AU7229500A - Antineoplastic methods and compositions employing optically pure (-)-fotemustine - Google Patents

Description

ANTINEOPLASTIC METHODS AND COMPOSITIONS EMPLOYING OPTICALLY PURE (-)-FOTEMUSTINE FIELD OF THE INVENTION This invention relates to pharmaceutical compositions containing (-)-fotemustine. In another aspect this invention relates to treatment of malignancies, including lymphomas, melanomas, gliomas and other brain tumors, lung cancer, liver metastases and hematological and gastrointestinal malignancies.

BACKGROUND OF THE INVENTION In the standard nomenclature of Chemical Abstracts, fotemustine is (2-chloroethyl)nitosoamino] carbonyl] amino] ethylphosphonic acid diethyl ester. Fotemustine is also known as 15 chloroethyl)-N-nitrosoureido] ethylphosphonic acid diethyl ester and as diethyl-1-[3-(2-chloroethyl)-3nitrosoureido] ethylphosphonate. Early references refer to fotemustine as S10036. The preparation of racemic fotemustine is described in U.S. Patent 20 4,567,169. The patent mentions that "the invention S..also relates to the optical isomers of the compounds...", but no such isomers are disclosed.

Racemic fotemustine has been used clinically to treat malignant melanoma, primary and metastatic brain tumors, colorectal cancer, soft tissue and bone sarcomas and hematological malignancies. [See Khayat et al. J. Nat'1. Cancer Inst. 80, 1407-1408 (1988); Khayat et al. Cancer Res. 47, 6782-6785 (1987); Jacquillat et al. Proc. Am. Assoc. Cancer Res. A1088 (1989); Rougier et al. Eur. J. Cancer 29A(2), 288-9 (1993); Kerbrat et al. Eur. J. Cancer 29A(1), 143-144 (1993)]. These clinical studies indicate that racemic fotemustine is clinically useful in treating these cancers, but 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 fotemustine is also believed to be mutagenic- [See Ashby et al. Mutation Res. 286, 101-109 (1993)].

Nausea is a problem in some cases.

It would be particularly desirable to find a 15 compound with the advantages of the racemic mixture of fotemustine but without the side effects enumerated above.

SUMMARY OF THE INVENTION ~It has now been discovered that the optically pure isomer of fotemustine is an effective agent for treating malignancies. The optically pure isomer of fotemustine provides this effective treatment while substantially reducing one or more adverse effects associated with administration of racemic fotemustine including, but not limited to, neutropenia, leucopenia and thrombocytopenia; renal, pulmonary and hepatic toxicity; mutagenicity; and nausea.

In one aspect the invention relates to a method of treating a malignancy which comprises -3administering to a mammal suffering from the malignancy, a therapeutically effective amount of (-)-fotemustine, substantially free of its stereoisomer.

In another aspect, the invention relates to a pharmaceutical composition which comprises fotemustine, substantially free of its stereoisomer, and a pharmaceutically acceptable carrier.

DETAILED DESCRIPTION. OF THE INVENTION The active compound used in the compositions and :"methods of the invention is the levorotatary or optical isomer of fotemustine. The absolute stereochemistry of the isomer is presently 15 unknown. Formula I indicates a single enanticmer of undetermined absolute stereochemistry, here presented arbitrarily as S': I 120 (1985). Thus reions involving wedge outlines and dotted or broken lines deno

I

The graphic representation of enantiomericaly pure fotemustine is taken from Maehr J. Chem. Ed. 62, 11- 120 (1985). Thus representations invoiving wedge outlines and dotted or broken lines denote enantiomerically pure compounds of indeterminate absolute configuration.

The present invention encompasses a method of treating a malignancy which comprises administering a therapeutically effective amount of (-)-fotemustine, substantially free of its stereoisomer.

