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  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
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  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
  • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents

Definitions

• Temozolomide or 8-carbamoyl-3-methylimidazo[5,1-dj- l,2,3,5,-tetrazin-4-(3H)-one, (CCRG 81045, NSC 362856) has been found to possess valuable antitumour properties, see Newlands et al., Br. J. Cancer. e>5_:287 (1992).
• temozolomide has shown activity against astrocytoma, glio as, malignant melanoma and mycosis fungoides. The drug is most useful when administered according to a repeat dose schedule.
• Methylated O ⁇ -al ylguanine e.g., from reaction with MTIC (the active methylating species of temozolomide)
• MTIC the active methylating species of temozolomide
• ATase protein o'-alkylguanine DNA alkyltransferase
• PCT published Application WO 91/13898 notes, for instance, a 3.8 fold decrease in the ED J0 for Me CCNU when combined with 0 6 -benzylguanine in SF767 cells.
• Moschel et al. show a general enhancement of the anti-neoplastic activity of an alkylating agent when used with a depletor of alkyltransferase.
• Applicants' invention which is surprising and unobvious in view of the earlier work, is that the chemotherapeutic effects of temozolomide can be dramatically potentiated (up to 300-fold for the MAWI cell line) by utilizing a particular dosing regimen which incorporates the administration of an ATase inhibitor.
• human cell cancers which were heretofore insusceptible or only mildly susceptible to temozolomide therapy can be treated by the combination of temozolomide with an ATase inhibitor.
• the present invention relates to the potentiation of temozolomide toxicity in human cancer cells using inhibitors of 0 6 -alkylguanine DNA alkyltransferase (ATase) . Further, a dosage regimen for optimal therapy, and methods of identifying potentially temozolomide- sensitive human cancer cells, are provided.
• ATase 0 6 -alkylguanine DNA alkyltransferase
• Figure 1 is a graph of the cytotoxicity (10 ⁇ ) of temozolomide ( ⁇ ) and CCNU (x) versus cellular Atase levels in the human tumour cell lines (in order of increasing ATase levels): ZR-75-1, U87MG, U373, LS174T, LOVO, MCF-7 and MAWI.
• Figure 2 is a graph of the cytotoxicity of temozolomide in pZipneoSV(X)1-transfected (o, ⁇ ) or phAT-transfected (T,A)XP-derived cell lines in the presence (»,A) or absence ( ⁇ , ⁇ ) of 10 ⁇ M BG. Error bars indicate +/- l s.d.
• Figure 3 is a graph of the cytoxicity ratio of repeated daily doses of temozolomide in MAWI (x) , MCF-7 (T) or
• Figure 4 is a graph of the effect of increasing concentrations of temozolomide on ATase levels in the human tumour cell lines: LOVO ( ⁇ ) , MAWI (x) , MCF-7 (T) , U373 (#) .
• Figure 5 is a graph of uptake of radiolabel at 4°C by cells treated with ,4 C-temozolomide. MAWI ( ⁇ ]), ZR-75-1 (x). DETAILED DESCRIPTION OF THE INVENTION
• temozolomide an antitumour agent useful in the treatment of human cancers
• a potentiator which is an inhibitor of the enzyme 0 6 -alkylguanine DNA alkyltransferase (ATase) .
• ATase inhibitors such as 0 6 -benzylguanine (BG)
• BG 0 6 -benzylguanine
• a parallel toxicity for temozolomide and lomustine (CCNU) (after 1 hour drug exposure) can be shown with a number of human tumour cell lines, correlating with their ATase content. Methylation of the O p position of guanine in DNA by temozolomide thus results in a cytotoxic lesion.
• CCNU lomustine
• sensitivity to temozolomide can be determined. A cell producing high amounts of ATase will thus be less sensitive to temozolomide alone than one producing minimal ATase levels.
• Pretreating cells with a single dose of BG causes a modest ( ⁇ 4 fold) increase in temozolomide toxicity.
