~n~ manner.
Example XI
Ihis example shows the effect of CRL-1095 on modulation of colchicine .~ ~ce on MDR KB 8-S cells in vitro. 5x105 cells/well were plated in the presence or absence of modulating agent in comhin~ion with colrhi~inr~ and incubated for 96 hours. "Fold Modulation" is equal to IC50 of drug alone/IC50 of drug with resistance modifying agent (RMA).
Figure 3 shows that CRL-1095 modulates colchicine resistance in ~) 2 1 90636 ~
~-- WO95/31981 r~ 'l67 a dose dependent tnanner and CRL-1095 exhibits greater effective mo~ t~lry activity than either cyclosporine or verapamil at equal CO~ n~
S Example XlI
This example shows the effect of CRL-1095 on modulation of VP-16 ~ ,C on MDR KB 8-5-11 cells in vitro.
5~10' cells/well were plated in the presence or absence of CRL, 1095 in comhin~ion with VP-16 and inc~lb~d for 96 hours.
IC50 is equal to the drug cnn~ inn that reduces cell proliferation to 50% of untreated controls using starldard MTI
cell proliferation assay. Figure 4 shows that in KB 8-5-11 cells (resistant), CRL-1095 (I ~Lg/rs~l) completely reverses VP-16 resistance to levels sirnilar in KB 3-1 (sensitive) cells.
Example XIII
This example shows the effect of CRL-1095 on modulation of doxorubicin resistance on MDR KB 8-5-11 cells in vitro. 5xlO' cells/well were plated in the presence or absence of CRL-1095 in combination with Doxorubicin and incubated for 96 hours. IC50is equal to the drug co"~ l".~iorl that reduces cell proliferation to 50% of untreated controls using standard Ml-r cell proliferation assay. Figure 5 shows the IC50 of do~corubicin (dox.) in KB 8-5-11 (resistant) cells in the presence of CRL-1095 (I llg/ml) is similar to that in KB 3-1 (sensitive) cells.
Example XIV
This example shows the effect of CRL- 1095 on modulation of actinomycin resistdnce on MDR KB 8-5-11 cells in vitro. 5x103 cells/well were plated in the presence or absence of CRL-1095 in combination with actinomycin and incllh ~ d for 96 hours. IC50 is equal to the drug c.~ncc~dLion that reduces cell proliferation to 50qO of untreated controls using standard Ml-r cell proliferation assay. Figure 6 shows the IC50 of actinomycin ,~ 2 1 ~0636 ~
WO 95/31981 r~l,. S'~fl~7 D (Act.D) in KB 8-5-11 (resistant) cells in the presence of CRL
1095 (1 llglml) is similar to that in KB 3-1 (sensitive) cells.
F,Y-~rl~ xv This e:~ample shows the effect of CRL-1095 on mo~ lAti.^,n of cQl^hi~ine resistance on MDR KB 8-5-11 cells in vitro. 5~;103 cells/well were plated in the presence or absence of CRL-1095 in comhin~tion with colrhinin~ and inrl~ for 96 hours. IC50 is equal to the drug c~J~ ion that reduces cell proliferation to 50% of untreated controls using the standard MTT cell proliferation assay. Figure 7 whows that the ICso of Colchicine in KB 8-5-11 (resistant) cells in the presence of CRL-1095 (I ~glml) is similar to that in KB 3-1 (sensitive) cells.
Example XVI
Ihis e~cample shows the effect of CRL-1095 on modulation of Taxol l~iakul~e on MI)R KB 8-5-11 Cells in vitro.
5x103 cells/well were plated in the presence or absence of CRL-1095 in combination with Taxol and innuh it~d for 72h. IC50 is equal to the drug con~e~ aLion that reduces cell proliferation to 50% of untreated controls using standard Ml'r cell proliferation assay. Figure 8 shows the IC50 f Taxol in KB 8-5-11 (resistant) cells in the presence of CRL-1095 (I ~gtml) is 5-6-fold reduced.
Example XVII
This e~ample shows the effect of CRL-1095 on modulation of daunorubicin resistance in MDR erythro-leukemic K562/III cells. 5x103 K562/III erythro-leukemic cells /well were plated in the presence or absenoe of CRL-1095 plus increasing arnounts of daunorubicin, and inollhAt~d for 72h. "Fold modulation" is equal to IC50 of drug alone/IC50 of drug with modulator. IC50is equal to the drug ~onr~ ",,.~ion that reduces cell proliferation to 50riiO of untreated controls using standard MTT
cell proliferation assay. Cyclo~ ulille was cytotoxic at 10 ~Lg/ml, and fold modulation was not determined (ND). Figure 9 shows ...... . . .. ..

