Pharmaceutical composition with anti-tumour activity and its preparation


(57) Abstract:

Pharmaceutical composition with anti-tumour activity contains as the active ingredient temozolomide-8-carbarnoyl-3-methylimidazo[5,1-d] -1,2,3,5-tetrazine-4-(3H)-he and the inhibitor of O6-alkylguanine-DNA alkyltransferase in the ratio from 1 : 1 to 1 : 20000. The pharmaceutical composition also contains at least one pharmaceutically acceptable carrier. Inhibitor selected from the group comprising O6-alkylguanine, O6-ariguani and O6-benzylidene the guanine, guanosine and 2'-deoxyguanosine. The method of obtaining the pharmaceutical composition includes a mixture temosolomida with inhibitor of O6-alkylguanine-DNA alkyltransferase and a pharmaceutically acceptable carrier. The composition provides enhanced antitumor activity. 2 S. and 2 C.p. f-crystals, 5 Il., table 2.

The invention relates to the field of treatment of cancer in human cells using derived tetrazinni, in particular to pharmaceutical compositions with anti-tumour activity and the way it was received.

Known application temosolomida, representing 8-carbarnoyl-3-methylimidazo[5,1-d] -1,2,3,5-tetrazine-4-(3H)-he, as antipodality antitumor activity temosolomida.

This task is solved by the proposed pharmaceutical composition with anti-tumour activity, containing temozolomide and optionally an inhibitor of O6-alkylguanine-DNA alkyltransferase in the ratio from 1 : 1 to 1 : 20000, and at least one pharmaceutically acceptable carrier.

The proposed composition was prepared by mixing temosolomida and the specified inhibitor in a specified ratio with at least one pharmaceutically acceptable carrier. This method is an additional object of the invention.

According to the invention toxicity temosolomida can be vastly improved by its use in combination with the amplifier, representing an inhibitor of O6-alkylguanine-DNA alkyltransferase (hereinafter: Atasa). In particular, through the use of inhibitors Atasi, for example, O6-benzylguanine (hereinafter: BG), you can increase the toxicity temosolomida, for example, in cell lines MAWI, up to 300 times higher if it is used in the following doses according to the present invention that allows the use of temozolomide to hemoterapia types of cancer in man, which still were not sensitive in respect of such therapy or only ment for one hour interconnected with their contents Atasi. Thus, methylation of temozolomide provisions O6guanine in DNA leads to cytotoxic damage. The sensitivity of cancer cells to temozolomide you can determine by examining their relative production Atasi. The cell that produces a large number of Atasi, is less sensitive to temozolomide than the cell that produces the minimum number of Atasi.

By pre-treatment of cells with one dose of BG is possible to achieve a small increase (less than 4 times) toxicity temosolomida. In experiments with colonies revealed that human fibroblasts transfected with the cDNA Atasi, after pre-processing using the BG are more resistant to temosolomida than control transfected fibroblasts, despite the destruction of Atasi. Incredibly, that the reason for this lies in the different transport temosolomida. This phenomenon may be a sign of the resynthesis Atasi phAT-fibroblasts to reduce the effect of pre-treatment with the inhibitor.

However, in cell lines MAWI suddenly there was a significant potentiation of the toxicity of temosolomida (approximately 300 times the quantity) way is that also contain a large number of Atasi, but only a small effect showed U373 cells containing a small amount of Atasi. Based on studies of cell lines MAWI and MCF-7, you can assume that the long-term presence of the inhibitor Atasi increasingly damaged DNA.

The cottage inhibitor Atasi preferably repeated for several or many days, and preferably it is carried out before giving temosolomida. Dose can be given within 1, 2, 3, 4 or 5 days, preferably the treatment is carried out for 4 or 5 days.

In addition, preferably temozolomide give the patient again in a few days, and before giving each dose temosolomida give inhibiting Atasu amount of inhibitor Atasi, which is expressed increasing toxicity temosolomida in cancerous human cells, for example, about 300 times in the case of cell lines MAWI.

According to a further preferred variant of the invention the inhibitor of Atasi give enough for sensitization of tumor in vivo, without causing not desired sensitization healthy tissue, when the inhibitor Atasi use together with temozolomide.