The term "substantially free of its stereoisomer" as used herein means that the compositions contain at least 90% by weight of fotemustine and 10% by weight or less of fotemustine. In a more preferred embodiment the term "substantially free of the isomer" means that the composition contains at least 99% by weight of fotemustine, and 1% or less of fotemustine. In S" 15 the most preferred embodiment, the term 99 "substantially free of its stereoisomer" as used herein means that the composition contains greater than 99% by weight of (-)-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 fotemustine" and "optically pure (-)-fotemustine" are S 25 also encompassed by the above-described amounts.

The term "treating a malignancy" as used herein means treating, alleviating or palliating such condition, suppressing the growth of cancerous tissue and thus providing increased survival time.

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 LO 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.

15 In the method of the invention a therapeutically effective amount may be administered in a single dose, or a fraction of a therapeutically effective amount may be administered in each of several doses.

Likewise a pharmaceutical composition of the invention may contain a therapeutically effective amount of (-)-fotemustine or it may be necessary to administer several individual compositions in order to achieve therapeutic effectiveness.

I 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 frequency 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 (-)-fotemustine for the conditions described -6herein is from about 20 mg/kg to about 150 mg/kg by intravenous or intraarterial infusion. Preferably a single dose range should be about 20 mg/m 2 to about mg/m 2 Generally, dosing is carried out periodically weekly) for a period of several weeks 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/m 2 to about 40 mg/m 2 and increased up to about 40 mg/m? or higher depending on the patient's global response.

It is further recommended that patients over 65 years of age and those with impaired renal or hepatic function initially receive low doses and that they be titrated based on individual response(s) and blood S, 15 level(s) 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 sufficient to suppress cancer but insufficient to cause said adverse effects" is encompassed by the above-described dosage amounts and dose frequency schedule.

25 Any suitable route of administration-may be employed for providing the patient with an effective dosage of (-)-fotemustine, but parenteral (particularly intravenous and incraarterial) forms of administration are preferred. Dosage forms include dispersions, suspensions, solutions, and the like.

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, excipients, or therapeutic ingredients. Compositions suitable for parenteral administration may be presented as an aqueous solution, optionally containing conventional adjuvants, and excipients.

Such compositions may be prepared by methods well known to those skilled in the art of pharmacy. All methods include the step of 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 of fotemustine may then be resolved by chromatography on a chiral medium. Alternatively, (-)-fotemustine may be obtained by resolution of the enantiomers of S 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, Stereochemistry of Carbon Compounds, McGraw Hill (1962) and [Wilen and Lochmuller, "Tables of Resolving Agents", Journal of Chromatoaraphv 113, 283-302 (1975) Relative activity, potency and specificity of opcically pure fotemustine and racemic fotemustine as an antineoplastic agent can be determined by pharmacological studies in vitro and in vivo.

-8- 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 and published by R.I. Geran et al. in Cancer Chemotherapy Reports, part III, Vol. 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 15 the start of treatment. The extent of this decrease is measured after treatment with one dose of N-N'bis (2-chloroethyl) -N-nitrosourea ("BCNU") used as a reference [Tang and Eisenbrand, Arch. Pharm. 314, 910 (1981)] and is compared to the cell count after 20 racemic fotemustine and fotemustine.

Hepatic toxicity is assessed according to Wroblewski's method by measuring the pyruvic glutamic transaminase activity -in serum from Long Evans rats treated with an intraperitoneal injection of doses of 25 racemic, and fotemustine.

Mutagenicity is assessed by 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 -9maintained as described by Kohn et al. [Cancer 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 fetal bovine serum, 10-sM 2-mercaptoethanol and standard antibiotics. All cultures are grown at 380 in a humidified atmosphere of 5% CO 2 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 before 15 use. Convenient standard stock solutions are 35 mM.

