JPS63246334A - Pharmaceutical composition with antineoplastic activity - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a pharmaceutical composition comprising an anti-cancer drug and reduced glutathione (GSH) as a combination agent to be taken simultaneously, separately or sequentially in an anti-cancer drug.

Typical examples used in anticancer chemotherapy are: cis-diamine-dichloroplatinum, i.e. e.g. CDDP or cis-platinum, cyclobosphamide, 5-fluorouracil, doxorubicin (Adriamycin), metaxane. Thorone, methotrexate, etoposide, vincristine and pleomycin; they are characterized by different mechanisms of action and different dosing regimens.

A few cancer chemotherapeutic agents are effective as single agents; ie, many are used, often in combination with other anticancer drugs, to take advantage of a variety of different functions in killing cancer cells.

Polychemotherapeutic anticancer treatment is aimed at achieving the highest possible mitigation factor for the disease and preventing the emergence of resistance to single chemotherapeutic agents in cancer types that are resistant to single chemotherapeutic agents. (often occurring in certain solid cancers).

Unfortunately, all cancer chemotherapeutic agents have side effects, the toxicity of which is strong enough to prevent the continuation of treatment and make the ultimate success of the treatment plan impossible. According to the usual practice of chemotherapy, combinations of anticancer drugs each result in a duplicate appearance of all the side effects of the single compound.

For example, when two or more compounds are used in combination with other anthracyclines (e.g., adriamycin) or anthraquinone compounds that have myocardial toxicity as another side effect, their most serious It has a virulence that is a combination of side effects.

Attempts to improve the pharmaceutical potential in cancer treatment have focused on finding new drugs that are less toxic and more active (e.g., novel cis-platinum complexes, i.e., N5C311).
058, N5C241240 and N5C258927
Alternatively, it has been done by increasing the therapeutic index of currently available compounds in a more direct manner by means of concurrent treatment methods with neoplastic agents.

To date, the latter approach has been used to modify and limit the side effects that are inevitable in anti-cancer treatments themselves.

That is, for example, urinary N-acetyl-glucosaminidase (NAG) and alanine-aminopeptidase (AA
Notable tubes (tubu
lar) cis-platinum with failure, 80-90
To limit the dose-dependent nephrotoxic effects that occur in % of cases, patients are subjected to forced hydration therapy with chronic intravenous infusions of saline, glycose, or mandol solutions.

In any case, protective measures, which are not comfortable for the patient and are sometimes insufficient, are required, especially when large doses of platinum complexes have to be administered. There is.

Moreover, the protective treatment can be dangerous as the large volumes of fluids administered may be necessary if renal insufficiency and/or cardiovascular disorders are present.

Thus, for example, EP2
495 by the combined administration of mercapto-alkane-sulfonic acids.

On the other hand, animal studies have shown that prophylactic and continuous administration of natural thiol compounds as well as reduced glutathione (γ-glutamyl-glycinylcysteines) is induced by anticancer drugs namely cyclophosphamide and cis-platinum, respectively. This appears to suggest that it may be useful in the prevention of nephrotoxicity and uremia (e.g. Tumor
LLl (1983) and Ca5cer, Chemo
ther,, Pharmac, 44.188, 19
85). Unfortunately, in the experimental studies mentioned above, the very crystalline doses of GSH (divided doses from 200 g to 4 g/Kg) used to achieve somewhat beneficial effects were
This precludes any usefulness of this natural thiol in clinical practice.

Useful doses in men require administration of 15-280 g of GSH. It poses a significant practical problem for people already undergoing hydration procedures.

Current knowledge calls for the reduction of intracellular GSH as a pharmaceutical objective to be pursued to advantage anti-cancer therapy (r
lde 1nfra) discourages the above approach.

On the contrary, reduced GSH has recently been used in doses ranging from 2.5 to 5 g in total to prevent side effects of platinum complexes and, in particular, cis-platinum, when administered for injectable treatments to prevent side effects. , has been proposed as an effective and clinically useful protective agent in men (Italian Patent Application No. 2059LA/85 of May 6, 1985).

This procedure reduces the amount of NA in urine without changing the hydration procedure.
It is claimed to limit tubular damage without significant damaging effects on anticancer efficacy, as shown by G and AAP enzyme measurements.

However, all those known treatments do not reduce the level of anti-cancer compounds that should be required for the particular conditions of natural or reduced resistance (e.g. in many cases of solid cancers, especially lung and colon cancers). do not allow increasing the dosage of the drug.