• the degree of enhancement for temozolomide and lomustine (CCNU) are a similar order of magnitude.
• human ATase cDNA-transfected fibroblasts pretreated with BG remained more resistant to temozolomide than control transfected fibroblasts, although the ATase protein was eliminated. This is unlikely to be due to differences in temozolomide transport and may simply reflect resynthesis of ATase by the phAT fibroblasts to diminish the effect of pretreatment with the inhibitor.
• the present invention thus provides a method of potentiating the toxicity of temozolomide in human cancer cells by administering an ATase inhibiting amount of an ATase inhibitor, and a product comprising temozolomide and an ATase inhibitor as a combined preparation for simultaneous, separate or sequential administration in said treatment of human cancer cells.
• this administration of an ATase inhibitor is repeated over a period of several or multiple days, and is prior to the administration of the doses of temozolomide.
• Repeat doses can be administered at 1, 2, 3, 4 or 5 days, with 4 or 5 days being the preferred period of therapy.
• the temozolomide is administered to a patient in repeat doses over a period of days, and an ATase inhibiting amount of an ATase inhibitor is administered prior to each dose of temozolomide, resulting in a markedly increased toxicity of the temozolomide to human cancer cells, e.g., about 300 fold for the MAWI cell line.
• the ATase inhibitor is administered in an ATase inhibiting amount, i.e., an amount sufficient to sensitize the tumour in vivo without causing undue sensitization of normal tissue, when the ATase inhibitor is used concurrently with temozolomide.
• the amount of ATase inhibitor employed in the present invention to be used varies according to the degree of the effective amount required for treating tumour cells.
• a suitable dosage is that which will result in a concentration of the ATase inhibitor in the tumour cells to be treated which results in the depletion of the ATase activity, e.g., about 1-2000 mg/kg, and preferably about 10-800 mg/kg, prior to chemotherapy.
• neoplasms for which temozolomide is a particularly suitable treatment include carcinomas, melanomas, sarcomas, lymphomas and leukaemias, with specific utility for astrocytoma, gliomas, malignant melanoma, and mycosis fungoides, Ewings sarcoma, chronic lymphocytic leukaemia, and lung and breast tumours.
• astrocytoma gliomas, malignant melanoma
• mycosis fungoides Ewings sarcoma
• chronic lymphocytic leukaemia and lung and breast tumours.
• Particularly dramatic enhancement of the temozolomide activity with an ATase inhibitor is found in breast, astrocytoma and colorectal tumour cells.
• Typical dosage ranges of temozolomide are generally between 0.1 and 200, preferably between 1 and 20, mg/kg body weight per day, or expressed in terms of body surface area, about 40-400, and preferably about 150-300 mg/m 2 per day.
• the amount of potentiation by the ATase inhibitor is dependent upon the amount of ATase normally present in the particular cancer cell type. A cancer cell having higher levels of ATase will be potentiated more dramatically by the preadministration of the ATase inhibitor.
• the ATase inhibitor utilizable in the present invention are those known to possess such activity, for instance, the 0 6 -alkylguanines such as 0 6 -methylguanine, the alkenylguanines such as 0 6 -allylguanine and the 0 6 - arylguanines, such as 0 6 -benzylguanine and the O 6 - benzylated guanine, guanosine and 2 , -deoxyguanosine compounds described in PCT International Application WO 91/13898 (published September 19, 1991). Particularly suitable for use in the present invention is 0 6 - benzylguanine.
• the particular dosage of ATase inhibitor depends upon the amount of ATase normally found in the cancer cell being treated, the age and condition of the patient, and the particular ATase inhibitor being utilized.
• Temozolomide has been found to be most preferably administered in repeat dosages on consecutive days, and the dramatic potentiation effects of the present invention are realized in the highly preferred regimen involving the administration of an ATase inhibiting dose of the ATase inhibitor prior to, or concurrent with, the administration of each dose of temozolomide administered.