For radioisotope labelling and X-irradiation 2.5X 10 P388 cell/mL are grown in medium containing either 0.05ACi/mL of 1 C)-thymidine or 0.5fCi/mL of 3

H)-

thymidine. After labelling for 20 hours, medium is 20 removed and cells washed with cold PBS. Standard chase treatments using 10iM non-radioactive thymidine for 4 hours are employed. Tritiated cells are S exposed, on ice, to 300 rads of X-irradiation as an internal standard. The alkaline elution procedure is described by Kohn et al. ["Measurements of strand breaks and cross links by alkaline elution.." In: Frieberg E. and Hanawalt P. (eds), DNA repair: A laboratory manual of research procedures, New York, Dekker, 1: 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 fetal calf serum and incubated at 370 for 1 to 500 minutes. P388 cells are resuspended in the medium diluted to reach the appropriate drug concentrations. After 3 days incubation at 5% CO,, cells are counted.

Induction of single strand breaks in P388 cells exposed to racemic fotemustine, fotemustine and fotemustine is compared. Cells (5X10 s labelled hours by ("C)-thymidine are treated for 2 hours with various concentrations of fotemustine and mixed with a similar number of 3 H)-thymidine labelled reference cells. These cells are collected on 2.0 pm pore size polycarbonate filters and lysed with 5 mL of 2% SDS/0.025 M Na 4 EDTA (pH9.7) in the presence of proteinase K. Elution is carried out in the dark at pH 12.1 as described by Kohn. DNA single strand break frequencies are calculated on the basis of S" first order elution kinetics.

Induction of DNA-Protein cross-links is examined in P388 cells treated for various times in the presence of racemic fotemustine, fotemustine or fotemustine. ("1C)-Thymidine labelled drug treated cells are irradiated with 600 rads of X-rays and 3

H)-

thymidine labelled reference cells are irradiated with 300 rads of X-rays. These cells are collected 25 on 2.Om pore-size polycarbonate filters 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 P 388 cells at various times after exposure to concentrations of racemic fotemustine and its enantiomers causing equivalent DNA damage can also be -11examined. The ("C)-thymidine labelled cells initially exposed to drug treatment for 2 hours are washed and resuspended in drug-free medium. After different times of incubation at 370, these cells are mixed with an equal number of (H)-thymidine labelled reference cells, collected on the polycarbonate filters and analyzed by alkaline elution.

The foregoing tests provide an estimate of relative activity, potency and selectivity.

Emesis in Ferrets. Experiments are performed on adult male ferrets. The animals are first 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- 15 harness, each animal receives a surgically implanted catheter in its right jugular vein. The catheter is flushed daily with heparinized sodium chloride. The drug studies are conducted 1- week after the surgical procedure. Tethered animals are individually housed.

20 Eight to eleven animals 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 fotemustine, (+)-fotemustine or fotemustine. At least three dose levels of each test compound are evaluated.

Individual animals are observed for 30 minutes following administration of the test substance. The frequency of, and latency to all expulsions, retches -12and defecations are recorded. Data obtained from dose-response curves is tested for statistical significance by chi-square analysis. ED,0 values are determined for each compound. The test provides an estimate of relative liability to nausea and vomiting.

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Claims (7)

1. A method of treating a malignancy which comprises administering to a mammal suffering from said malignancy, a therapeutically effective amount of (-)-fotemustine, substantially free of its stereoisomer.

2. The method of claim 1 wherein said malignancy is chosen from the group consisting of ,lymphomas, melanomas, gliomas and other brain tumors, lung cancer, liver metastases and hematological and gastrointestinal malignancies.

3. The method of claim 1 wherein fotemustine is administered intravenously or intraarterially. S:

4. The method of claim 3 wherein the amount of (-)-fotemustine administered is from about 20 mg/m 2 to *6 about 150 mg/m 2

5. The method of claim 1 wherein the amount of (-)-fotemustine is greater than approximately 90% by weight of the total weight of fotemustine.

6. A pharmaceutical composition which comprises (-)-fotemustine substantially free of its stereoisomer, and a pharmaceutically acceptable carrier.

7. The composition according to claim 6 adapted for parenteral delivery. DATED THIS FOURTEENTH DAY OF DECEMBER 2000. SEPRACOR INC. BY PIZZEYS PATENT TRADE MARK ATTORNEYS