Recently, great efforts have been made to discover substances that are able to restore the sensitivity of resistant cells to drugs such as doxorubicin, vincristine, cis-platinum and to increase their cytotoxicity as much as possible.

For example, calmodulin, a ubiquitous calcium-binding protein closely related to many intracellular actions of calcium, has recently been proposed as an important target for cancer chemotherapeutic agents (W, H, alt J, of Cl1
n, Onco+, 4.994 (1986). A few carmodulin antagonists are themselves cytotoxic agents, and various studies have demonstrated that their addition to conventional bacteriotoxic chemotherapeutic treatments extends the efficacy of currently available drugs. ing.

Recent research has focused on the use of calcium channel blockers, namely in combination with classical cancer chemotherapeutic agents.

On the other hand, L-phenylfuranine mustard, chloramppyl, CDDP, mechlorethamine, carboplatinum, dichloroethylenediamine-platinum (n), 1.2-'
) The use of pthionine sulfoximine as a specific agent to deplete GSH from cells was proposed by Antrius et al. to increase the cytotoxic effects of compounds such as amino-cyclohexylplatinum(n)-malonate. (Cancer Res, niece, G250.1985)
.

Now, surprisingly and in contrast to the above teachings, we have demonstrated that periocellular carcinoma in combination with anticancer drugs selected from the group consisting of platinum compounds, oxazophosphorine compounds, 5-fluorouracil, methotoxate and anthracyclines. With GSHM having no effect, treatment alone with mono- or polychemotherapy not only prevents the development of the u11 effect of the drug, but also allows an increase in response rate, ie palliation, and increases recovery. (relief that lasted for two years from the end of the treatment) was found.

It is therefore an object of the invention to provide a pharmaceutical formulation comprising the above-mentioned reduced glutathione and at least one anti-cancer drug as a combination for simultaneous, separate or sequential use in anti-cancer treatment. It is.

In a preferred embodiment, the present invention provides a package in which the GSH and anticancer drug are contained in separate containers and can be administered according to the most appropriate dosing regimen.

The term "platinum compound" according to the invention preferably includes cis-dichloro-diamine-platinum. Other compounds are e.g. carboplatinum, dichloro-
Ethylenediamine-platinum (n), 1,2-diamino-cyclohexyl-platinum (■)-malonate or sulfate, diisopropylamino-trans-dihydroxy-cis-dichloro-platinum (■).

Oxazophosphoric compounds are the well-known cyclophosphamide and ifosfamide CIphosp.
It is ha■fde.

Finally, the anthracycline compound preferably includes doxorubicin (Adriamycin). Other compounds that can be used in the present invention include evirubicin, daunorubicin, carmamycin, midoxantrone alone.

The above compounds are usually used in combination according to an appropriate protocol for polychemotherapy.

It is thus possible to administer antineoplastic agents, ie platinum (II)- or platinum (IV), together with cyclophosphamide and methotrexate.

Of course, any other possible formulations of the antineoplastic agents described herein are possible and within the knowledge of those skilled in the art.

The administration of GSH is particularly advantageous in combination with platinum complexes.

For example, according to the present invention, administering cis-platinum together with glutathione not only dramatically reduces the side effects observed when administering cis-platinum alone, such as nephrotoxicity and neurotoxicity, but also Surprisingly, it has been pointed out that the anticancer effect expressed as a palliative rate and the life expectancy free from disease increase.Furthermore, administration of large doses of cis-platinum is particularly effective. The administration of high doses of cis-platinum is particularly advantageous when administration of high doses of cis-platinum is required to adequately treat terminal cancers. It is also surprising that large amounts of cis-platinum in each course can be used cumulatively, so that high efficacy can be achieved even in the late stages, since cis-platinum alone is administered. However, this is not possible due to the high toxicity and serious side effects associated with it.

In fact, the maximum possible dose is approximately 1000 mg/m2, which is an increase of 600-400 mg/m2 compared to the maximum dose of 400-800 mg/m2 when cis-platinum is administered without glutathione. It means.

In the latter case, serious side effects must be considered.

Similarly, as mentioned above, the premyocardial effects associated with the use of doxorubicin are reduced when administered with glutathione. Comparable effects (relief of myelotoxicity or prevesical) were also observed when administering cyclophosphamide or methotrexate.

