Presence Of Aminopyrine Research Articles

Redox mediation in the peroxidase-catalyzed oxidation of aminopyrine: possible implications for drug-drug interactions.

Many drugs, industrial pollutants, and other xenobiotics are known to be oxidized by peroxidases to potentially harmful free-radical intermediates. We have examined the possibility that certain compounds, acting as efficient peroxidase substrates, may stimulate the formation of reactive free radicals by acting as mediators of electron transfer reactions (redox mediators). To explore this hypothesis, we have investigated the interaction of two well-known peroxidase substrates, chlorpromazine and aminopyrine. As shown by ESR and UV-visible spectroscopy, chlorpromazine radical was able to oxidize aminopyrine to aminopyrine cation radical. The rate constant for this rapid, pH-dependent, reaction was estimated to be 1 x 10(7) M-1 s-1 at pH 4.5. Transient-state and steady-state kinetic studies both showed that rate constants for chlorpromazine oxidation to its cation radical by horseradish peroxidase (HRP) were about 100-fold greater than for the corresponding HRP-catalyzed oxidation of aminopyrine to its cation radical. When both aminopyrine and chlorpromazine were present with HRP and H2O2, aminopyrine cation radical formation was stimulated 100-fold. Concomitantly, the accumulation of chlorpromazine cation radical was completely inhibited in the presence of aminopyrine. Similar results were obtained when lactoperoxidase, myeloperoxidase, or the myeloperoxidase mimic HOCl were substituted for HRP. These data suggest that chlorpromazine can act as a redox mediator for peroxidase-catalyzed oxidation of aminopyrine and other chemicals. We suggest that some peroxidase substrates, acting as redox mediators, may stimulate the production of toxic free-radical intermediates from various drugs and other xenobiotics. As such, this may have implications for a number of adverse effects caused by these xenobiotic chemicals.

Effects of oxygen on the relative photodissociability of cytochrome P-450.cntdot.carbon monoxide complex in rat liver microsomes

Flash photolysis of cytochrome P-450 in phenobarbital-induced rat liver microsomes was examined by observing the decay of absorbance change, A(t), at 450 nm after photolysis of the heme.CO complex by a depolarized laser flash. The relative photodissociability, A(0)/Ab, is significantly decreased upon an increase in oxygen concentration. A(0)/Ab is 96%, 86%, and 51% at oxygen concentrations of 90, 115, and 155 microM when compared with A(0)/Ab at 0 microM O2. In the presence of aminopyrine, the oxygen-dependent decrease in the relative photodissociability was enhanced about 2 times. The rate of CO recombination with cytochrome P-450 was decreased by increasing O2 concentration. By going from 0 to 155 microM O2, about 17% decrease and 58% decrease in the recombination rate were observed in the absence and presence of aminopyrine, respectively. In the absence of CO, no absorbance change A(t) at 450 nm was observed even at 155 microM O2, excluding a possible contribution of P-450.O2. The present observation leads to the speculation that oxygen molecules around the heme.CO complex affect the electronic state of the heme, resulting in an increase in Fe-CO bond strength.

Cytochrome P-450 deficiency and resistance to t-butyl hydroperoxide of hepatoma microsomal lipid peroxidation

Lipid peroxidation of microsomes from rat liver and Morris hepatoma 9618A was induced by means of tert-butyl hydroperoxide ( t-BuOOH). In rat liver microsomes t-BuOOH stimulated an early formation of lipid hydroperoxides (LOOH) and an increasing accumulation of malondialdehyde; t-BuOOH was completely consumed and Cytochrome P-450 was rapidly destroyed. In hepatoma microsomes (60% deficiency of cytochrome P-450) a remarkable inhibition of both malondialdehyde and LOOH was observed; t-BuOOH was consumed only partially and cytochrome P-450 was destroyed slowly. In the presence of aminopyrine, malondialdehyde production was inhibited to the same extent (about 70%) in normal and tumour microsomes. The concentration of t-BuOOH required to achieve half-maximal velocity of malondialdehyde accumulation was comparable in the two microsome types. It is proposed that the deficiency of cytochrome P-450 limits the activation of t-BuOOH to the free radical species which initiate lipid peroxidation. Low cytochrome P-450 content would also affect the LOOH-dependent propagation of lipid peroxidation.

