HK1016888A - 1,2,4-benzotriazine oxides formulations - Google Patents

Description

1, 2, 4-benzotriazine oxide formulations

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Background

Technical Field

The present invention relates to the treatment of cancer (cancer tumor). In particular, the invention relates to the treatment of cancer tumors with a buffered aqueous vehicle containing 1, 2, 4-benzotriazine oxide.

Reported research progress

1, 2, 4-benzotriazine oxides are known compounds. US3980779 discloses a composition for promoting growth in livestock comprising 3-amino-1, 2, 4-benzotriazine-1,antibacterial compositions of 4-dioxides

Wherein

R and R¹One of which is hydrogen, halogen, lower alkyl, halo (lower alkyl), lower alkoxy, carbamoyl, sulfonamido, carboxy or lower alkoxycarbonyl, R and R¹Is halogen, lower alkyl, halo (lower alkyl), lower alkoxy, carbamoyl, sulfonamido, carboxy or lower alkoxycarbonyl.

US5175827 published 29.12.1992 discloses the use of 1, 2, 4-benzotriazine oxides in combination with radiation therapy for the treatment of tumors. 1, 2, 4-benzotriazine oxide sensitizes tumor cells to radiation therapy and makes patients more amenable to the treatment regimen.

Holden et al (1992) in "enhancing the Activity of alkylating agents in FSaIIC murine fibrosarcoma with SR-4233" (JNCI 84: 187-193) disclose the use of SR-4233 (i.e., 3-amino-1, 2, 4-benzotriazine-1, 4-dioxide, a compound known and sometimes referred to hereinafter as tirapazamine) in combination with an anti-tumor alkylating agent. Four anti-tumor alkylating agents, namely cisplatin, cyclophosphamide, carmustine and melphalan, were tested to determine the ability of the tipazamine to overcome the resistance of hypoxic tumor cells to the anti-tumor alkylating agents. The tirapazamine itself and its binding to varying amounts of each anti-tumor alkylating agent were tested. When SR-4233 was administered just prior to a single dose of cyclophosphamide, carmustine and melphalan, it was found that the dose increase resulted in a synergistic cytotoxic effect on tumor cells.

International application PCT/US89/01037 discloses 1, 2, 4-benzotriazine oxides for use as radiosensitizers and selective cytotoxic agents. Other related patents include: US3868372 and 4001410, wherein the preparation of 1, 2, 4-benzotriazine oxides is disclosed; and US3991189 and 3957799, in which derivatives of 1, 2, 4-benzotriazine oxides are disclosed.

It has been found that 1, 2, 4-benzotriazine oxides are effective in the treatment of cancer when combined with radiation and chemotherapy.

Radiation therapy, chemotherapy and surgery are the three main methods of treating cancer. As an alternative to surgery, radiotherapy and chemotherapy are primarily used to control tumor changes, whereas surgery is limited to anatomical aspects. It has been shown that if the effects of radiotherapy and chemotherapy are increased, the cure rate of cancer patients is higher and the quality of life is better.

One way to increase the effectiveness of radiation or chemotherapy is to take advantage of the hypoxic nature of tumors-one of several available differences between normal and tumor tissues. Abnormal growth of blood vessels is characterized by a large number of solid tumors. Abnormal capillary systems typically result in temporary or permanent anoxic zones. Generally, hypoxia increases the tolerance of normal or cancer cells to treatment. Methods to enhance killing of hypoxic tumor cells (or to limit radiation damage to normal tissue) will increase the therapeutic index of radiotherapy or chemotherapy.

Benzotriazine compounds have been developed that take advantage of the relative lack of oxygen in tumor cells. The tirapazamine in the most promising series of benzotriazines to date is bioreduced under hypoxic conditions to an active intermediate. The active intermediate can cause DNA damage, thereby enhancing the effect of radiotherapy or chemotherapy, and has cytotoxicity peculiar to itself. Since the adjacent normal tissue is not hypoxic, the bioreduction has a selective cytotoxic effect on hypoxic tumor cells.

In vitro studies have shown that the benzotriazine class of compounds are actually superior to nitroimidazole radiosensitizers and other bioreductive agents, as shown in table I.

TABLE I

In vitro hypoxic cytotoxicity ratio to various bioreductive Agents

Bioreductive agents (and type)	Hypoxic cytotoxic ratioa
			Rodent animal	Human being
	Tirapazamine (benzotriazine di-N-oxide) RSU-1069 (nitroimidazole/aziridine) etherol nitroimidazole porphyrinomycin (quinone) diamine nitracridine (nitroaziridine) mitomycin C (quinone)	75-200 15-10075-100 10-2010-15 155-10 ～107 --1-5 1-2

^aHypoxic cytotoxicity ratio-the ratio of drug concentrations required to kill the same amount of cells under aerobic conditions as compared to hypoxic conditions.