W0 95131981 r~ 6lc7 the CRL-1095 t~ ,ly modulates Daunorubicin resistance in a dose-~ manner. In addition, CRL-1095 exhibits a 4-fold higher mo~ .J.y effect than verapamil at 10 llg/rnl. At sirnilar doses (I llg/ml) mn~ it)n by CRL-1095 is more effective than S verapamil arld ~,y~
Example XVIII
This e~ample shows the cytotoxicity of CRL-1095 on KB 3-1, KB 8-5, KB 8-5-11, and K562/III cells. As shown itl Figure 10, cells are treated w~th increasing c~ ;ons of CRL-1095 for 72 h. IC50is equal to the drug corl, ~utla~ion that reduces cell proliferation to 50qo of untreated controls using standard MTT cell proliferation assay. Growth is related as % relative to untreated cells. As shown in Figure 10, CRL-1095 is tolerated at relatively high doses in vitro. A comr~ricon of CC50 levels for parental and drug resistant tumor cell lines is shown in Table VI.
CRL-1095 is 4-19 times less cytoto~ic than verapamil or cyclosporine.
TABLE VI
C~ll Line CC~ g/ml) CRI~ 095 Ver~ mil Cyt', ' ~
KB 3- . 34 4-J SO
KB ~ 19 ~D
~8-5- 1 >2~ ~ 42 K562/r: 14 ~ 22 Example XIX
This e~ample shows the acute toxicity of CRL-1095 in C57BU6 mice. C57BV6 mice were ~rlminict~red via i~ aY~;nuus route a bolus injection at conc~ntr~tionc indicated in Table VII, or . with vehicle control. Survival was morlitored through 14 dàys post injection. As seen in Table VII, in vivo studies in C57BV6 mice indicate that CRL-1095 is tolerated at relatively high doses.
MaYimum tolerated dose (MTD) of CRL-1095 in which all mice ,~ 21 90b36 ~3 WO 9513 198 1 ~ '7 survived is S00 mg/kg. MTD of Verapamil is 75 mg/kg (Horton, et aL, 1989) and Cy~lrl~r~lrine is 30 mglKg (personnel c-""~
with Dr. plowman at NCI).

TABLE VII
Dose tnlg/k~) Sun~iv-l RMA CRL~1095 P.Ys-mrle XX
This example shows the effect of CRL-1095 on vincristine arcllmlll~tion in ECB 8-5 human xenograft hlmor tissue in mice. Nude mice carrying the human ~enograft tumor, KB 8-S, were given 3H-Vincristine (VCR) (2 mglkg, i.p.) as a single injection with or without CRL-1095 (50 mglkg, i.v.). At various ime points following drug ~rlminic~ion tissues were removed and counted for radioactivity. Figure 12 shows that ~lld~,,lOUS
(IV) ad~ L,aLion of CRL-1095 increases ~r~ alon of H-Vincristine 4 - 6 fold in human xenograft tumor tissue in mice.
Example XXI
Examples XXI through XXV are directed to CRL-1605. This molecule has the following structure:
HO(C3H60)a(C2H40)b(C3H60)aH
wherein the average of "a" is d~ a~t:ly 40 and "b" is approximately 20 and is ~l~ci~n~d CRL 1605. This molecule is available from CytRx Corporation, Atlanta, GA.

2 1 90636 =
.--. ~ '.~
W0 95131981 ~ _.'C '1 67 In a 2 liter stainless steel pressure reactor, 3.7 grams of dry cesiurn hydro~ide ~ uo~ dLdle and 14.5 grarns of ethyle glycol are rni~ced and the reactor is eYaculated. The contents are heated at 100C and 200 gra~ns of ethylene o~ide is slowly S allowed to react to produce a pol~,;hyl~,e glycol polymer of molecular eight of about 875 daltons. 1084 grarns of propylene oxide then is slowly allowed to react with the contents in the reactor to produce a block copolymer with a m~'.e l~r weight of about 5040 daltons. The contents are heated with 18.4 grarns of m~n~ci~lm silicate and 1.8 grams of silica at 100C for 6 hours to adsorb the catalyst. The block copolymer product is. isolated by filtration. The final product has a cloud point of 11.3C.
This example shows the effect of the CRL-1605 o~
modulation of Taxol resistance on MDR KB 8-5 cells. 5XI03 cells/rnl were plated in the presence or absence of CRL-1605 in ~o.l.l,il.~Lion with Taxol and in~ b~t.od for 72h. Figure 12 shows the IC50 of Taxol in KB 8-5 (R) cells is 2-fold reduced in the presence of CRL-1605 (I ~lg/ml).
Example XXII
This example shows the effect of CRL~1605 on mo~ tion of daunorubicin resistance on MDR K562/III cells.
5XI03 cells/rnl were plated in the presence or absence of CRL-1605 in combination with Daunorubicin and ;,., ~ l- t~ d for 72h.
Figure 13 shows the ICso of Daunorubicin in K562/III (R) is 40-fold reduced in the presence of CRL-1605 (10 ~g/rnl).
Example XXIII
This example shows the effect of CRL-1605 on vincristine accllm~ ti-n in KB 8-5 hu}nan xenograft tumor tissue in mice. Nude mice carrying the human xenograft tumor, KB 8-5, were given 3H-Vincristine (VCR) (2 mg/kg, i.p.) as a single injection with or without CRL-1605 (50 mg/kg, i.Y.). At various time points following drug administration, tissues were removed and counted for radioactivity. Figure 14 shows that the .. ..