The most surprising aspect of the present invention is that even though some of the above scientific literature teaches that depletion of intracellular GSH is desirable to enhance anticancer effects, GSH and the above anticancer drugs provided by the synergistic effect of

Moreover, the synergistic effect is particularly surprising in view of the fact that GSH has no anticancer effect when administered alone.

Their unexpected effects, which can therefore be described as "synergistic", are due to the selected dose treatments, i.e. 2.5-5.
g of glutathione (which is 1500mg/m2 of GSH)
-3000mg/m2, however, the average body surface for women is about 1.4m2 and for men about 1.7m2).On the other hand, each anticancer drug is usually used for treatment. This is evident when administered at a known dose or in higher doses. This is because the administration of GSH makes it possible to increase the dose of anticancer drugs considerably without causing toxic symptoms. This often results in significant improvements in overall therapeutic efficacy.

G that is known to be inconvenient for patient administration.
It was found that higher doses of SH were accompanied by a possible reduction in anti-cancer efficacy. However, lower doses result in only a marginal improvement in the tolerability of anticancer drugs.

Another important advantage of the present invention that is apparent when using platinum compounds is that unavoidable hydration is dramatically reduced when pretreating the patient with GSH.

Dosages for anticancer drugs according to the invention are well known in the art. C For example, Goodtan &
Gllman, Pharmaceutical Basics of Therapeutic Agents Edition Mac M
lllan Pub, Co, N, Y, 1985.1
240-130G ω For example, as far as platinum complexes are concerned, and exemplifying cis-diamine platinum, two dose treatments are widely used.

That is, the first one was to administer 90 mg/peng 2 on the first day, then 3a wash out and 4 more doses.
Always treated 4 times, each 3 weeks, thus giving a total dose of 450 mg/e2.

Other treatments were 40 mg/m2 CDDP daily for 5 days.
(Total amount 200g/■2) Shi, total amount of this treatment 1g/
Continue until reaching s2.

However, in therapeutic practice, this treatment usually has 2- or 3-week cycles (400 Peng/ie2 or 600 Peng/12
) is normally interrupted.

The simultaneous administration of 1, 5 g/m2 or 3 g/m2 of GSH produces advantageous effects, in each case making it possible to complete the treatment up to the highest permissible dose.

When using cyclophosphamide or phosphamid, 0.5 to 4 g of GSH per 1 g of cyclophosphamide is sufficient to obtain the effect of the present invention. In the case of anthracyclines, see doxorubicin. , the GSH/doquinrubicin ratio ranges from 2 mg to 50 mg of GSH per gram of anthracycline.

Anthracyclines are usually 30 to 60 g/m2
The GSH dose is 1 to 5 g.

A particularly surprising effect obtained by administering GSH together with anthracyclines is the premyocardial protection that is a typical side effect of such drugs. Moreover, it is noteworthy that the combination of anthracyclines and GSH is effective against the resistance tendency of cancer cells to the anthracyclines themselves, as animal studies report below: It is possible that the resistance of cancer cells that resist other chemotherapeutic agents can be alleviated.

The therapeutic consequences of such efficacy are enormous. This is because resistance phenomenon is a serious problem in anticancer chemotherapy.

It should be noted that clinical doses can vary significantly as far as methotrexate is concerned.

An intravenous dose of 50/250 mg/s2 is considered a "low" dose, an intravenous dose of L-2 g/ya2 is considered a "moderate" dose, and 10 g/ya2 is considered a "high" dose. Under these conditions, the ratio of GSH to methotrexate is, for example, 1-5 g of total GSH, 1 g of methotrexate,
It can be changed between 0.3 and 15 g of GSH per g.

The average weight ratios for selected anti-cancer drugs are summarized below.

Anticancer drug Ig of GSH per gram of anticancer drug Cis-dichlorodiamine platinum 20-100 cyclophosphamide 0.5-4 Doxorubicin 20-50 Methotrexer) 0.3-15 The composition of the present invention can be prepared by a conventional method or Auxiliary drug”Remlmgto
n's Pharmaceutical 5cl
ences ■andbook, Fack Pub
Co, Ne, w York, USA”.

The route of administration is that commonly used for anti-neoplastic therapy, ie, intravenous route (porus or infusion), but in the case of methotrexate, oral route may also be used.

However, the most common route is infusion with dosage regimens already established in medical practice and well known to the expert.

