METHODES ET COMPOSITIONS POUR REDUIRE LA RESISTANCE POLYMEDICAMENTEUSE

METHODS AND COMPOSITIONS FOR REDUCING MULTI-DRUG RESISTANCE

Abrégé anglais

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.

Revendications

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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.

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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.

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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.

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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.

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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.

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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:
<IMG>
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.

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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:
<IMG>
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.

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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:
<IMG>
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.

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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:
<IMG>
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.

Description

WO 93/11668 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

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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

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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.

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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.

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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
N H2C -CH2 N
(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:

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(C2H40)a~c3H6o)b~ / (C3H6)b(C2H4)a
N H2C -CH2 N
(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.

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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
: : ~.

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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

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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.
.:

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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
2125279
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

WO 93/11668 2 1 2 5 2 7 9 PCI/US92/10563
- 14-
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 ~ :
- 15-
(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
NH2C-CH2 N
(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
- 16-
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.

WO 93/1 1668 PCI /US92/10563
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
2125279
- 18 -
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
- 19 -
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 ,~

- - WO 93/1 1 668 PCI /US92/10563
2125279
- 21 -
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
- 23 -
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.