However, the tirapazamine has the disadvantage of having insufficient solubility in pharmaceutical carriers for parenteral administration and is unstable in these carriers. It has been found that the solubility of tirapazamine in water is about 0.81mg/ml, which requires the administration of large volumes of about 1 liter solution to a patient to provide a suitable dosage. Attempts have been made to increase solubility with surfactants such as tween 80 and polymers such as Pluronic F68, pyrrolidone and albumin, but have not been successful, only minimally increasing solubility. Increasing solubility with co-solvents was more successful, however, the proportion of co-solvent necessary to achieve dissolution of the expected minimum tolerated dose of tirapazamine would mean that large amounts of co-solvent, for example up to 120 ml of propylene glycol, in the form of a 50% v/v propylene glycol/water solution would need to be infused. Such large volumes of co-solvent are undesirable for injectable formulations and pose undesirable clinical risks to patients.

Tirapazamine also lacks shelf stability: complete degradation is achieved in 0.1N NaOH at reflux for less than 4 hours.

The main object of the present invention is to provide an injectable/infusible aqueous formulation which contains a sufficient amount of an anticancer agent and is shelf stable. Extensive clinical studies on tirapazamine have shown that this very promising drug, which does not have sufficient solubility and stability, is not helpful for countless patients with carcinomas.

Summary of The Invention

The present invention provides a parenteral aqueous formulation for the treatment of cancer comprising:

a cancer tumor treating effective amount of a compound of formula (I) or a pharmaceutically acceptable salt of said compound in a parenterally acceptable buffer solution at a concentration of about 0.001M to about 0.1M

Wherein X is H; a hydrocarbon group of 1 to 4 carbon atoms; by OH, NH₂NHR or NRR substituted hydrocarbyl of 1 to 4 carbon atoms; halogen; OH; alkoxy of 1 to 4 carbon atoms; NH (NH)₂(ii) a NHR or NRR; wherein each R is independently selected from the group consisting of lower alkyl of 1-4 carbon atoms and lower acyl of 1-4 carbon atoms, and is substituted with OH, NH₂Secondary alkyl amino of 1 to 4 carbon atoms and tertiary dialkyl amino of 1 to 4 carbon atoms, alkoxy of 1 to 4 carbon atoms or lower alkyl of 1 to 4 carbon atoms substituted by halogen and lower acyl of 1 to 4 carbon atoms; and when X is NRR, both R are joined directly or through an oxygen bridge to form a morpholino, pyrrolidino, or piperidino ring;

n is 0 or 1; and is

Y¹And Y²Are independent of each otherH; a nitro group; halogen; hydrocarbyl of 1 to 14 carbon atoms, including cyclic and unsaturated hydrocarbyl, optionally substituted with 1 or 2 substituents selected from halogen, hydroxy, epoxy, alkoxy of 1 to 4 carbon atoms, alkylthio of 1 to 4 carbon atoms, primary amino (NH)₂) Secondary alkyl amino of 1 to 4 carbon atoms, tertiary dialkyl amino of 1 to 4 carbon atoms (in tertiary dialkyl amino of 1 to 4 carbon atoms, two alkyl groups are linked together to form morpholino, pyrrolidino or piperidino), acyloxy of 1 to 4 carbon atoms, acylamino of 1 to 4 carbon atoms and thio-analogues thereof, alkyl of 1 to 4 carbon atoms of acetylamino, carboxyl, alkoxycarbonyl of 1 to 4 carbon atoms, carbamoyl, alkylcarbamoyl of 1 to 4 carbon atoms, alkylsulfonyl of 1 to 4 carbon atoms or alkylphosphoryl of 1 to 4 carbon atoms, wherein the hydrocarbon groups may optionally be interrupted by monoether (-O-) bonds; or wherein Y is¹And Y²Independently of one another, morpholino, pyrrolidino, piperidino, NH₂NHR ', NR ' R ' O (CO) R ', NH (CO) R ', O (SO) R ' or O (POR ') R ', wherein R ' is a hydrocarbon group of 1 to 4 carbon atoms which may be substituted by OH, NH, or₂Secondary alkyl amino of 1-4 carbon atoms, tertiary dialkyl amino of 1-4 carbon atoms, morpholino, pyrrolidino, piperidino, alkoxy of 1-4 carbon atoms or halogen substituent.

In particular, the parenteral formulation of the invention for the treatment of cancer or neoplasia comprises:

from about 0.500 to about 0.810 grams of a compound of formula (I);

about 0.100 to about 9.000 grams of sodium chloride;

about 0.1 to about 10.00 grams of citric acid;

about 0.02 to about 3.00 grams of sodium hydroxide; and

adjusting pH to 3.0-5.0 in water, and adding water to 1000 ml.

The preferred anticancer tumor compound of the present invention is tirapazamine, 1, 2, 4-benzotriazin-3-amine 1, 4-dioxide, and the structural formula is as follows

Its molecular weight is 178.16, and its melting point is 220 deg.C decomposition.