Partial inhibition of hepatic microsomal aminopyrine N-demethylase by caffeine in partially purified cytochrome P450

Cytochrome P-450 substrate interactions were studied with cytochrome P-450 partially purified from livers of untreated, phenobarbital-treated, benzo[ a]pyrene-treated and caffeine-treated rats. Partial inhibition of aminopyrine N-demethylase in presence of in vitro caffeine observed with intact microsomes was further investigated in a reconstituted system composed of partially purified cytochrome c reductase. Caffeine addition (in vitro) to partially purified cytochrome P-450 altered the hexobarbital, aniline and ethylisocyanide induced spectral change, and decreased NADPH oxidation in presence of substrates aminopyrine and acetanilide. NADPH oxidation was found to be increased in presence of aminopyrine and unaltered in presence of acetanilide in reconstituted system having partially purified cytochrome P-450 from caffeine-treated rats. Our studies suggest that caffeine acts as a true modifier of cytochrome P-450 and is possibly responsible for the formation of abortive complexes with aminopyrine.

Pyridine nucleotide involvement in rat hepatic drug metabolism. The influence of NADH on the NADPH kinetics of phenobarbital pre-treated rat microsomes.

Apparent kinetic constants for NADPH have been calculated for NADPH-cytochrome P450 reductase and demethylase activity in the presence of aminopyrine and ethylmorphine with hepatic microsomal preparations from phenobarbital pre-treated rats. The presence of NADH changed Km values but not the V values for cytochrome P450 reductase. In addition, NADH reduced Km values for demethylase activity and had a biphasic effect on V values; elevating them at high and depressing them at low concentrations. The V values at different NADH concentrations appeared to correlate with the concentration of reduced cytochrome b5 that the added NADH could sustain during the incubation period. Tetrahydronicotinamide adenine dinucleotide (NADH3), a structural analogue of NADH, reduced demethylase activity without affecting NADPH-cytochrome P450 reductase activity or the concentration of reduced cytochrome b5. As this analogue cannot reduce biological systems, the depressed V values for demethylation suggest that in the presence of NADH3 and NADPH there exists an alternative mechanism for the supply of electrons from NADPH to the partially reduced oxygenated cytochrome P450-substrate complex.

Pyridine nucleotide involvement in rat hepatic microsomal drug metabolism—IV: Inhibition of aminopyrine and ethylmorphine- N-demethylases by 2,6-dihydroxy-acetophenone

Pyridine nucleotide kinetics for both aminopyrine and ethylmorphine- N-demethylases were evaluated in the presence of 2.6-dihydroxyacetophenone. (DiHAP) which inhibits certain microsomal mixed function oxidase reactions. The nature of the inhibition in the presence of varying NADPH concentrations was shown to be slope-linear, intercept-linear non-competitive. DiHAP was shown to combine preferentially with the ferric cytochrome P 450-substrate complex in the presence of aminopyrine, but had a greater affinity for the ferrous cytochrome P 450-substrate complex when ethylmorphine was present. There were changes in the values of K ii and K is without changes in the type of inhibition of aminopyrine- N-demethylase in the presence of both NADH and DiHAP, whereas with ethylmorphine- N-demethylase, the nature of the inhibition kinetics changed to slope-hyperbolic, intercept-hyperbolic non-competitive inhibition in the presence of both NADH and DiHAP. These results are consistent with the proposal that NADH interacts with the ferric cytochrome P 450-substrate complex prior to the reduction of this complex by an electron from NADPH.

Pyridine nucleotide involvement in rat hepatic microsomal drug metabolism—II. Evidence for a co-operative interaction between NADPH and NADH

There was a significant reduction in apparent K m (NADPH) values for both aminopyrine and ethylmorphine demethylases when the kinetic constants for NADPH were determined in the presence of constant NADH concentrations. NADH was also shown to significantly stimulate apparent V for NADPH cytochrome P450-reductase (in the presence of aminopyrine) without changing the apparent K m (NADPH) value. Further, NADH stimulated the initial rapid phase of the biphasic reduction kinetics of NADPH cytochrome P450-reductase in the presence of aminopyrine. These findings suggest that in the presence of both pyridine nucleotides, there has been a change in the rate limiting step which, with NADPH alone, is generally accepted to be the reduction of the cytochrome P450-substrate complex. It has been necessary to make certain modifications to a previously proposed mechanism to explain the results obtained in the present study.

Drug oxidations dependent on cytochrome P-450 in isolated hepatocytes. The role of the tricarboxylates and the aminotransferases in NADPH supply.