The infusion solution is a physiologically acceptable solution, in particular an isotropic solution, which contains the required amount of salts, i.e. sodium chloride, etc. or glycose and suitable viscosity-adjusting compounds, e.g. polyethylene gelicol, polypropylene glycol, etc. can be included.

As far as GSH is concerned, the preferred dilution is 1 g GSH in 4011 of physiological saline or physiological glucose solution;
It is administered 15 minutes to 2 hours before the administration of anticancer drugs.

Oral administration is also suitable for GSH and methotrexate. Suitable pharmaceutical layers in this case are capsules, tablets, granules, syrups, etc.

Glutathione and anticancer drugs can be combined into unit dosage forms. Separate unit doses are however preferred. This is because, as a result, there is a flexible effect (treatment planning is possible).

The following examples illustrate, but do not limit, the invention and evaluate the effectiveness of this treatment and the severity of side effects.

Example 1 A composition of cis-platinum + cyclophosphamide and 1-GSH was used for advanced ovary II without prior treatment! 1g
It was administered to seven patients suffering from s. The treatment plan was as follows; cis-platinum 90n/l/infusion in 250al saline over 30 minutes, GSHl 5g
/j, cyclophosphamide 800 mg/rIP1 was injected intravenously in 100 liters of physiological saline 15 minutes before administration of cis-platinum. Repeat this plan 5 times every 3 weeks. - One patient was excluded from the evaluation because he was affected by another tumor 11 (colon cancer). The remaining 6 patients (1 patient in stage ■, 4 patients in stage m, 1 patient in stage
subjects) received a standard pre-, post-hydration intravenous injection consisting of 2000 liters of fluid. Diuretics were not used on a regular basis. Patients received a total of 28 courses at G581500g/7 dose levels.

No symptoms of nephrotoxicity, medullary cytotoxicity, or neurotoxicity were observed on the floor. Side effects such as nausea and nausea were very minimal compared to observed patients without adjunctive GSH treatment. There were no signs of severe bone marrow depression.

First of all, these results demonstrate that there is no GSH-negative interference with the therapeutic effect of cis-platinum. In fact, 6 of 6 patients with advanced disease had complete remission (pathologically proven); 5 remained in remission after more than 24 months of follow-up; The patient relapsed after 20 episodes of no symptoms.

Although a small number of patients have been treated, it should be pointed out that in similar cases, the rate of complete remission within 2 years after the end of treatment is only 5-10%.

Example 2 Nine patients with ovarian cancer at high risk of recurrence after surgery were treated with a composition of cis-platinum + cyclophosph 1 mid-10 SH. All patients had not been previously treated with other antineoplastic agents.

The treatment plan was as follows; cis-platinum (90 mg/♂, intravenous injection in saline 280
minutes), cyclophosphamide (800 g/m, i.v.), and GSH (3 g/m). This plan was repeated every 3 weeks.

All patients (6 patients in the 1st period, 2 patients in the ■ period, 1 patient in the ■ period)
Reduced hydration (1000 μl of fluid) and GSH (3 g//
) In 20 Osl of saline, intravenously injected, at least 15 minutes before each cis-platinum administration.

Diuretics were not administered on a routine basis.

No symptoms of nephrotoxicity or neurotoxicity were observed, and there were no signs of severe bone marrow depression.

The reduced hydration increased patient compliance and avoided any potential side effects that could occur due to changes in the amount of fluid administered, while the side effects were very mild following the pre-treatment protocol.

The patients have received up to 44 courses to date; 5 have no signs of disease after the completion of 5 courses and 4 still have
He is undergoing treatment.

This result indicates that GSH is a cis-platinum anti-I! The use of GSH does not reduce tumor activity, and platinum-
It strongly suggests that this is a safe alternative method to enable administration of high doses of the complex and also to increase the rate of relaxation tremendously.

Example 3 Ovarian Il previously treated with cis-platinum and relapsed and progressed]! Treat patients with ulcers with sis-platinum and GSH
The combination was treated every 3 weeks. Sith Platinum,
Weekly dose 200 g/IIi' (40 mg/j
GSH
was administered at a dose of 35 mg/gmg of cis-platinum in saline 30 minutes before administration of cis-platinum. This patient had an unusual clinical response with resolution of significant peritoneal cancer tumor ascites after one cycle of treatment.

Usually, the dose of cis-platinum 200+wg/I/ is considered the maximum dose limit and is usually associated with severe side effects after 2 or 3 weeks. The above patient did not show any side effects from the treatment after 5 weeks of treatment and the patient is still alive 8 or 8 days after treatment.