Most preferred intravenous formulations are those containing from about 0.7 to about 0.81mg/ml tirapazamine per ml of solution in isotonic citrate buffer at a pH of from about 3.7 to about 4.3.

The invention also relates to a method of treating cancer in a patient suffering from cancer, which method comprises administering to said patient an amount of the formulation effective to treat cancer.

Detailed Description

Antitumor agent

The present invention provides compositions and methods for treating mammalian, including human, cancers, particularly solid tumors. For this aspect of the invention, an effective amount of a compound of formula I contained in citrate buffer is administered to a mammal having a cancer and in need of treatment, and after about 1.5 to about 24 hours, a tumor-sensitive effective amount of a chemotherapeutic agent is administered to the mammal. Compounds of formula I and test compounds are described in U.S. patent application 125609, filed on 9/22/1993, the disclosure of which is incorporated herein by reference in its entirety.

In the preparation of the formulations of the present invention, extensive studies have been made on the sufficient solubility of the cancer compound and the shelf-stability of the formulations, and the results are as follows.

The invention will be described with particular reference to tirapazamine formulations, but it will be understood that other compounds encompassed by formula (I) are also encompassed within the scope of the claims of the present invention.

Solubility of Tirapazamine

The solubility of Tirapazamine in water and various carriers is shown in table II.

TABLE II

Solubility of Tirapazamine in aqueous media

Solvent temperature deg.C mg/ml Water for injection 201.43 Water for injection 150.85 physiological saline 150.85 citrate buffer 0.05M pH4 (isotonic) 150.81 lactate buffer 0.1M pH4 (isotonic) 150.90 Tween 800.2% w/v 150.9 Tween 8020% w/v 151.02 Pluronic F6820% w/v 151.08 pyrrolidone (Kollidon 12PF) 10% w/v 150.95 albumin 4.5% w/v 201.33 albumin 20% w/v 201.71 glycerol 50% v/v aqueous solution 152.93 glycerol 154.59 propylene glycol 50% v/v 152.58 aqueous solution propylene glycol 153.27 PEG 40050% v/v aqueous solution 151.60 PEG 400155.12 dimethylformamide 25% v/v aqueous solution 151.831% benzyl alcohol: 10% ethanol: 89% Water v/v 151.23 ethanol 10% v/v aqueous solution 150.93 ethanol 50% v/v aqueous solution 152.32 ethanol 65% v/v aqueous solution 152.84 ethanol 85% v/v aqueous solution 151.71 ethanol 150.47

The limited solubility of 0.81mg/ml makes it necessary to infuse 1 litre of liquid, and therefore to minimise the volume of liquid, the solubility needs to be increased. Attempts have been made to increase solubility by using surfactants (tween 80) and polymers (Pluronic F68, pyrrolidone, albumin), but without success, there was only a minimal increase in solubility.

Increasing solubility with co-solvents has been successful, however, the proportion of co-solvent necessary to dissolve the maximum tolerated dose of tirapazamine (-700 mg) would mean that large amounts of co-solvent (e.g. up to 120 ml of propylene glycol, infused as a 50% v/v propylene glycol/water solution) would need to be infused.

The biochemical properties of Tirapazamine suggest that the molecule is neither highly polar nor highly lipophilic. This property is illustrated by (i) partition coefficient (octanol/water) 0.15(logP-0.82) and (ii) melt decomposition at 200 deg.C, which indicates that the crystal structure of tirapazamine is strongly bonded by intermolecular forces. The planar (planar) nature of the molecule allows ordered packing of the crystals due to intermolecular attraction (charge transfer) between each plane by nitrogen and oxygen of the N-oxide. The tirapazamine may be in a hydrated form if the water molecule is hydrogen bonded to the oxygen containing moiety.

In order to predict the solubility of compounds in water-solvent mixtures, attempts have been made to classify organic solvents by parameters such as dielectric constant, solubility parameters, surface tension, interfacial tension, hydrogen bond donor and acceptor density, and octanol-water partition coefficient. The various solvents selected for the tirapazamine solubility study are shown in table III. These parameters have been used accurately to predict the solubility of non-polar solutes by correcting them with the slope of a solubility map plotted from experimental data. Those parameters that reflect solvent cohesion, such as solubility parameters and interfacial tension, are most correlated with slope, and therefore, the hydrogen bonding capability of the pure co-solvent is expressed in terms of the density of the proton groups or accepting proton groups.