1. Isolated hepatocytes from male rats pretreated with phenobarbital were used to study metabolic effects associated with monooxygenase activity dependent on cytochrome P‐450. The hepatocytes metabolized an added drug substrate, [dimethylamino‐14C]aminopyrine (1 mM) at an N‐demethylation rate of 1.31 ± 0.1 μmol × min−1× g (dry weight of hepatocytes)−1. The overall ATP/ADP ratios were 5.0 in the presence of aminopyrine, similar to the controls.2. The levels of citrate and isocitrate decreased, and the citrate/2‐oxoglutarate ratio decreased to 55% of the control in the presence of drug substrates (aminopyrine, ethylmorphine, hexobarbital), correlating well with the decrease in the overall NADPH/NADP+ ratio observed earlier with the perfused liver. It is concluded that supply of NADPH by extramitochondrial isocitrate dehydrogenase accompanies the NADPH supply by the pentosephosphate cycle, so that in spite of the larger capacity of the latter a portion of the reducing equivalents is derived from the mitochondria via the tricarboxylates even in the fed state. This results from the necessity of relieving the inhibition of glucose‐6‐phosphate dehydrogenase by NADPH for activation of the pentosephosphate cycle.3. Subfractionation of hepatocytes from fed rats with the digitonin method revealed decreases of the citrate/2‐oxoglutarate ratios both in the particulate (mitochondrial) and the supernatant (cytosolic) fractions, corresponding to a positive shift in the NADPH redox potential of about 10 mV (or to a decrease of the free [NADPH]/[NADP+] ratio to one‐half) in both compartments as calculated from the combined equilibria of NADP+‐linked isocitrate dehydrogenase and aconitate hydratase.4. Only in cells from fasted, not from fed rats, the rate of aminopyrine N‐demethylation was diminished to 2/3 in the presence of aminotransferase inhibitors, suggesting a requirement for transamination steps in the transfer of NADPH‐reducing equivalents from mitochondria to cytosol under these conditions.

Interaction between antipyrine and aminopyrine

Aminopyrine administered to normal human volunteers in an oral dose of either 9 mg/kg or 4.5 mg/kg prolonged the plasma half-life and reduced the metabolic clearance rate of antipyrine (18 mg/kg, orally) without changing its apparent volume of distribution. By contrast, this same oral dose of antipyrine given simultaneously with 9 mg/kg aminopyrine failed to alter aminopyrine disposition. Thus, antipyrine and aminopyrine should not be administered simultaneously to measure different steps in hepatic drug oxidation, although in man aminopyrine can be given for this purpose 24 hr after antipyrine. Antipyrine elimination was prolonged to the same extent when aminopyrine was given 5 hr before antipyrine as when the drugs were given simultaneoulsy. Since in man aminopyrine has a biologic halic-life of approximately 2.7 hr, the marked inhibitory effects observed 5 hr after aminopyrine administration may be due to its major metabolite, 4-aminoantipyrine. To define mechanism by which aminopyrine affects antipyrine disposition in vivo, hepatic microsomes were prepared from rats, mice, and dogs, and rates of antipyrine hydroxylation were measured in vitro both in the absence and in the presence of aminopyrine. In these species in vitro inhibition of antipyrine hydroxylation by 4-aminoantipyrine was of a mixed type; antipyrine inhibited competitively aminopyrine N-demethylation in vitro in rats, mice, and dogs. There were some sex and species differences in the Km' V max' and Ki for aminopyrine and antipyrine.

The effects of substrates of mixed function oxidase on ethanol oxidation in rat liver microsomes.

The effects of mixed function oxidase substrates, aminopyrine and ethylmorphine, on the NADPH and O2 dependent rate of ethanol oxidation have been examined. Aminopyrine like ethylmorphine exerts different effects on the rate of acetaldehyde formation depending upon the ethanol concentration used. At saturating ethanol concentration V max increases. Inhibition is observed at low concentrations of ethanol. Plots of acetaldehyde formation versus ethanol concentration reveal, in the presence of aminopyrine, curves which indicate the simultaneous action of two enzymes functional in ethanol oxidation. These data provide support for the existence of a microsomal ethanol oxidizing enzyme system (Orme-Johnson et al., 1965; Lieber et al., 1970), in addition to the well documented azide sensitive and H2O2 dependent pathway for ethanol oxidation (Thurman et al., 1972; Feytmans et al., 1973).

Energy coupling and extramitochondrial electron transport in slices of Morris hepatoma 3924 A.