Example 4 Six patients with advanced brain tumors who had undergone radiotherapy and surgery and whose symptoms had worsened were treated with cis-platinum (100 g/g/g in physiological saline 100 g/l, intravenously injected). +
5-Fluorouracil (Ig // Physiological saline 500
+1 medium, intravenous) combination for 2-4 courses every 2a
It was treated for a while. GSH was administered at all doses ff15 at the end of treatment.
g/m were administered simultaneously (saline 10
0ml, intravenous injection).

All patients achieved the desired significant response, which occurred within a few days of treatment. In all patients, one year after treatment, there was a reduction and/or disappearance of the tumor disease, with a surprisingly significant reduction in all pathologies. No side effects were recorded.

Example 5 Various animal tests were conducted to demonstrate the effects of administering GSH in combination with an anthracycline (doxorubicin) and mentrexate.

1) Sorubicin deno')
ni Run's weight 150
-200g of normal diet and water
Four groups of 6 animals each (A, B, C, D) consisted of 24 Sprague Dawley ants reared in the intestines.
randomly divided into.

Heltonia' (GSH + Doquinrubicin): Six animals were treated with intravenous injection of glutathione (Ig/kg) dissolved in saline in the morning (10 am) (total injection volume, 11/1).
kg). Thirty minutes later, the animals were treated intravenously with doxorubicin (3 mg/kg) dissolved in saline (total injection volume, Igtl/kg).

The treatment was then repeated 7 days later, for a total of 4 consecutive hours.

Dangugoji A (doxorubicin): 6 animals were treated as in group A. However, glutathione was omitted and replaced only with physiological saline.

Treatment was performed.

ΩI Fire 2 (GSH): 6 animals were treated in the same way as Group A. However, doxorubicin was omitted and replaced only with physiological saline.

, Yukugonige (control): 6 animals were treated in the same way as Group A. However, both drugs were omitted and replaced with physiological saline.

The first dose was designated as "day zero" and all animals were evaluated weekly for weight gain throughout the experiment starting at time zero of the experiment. Electrocardiogram between Q-T and S-T. After 8 weeks of experiment, the degree of myocardial disease expressed as the average of vacuolated cells in 1011 fields at *It(200x) was determined for each animal by anatomical experiments. was able to evaluate.

Although treatment with GSH alone did not induce significant changes in the different parameters in the experiment when compared with the control group, in all animals of group A, prophylactic treatment with GSH was more effective than with doxorubicin. It was found that the toxic and cardiac effects seen in the treated B group (reduction in body growth curves, ECG recordings, cavitated cell numbers) could be corrected.

Comparable results were observed in subsequent experiments in which 0.5 g/kg of glutathione was administered intravenously 30 minutes before doxorubicin administration (3@g/kg).

Results obtained in CD2Fl mice treated with injection and administered doxorubicin (DX) (7° i.p.) on day 1 are reported.

GSH was administered 30 minutes before DX.

The GSH+DX combination experiment was repeated with two groups of animals. [See Table 1 at the end. TMS in the table means survival period (days), and T/C
% refers to the % response of drug-treated animals compared to control (taken as 100).

TOX refers to the number of treated animals that died before control animals. LTS indicates the number of animals that survived for a long period of time (at least 2 months).

It is emphasized that the test using the experimental model, P388 leukemia, is characterized by the growth of intraperitoneal tumors exclusively within the peritoneal cavity, during which sequestration of tumor cells takes place.

The results reported in Table 1 confirm that glutathione alone lacks direct antitumor activity and does not increase the natural defenses that M tumor engraftment protects animals from tumor attack. are doing.

On the contrary, prophylactic treatment with GSH, compared to subsequent treatment with an anthracycline (e.g. doxorubicin), shows the drug's anti-inflammatory effects, expressed as a significant increase in TMS, T/C% and LTS values. causing an unexpected increase in tumor activity.

3) Doxolpici in mice The experimental conditions are similar to those of the previous test.

In these tests, glutathione was administered intravenously 30 minutes before any intraperitoneal administration of the anthracycline, doxorubicin.

GSH-treated animals received the same drug (1500 mg/
kg) was processed. Doxorubicin-treated animals received the anthracycline scalar Wk (scalar amou).
nts).