The amount of inhibitor Atasi given in the framework of this the cells. Suitable dose is the dose that leads to the concentration of inhibitor Atasi in treated tumor cells, causing the disappearance of activity Atasi, this dose may be, for example, about 1 to 2000 mg / kg body weight, preferably about 10 to 800 mg per kg of body weight, if the inhibitor is given before chemotherapy.

As tumors, for processing which temozolomide particularly suitable, can be called carcinoma, melanoma, sarcoma, leukemia and lymphoma, in particular, astrocytomas, gliomas, malignant melanoma, pogodno granuloma, sarcoma of Wings, chronic limfotsity leukemia and tumors in the lungs and chest. A particularly strong increase in the activity temosolomida by using inhibitor Atasi observed in tumor cells in breast cells by astrocytomas, and in tumors of the colon and rectum.

The usual dose temosolomida is 0.1 to 200, preferably 1-20 mg / kg of body weight per day, or in relation to the body surface, about 40 to 400, preferably about 150 to 300 mg / m2a day.

The degree of potentiation inhibitor Atasi depends on the number Atasi commonly found in cancer cells of a particular type. Activity temosolomida in relation R. the Torah Atasi, than for cells containing fewer Atasi.

In the framework of the present invention is suitable inhibitors Atasi, which have such activity, for example, O6-alkylguanine as, for example, O6-methylguanine, alkenylamine as, for example, O6-Allegany,6-ariguani as, for example, O6-benzylguanine, and6-benzylidene guanine, guanosine and 2'-deoxyguanozine. For use within the present invention, in particular suitable ON6-benzylguanine.

In case the dose of the inhibitor Atasi depends on the number Atasi usually available to be processed cancer cells, age and condition of the patient and used inhibitor Atasi.

Temozolomide particularly preferably given in the form of repeated doses in consecutive days, and the effect of strong potentiation according to the invention is achieved by a preferred mode according to which the inhibitory Atasu dose of the inhibitor Atasi give before giving each dose temozolomide or simultaneously with it. Preferably as an inhibitor Atasi use OF6-benzylguanine, which give together with temozolomide, the latter give the number Stateline each dose of the inhibitor Atasi give 2 - 8 hours before giving the next dose temosolomida than achieving the strong potentiation of toxicity temosolomida that leads to the most effective treatment of the patient's tumor. Particularly preferably, such processing is repeated after approximately four weeks.

An alternative mode villas temosolomida with O6-benzylguanine is a continuous mode, both of the active substance given daily for four or more days. Such combination therapy can be extended continuously as needed to reduce swelling.

In addition, through research production Atasi in specific cancer cell can be determined by the potentiation of toxicity temosolomida in this cell. According to a preferred variant of the invention, the content of Atasi in certain cell lines to determine, for example, by the method described by Lee and others (Cancer Res. , 51, page 619, 1991), and explore possible sensitivity to temozolomide. On the basis of this research then, you can find suitable ratio temosolomida and inhibitor Atasi that give under the relevant mode.

The proposed pharmaceutical composition may emetics acceptable carrier.

As solid preparations include, for example, powders, tablets, measurable dispersion of the granules, capsules, including starch capsules, and suppositories. The powders and tablets may contain about 5-70% of the active substance. Suitable solid carriers are known. As such carriers include, for example, carbonate, and magnesium stearate, talc, sugar and lactose. Tablets, powders, starch and other capsules can contain one dose of the active substance, and they are suitable for oral testimony.

To obtain suppositories first melt the wax with a low melting point, for example, a mixture of glycerides of fatty acids or cocoa butter, and then it is homogeneous dispersed active substance by mixing. The molten homogeneous mixture is poured into molds to normal size, allow to cool, and the product solidifies.

As liquid preparations include, for example, solutions, suspensions and emulsions, such as solutions in water or a mixture of water and propylene glycol, are suitable for parenteral injection.

Also suitable solid preparations intended for transfer to liquid medication directly in front of the cottage, in particular, oraln the/P> The proposed connection may also be transdermal, and as preparations suitable for transdermal villas include creams, lotions, aerosols and emulsions, which can also be used in known standard transdermal patches or transdermal patches for prolonged action.

Preferably the pharmaceutical composition is contained in the dosing units. Each dosing unit includes a separate doses containing appropriate quantities of the active substance, for example, an effective amount for achieving the desired effect.