TABLE III

Polarity index of solvent

(Rubino, j.t. and Yalkowsky, s.h., cosolubility and cosolvent polarity, pharmaceutical

Research (Pharmaceutical Research), 4(1987)220-

Aqueous DMSO DMF DMA GLYC PG PEG400 dielectric constant 78.546.736.737.842.532.013.6 solubility parameter 23.412.012.110.817.712.611.3 interfacial tension (dyne/cm) 45.60.96.94.632.712.411.7 surface tension (dyne/cm) 72.744.036.835.760.637.146.0 logP-4.0-1.4-0.85-0.66-2.0-1.0- - -hydrogen bond donor density 111.00.00.00.041.127.45.6 hydrogen bond acceptor density 11.028.238.732.382.254.450.8

Wherein:

DMSO ═ dimethyl sulfoxide

DMF ═ dimethylformamide

DMA ═ dimethyl acetamide

GLCY ═ glycerol

PG ═ propylene glycol

PEG400 ═ polyethylene glycol 400

Large volume fractions of aprotic solvents such as dimethyl sulfoxide (DMSO), Dimethylformamide (DMF) and Dimethylacetamide (DMA) disrupt the structure of water due to dipolar action and hydrophobic interaction. Amphoteric solvents such as glycerol, PEG400 and Propylene Glycol (PG) can associate with themselves and pass hydrogen bonding with water, and therefore, the solvents are not practically suitable for solutes that do not participate in hydrogen bonding. The partition coefficient of the solute is an indicator of whether the co-solvent is effective or not. The solubility in various solvent systems can be predicted successfully using the following equation:

logCs＝logCo＝f(logR+0.89logP+0.03)

wherein C is_sAnd C_oIs the solubility in the solvent mixture and water, respectively, f is the co-solvent coefficient, R is the relevant solubility capacity (typical values: DMF ═ 4, glycerol ═ 0.5), and P is the partition coefficient. When P goes to 1(logP  0), the solubility cannot be increased because of

logCs＝logCo

Since tirapazamine has a logP value of-0.8, this equation indicates that the co-solvent does not have a significant effect on water solubility as might be expected. The results of experiments performed with these co-solvents indicate that these co-solvents do not significantly increase the solubility of tirapazamine.

Stability of

Stress experiments were performed at 121 ℃ using multiple 21 minute autoclave cycles. These studies indicate that tirapazamine is more stable in an acidic solution of physiological saline or in a solution buffered with 0.05M citrate or 0.1M lactate buffer. The tirapazamine is unstable in the presence of phosphate buffer at pH5.9 and in citrate buffer at pH 6. After 8 autoclave cycles, the pH of the physiological saline formulation changed from 4.5 to 4.9, so the formulation required some degree of buffering.

Storage at elevated temperatures of 50 ℃ and 70 ℃ also stressed the formulation after a single 21 minute autoclave cycle at 121 ℃. Tirapazamine has been found to be unstable after storage at 70 ℃ in the presence of lactate buffer. This instability did not occur over multiple autoclave pressures. It was found that the formulation was most stable for 0.05M citrate at pH4.

Therefore, tirapazamine formulations were prepared using citrate buffer. The solubility of tirapazamine at 15 deg.C requires a reduction in concentration from 1mg/ml to 0.5 mg/ml. The stress response in citrate buffer at pH3.5, 4.0 and 4.5 was also measured for pH limits in the same way. Based on the data from this study, the pH should be limited to 4.0. + -. 0.3.

According to the stability data obtained, the most stable tirapazamine formulation was in citrate buffer at pH4. the solubility of tirapazamine in citrate buffer was 0.81mg/ml at 15 ℃. Therefore, to limit the volume of infusion fluid, the maximum concentration used in further studies was 0.7 mg/ml.

The effect of buffer concentration (0.05 or 0.005M) on stability was evaluated by applying pressure to a 2 x 10 liter batch of stable tirapazamine in citrate buffer at pH 4.0.

Tirapazamine in 0.005M and 0.05M citrate buffer was stable after 2 months at 50 ℃. At 70 ℃, 0.05M citrate formulations were unstable, therefore, lower citrate concentrations (0.005M) were chosen to develop clinical formulations. Clinical formulations for chemical studies will be discussed below:

tirapazamine 0.700 g

8.700 g of sodium chloride

0.9605 g of citric acid

0.2500 g of sodium hydroxide

Adding water to 1000 ml, adjusting pH to 4.0.

The tirapazamine was stored in 20 ml clear glass ampoules containing 0.7 mg/ml (14 mg) of tirapazamine in isotonic citrate buffer. The ampoules were stored in light-tight packaging at 15 ℃ and 30 ℃.

Administration of drugs

Acute tolerance studies, single or multiple dose studies, and in vitro spinal cord inhibition studies were performed in rats and dogs using the formulations of the present invention.

In acute tolerance studies in mice, the LD of tirapazamine₁₀And LD₅₀Values were 98 and 101 mg/kg, respectively.

Single, 2 week and 2 month multiple dose studies were performed in rats and dogs. Clinical signs and symptoms observed in both animals and in each protocol included salivation, a decrease in the amount of blood leukocytes (including lymphocyte counts in dogs), and a decrease in the amount of blood erythrocytes.