Animals that received GSH and DX sequentially were treated with the two drugs at a ratio of GSHIOo, 125, 150 and 200 mg per mg of DX.

The experimental results reported in Appendix 2 confirm that GSH has no direct or indirect tumor growth effects and no toxic effects when administered intravenously.

Treatment with DX is, in this experimental model, lomg/kg
The dose showed efficacy with T/C% value of 170 and 7.5++
The mg/kg dose also shows little activity, but higher doses show immediate toxic effects. [See Table 2 at the end] Combined treatment of GSH/DX shows the protective effect of the combination against the toxicity of anti-tumor drugs at all weight ratios used.

Indeed, at the highest DX dose, the relatively low GSH/DX ratio (100 g/kg) indicates a reduction in TOX parameters from 8/8 to 7/8 after pretreatment with GSH.

On the other hand, for the lowest DX dose, a relatively high GSH/DX weight ratio, such as 200 mg/mg, appears to indicate a decrease in the efficacy of the anti-N-cancer drug.

T/C% is decreased from 155 to 149th for DX treatment only (7,51 g/kg), although some protective effect is inferred from the survival of 178 animals after 60 days.

This experiment was conducted in the same manner as the experimental design for Tests 2 and 3 above. Glutathione was administered intravenously 30 minutes before administration of doxorubicin. In this test, the experimental data are reported in Tables 3 and 4 below, doxorubicin was administered intraperitoneally at the suboptimal dose (for the treatment regimen used) to demonstrate the synergistic effect of glutathione treatment.

In the tests in Table 3, using various doses of glutathione seeded with P388 leukemia cells, the DX was 7.5.
Fix the GSH/DX ratio to DX1m.
The GSH content was adjusted to 20 and 50 mg per gram (no change in content), respectively.

Table 3 attached at the end shows that CD2Fl mouse 1'(7)P3
This is the effect of GSH and doxorubicin on 88 leukemia.

When inoculated with P388/DX-resistant leukemia cells, a moderate dose of glutathione was administered at a GSH/DX ratio of 25 mg
/mg (for total GSH, 1187.5).

Table 4 at the end of the attachment shows I) 3 in CD2Fl mice.
This figure shows the effects of GSH and doxorubicin on 88/D'X Atria-resistant leukemia.

The results demonstrate synergistic effects on both sensitive and resistant species.

Human HT-29 cells (colon cancer) were used;
Pretreated with GSH at concentrations of 20 and 40 mg/ml 5
They were then exposed to MTX for 24 hours at concentrations of 0.05.0, 1, 0.5 and 1/ml.

The concentration that kills 50% of cells is 0.1 in the case of GSH deficiency.
2 mg/1 ml, and in the presence of GSH, approximately 0.04-0.06 mg/+lt'. The cells were cultured in RPM1164n ↓1(V, skewered fetal serum).

Table 2

Claims (8)

[Claims] (1) consisting of an effective amount of one or more anticancer drugs selected from the group consisting of platinum complexes, oxazophosphorines, anthracyclines, 5-fluorouracil, and methotrexate and 2.5-5 g of glutathione (GSH); Synergistic anti-cancer preparations in the form of combinations for simultaneous, separate or sequential use for anti-cancer treatment (2) The pharmaceutical preparation according to claim 1, wherein the anticancer drug is cis-dichlorodiaminoplatinum, cyclophosphamide, methotrexate, doxorubicin (Adrimycin) or 5-fluorouracil. (3) The anticancer preparation according to claim 1 or 2, wherein the anticancer drug and glutathione are separate single agents. (4) The anticancer preparation according to claim 1 or 2, wherein the anticancer drug and glutathione are in the form of a unit dose. (5) A pharmaceutical formulation according to any one of the preceding claims, wherein the anticancer drug is cis-dichlorodiaminoplatinum and the GSH is present in an amount of 20-100 mg of the anticancer drug. (6) A pharmaceutical formulation according to any one of the preceding claims, wherein the anticancer drug is cyclophosphamide and GSH is present in an amount of 0.5 to 4 mg of the anticancer drug. (7) A pharmaceutical formulation according to any one of the above claims, wherein the anticancer drug is doxorubicin and GSH is present in an amount of 20 mg to 50 mg of the anticancer drug. (8) The pharmaceutical formulation of any one of the preceding claims, wherein the anticancer drug is methotrexate and GSH is present in an amount of 0.3 mg to 15 mg of the anticancer drug.