The number of active substances in a single dose may be about 0.1 - 1000 mg, preferably about 1 to 500 mg, depending on the specific application.

Specific applied dose can vary depending on the patient and the severity of his condition. The specialist is able to determine the suitable dose in a particular case. If desired, the total daily dose can be given in the form of several individual doses.

Temozolomide can be given by known methods described, for example, Wassermann and others (Cancer, 36, pp. 1258-1268, 1975). In cases where suitable oral giving, particularly 4 - 5 doses, preferably for 4 to 5 consecutive days. For continuous therapy is suitable intravenous giving 25 - 250 mg/m2in day. Oral giving suitable scheme of re-giving.

As already mentioned, the inhibitor Atasi you can give or separately before temozolomide, or simultaneously with it. In cases where the desired simultaneous house, inhibitor Atasi and temozolomide may be present in combined form to facilitate giving. This fit the above drugs, and they prefer drugs intended for oral or intravenous villas.

Temozolomide and inhibitor Atasi can be included in one set, in which temozolomide and inhibitor Atasi are in the form of single compounds to give certain way, with attached instructions for use contained in the set of drugs. For example, a set of drugs intended for oral villas, contains suitable for oral giving the drug temosolomida, and, separately, suitable for oral giving the drug inhibitor Atasi.

The doctor will be able to vary the size of the dose within hemoterapia on a specific patient.

The following examples are some of the who's who but.

Example 1. Intended for oral giving the drug, mg per capsule:

Temozolomide - 100

Lactose - 213

Microcrystalline cellulose - 30

Sodium dodecyl sulfate - 20

Corn starch - 25

Magnesium stearate 2

Stir temozolomide, lactose, microcrystalline cellulose, sodium lauryl sulphate and corn starch, the mixture was passed through sieve # 80, add magnesium stearate, re-stir and serve in a composite gelatin capsules of suitable size.

Example 2. Intended for oral giving the drug, mg per capsule:

ABOUT6-benzylguanine - 100

Lactose - 213

Microcrystalline cellulose - 30

Sodium dodecyl sulfate - 20

Corn starch - 25

Magnesium stearate - 2

Stir ABOUT6-benzylguanine, lactose, microcrystalline cellulose, lauric sodium sulfate and corn starch, the mixture was passed through sieve # 80, add magnesium stearate, re-stir and serve in a composite gelatin capsules of suitable size.

Example 3. Intended for oral giving the drug, mg per capsule:

Temozolomide - 100

ABOUT6-benzylguanine - 100

Lactose - 213

M is fester - 2

Stir temozolomide,6-benzylguanine, lactose, microcrystalline cellulose, sodium lauryl sulphate and corn starch, the mixture was passed through sieve # 80, add magnesium stearate, re-stir and serve in a composite gelatin capsules of suitable size.

Example 4. Intended for intravenous giving the drug, mg / ml:

Temozolomide - 100

Sodium bisulfite - 3,2

The disodium salt of ethylendiaminetetraacetic acid - 0,1

Water for injections To 1 ml

Example 5. Intended for intravenous giving the drug, mg / ml:

Temozolomide - 100

ABOUT6-benzylguanine - 100

Sodium bisulfite - 3,2

The disodium salt of ethylendiaminetetraacetic acid - 0,1

Water for injections To 1 ml

The positive effect is illustrated by the following example.

Example 6.

Material and method

Use cultural environment sold by ICN Biochemicals Ltd. in High Wycombe, United Kingdom), and fetal calf serum company Gibco Ltd. (, Paisley, United Kingdom). ABOUT6-benzylguanine (BG) put other R. C. Moschel (NCI - Frederick Cancer Research & Development Center, Frederick, Maryland, USA). Temozolomide produced May and Baker L -70oC. All other chemicals manufactured by Sigma Chemical Co. Ltd. (Poole, United Kingdom).