~ -, 219063~ ~
Wo 9s/31s81 ~ ,,5~ 'l67 intravenous (IV~ A~minictr~tinn of CRL-1605 increases af,,...,...lA~;f~n of H-Vincristine 4 - 6 fold in human xenogrdft tumor tissue in mice.
F~ XXIV
This example shows the acute toxicity of CRL-1605 in C57BI/6 mice. In vivo studies in C57BI/6 mice indicate that CRL-1605 is tolerated at relatively high doses. As shown in Table VI~, the IIIA~;IIIIIIII tolerated dose (MTD) of CRL-1605 is 350 mg/kg. At 400 mg/Kg mice were slightly ill. MTD of Verapamil is 75 mglkg (Horton, et al., 1989) and the MTD of cyclf)crorin~ is 30 mg/Kg (personnel comm~nicAtif~n with Dr. Plowman at National Cancer Institute).
TABLE VIII
Do~e (mglkg) Survivsl RMA CRI,1095 loo ~5 200 s/s 400 3n soo ol5 c40 o/s Example XXV
This example shows the mo~ tlnn of tumor growth in KB 8-5 human xenograft model by CRL-1605. Hurnan KB 8-S
nlmors (2 rnm~) were imrl~An~f-d on both sides of the hind fldnk in nude ~uce (strain NCRlNu/NCI). Two weeks following implantation, Ll~dnLI~ was ~.l.";";cl ~d on days 1, 5, and 23 as follows: Doxorubicin only animals received doxorubicin 8 mg/kg. Doxorubicin + vehicle animals received the same dose of do~orubicin plus the equivalent volume of CRL-1605 vehicle.
Doxorubicin + CRL-1605 animals received the sdme dose of ,-~ 2 1 90636 W0 95131981 P. ~ S~C7 do~orubicin plus a total daily dose of 100 mgAcg CRL~1605 slAmini ~,d as 4 individual doses of 25 mg/kg at 2 hour intervals. Tumor volume was rneasured el~ lly using calipers. Each point ~ GIlla the average of 4-8 tumors.
It should be ~lntl~rstoo~ of course, that the f~lG~iUg relates only to a preferred ~ ,ol~ of the present inventio~
and that ~U~ Jua mrltlifir~ionc or ~lr,~ .ol~ may be n~ade therein without departing from the spirit and the scope of the invention as set forth in the 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 administration to the human or animal with cancer an effective amount of a compound with the following general formula:
H3C(CH2)aXH2CCH2O(H2CCHO)bH2CCH2X(CH2)aCH3 wherein the average of "a" is between approximately 10 and 30 and "b" is between 4 and 100 and X=CO-O (ester), CO-NH
(amide) or CH2-O (ether).

2. The method of Claim 1, wherein the polyethylene glycol has between approximately 8 and 60 ethylene oxide units.

3. The method of Claim 1, wherein the polyethylene glycol has between approximately 15 and 60 ethylene oxide units.

4. The method of Claim 1, wherein the polyethylene glycol has approximately 20 ethylene oxide units.

5. 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.

6. The method of Claim 1, wherein the fatty acid is selected from the group consisting of stearic acid, 12-hydroxystearic acid, oleic acid, palmitic acid, and ricinoleic acid.

7. The method of Claim 1, wherein the fatty acid has between approximately 8 and 60 carbon atoms.

8. The method of Claim 1, wherein the fatty acid has between approximately 12 and 50 carbon atoms.

9. The method of Claim 1, wherein the fatty acid has approximately 18 carbon atoms.

10. A method for potentiating the cytotoxicity of a chemotherapeutic agent in a human or animal with a cancer exhibiting multidrug resistance comprising the administration to the human or animal with cancer an effective amount of a compound with the following general formula:
H3C(CH2)aXH2CCH2O(H2CCHO)bH2CCH2X(CH2)aCH3 wherein "a" is between approximately 10 and 30 and "b" is between 4 and 100 and X=CO-O (ester), CO-NH (amide) or CH2-O (ether).