Pharmacology of

The effect of tirapazamine on various aerobic and anoxic cells was studied in culture to determine the selectivity of tirapazamine cytotoxicity. Tirapazamine (20 μ M) is a potent and selective hypoxic cell in vitro killing agent with hypoxic cytotoxicity ratios of 150, 119 and 52 (1-2 orders of magnitude greater than radiosensitizers such as nitroimidazoles, mitomycin C and methylmitomycin), respectively, on hamster, mouse and human cell lines. Under oxygen pressure (1% -20% O)₂(ii) a Mainly 1% -4% of O₂) This cytotoxicity was also observed within the range.

In smallIn vivo in murine tumor models, when tirapazamine is administered at 0.30 mmole/kg (160 mg/m)²) Single dose or 0.08 mmole/kg (43 mg/m)²) The multiple doses of (2.5 Gy. times.8) were also effective when used with fractionated radiation. 0.30 mmole/kg (160 mg/m) when used simultaneously with a single bolus (20Gy) of radiation²) Single doses of tirapazamine are also effective. Before each radiotherapy (2.5 Gy.times.8), at 0.08 mmole/kg (43 mg/m)²) Multiple doses of tirapazamine were most effective, curing SCCVII tumors in several mice; tirapazamine is least effective if not treated with radiation, and generally has a log of cell kill of less than 1. When used with fractionated radiation therapy, the tirapazamine produced the same effect as predicted if it acted on a single cell population (hypoxic cells) as compared to radiation therapy on aerobic cells.

The mechanism of action of tirapazamine, which is closely related to drug metabolism, has been studied in detail. The following describes a possible mechanism of action of the tirapazamine to generate free radicals during reduction of the mono-N-oxide, which cause single or double strand breaks in DNA. Under anoxic conditions, tirapazamine is metabolized to 2-electron reduction product WIN64102 (mono-N-oxide; SR4317) and then to 4-electron reduction product WIN60109 (O-N-oxide; SR 4330). Several studies examining DNA damage repair after treatment with tirapazamine showed that DNA repair inhibition is dose-related and similar to X-ray production.

Extensive in vitro and in vivo studies of the benzotriazine di-N-oxide tirapazamine were performed to determine and quantify its efficacy and elucidate its mechanism of action.

In vitro

The effect of tirapazamine on various aerobic and anoxic cells was studied in culture to determine the selectivity of tirapazamine cytotoxicity. The study used Chinesian ovarian cells (CHO-HA-1), mouse cells (C3H 10T1/2, RIF-1, and SCCVII), and human cell lines (HCT-8, AG1522, A549, and HT 1080). As shown in Table 4, tirapazamine (20. mu.M) is a potent and selective hypoxic cell killing agent in vitro.

TABLE 4

The pair of tirapazamines being cultured under aerobic or anoxic conditions

In vitro cytotoxicity of 8 cell lines

Cell lines	Sensitive IC50 cIndex of refractionb (μM)	Hypoxic cytotoxic ratioa
			Name of species		Cell line average number of the species
Hamster CHO-HA-1 (Normal)d)	48 5	100-200 150
			Mouse RIF-1 (tumor) SCCVII (tumor) C3H 10T1/2 (normal)	30 339 4118 12	80-100160-200 11975-100
Human HCT-8 (tumor) A549 (tumor) AG1522 (normal) HT1080 (tumor)	94 10280 15190 1322	15-4025-5050 52100

^aHypoxic cytotoxicity ratio-the concentration of tirapazamine in air/concentration of tirapazamine in nitrogen that yields approximately the same survival.

^bSensitivity index of 10 under anoxic conditions and at a concentration of 20. mu.M^-2(1%) time required for survival (min).

^cIC₅₀Concentration required to inhibit 50% of cell growth when cultured for 1 hour under hypoxic conditions.

^dNormal is no tumorigenesis.

In vivo

Treatment with tirapazamine alone

When mice were studied in vivo with only a single dose of tirapazamine, it was expected that only a relatively small number of cells would be killed, corresponding to the percentage of hypoxic tumor cells. Experimental data show that in this case, the log value of the killed cells is generally lower than 1 (survival fraction is more than or equal to 1-10)^-1). For example, the maximum cell kill observed with a single dose of treatment is in SCCVII tumors (survival score of 5 · 10)^-1) And only a small delay in tumor growth in FSaIIC fibrosarcoma of 3 days.

Multiple doses of tirapazamine treatment without radiation therapy would be expected to produce slightly greater cell killing than single dose treatments, even when the tirapazamine dose is lower. However, the lowest survival score among the four different mouse tumors was 5 · 10^-1Whereas in the 5 th mouse tumor (RIF-1 tumor) the value decreased to 5.10^-2。

Tirapazamine in combination with radiation therapy

In the various models described below, tirapazamine potentiated the anti-tumor effect of radiation therapy, presumably due to killing cells or delaying tumor growth. Experimental tumors included FSaIIC, SCCVII, RIF-1, EMT6, and KHT. The tirapazamine potentiates cell killing when the tirapazamine is treated in a single dose or multi-dose regimen, or when the drug is combined with a single dose of radiation therapy or fractionated radiation therapy.

