Note: Descriptions are shown in the official language in which they were submitted.
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WO97/1881g PCT~S96/18695
ANTINEOP~ASTIC METHODS AND COMPOSITIONS EMPLOYING
OPTICA~LY PURE (-)-FOTEhu~ 'l'LN~
FIE~ OF T~E INVENTIQN
This invention relates to pharmaceutical
compositions cont~'n~ng (~ otemustine. In another
aspect this invention relates to treatment o~
malignancies, including lymphomas, melanomas, gliomas
and other brain tumors, lung cancer, liver metastases
and hematological and gastrointestinal malignancies.
BACKGROUND OF TH~ INVENTION
In the standard nomenclature o~ Chemical
Abstracts, ~otemustine is [l-[[[~2-chloroethyl)
nitosoamino~carbonyl~amino]ethylphosphonic acid
diethyl ester. Fotemustine is also known as 1-[N-(2-
chloroethyl)-N-nitrosoureido~ethylphosphonic acid
diethyl ester and as diethyl-1-[3-(2-chloroethyl)-3-
nitrosoureido]ethylphosphonate. Early re~erences
re~er to ~otemustine as S10036. The preparation o~
racemic ~otemustine is described in U.S. Patent
4,567,169. The patent mentions that "the invention
also relates to the optical isomers o~ the
compounds...", but no such isomers are disclosed.
Racemic rotemus~ine has been used 'clinically to
treat malignan~ melanoma, ~rimary and metastatic
brain tumors, colorectal cancer, so~t tissue and bore
sarcomas and hematological malisnancies. [See .~hayat
et al. J. Nat'l. Cancer Inst. 80, 1407-1408 (1988);
Khayat et al. Cancer Res. 47, 6782-6785 (1987);
Jacquillat et al. Proc. Am. Assoc. C~cer Res. 30,
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WO97/18819 PCT~S96/18695
--2--
A1088 (1989); Rougier et al. Eur. J. Cancer 2~A~2),
288-9 (1993); Kerbrat et al. Eur J. Cancer 2~Arl),
143-144 ~1993)]. These clinical studies indicate
that racemic fotemustine is clinically use~ul in
treating these cancers, ~ut its use is accompanied by
delayed, cumulative toxicity in bone marrow. The
dose-limiting toxicity is usually myelosuppression
leading to neutropenia, leucopenia and
thrombocytopenia. Renal, pulmonary and hepatic
toxicity are observed with a lesser frequency.
Racemic ~otemustine is also believed to be mutagenic
~See Ashby et al. Mutation Res. 286, lO1-109 (~993)].
Nausea is a problem in some cases.
It would be particularly desirable to find a
compound with the advantages of the racemic mixture
of fotemustine but without the side effects
enumerated above.
SUMMARY OF THF lN V~N 1 ION
It has now been discovered that the optically
pure (-) isomer of ~otemustine is an ef~ective agent
~or treating malignancies. The optically pure (-)
isomer of fotemustine provides this effective
treatment while substantially reducing one or more
ad~erse ef~ects associated with administration of
racemic ~otemustine including, but not limited to,
neu~ropenia, leucopenia ard thrombocytopenia; renal,
~ulmonary and hepatic toxicity; mutagenicity; and
nausea.
In one aspect the invention relates to a method
o~ treating a malignancy which comprises
.
.
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WO97/18819 PCT~S96/1869
administering to a ~mm~ 1 su~ering ~rom the
malignancy, a therapeutically e~ective amount o~
c (~ otemustine, substantially ~ree o~ its (+)
stereoisomer.
In another aspect, the invention relates to a
pharmaceutical composition which comprises (-)-
~otemustine, substantially free of its (+)
stereoisomer, and a pharmaceutically acceptable
carrier.
DET~ILED DESCRIPTION OF THE INVENTION
The active compound used in the compositions and
methods o~ the invention is the levorotatary or (-)
optical isomer of ~otemustine. The absolute
stereochemistry o~ the (-) isomer is presently
unknown. Formula I indicates a single enanticmer o~
undetermined absolute stereochemistry, here presented
arbitrarily as S~:
E t 3 3~ ~ ~l o
E; C~ ~ N~
The sraphic representation o~ enantiomerically pure
~otemustine is taken from Maehr J. Chem. E-. 62, ll~-
120 (1985). Thus representations involvins wedge
outlines and dotted or broken lines denote
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WO97/18819 PCT~S96/18695
enantiomerically pure compounds of indeterminate
absolute configuration.
