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Effect of doxapram on the action of other drugs and the hepatic drug-metabolizing system in mice.

Effects of doxapram, a respiratory stimulant, on the action of other drugs and the activity of the hepatic drug-metabolizing enzyme were studied in mice. The hypothermic effect induced by aminopyrine and the muscle relaxant effect induced by meprobamate were potentiated by the pretreatment with doxapram 60 min before. Furthermore, doxapram significantly enhanced the lethalities of picrotoxin and strychnine and the analgesic actions of aminopyrine and morphine. The plasma concentration of aminopyrine or pentobarbital in doxapram-treated mice was higher than those in untreated mice, and the plasma concentration of normustard related to an active metabolite of cyclophosphamide after the administration of cyclophosphamide was lower in doxapram-treated mice. On the other hand, doxapram (50 mg/kg, i.p.) reduced remarkably the activities of aminopyrine N-demethylase and aniline hydroxylase in the hepatic 9,000xg supernatant fraction, and also reduced the cytochrome P-450 contents in hepatic microsomes. However, no significant alteration by doxapram was observed on the activities of NADH-ferricyanide reductase and NADPH-cytochrome c reductase and cytochrome b5 contents. It seems likely that the mechanisms of the interaction between doxapram and combined drugs involved the depression of the hepatic drug-metabolizing system in microsomes and a subsequent variation of drug level in the plasma.

Abstract-Effects of doxapram, a respiratory stimulant, on the action of other drugs and the activity of the hepatic drug-metabolizing enzyme were studied in mice. The hypothermic effect induced by aminopyrine and the muscle relaxative effect induced by meprobamate were potentiated by the pretreatment with doxapram 60 min before. Furthermore, doxapram significantly enhanced the lethalities of picrotoxin and strychnine and the analgesic actions of aminopyrine and morphine. The plasma concentration of aminopyrine or pentobarbital in doxapram-treated mice was higher than those in untreated mice, and the plasma concentration of normustard related to an active metabolite of cyclophosphamide after the administration of cyclophosphamide was lower in doxapram-treated mice. On the other hand, doxapram (50 mg/kg, i.p.) reduced remarkably the activities of aminopyrine N-demethylase and aniline hydroxylase in the hepatic 9,000xg supernatant fraction, and also reduced the cytochrome P-450 contents in hepatic microsomes. However, no significant alteration by doxapram was observed on the activities of NADH-ferricyanide reductase and NADPH cytochrome c reductase and cytochrome b5 contents. It seems likely that the mechanisms of the interaction between doxapram and combined drugs involved the depression of the hepatic drug-metabolizing system in micro somes and a subsequent variation of drug level in the plasma.
Doxapram has been reported to be a potent respiratory stimulant (1,2) and to produce marked arousal effects in barbiturate induced sleep (3)(4)(5)(6). However, it was shown earlier that doxapram potentiated the hypnotic action induced by amobarbital and pen tobarbital in rats (7) and mice (8).
Previously, we demonstrated that pen tobarbital sleeping time was significantly prolonged 60 min after the intraperitoneal administration of doxapram in mice and that the level of pentobarbital in plasma was higher than that after the administration of pentobarbital alone (9). We found also that doxapram had little influence on the sensitivity of the central nervous system to pentobarbital (9).
The drug interaction is related to changes of drug metabolism in the majority of cases. It was known that the action and toxicity of some combined drugs were changed by SKF-525A, a representative drug which is a hepatic enzyme inhibitor (10). Although doxapram is a respiratory stimulator, it has an inhibitory action on the hepatic enzyme that is similar to SKF-525A. Therefore, the effect of doxapram on the actions of combined drugs is of interested in connection with the drug metabolism.
The present investigation was designed to determine whether there were some inter actions between doxapram and the other drugs in mice and to clarify possible mecha nisms for this interaction. The drugs which had activities that were easily changed by a variations of their metabolisms were selected for combination with doxapram.