In one study, the antitumor effect of tirapazamine in combination with radiation therapy exceeded the additive effect of the two treatments. The activity of the tirapazamine is potentiated when the tirapazamine is administered 2.5-0.5 hours after radiation therapy or up to 6 hours after. In addition to its effect on hypoxic cells, a tirapazamine can radiosensitize aerobic cells in vitro if the aerobic cells are contacted with the tirapazamine under hypoxic conditions, either before or after radiation therapy.

In one study, treatment with tirapazamine potentiated the anti-tumor activity of radiation therapy to a greater extent than the hypoxic cell sensitizer etanidazole.

the oxygen concentration/cytotoxicity profile of tirapazamine indicates that the drug is particularly suitable for use in combination with radiation therapy. Cells below about 30torr (mmhg) have increased resistance to the damaging effects of radiation. However, the nitroaromatic and quinone antibiotic radiosensitizers are only most effective at very low oxygen levels. Therefore, they are not toxic to the moderately hypoxic radiation resistant cells present in tumors. In contrast, the cytotoxicity of tirapazamine remained relatively constant over the entire range of oxygen concentrations that radiotolerance had.

Unlike other radiosensitive substances studied to date, tirapazamine toxicity is reduced at high oxygen concentrations (i.e., as observed in normal tissues). In an in vitro system, the toxicity of the tirapazamine is at least 50 to > 2000 times greater under anoxic conditions than under 100% oxygen vapor conditions. Because the drug has activity on various radiation-resistant tumor cells, but is nontoxic to normal cells with high oxygen content, the tirapazamine has selectivity on the toxicity of hypoxic tumor cells.

Tirapazamine in combination with chemotherapy

When tirapazamine (25-75 mg/kg, IP 83.3-250 mg/m) is administered to mice with FSaIIC fibrosarcoma²) When this was done, some tumor cells were observed to be killed directly. Tirapazamine (50 mg/kg, IP 167 mg/m)²) With cyclophosphamide (150 mg/kg, IP 500 mg/m)²) Phenylalanine mustard (10 mg/kg, IP 33 mg/m)²) Or cisplatin (10 mg/kg, IP 33 mg/m)²) In combination, the delay in tumor growth increased 1.6-5.3 fold in this model.

Effects on Normal tissue

Female C3H/Km mice were used in the following two experiments to determine the effect of tirapazamine on the possible presence of normal tissue that is sensitive to ionizing radiation. Normal skin reaction experiments and leg (thigh) spasm experiments were performed using fractionated irradiation. In both experiments, tirapazamine had no effect on tissue.

To determine whether tirapazamine might affect normal tissue, the right hind limb of female C3H/Km mice was irradiated for 4 days at 8 (3, 4, 5 or 6 Gy) with one irradiation change every 12 hours. Mice were injected with saline or tirapazamine (0.08 mmol/kg 43 mg/m) 30 minutes before or immediately after each stage of irradiation²). Skin reactions of the irradiated thighs were graded three times a week from day 10 to day 32 after the first irradiation. Mice were identified as "blind" according to a similar grade specification as previously studied mice-treatment unaware groups [ Brown JM, Goffinet DR, Cleaver JE, Kallman RF, "chronic intra-arterial infusion of halogenated pyrimidine analogs preferentially radiosensitizing mouse sarcomas relative to normal mouse skin", JNCI (1971)47, 77-89]. Skin reaction assays showed that tirapazamine did not produce radiosensitizing effects or additional toxicity in combination with radiation therapy.

The invention has been described above with reference to specific examples, it being understood that any modification within the scope of the invention will be apparent to a person skilled in the art.

Claims (11)

1. A parenteral aqueous formulation for the treatment of cancer comprising:

a cancer tumor treating effective amount of a compound of formula (I) or a pharmaceutically acceptable salt of said compound in a parenterally acceptable buffer solution at a concentration of about 0.001M to about 0.1M

Wherein X is H; a hydrocarbon group of 1 to 4 carbon atoms; by OH, NH₂NHR or NRR substituted hydrocarbyl of 1 to 4 carbon atoms; halogen; OH; alkoxy of 1 to 4 carbon atoms; NH (NH)₂(ii) a NHR or NRR;wherein each R is independently selected from the group consisting of lower alkyl of 1-4 carbon atoms and lower acyl of 1-4 carbon atoms, and is substituted with OH, NH₂Secondary alkyl amino of 1 to 4 carbon atoms and tertiary dialkyl amino of 1 to 4 carbon atoms, alkoxy of 1 to 4 carbon atoms or lower alkyl of 1 to 4 carbon atoms substituted by halogen and lower acyl of 1 to 4 carbon atoms; and when X is NRR, both R are joined directly or through an oxygen bridge to form a morpholino, pyrrolidino, or piperidino ring;