The study of the respiration of solid tumours performed with slice preparations may provide opportunities to evaluate the mitochondrial energy efficiency in a more suitable manner than by using isolated mitochondria. Moreover, such an experimental approach affords a method for estimating at which extent systems of extramitochondrial electron transport can contribute to the total oxygen consumption of the whole cell. In this work we have studied an experimental tumour of the rat, the Morris hepatoma 3924A. Experiments have been also carried out comparatively with rat liver of the ACI/T and Wistar strains. The endogenous respiration of hepatoma slices was stimulated by 27% by 2,4-dinitrophenol and inhibited by 47% by oligomycin. The oligomycin inhibition was not completely released by 2,4-dinitrophenol. A similar behaviour is shown by rat-liver slices. Succinate, but not the NAD-linked substrates, enhanced the endogenous respiration of hepatoma slices. A 17% inhibition by ouabain of the respiration in the presence of glucose was observed in hepatoma slices preincubated 120 min at 1°. Electron microscopic examination of tumour slices showed that mitochondria are in the orthodox conformation. They changed to the condensed conformation following inhibition of respiration at 1°; at 38° they underwent transition to the orthodox conformation. Rotenone, antimycin A and cyanide inhibited the endogenous respiration of hepatoma slices by 85–90% and that of rat liver slices (ACI/T strain) by 57, 68, and 74%, respectively. Treatment of rats of the Wistar strain with phenobarbital did not modify the endogenous respiration of rat-liver slices. However, the respiration was stimulated by 15–20% in the presence of aminopyrine, and inhibited by 48.5% by antimycin A and by 30% by antimycin A plus aminopyrine. Phenobarbital administration to tumour-bearing rats of the ACI/T strain did not influence the tumour respiration. These results provide evidence that the respiration of the hepatoma 3924 A slices is coupled to energy conservation reactions. They further suggest that mitochondria within the slices are in a state intermediate between States 3 and 4. The inhibitor-insensitive oxygen consumption may be ascribed to the operation of electron transport pathways located in membrane systems of the extramitochondrial compartment of the cell.

Interaction of mixed-function oxidation with biosynthetic processes. 2. Inhibition of lipogenesis by aminopyrine in perfused rat liver.

Lipogenesis from glucose was studied in perfused livers from normal and phenobarbital‐pretreated rats by measurement of the incorporation of tritium from tritiated water and 14C from from [U‐14C]glucose into the lipid fraction. In the presence of aminopyrine, a substrate for NADPH utilizing mixed‐function oxidations, rates of tritium incorporation decreased to 82 and 57% of control in livers from normal and phenobarbital‐pretreated animals, respectively. Aminophenylpyrazolone, a demethylated product of aminopyrine metabolism, had no effect implicating that inhibition of lipogenesis by aminopyrine is due to the process of mixed‐function oxidation. It is concluded that both mixed‐function oxidation and fatty‐acid synthesis compete for extramitochondrial NADPH. These data indicate that NADPH supply may, under the extreme conditions of enhanced mixed‐function oxidation, be the rate‐limiting step for lipogenesis in liver.

Detection of serum antibodies to amino-pyrine or its derivatives by a passive hemagglutination assay method.

An attempt has been made to detect antibodies to aminopyrine or its derivatives ( pyrazolone derivatives) with a passive hemagglutination assay system. Formalinized red blood cells were sensitized by coupling diazotized 4-aminoantipyrine to the surface membrane. With this method, circulating antibodies were detected in a patient with aminopyrine-induced agranulocytosis. Serial determinations revealed that the concentration of antibody increased rapidly after the onset of clinica l manifestations, reaching a maximum in about a week, and thereafter began to fall off gradually. The presence of antibody was found in 32 sera of 75 patient s with a history of skin reaction due to aminopyrine or its analogues and in 15 sera of 62 control subjects having no such history. The highest hemagglutination titer was 1 : 16. Adverse reactions to drugs occur commonly in clinical practice and constitute a problem of considerable importance. Some of these reactions are judged to be allergic in mechanism t) -s). For example, the administration of aminopyrine or its derivatives (pyrazolone derivatives) has been known to induce agranulocytosis or skin reactions4l. Moeschlin and Wagner5l demonstrated that agranulocytosis was elicited in volunteers to whom blood from a patient with aminopyrine-induced agranulocytosis had been given intravenously. They also found a substance in the plasma of the patient at the height of disease which produced in vitro agglutination of homologous and heterologous leukocytes. In addition to this finding, Thierfelder et al.6l succeeded in demonstrating in the serum of a patient suffering from agranulocytosis due to aminopyrine an antibody-like principle able to agglutinate donor leukocytes in the presence of aminopyrine. The detection and quantitative measurement of such fi::j'i: JJI{::, tifr:j'i: {~, fi:~51JlA, li'i!JIIliifl\'t Received for publication October 22, 1968. * Presented in abstract form at the 9th Annual Meeting of the Japanese Society of Clinical Hematology, Nara, October 31, 1967. ** Present Address: Professor, Second Department of Internal Medicine, Nagoya City University Medical School, Nagoya.