• 0 6 -benzylguanine is utilized as the ATase inhibitor, along with temozolomide administered at a daily rate of 150-300 mg " 2 in four or five divided doses over four or five consecutive days (total dose 750-1500 mg/m 2 ) .
• each dose of the ATase inhibitor is administered 2-8 hours prior to each dose of the temozolomide. This affords the greatest potentiation of temozolomide toxicity, and results in the most effective treatment of the patient's particular neoplasm. Most preferably, this regime is repeated after an interval of about four (4) weeks.
• An alternate dosage schedule for the administration of temozolomide with 0 6 -benzylguanine is a continuous schedule wherein the two drugs are administered on a daily basis for a period of four (4) or more days. This combination therapy can be extended as needed on a continual basis until remission is attained.
• the ATase production of a particular human cancer cell can be utilized as a screening method to determine potentiation of temozolomide toxicity to said cell.
• the ATase content of the particular cell line is assayed, e.g., by the method described by Lee et al., Cancer Res.. 5.1:619 (1991), and the potential sensitivity to temozolomide determined. Such a determination then enables the appropriate combination of temozolomide and ATase inhibition to be administered in a therapeutic regimen.
• inert, pharmaceutically acceptable carriers can be either solid or liquid.
• Solid form preparations include powders, tablets, dispersible granules, capsules, cachets and suppositories.
• the powders and tablets may be comprised of from about 5 to about 70 percent active ingredient.
• Suitable solid carriers are known in the art, e.g. , magnesium carbonate, magnesium stearate, talc, sugar, lactose. Tablets, powders, cachets and capsules can be used as solid dosage forms suitable for oral administration.
• a low melting wax such as a mixture of fatty acid glycerides or cocoa butter is first melted, and the active ingredient is dispersed homogeneously therein as by stirring. The molten homogeneous mixture is then poured into convenient sized molds, allowed to cool and thereby solidify.
• Liquid form preparations include solutions, suspensions and emulsions. As an example may be mentioned water or water-propylene glycol solutions for parenteral injection.
• solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for either oral or parenteral administration.
• liquid forms include solutions, suspensions and emulsions.
• the compounds of the invention may also be deliverable transdermally.
• the transder al compositions can take the form of creams, lotions, aerosols and/or emulsions and can be included in a transdermal patch of the matrix or reservoir type as are conventional in the art for this purpose.
• the pharmaceutical preparation is in unit dosage form.
• the preparation is subdivided into unit doses containing appropriate quantities of the active component, e.g., an effective amount to achieve the desired purpose.
• the quantity of active compound in a unit dose of preparation may be varied or adjusted from about 0.1 mg to 1000 mg, more preferably from about 1 mg to 500 mg, according to the particular application.
• the actual dosage employed may be varied depending upon the requirements of the patient and the severity of the condition being treated. Determination of the proper dosage for a particular situation is within the skill of the art. For convenience, the total daily dosage may be divided and administered in portions during the day, if desired. Temozolomide may be administered using conventional techniques such as those described in Wasserman et al., Cancer. 3,6:1258-1268 (1975). Where appropriate, oral administration at a rate of 40-400 mgm" 2 per day, and preferably 150-300 mgm "2 per day, in 1-5, and preferably 4-5 doses, over 1-5, and preferably 4-5, consecutive days is highly preferred. Intravenous administration at a daily dose of 25-250 mgm" 2 is preferable for a continuous dosing therapy regimen. Oral administration can be utilized for a repeat dosing regimen.
• the ATase inhibitor can be administered separately prior to, or concurrent with, the temozolomide. Where it is desirable to do so, both the ATase inhibitor and temozolomide can be combined into a unit dosage form to facilitate patient dosing.
• Such combination dosage forms may be in any of the above-described dosage forms, but, as noted above, are preferably in oral or intravenous forms.
• the temozolomide and the ATase inhibitor can be packaged in a kit form.
• the temozolomide and the ATase inhibitor would be individually formulated into particular dosage forms for the particular route of administration, and contain instructions for the administration of the contents.