11. The method of Claim 10, wherein the polyethylene glycol has between approximately 8 and 60 ethylene oxide units.

12. The method of Claim 10, wherein the polyethylene glycol has between approximately 15 and 60 ethylene oxide units.

13. The method of Claim 10, wherein the polyethylene glycol has approximately 20 ethylene oxide units.

14. The method of Claim 10, 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.

15. The method of Claim 10, wherein the fatty acid is selected from the group consisting of stearic acid, 12-hydroxystearic acid, oleic acid, palmitic acid, and ricinoleic acid.

16. The method of Claim 10, wherein the fatty acid has between approximately 8 and 60 carbon atoms.

17. The method of Claim 10, wherein the fatty acid has between approximately 12 and 50 carbon atoms.

18. The method of Claim 10, wherein the fatty acid has approximately 18 carbon atoms.

19. A composition for treating multidrug resistant human cancer cells in a human or animal comprising a compound with the following general formula:
H3C(CH2)aXH2CCH2O(H2CCHO)bH2CCH2X(CH2)aCH3 wherein "a" is between approximately 10 and 30 and "b" is between 4 and 100 and X=CO-O (ester), CO-NH (amide) or O
(ether).

20. The composition of Claim 19, wherein the polyethylene glycol has between approximately 8 and 60 ethylene oxide units.

21. The composition of Claim 19, wherein the polyethylene glycol has between approximately 15 and 60 ethylene oxide units.

22. The composition of Claim 19, wherein the polyethylene glycol has approximately 20 ethylene oxide units.

23. The composition of Claim 19, 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.

24. The composition of Claim 19, wherein the fatty acid is selected from the group consisting of stearic acid, 12-hydroxystearic acid, oleic acid, palmitic acid, and ricinoleic acid.

25. The composition of Claim 19, wherein the fatty acid has between approximately 8 and 60 carbon atoms.

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

27. The composition of Claim 19, wherein the fatty acid has approximately 18 carbon atoms.

28. A composition for reversing multidrug resistance comprising:
HO(C3H6O)a(C2H4O)b(C3H6O)aH
wherein "a" is between approximately 20 through 60 and "b" is between approximately 10 and 30.

29. The composition of Claim 28 wherein "a" is between approximately 30 and 50 and "b" is between approximately 15 and 25.

30. The composition of Claim 28, where "a" is approximately 40 and "b" is approximately 20.

31. 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(C3H6O)a(C2H4O)b(C3H6O)aH
wherein a is between approximately 20 through 60 and b is between approximately 10 and 30.

32. The method of Claim 31, where "a" is approximately 40 and be is approximately 20.

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

34. A method for treating a human or animal with an infection caused by a microorganism exhibiting multidrug resistance comprising the administration to the human or animal with the infection an effective amount of a compound with the following general formula:
HO(C3H6O)a(C2H4O)b(C3H6O)aH
wherein a is between approximately 20 through 60 and b is between approximately 10 and 30.

35. The method of Claim 34, where "a" is approximately 40 and be is approximately 20.

36. A method for treating a human or animal with an infection caused by a microorganism exhibiting multidrug resistance comprising the administration to the human or animal with the infection an effective amount of a compound with the following general formula:
H3C(CH2)aXH2CCH2O(H2CCHO)bH2CCH2X(CH2)aCH3 wherein "a" is between approximately 10 and 30 and "b" is between 4 and 100 and X=CO-O (ester), CO-NH (amide) or O
(ether).

37. The method of Claim 36, wherein the polyethylene glycol has between approximately 8 and 60 ethylene oxide units.

38. The method of Claim 36, wherein the polyethylene glycol has between approximately 15 and 60 ethylene oxide units.

39. The method of Claim 36, wherein the polyethylene glycol has approximately 20 ethylene oxide units.

40. The method of Claim 36, 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.

41. The method of Claim 36, wherein the fatty acid is selected from the group consisting of stearic acid, 12-hydroxystearic acid, oleic acid, palmitic acid, and ricinoleic acid.

42. The method of Claim 36, wherein the fatty acid has between approximately 8 and 60 carbon atoms.

43. The method of Claim 36, wherein the fatty acid has between approximately 12 and 50 carbon atoms.

44. The method of Claim 36, wherein the fatty acid has approximately 18 carbon atoms.

45. A method of increasing the tissue level of tetracycline in a human or animal comprising the administration to the human or animal of tetracycline and a compound with the following general formula:
H3C(CH2)aXH2CCH2O(H2CCHO)bH2CCH2X(CH2)aCH3 wherein "a" is between approximately 10 and 30 and "b" is between 4 and 100 and X=CO-O (ester), CO-NH (amide) or O
(ether).

46. A method of increasing the tissue level of tetracycline in a human or animal comprising the administration to the human or animal of tetracycline and a compound with the following general formula:
HO(C3H6O)a(C2H4O)b(C3H6O)aH
wherein a is between approximately 20 through 60 and b is between approximately 10 and 30.