The present invention encompasses a method of
treating a malignancy which comprises administering a
5 therapeutically effective amount of ~-)-fotemustine,
substantially free of its (+) stereoisomer.
The term "substantially ~ree of its (+)
stereoisomer" as used herein means that the
compositions contain at least 9O~ by weight of (-)-
~otemustine and lO~ by weight or less of (+)
~otemustine. In a more preferred embodiment the term
llsubstantially free of the (+) isomer" means that the
composition contains at least 99~ by weight of (-)-
fotemustine, and l~ or less o~ (+) fotemustine. In
the most preferred embodiment, the term
"substantially free o its (+) stereoisomer" as used
herein means that the composition contains greater
than 99~ by weight o~ fotemustine. These
percentages are based upon the total amount of
fotemustine in the composition. The terms
"substantially optically pure (-) isomer of
fotemustine" or "substantially optically pure (-)-
fotemustine" and "optically pure (-) isomer of
fotemustinen and ~loptically pure (-)-fotemustine" are
also encompassed by the abov~-described amounts.
The term "treatlng a malignancyl~ as used herein
means treating, alleviating or palliating such
condition, suppressing the growth o~ cancerous tissue
and thus providing increased survival time.
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The term "therapeutically effective amount"
refers to that amount of ~-)-fotemustine which is
sufficient to treat a malignancy. Malignancies
against which (-) fotemustine is active include
lymphomas, melanomas, gliomas and other brain tumors,
lung cancer, liver metastases and hematological and
gastrointestinal malignancies.
The method of the invention reduces the
concomitant liability of adverse effects associated
with the administration of racemic fotemustine. The
term "adverse effects" includes, but is not limited
to, neutropenia, leucopenia and thrombocytopenia;
renal, pulmonary and hepatic toxicity; mutagenicity;
and nausea.
In the method of the invention a therapeutically
e~fective amount may be ~m;n~ stered in a single
dose, or a ~raction of a therapeutically effective
amount may be ~mi n~ stered in each of several doses.
hikewise a pharmaceutical composition of the
invention may contain a therapeutically e~fective
amount of (-)-fotemustine or it may be necessary to
~mi n, ster several individual compositions in order
to achieve therapeutic effectiveness.
The amount of (-)-fotemustine that constitutes a
therapeutically effective amount varies with the
severity and nature of the condition to be treated
and the route of administration. Dose size and dose
~requency may also vary according to the age, body
weight and response of the individual patient. In
general, the dose range for a single administration
of (-)-~otemustine for the conditions described
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herein is from about 20 mg/kg to about 150 mg/kg by
intravenous or intraarterial in~usion. Preferably a
single dose range should be about 20 mg/m2 to about 80
mg/m2. Generally, dosing is carried out periodically
(e.g., weekly) ~or a period o~ several weeks (e.g.,
about two to about to about eight weeks).
In managing the patient, therapy should be
initiated at a lower dose, perhaps at about 20 mg/m2
to about 40 mg/m2, and increased up to about 40 mg/m~
or higher depending on the patient's global response.
It is ~urther reco~m~n~ed that patients over 65 years
of age and those with impaired renal or hepatic
~nction initially receive low doses and that they be
titrated based on individual response(s) and blood
level (8) . It may be necessary to use dosages outside
these ranges in some cases, as will be apparent to
those skilled in the art. Further, the clinician or
treating physician will know how and when to
interrupt, adjust, or terminate therapy in
conjunction with individual patient response. The
term "an amount su~ficient to suppress cancer but
insu~icient to cause said adverse e~ects" is
encompassed by the above-described dosage amounts and
dose ~requency schedule.