Studies on the cytotoxicity

Cell lines were grown with conventional method as monolayers in a buffered environment DMEM containing 10% fetal calf serum, 25 mmol HEPES, glutamine and penicillin/streptomycin. Cytotoxicity was investigated in free HEPES environment in an atmosphere of 5% carbon dioxide. 96 tiles with grooves, filed for 750 - 1000 cells per cavity, incubated overnight, and within two hours was treated with 33 µmol BG or without him. Then for hours in the same environment added temozolomide, and the final concentration of DMSO did not exceed 1%. Cells were grown for 7 further days in fresh medium, after which the protein content was determined using the analysis according to Skehan and others (J. Natl. Cancer Inst., 82, page 1107, 1990). Studies of growth revealed that during the experience of the cells were in the logarithmic phase of growth. In the framework of the re-villas temosolomida cells were treated consistently for 24 hours a day, every day using a fresh environment. The experiments were performed at least twice.

At a minimum support the entrances xeroderma retinitis) [see Fan and others, Nucleic Acid Res., 18 p. 5723, 1990], and in deepening tiles filed in 1000 cells. Incubated for three hours, then added temozolomide, sweerstroy in minimal supportive environment, and tiles were incubated for five days. Cells that have experienced, researched by experience Morten and others (Carcinogenesis, 13, page 483, 1992). In experiments using BG thrice filed on 300 cells in deepening the tiles and left in contact for five hours. Then three hours before being processed by temozolomide, swierzbinski in a minimum maintenance medium containing 10 µmol BT, added BG (10 µm in minimum support environment). After seven days the colonies were analyzed by known techniques and subjected to reading.

Experience with the use OF6-alkylguanine-DNA transferase

This experience was carried out according to the method described by Lee and others (Cancer Res. , 51, page 619, 1991). Thus different amounts of cell extracts at a temperature of 37oC for 2 hours, incubated with DNA containing ABOUT6-methylguanine labeled with [3H] methyl group, 300 ál of buffer 1 containing 1 mg / ml bovine serum albumin. After incubation consistently fast DOB. dobavlaut another 2 ml of 1 M perchloric acid and the resulting mixture is heated at a temperature of 75oC for 40 minutes for the conversion of DNA to acid-soluble material. The protein containing the methylated Atasu, then harvested by centrifugation and washed with 4 ml of 1 M perchloric acid, and then re-suspended in 300 ál of 0.01 m of sodium hydroxide, dissolved in 3 ml of aqueous scintillation medium (brand Ecosint A firm National Diagnostics) and subjected to reading. Protein content in the cells was determined using the set BioRad using as a standard of bovine serum albumin. Activity Atasi expressed in fmol translated into protein bromide per mg total protein content in the extract.

Uptake by cells labeled with [14C] temosolomida

8-carbarnoyl-3-[14C] methylimidazo[5,1-d] -1,2,3,5-tetrazine-4-(3H)-he (specific activity 26,3, mcurie/mmol) was supplied other John Slack (Aston Molecules Ltd., , Birmingham, United Kingdom). Cell suspension containing cells at a concentration of 5 x 106on ml, balanced at a temperature of 4oC and were processed using 200 µmol labeled medicines. 106 cells pipette filed in Eppendorf tubes and centrifugal layer was aeronavali and the oil layer was carefully washed using 300 μl of saline solution. After centrifugation were aeronavali both layers containing cells the residue was dissolved in a suitable solubilizer, for example the commercial product Protosol, representing the Quaternary ammonium hydroxide in toluene, and was filed in scintillation tubes containing Optiphase, representing 95-99% diisopropylnaphthalene.

The results of experiments on determination of cytotoxicity

The results are shown below in table. 1.

Cells in the culture medium fabric before treatment single dose temosolomida subjected or not subjected to the influence OF6-benzylguanine (BG).

1. CT50[-BG]/KT50[+BH]

2. The results obtained by analysis according to Wassermann and other (Int. J. Radiat. Oncol. Biol. Phys., 15, p., 699, 1988).

The data table. 1 graphically shown in Fig. 1, in which the tumor cell line person listed in order of increasing level Atasi: ZR-75-1, U87MG, U373, LS174T, LOVO, MCF-7 and MAWI. These data show a correlation between sensitivity (defined through the concentration leading to 50% increase growth, or KT50) tumor cell lines to temozolomide (r = coefficient of correlation = 0,87) and their contents Atasi.

Cell line, preformed obrabotannoi untreated cell lines.