MATERIALS AND METHODS
Drugs: The drugs used were obtained from the following commercial sources: Muscle relaxative action: Muscle relaxative actions in mice were examined by the rotarod test (11) and the traction test (12).
Analgesic action: Analgesic action of aminopyrine or morphine was examined by the pressure method (13).
Tumor cells: Ehrlich ascites carcinoma cells maintained in the peritoneum of ddY mice were used. Seven days after the cell inoculation (1 x 107/mouse), the cells were collected from the mice.
Antitumor activity: Cyclophosphamide was injected once intraperitoneally at 24 hr after inoculation of tumor cells (1 X106 cells) into the right thigh. The antitumor activity of cyclophosphamide was estimated by com parison with the control by weighing the solid tumor on the 15th day after cell inoculation.
Determination of the level of hexobarbital, pentobarbital, cyclophosphamide, and amino pyrine in plasma: Hexobarbital (80 mg/kg) or pentobarbital (50 mg/kg) was intraperi toneally administered, and the plasma was collected 30 min after the injection of the barbiturate. Cyclophosphamide or amino pyrine was intraperitoneally administered at 100 mg/kg, and the plasma was collected at the indicated time. Doxapram was admin istered 60 min before these four drugs. The observations on the doxapram-pretreated group and the corresponding control were carried out at the same time under the same conditions. The level of hexobarbital, pen tobarbital, and aminopyrine in the plasma were assayed by the methods of Cooper and Brodie (14), Brodie et al. (15), and Brodie and Axelrod (16), respectively. The metabolite of cyclophosphamide was estimated ac cording to the method of Sladek (17).
Enzyme assays: The excised livers were placed in ice-cold 1.15% KCI solution. All further procedures for the preparation of the enzyme were carried out at 0-4'C. A 20% homogenate fraction was prepared in 1.15% KCI solution using a Potter-Elvehjem type homogenizer. The homogenate was trans ferred to polypropylene centrifuge tubes and centrifuged at 9,000xg for 20 min to obtain the 9,000 x g supernatant fraction. M icrosomal pellets were prepared by centrifuging the 9,000xg supernatant fraction in an ultracen trifuge at 105,000xg for 60 min at 4°C and suspending the resulting pellet in 0.05M phosphate buffer (pH 7.4) so that 1 ml of the suspension contained microsomes obtained from 200 mg of liver. The activities of amino pyrine N-demethylase and aniline hydroxylase in the 9,000 x g liver supernatant fraction were assayed by a slight modification of the method of Kitagawa et al. (18). The activity of pentobarbital oxidase in the 9,000xg liver supernatant fraction was assayed by the method of Brodie et al. (15). Microsomal cytochrome P-450 and cytochrome b5 con tents of the liver were determined according to the method of Omura and Sato (19) in a spectrophotometer (Shimazu UV 300). The activity of UDP-glucuronyl transferase in the 9,000xg liver supernatant fraction was deter mined by the modified method of Hollman and Touster (20). The activities of NADPH cytochrome c reductase and NADH-fer ricyanide reductase were determined by the method of Williams and Kamin (21) and Mihara and Sato (22), respectively.
Protein was determined by the method of Lowry et al. (23) with bovine serum albumin as the standard.
The statistical significance of the results was determined by the Student's t-test.