2s Any sui~able route of administration may be
employed ~or providing the patient with an efIective
dosage of (-)-fotemustine, but parenteral
(particularly intravenous and intraarterial) forms of
adminis~ration are preferred Dosage forms ir.clude
dispersions, suspensions, solutions, and the like.
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The pharmaceutical compositions of the present
invention comprise (-)-fotemustine as the active
ingredient, and may also contain a pharmaceutically
acceptable carrier, and optionally, other adjuvants,
5 excipients, or therapeutic ingredients. Compositions
suitable for parenteral administration may be
presented as an a~ueous solution, optionally
containing conventional adjuvants, and excipients.
Such compositions may be prepared by methods well
10 ~nown to those s~illed in the art of pharmacy. All
methods include the step o~ bringing into association
the active ingredient with the carrier which
constitutes one or more necessary ingredients.
The chemical synthesis of the racemic mixture of
15 fotemustine can be performed by the method described
in U.S. Patent 4,567,169 cited above. The (-) isomer
o~ fotemustine may then ~e resolved by chromatography
on a chiral medium. Alternatively, (-)-fotemustine
may be obtained by resolution of the enantiomers of
20 the amine precursor to fotemustine using fractional
crystallization or chromatography of diastereomeric
esters of chiral acids. Other standard methods of
resolution known to those skilled in the art can also
be used. (See for example, E.L. Eliel,
25 Stereochemistry of Carbon Compounds, McGraw Hill
~19~2) and [Wilen and Lochmuller, "Tables of
Resolving Agents", Journal of ChrQmatoqra~h~ 113 t
28~-302 (1975)].
,,
Relative activity, potency and specificity of
r 30 optically pure fotemustine and racemic fotemustine as
an antineoplastic agent can be determined by
pharmacological studies in vi tro and in vivo .
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The compounds are tested for their capacity to
increase the life-span of mice bearing tumorous cells
inoculated by intraperitoneal or intramuscular route
in accordance with the protocols established by the
National Cancer Institute (U.S.A.) and published by
R.I. Geran et al. in ~ancer ChemotheraPY Re~orts,
part III, Vol. 3(2), 1-87 (1972).
Hematopoietic toxicity_is assessed, in animals
treated with one or several administrations of the
compound according to the invention, by the count of
the peripheral blood cells and of the bone marrow,
and the stem cells contained in the bone marrow [Till
and McCulloch, Radiation Res. 14, 213 (1961)]. The
lowest cellular concentrations are noted 3 days after
the start of treatment. The extent of this decrease
is measured a~ter treatment with one dose of N-N~-
bis(2-chloroethyl)-N-nitrosourea ("BCNU") used as a
reference tTang and Eisenbrand, Arch. Pharm. 314, 9lO
(1981)~ and is compared to the cell count after
racemic fotemustine and (+) fotemustine.
Hepatic toxicity is assessed according to
Wroblewski's method by measuring the pyruvic glutamic
trans~ se activity in serum from Long Evans rats
treated with an intraperitoneal injection o~ doses of
racemic, ~+) and (-) ~otemustine.
Mutagenicity is assessed Dy the method of
Tapiero et al. ~Anticancer Research 9, 1617-1622
(1989)] as follows:
Human lung carcinomas A427 and A549 and human
colon carcinomas BE and HT29 are obtained and
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maintained as described by Kohn et al. rCancer
Chemother. Pharmacol 19, 291-295 (1987) and Cancer
Res. 48, 3622-3625 (1988)]. Murine leukemia cell
line P388 is obtained from NCI. Cells are grown and
maintained in RPMI 1640 medium supplemented with lO~
~etal bovine serum, lO-sM 2-mercaptoethanol and
standard antibiotics. All cultures are grown at 38~
in a humidified atmosphere o~ 5~ CO2. Cell growth
inhibition studies are carried out on exponential
cultures in the continuous presence of drug.
Resultant cell numbers are estimated on a Coulter
Counter model ZBI, Coulter Electronics, Hialeah, Fl.
Racemic, (+~ and (-) fotemustine are
individually dissolved in ethanol immediately be~ore
use. Convenient standard stock solutions are 35 mM.