Sensitivity control cells XP (cells xeroderma retinitis, transfected pZipneoSV(X)1) (Fan and others, Nucleic Acids Res., 18 p. 5723, 1990), exprimarea almost not determine the content of Atasi, temozolomide 4 - 5 times higher than the sensitivity of the transfected cDNA Atasi human cells to temozolomide (see tab. 1). In the experiment, including the formation of colonies, cytotoxicity temosolomida (see Fig. 2, showing the curve of cytotoxicity temosolomida in cell lines transfected pZipneoSV(X)1. or phAT (,) and derived from XP, in the absence of BG, respectively, the presence of (,) 10 µmol BG, marks errors indicate standard deviation of +/-1) pre-processing BG resulted in a similar degree of potentiation of cell XP person, transfected with Atasoy as in tumor cells, but had no measurable effect on control cells XP, not exprimarea Atasu. Although BG has reduced activity Atasi in transfected cells (see below), they were more resistant to temozolomide than control fibroblasts transfected pZip.

The data obtained in the repeat mode villas are set forth below in table. 2.

Before giving repeated daily doses temosolomida cells in tissue cultures, for example, toxicity temosolomida in cells MAWI and MCF-7 (see also Fig. 3, showing the curve of the ratio of cytotoxicity daily repeated doses temosolomida in tumor cell lines of human MAWI (x), MCF-7 (a) and U373 (#) in giving only the drugs (KT50'That +BT) compared to pre-incubation with BG (KT50', +BH).

After giving five doses every 24 hours, the sensitivity of the cell lines MAWI exposure temosolomida had more than 300 times the size in the presence of BG. When you re giving only temosolomida toxicity of not more than one dose in 24 hours. In a similar experiment on the U373 cells having lower contents Atasi, the presence of BG has only led to a threefold increase after four doses in 24 hours.

Content alkyltransferase

The quantities used BG quickly lowered initially high content of Atasi in cells MAWI and transfected with the cDNA Atasi fibroblasts XP person to undetectable levels. In the liquid chromatography high resolution was that BG was sustainable in the cultural environment of the fabric at least 24 hours at a temperature of 37oC.

It also turned out that after incubation for three hours temozolomide leads to 0 µmol observed 50% reduction (see Fig. 4, which shows a curve indicating the effect of increasing concentrations temosolomida on the contents Atasi in neoplastic human cell lines: LOVO () , MAWI (x), MCF-7 () , U373 (#), despite the 3 - to 4-fold difference cytotoxicity temosolomida when giving the individual dose between MCF-7 and cell lines, colon and rectum (LOVO and MAWI)). Similar declines were observed in a more sensitive line U373, although the contents Atasi was on the verge of obnaruzhili experience.

To exclude the possibility of differences in transport temosolomida investigated the uptake of [14C]-labeled compounds are the most sensitive and the most resistant cell lines [ZR-75-1 (x), respectively MAWI () ]. The results, shown in Fig. 5, show that the fast absorption was carried out at a temperature of 4oC, and in both cell lines, it was completed within five minutes. In both cell lines found a similar number of drugs in the study of the concentration of the protein. Fast absorption at a temperature of 4oC consistent with passive diffusion temosolomida previously proven in two lymphoid cell lines (Sull and others, Biochem. Pharmacol., 36 p. 3215, 1987).

1. Pharmaceutical composition with one pharmaceutically acceptable carrier, characterized in that it further comprises an inhibitor of O6-alkylguanine-DNA alkyltransferase in the ratio from 1 : 1 to 1 : 20000.

2. The pharmaceutical composition according to p. 1, wherein the inhibitor is selected from the group comprising O6-alkylguanine, O6-alkenylamine, O6-ariguani and O6-benzylidene the guanine, guanosine and 2'-deoxyguanosine.

3. The pharmaceutical composition according to p. 2, characterized in that the inhibitor is a O6-benzylguanine.

4. A method of obtaining a pharmaceutical composition comprising mixing 8-carbarnoyl-3-methylimidazo[5,1-d] -1,2,3,5-tetrazine-4-(3H)-she, at least, one pharmaceutically acceptable carrier, characterized in that the 8-carbarnoyl-3-methylimidazo[5,1-d] -1,2,3,5-tetrazine-4-(3H)-it is additionally mixed with an inhibitor of O6-alkylguanine-DNA alkyltransferase in the ratio from 1 : 1 to 1 : 20000.


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