n is 0 or 1; and is

Y¹And Y²Independently of one another are H; a nitro group; halogen; hydrocarbyl of 1 to 14 carbon atoms, including cyclic and unsaturated hydrocarbyl, optionally substituted with 1 or 2 substituents selected from halogen, hydroxy, epoxy, alkoxy of 1 to 4 carbon atoms, alkylthio of 1 to 4 carbon atoms, primary amino (NH)₂) Secondary alkyl amino of 1 to 4 carbon atoms, tertiary dialkyl amino of 1 to 4 carbon atoms (in tertiary dialkyl amino of 1 to 4 carbon atoms, two alkyl groups are linked together to form morpholino, pyrrolidino or piperidino), acyloxy of 1 to 4 carbon atoms, acylamino of 1 to 4 carbon atoms and thio-analogues thereof, alkyl of 1 to 4 carbon atoms of acetylamino, carboxyl, alkoxycarbonyl of 1 to 4 carbon atoms, carbamoyl, alkylcarbamoyl of 1 to 4 carbon atoms, alkylsulfonyl of 1 to 4 carbon atoms or alkylphosphoryl of 1 to 4 carbon atoms, wherein the hydrocarbon groups may optionally be interrupted by monoether (-O-) bonds; or wherein Y is¹And Y²Independently of one another, morpholino, pyrrolidino, piperidino, NH₂NHR ', NR ' R ' O (CO) R ', NH (CO) R ', O (SO) R ' or O (POR ') R ', wherein R ' is a hydrocarbon group of 1 to 4 carbon atoms which may be substituted by OH, NH, or₂Secondary alkyl amino of 1-4 carbon atoms, tertiary dialkyl amino of 1-4 carbon atoms, morpholino, pyrrolidino, piperidino, alkoxy of 1-4 carbon atoms or halogen substituent.

2. A method of treating cancer in a patient having cancer, the method comprising administering to the patient an effective cancer treating amount of a formulation comprising:

a cancer tumor treating effective amount of a compound of formula (I) or a pharmaceutically acceptable salt of said compound in citrate buffer at a concentration of about 0.005M to about 0.05M

Wherein X is H; a hydrocarbon group of 1 to 4 carbon atoms; by OH, NH₂NHR or NRR substituted hydrocarbyl of 1 to 4 carbon atoms; halogen; OH; alkoxy of 1 to 4 carbon atoms; NH (NH)₂(ii) a NHR or NRR; wherein each R is independently selected from the group consisting of lower alkyl of 1-4 carbon atoms and lower acyl of 1-4 carbon atoms, and is substituted with OH, NH₂Secondary alkyl amino of 1 to 4 carbon atoms and tertiary dialkyl amino of 1 to 4 carbon atoms, alkoxy of 1 to 4 carbon atoms or lower alkyl of 1 to 4 carbon atoms substituted by halogen and lower acyl of 1 to 4 carbon atoms; and when X is NRR, both R are joined directly or through an oxygen bridge to form a morpholino, pyrrolidino, or piperidino ring;

n is 0 or 1; and is

Y¹And Y²Independently of one another are H; a nitro group; halogen; hydrocarbyl of 1 to 14 carbon atoms, including cyclic and unsaturated hydrocarbyl, optionally substituted with 1 or 2 substituents selected from halogen, hydroxy, epoxy, alkoxy of 1 to 4 carbon atoms, alkylthio of 1 to 4 carbon atoms, primary amino (NH)₂) Secondary alkyl amino of 1 to 4 carbon atoms, tertiary dialkyl amino of 1 to 4 carbon atoms, in which two alkyl groups are linked together to form morpholino, pyrrolidino or piperidino, acyloxy of 1 to 4 carbon atoms, acylamino of 1 to 4 carbon atoms and thio-analogues thereof, alkyl of 1 to 4 carbon atoms of the acetamido group, carboxyl, alkoxycarbonyl of 1 to 4 carbon atoms, carbamoyl, alkylcarbamoyl of 1 to 4 carbon atoms, alkylsulfonyl of 1 to 4 carbon atoms or alkylphosphoryl of 1 to 4 carbon atoms, wherein the hydrocarbon groups may optionally be interrupted by monoether (-O-) bonds; or wherein Y is¹And Y²Independently of one another, morpholino, pyrrolidino, piperidino, NH₂NHR ', NR ' R ' O (CO) R ', NH (CO) R ', O (SO) R ' or O (POR ') R ', wherein R ' is of 1 to 4 carbon atomsA hydrocarbon group which may be substituted by OH, NH₂Secondary alkyl amino of 1-4 carbon atoms, tertiary dialkyl amino of 1-4 carbon atoms, morpholino, pyrrolidino, piperidino, alkoxy of 1-4 carbon atoms or halogen substituent.