RESULTS
Effect of doxapram treatment on sleeping action of hexobarbital or thiopental and the hypothermic action of aminopyrine: The mean sleeping time±S.E. of the intraperitoneal administration of hexobarbital (80 mg/kg) or thiopental (60 mg/kg) was 31.9±2.5 and 24.7±4.1 min, respectively. Hexobarbital and thiopental sleeping times of the groups with 25 mg/kg, i.p., of doxapram 60 min before were 105.4±10.1 and 61.6±10.2 min, re spectively. Furthermore, the hypothermic action of aminopyrine (50 or 100 mg/kg, i. p.) in the group with 50 mg/kg, i.p., of doxapram 60 min before was stronger than that of the control group, continuing for a period of 90 min following aminopyrine.
Effect of doxapram treatment on the muscle relaxative action of meprobamate, the analgesic actions of aminopyrine and morphine, and the convulsive actions of strychnine and picrotoxin: As shown in Fig. 1, the muscle relaxative action of meprobamate (100 mg/kg, i.p.) was strongly potentiated by the pretreatment with doxapram (50 mg/kg, i.p.) 60 min before the examinations by the rotarod test and the traction test.
Furthermore, pretreatment with doxapram (50 mg/kg, i.p.) 60 min before enhanced the analgesic activities of aminopyrine (200 mg/ kg, i.p.) and morphine (5 mg/kg, i.p.) as observed by the pressure method. Figure 2 shows the effect of doxapram administered 60 min before on the convulsive action of two convulsants. Regarding the convulsion induced by picrotoxin (7.5 mg/ kg, i.p.), the pretreatment of doxapram 60 min before picrotoxin caused significant ac celeration of the onset of convulsion and death. Similarly, a potentiating effect of doxapram was seen in combination with strychnine: by the pretreatment with doxapram (50 mg/kg, i.p.), the lethality of strychnine at a dose of 0.9 mg/kg, i.p., or 2 mg/kg, i.p., considerably increased and the death time induced by strychnine at the same dose was shortened.  showed an inhibitory effect on tumor growth,   Groups of 12 mice were used. Drugs were injected intraperitoneally. a) The antitumor activity was evaluated by weighing the solid tumor on the 1 5th day after Ehrlich ascites cell inoculation (1 x 106 cells). Cyclophosphamide was injected once, 24 hr after the cell inocu lation. '1) Doxapram was injected 60 min before the injection of cyclophosphamide. °) Drugs were injected 30 min before the injection of cyclophosphamide. *P<0 .05: significantly different from the corresponding control group.  normustard was lower in doxapram-treated mice.
Effect of doxapram on the hepatic drug metabolizing system: Effect of doxapram on the activity of the drug-metabolizing enzyme in the hepatic 9,000xg supernatant fraction in mice was investigated and the results are shown in Fig. 5. The activities of aminopyrine N-demethylase, pentobarbital oxidase, and aniline hydroxylase decreased 60 min after the administration of doxapram (25, 50 or 100 mg/kg, i.p.); and the activity of UDP glucuronyl transferase was slightly inhibited by the injection of doxapram. As shown in Fig. 6, doxapram (50 mg/kg, i.p.) reduced remarkably the activities of aminopyrine N demethylase and aniline hydroxylase in the hepatic 9,000 x g supernatant fraction, 1-24 hr and 1-4 hr later, respectively. Doxapram (50 or 100 mg/kg, i.p.) also reduced cyto chrome P-450 contents in hepatic micro somes 1-4 hr later, but doxapram only slightly decreased the activities of NADPH cytochrome c reductase and NADH-ferricya nide reductase and cytochrome b5 contents.
In contrast, after the consecutive adminis tration of doxapram (50 mg/kg/day for 3 days or for 7 days, i.p.), the activity of aminopyrine N-demethylase was significantly higher than that of the control group, and the activities of aniline hydroxylase and NADPH-cytochrome  c reductase and cytochrome P-450 contents were slightly higher than that of the control group. However, no effects on the activity of NADH-ferricyanide reductase and cyto chrome b5 contents were observed in mice treated with doxapram consecutively (Fig. 7).

DISCUSSION
In the present experiment, it was examined how doxapram affects the action of combined drugs by changing the hepatic microsomal drug-metabolizing system in mice.
It has been known that the actions of many drugs were often affected by the combination of drugs. The previous work in our laboratory showed that doxapram has a biphasic effect, prolongation at first and shortening later, on the pentobarbital sleeping time in mice (9). The barbiturates are oxidized by a hepatic microsomal enzyme (24), and the duration of sleep induced by the barbiturate is affected by stimulation or depression of the enzyme activity (25).
Pretreatment with doxapram 60 min before produced a significant increment of the hypnotic action induced by hexobarbital and thiopental. This result was similar to that obtained when pentobarbital was admin istered with doxapram (9). The concen trations of pentobarbital and hexobarbital in plasma were markedly elevated 1-4 hr after the administration of doxapram and lowered 12-24 hr after as compared with those of untreated mice. Thus, the biphasic effect of doxapram as mentioned previously was also observed on the plasma levels of both drugs. Therefore, it may be considered that doxapram is classified as a SKF-525A-like agent, which has a biphasic effect of inhibition and induction on the drug metabolism (26).
The hypothermic action of aminopyrine, the muscle relaxative action of meprobamate, the analgesic actions of aminopyrine and morphine, and the lethalities of strychnine and picrotoxin were potentiated by the pretreatment with doxapram 60 min before. On the other hand, the antitumor activity of cyclophosphamide on Ehrlich solid tumors was inhibited by the pretreatment with doxapram 60 min before. The above results indicate that doxapram inhibited the me tabolism of combined drugs when admin istered 60 min before these drugs.
It has been well known that aminopyrine is metabolized to 4-methylaminoantipyrine and then to 4-aminoantipyrine which would be biologically inactive (16) and that cyclophosphamide, an alkylating agent, is inactive per se, but is activated by the hepatic microsomal enzyme (27,28). The potentiating effect of doxapram on the action of amino pyrine and the inhibitory effect on cyclo phosphamide were further proven by determi nation of the plasma levels of these com pounds. The concentration of aminopyrine in plasma was elevated by doxapram pretreat ment and the rate of decay of aminopyrine in plasma was less in the doxapram-treated group than that in the control group. In addition, the plasma level of normustard determined as an active metabolite of cyclophosphamide in the doxapram-treated group was remarkably lower than that in the control group.