• such a kit may be in the form of a blister package with separately formulated oral dosage forms of the temozolomide and the ATase inhibitor.
• Tissue culture medium was purchased from ICN Biomedicals Ltd. (High Wycombe, UK) and fetal calf serum from Gibco Ltd (Paisley, UK) .
• BG 0 6 -benzylguanine
• Temozolomide and its chloroethyl analogue, mitozolomide (8-carbamoyl-3-(2-chloroethyl)imidazo[5,l-d]-1,2,3,5- tetrazin-4-(3H)-one) were synthesized by May and Baker Ltd (Dagenham, UK) and stored as solutions in DMSO at -70°C. All other chemicals were purchased from Sigma Chemical Co. Ltd. (Poole, UK) .
• Q 6 -alkylquanine DNA alkyltransferase assay This assay was carried out as described by Lee et al., Cancer Res.. 5L:619 (1991). Thus, varying amounts of cell extracts are incubated with DNA which contains O 6 - methylguanine labelled with [ 3 H] in the methyl group, at preferably 37°C for 2 hours in a total volume of 300 ⁇ l of 1 mg/ml of bovine serum albumin in buffer 1. After incubation, bovine serum albumin (lOO ⁇ l of a 10 mg/ml solution in buffer 1) and perchloric acid (lOO ⁇ l of a 4 M solution) are added in rapid succession.
• IM perchloric A further 2 ml of IM perchloric is added and the mixture heated at 75°C for 40 minutes to degrade the DNA to acid soluble material.
• the protein, which contains the methylated ATase, is then collected by centrifugation, washed with 4ml of IM perchloric acid before being resuspended in 300 ⁇ l of 0.01M sodium hydroxide and dissolved in 3ml of aqueous scintillation fluid (Ecoscint A: National Diagnostics) , and counted.
• the protein content of the cells was determined with a BioRad protein assay kit using bovine serum albumin as a standard. ATase activity is expressed as fmol methyl transferred to protein per mg of total protein in the extract.
• Protosol® (quaternary ammonium hydroxide in toluene) and added to scintillation vials containing Optiphase® (95- 99% diisopropylnaphthalene) .
• BG +/- O 6 - benzylguanine
• control XP cells xeroderma pigmentosa cells transfected with pZipneoSV(X) 1 (Fan et al.. Nucleic Acids Res.. U3:5723, 1990), which express barely detectable levels of ATase, are 4-5 fold more sensitive to temozolomide or the CCNU-related agent mitozolomide than the human ATase cDNA-transfected cells (see Table I) .
• BG pretreatment showed a similar degree of potentiation for the human ATase- transfected XP cells as for the tumour cells, but had no measurable effect on the control XP cells, which do not express ATase. Although BG depleted the ATase activity in the former cells (see below) , they remained more resistant to temozolomide than the control pZip transfected fibroblasts.
• BG +/- O 6 - benzylguanine
• the MAWI cell line was over 300 fold more sensitive to temozolomide when BG was present. Multiple doses of temozolomide, by itself, were not more toxic than a single 24 hour dose in either cell line. In a similar experiment on U373 cells, which have a low level of ATase, the presence of BG caused only a 3 fold potentiation, after four 24 hour doses.
• BG concentration of BG used rapidly reduced to an undetectable level the initially high ATase content of MAWI cells and human ATase cDNA transfected XP fibroblasts. HPLC analysis showed that BG was stable in tissue culture medium for at least 24 hours at 37°C.
• Temozolomide following a three-hour incubation, was found to cause a decrease in ATase content of U373, MCF- 7, LOVO and MAWI cell lines. There was a 50% reduction at 50-100 ⁇ M for each line (see Figure 4) , despite a 3-4 fold difference in the single dose temozolomide cytotoxicity between MCF-7 and the colorectal lines (LOVO and MAWI) . A similar reduction was found in the more sensitive U373 line, although the ATase levels were close to the detection limit of the assay.

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