3. A parenteral aqueous formulation for the treatment of cancer comprising:

about 0.500 to about 0.810 grams of a compound of formula (I) or a pharmaceutically acceptable salt of said compound in a citrate buffer at a concentration of about 0.005M to about 0.05M

Wherein X is H; a hydrocarbon group of 1 to 4 carbon atoms; by OH, NH₂NHR or NRR substituted hydrocarbyl of 1 to 4 carbon atoms; halogen; OH; alkoxy of 1 to 4 carbon atoms; NH (NH)₂(ii) a NHR or NRR; wherein each R is independently selected from the group consisting of lower alkyl of 1-4 carbon atoms and lower acyl of 1-4 carbon atoms, and is substituted with OH, NH₂Secondary alkyl amino of 1 to 4 carbon atoms and tertiary dialkyl amino of 1 to 4 carbon atoms, alkoxy of 1 to 4 carbon atoms or lower alkyl of 1 to 4 carbon atoms substituted by halogen and lower acyl of 1 to 4 carbon atoms; and when X is NRR, both R are joined directly or through an oxygen bridge to form a morpholino, pyrrolidino, or piperidino ring;

n is 0 or 1; and is

Y¹And Y²Independently of one another are H; a nitro group; halogen; hydrocarbyl of 1 to 14 carbon atoms, including cyclic and unsaturated hydrocarbyl, optionally substituted with 1 or 2 substituents selected from halogen, hydroxy, epoxy, alkoxy of 1 to 4 carbon atoms, alkylthio of 1 to 4 carbon atoms, primary amino (NH)₂) Secondary alkyl amino of 1 to 4 carbon atoms, tertiary dialkyl amino of 1 to 4 carbon atoms, in which two alkyl groups are linked together to form morpholino, pyrrolidino or piperidino, acyloxy of 1 to 4 carbon atoms, acylamino of 1 to 4 carbon atoms and thio-analogues thereof, alkyl of 1 to 4 carbon atoms, carboxyl, alkoxycarbonyl of 1 to 4 carbon atoms, carbamoyl, 1-Alkylcarbamoyl of 4 carbon atoms, alkylsulfonyl of 1 to 4 carbon atoms or alkylphosphoryl of 1 to 4 carbon atoms wherein the hydrocarbon radicals may optionally be interrupted by monoether (-O-) linkages; or wherein Y is¹And Y²Independently of one another, morpholino, pyrrolidino, piperidino, NH₂NHR ', NR ' R ' O (CO) R ', NH (CO) R ', O (SO) R ' or O (POR ') R ', wherein R ' is a hydrocarbon group of 1 to 4 carbon atoms which may be substituted by OH, NH, or₂Secondary alkyl amino of 1 to 4 carbon atoms, tertiary dialkyl amino of 1 to 4 carbon atoms, morpholino, pyrrolidino, piperidino, alkoxy of 1 to 4 carbon atoms or halogen substituent;

about 0.100 to about 9.000 grams of sodium chloride;

about 0.9000 to about 10.00 grams of citric acid;

about 0.200 to about 3.000 grams of sodium hydroxide; and

adjusted to pH3.0-5.0 in water, and added with water to 1000 ml.

4. A method of treating cancer in a patient suffering from cancer, the method comprising administering to said patient an amount of the formulation of claim 3 effective to treat cancer.

5. A parenteral aqueous formulation for the treatment of cancer comprising:

an effective cancer treating amount of 1, 2, 4-benzotriazin-3-amine 1, 4-dioxide in a citrate buffer at a concentration of about 0.005M to about 0.05M.

6. The parenteral aqueous formulation of claim 5 wherein the pH of said citrate buffer is from about 3.7 to about 4.3.

7. A method of treating cancer in a patient suffering from cancer, the method comprising administering to the patient the formulation of claim 5 in an amount effective to treat the cancer.

8. A parenteral aqueous formulation for the treatment of cancer comprising:

from about 0.500 to about 0.810 grams of 1, 2, 4-benzotriazin-3-amine 1, 4-dioxide;

about 5.000 to about 9.000 grams of sodium chloride;

about 0.9000 to about 10.00 grams of citric acid;

about 0.200 to about 3.000 grams of sodium hydroxide; and

adjusting pH to 3.7-4.3 in water, and adding water to 1000 ml.

9. A method of treating cancer in a patient suffering from cancer, the method comprising administering to the patient the formulation of claim 8 in an amount effective to treat the cancer.

10. A parenteral aqueous formulation for the treatment of cancer comprising:

0.700 g of 1, 2, 4-benzotriazin-3-amine 1, 4-dioxide;

8.700 g of sodium chloride;

0.9605 g of citric acid;

0.2500 grams of sodium hydroxide; and

adjusted to pH4 in water and added with water to 1000 ml.

11. A method of treating cancer in a patient suffering from cancer, the method comprising administering to the patient the formulation of claim 10 in an amount effective to treat the cancer.