For radioisotope labelling and X-irradiation 2.5X lOs
P388 cell/mL are grown in medium cont~in;ng either
O.05~Ci/mL of (14C)-thymidine or 0.5~Ci/mL of (3H)-
thymidine. After labelling for 20 hours, medium is
removed and cells washed with cold PBS. Standard
chase treatments using lO~M non-radioactive th~midine
~or 4 hours are employed. Tritiated cells are
expo~ed, on ice, to 300 rads of X-irradiation as an
internal standard. The alkaline elution procedure is
described by Kohn e~ al. ~Measurements of strand
breaks and cross l~nkc by alkalire elut~on." In:
Frieberg E. and Hanawalt P. (eds), DNA repair: A
laboratory m~n1l~l of research procedures, New York,
' Dekker, l: 1981, pp. 379-401.] To determine the
relationship between preincubation of drug in
complete growth medium and loss of cytotoxic
activity, drugs are resuspended in medium containing
lO~ fetal calf serum and incubated at 37O for l to
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500 minutes. P388 cells are resuspended in the
medium diluted to reach the appropriate drug
concentrations. After 3 days incubation at 5~ CO2,
cells are counted.
Induction of single strand breaks in P388 cells
exposed to racemic fotemustine, ~+) fotemustine and
~ otemus~ine i5 compared. Cells (5XlO~) labelled
20 hours by (l4C)-thymidine are treated ~or 2 hours
with various concentrations of ~otemustine and mixed
with a similar number of (3H)-thymidine labelled
reference cells. These cells are collected on 2.0 ~m
pore size polycarbonate ~ilters and lysed with 5 mL
o~ 2~ SDS/0.025 M Na4EDTA ~pH9.7) in the presence o~
proteinase K. Elution is carried out in the dark at
pH 12.l as described by Kohn. DNA single strand
break ~requencies are calculated on the basis of
~irst order elution kinetics.
Induction of DNA-Protein cross-links is examined
in P388 cells treated ~or various times in the
presence of racemic fotemustine, (+) ~otemustine or
~-) fotemustine. (l4C)-Thymidine-labelled drug treated
cells are irradiated with 600 rads o~ X-rays and (3H)-
thymidine labelled reference cells are irradiated
with 300 rads of X-rays. These cells are collected
on 2.0~m pore-size polycarbonate ~ilters and lysed at
pH 9.7 in the absence of proteinase K. Elution is
the same as above.
Removal of DNA single strand breaks in ~ 388
cells at various times after exposure to
concentrations of racemic fotemustine and its
enantiomers causing equivalent DNA damage can also be
_
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--11--
m; n~d. The (l~C)-thymidine labelled cells
initially exposed to drug treatment ~or 2 hours are
washed and resuspended in drug- f ree medium. A~ter
di~ferent times of incubation at 37~, these cel~s are
mixed with an equal number of ~3H)-thymidine labelled
reference cells, collected on the polycarbonate
~ilters and analyzed by alkaline elution.
The ~oregoing tests provide an estimate o~
relative activity, potency and selectivity.
Emesis in Ferrets. Experiments are performed on
adult male ~errets. The ~im~l S are ~irst adapted to
wearing a nylon jacket connected to a stainless-steel
cable, which in turn is attached to a brass swivel at
the cage top. After habituation to the tether-
harness, each ~n;m~l receives a surgically implanted
catheter in its right jugular vein. The catheter is
~lushed daily with heparinized sodium chloride. The
drug studies are conducted l week a~ter the surgical
procedure. Tethered animals are individually housed.
Eight to eleven ~nim~l s are used to evaluate
each dose of each test compound. Individual animals
are weighed weekly and randomly given, at greater
than 48 hour intervals, a single i.v. or p.o. dose of
racemic ~otemustine, (+)-~otemustine or (-)-
~otemustine. At least three dose levels o~ each test
compound are evaluated.
Individual animals are observed ~or 30 minutes
~ollowing administration o~ the test substance. The
~requency of, and latency to all expulsions, retches
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and de~ecations are recorded. Data obtained ~rom
dose-response curves is tested ~or statistical
significance by chi-square analysis. EDso values are
determined ~or each compound. The test provides an
estimate o~ relative liability to nausea and
vomiting.
