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Inhibition of ethinyloestradiol and tolbutamide metabolism by quinoline derivatives in vitro
Chem.-Eiol. Elsevier
Interactions,
Scientific
INHIBITION METABOLISM
JUDITH
59 (1986)
Publishers
Ireland
OF ETHINYLOESTRADIOL AND BY QUINOLINE DERIVATIVES
H. RIVIERE
and
D.J.
Department of Pharmacology Liverpool L69 3BX (U.K.) (Received (Revision (Accepted
301
301-308 Ltd.
April 21st, 1986) received July 3rd, July 21st, 1986)
TOLBUTAMIDE IN VITRO
BACK*
& Thempeutics.
University
of
Liverpool,
P.O. Box
147,
1986)
SUMMARY
The effects of the quinoline derivatives amodiaquine (AQ), chloroquine (CQ), mefloquine (MQ), primaquine (PQ), quinine (Q) and quinidine (QD) on in vitro hepatic metabolism has been studied using as substrates ethinyloestradiol (EE,) and tolbutamide (TOL). The 2-hydroxylation of EE2 and the hydroxylation of TOL were determined in the presence of variable concentrations of each compound. MQ, PQ, AQ and Q significantly inhibited EE, metabolism at each of the concentrations studied (0.1, 0.2 and 0.5 mM) as shown by an increase in the percentage of unmetabolised EE,. QD significantly inhibited metabolism at 0.2 and 0.5 mM but CQ was without effect. In terms of recovery of 2OHEE,, PQ was the most potent inhibitor. At an inhibitor concentration of 0.5 mM the order of potency was PQ>MQ> Q> &Da AQ> CQ. TOL hydroxylase activity in control microsomes was 1.52 f 0.33 nmol. min-’ - mg protein-‘. The order of potency of the inhibitors (0.5 mM) was PQ> MQ> Q> QD> AQ> CQ. These data provide further evidence of the inhibitory potential of some of the quinoline derivatives. PQ, MQ, and to a lesser extent Q produce the most marked inhibitory effects. QD and AQ are of intermediate potency and CQ is essentially non-inhibitory. -
Key words: Enzyme inhibition - Hepatic microsomal line derivatives - Ethinyloestradiol - Tolbutamide
*To whom correspondence should be sent. Abbreviations: AQ, amodiaquine; CQ, chloroquine, EE,, quine; 2-OHEE,, 2-hydroxyethinyloestradiol; OH-TOL, primaquine; Q, quinine; QD, quinidine; TOL, tolbutamide.
0009-2797/86/$03.50 o 1986 Printed
Elsevier Scientific Publishers and Published in Ireland
Ireland
Ltd.
metabolism
ethinyloestradiol; hydroxytolbutamide;
- Quino-
MQ,
mefloPQ,
302 INTRODUCTION
There is increasing evidence from both in vitro and in vivo studies in animals [l-5] and in vivo studies in man [6,7] of the potential of some quinoline drugs to inhibit hepatic microsomal drug oxidation. Using aminopyrine as substrate in vitro, Murray [2] found that PQ, AQ and Q were relatively potent inhibitors of the N-demethylase activity, but that QD and CQ were essentially non-inhibitory. These findings were in agreement with our previous data which indicated that PQ inhibited aminopyrine N-demethylation at very much lower concentrations that CQ [l]. More recently we have shown that MQ inhibits hepatic microsomal enzymes both in vitro and in vivo and produces comparable inhibition in vitro to that seen with PQ [51* The present study was designed to investigate the relative inhibitory potential of six quinolines (AQ, CQ, MQ, PQ, QD and Q, Fig. 1) on two substrates, EE2 and TOL in vitro in the rat. There is evidence that steroids are oxidised by enzymes distinct from those responsible for the oxidation of drugs [B-lo] and that the specific cytochrome P-450 responsible for tolbutamide metabolism is distinct from other isoenzymes [ 111, hence these substrates were chosen for study to give information on two distinct forms of cytochrome P-450. MATERIALS
AND
METHODS
Materials Male Wistar rats (200-250 g) were from Bantin and Kingman, Hull, U.K. PQ diphosphate, CQ diphosphate, QD hydrochloride, Q hydrochloride and chlorpropamide were obtained from Sigma. TOL and hydroxytolbutamide (OH-TOL) were obtained from Hoechst. AQ hydrochloride was from Parke, Davis and Co., and MQ hydrochloride was a gift from Hoffman-La-Roche, Basel, Switzerland. [6,7,13H] 17a-EE2 (spec. act., 45.8 Ci/mmol) was from the New England Nuclear Corp. (F.R.G.) and EE, was a gift from Schering A.G. (Berlin, F.R.G.). All other reagents were obtained from B.D.H. Ltd. Preparation of microsomes Microsomes were prepared from rat liver as described Microsomal protein content was determined by the method
previously of Lowry
[5]. et al.
[=I. Analysis of [3H] EE2 metabolites in microsomal incubations The following reaction mixture was used: [3H]EEz (1 PCi), EE2 (0.01 mM), ascorbic acid (1 mM), AQ, CQ, MQ, PQ, QD and Q (0.1, 0.2 and 0.5 mM), microsomes (1 ml of 6 mg - ml-’ ) and NADPH (0.6 mM). Incubations were performed at 37°C for 30 min in an agitating water bath. Microsomal reactions were stopped by extracting twice with ether (4 ml and 3 ml). Radiolabelled metabolites present in the ether extracts were analysed by
303 HPLC substantially according to the method described by Maggs et al. [ 131. A Spectra-Physics SP 8700 solvent delivery pump was used connected to an LKB 2112 Redirac fraction collector. A stainless-steel column was used packed with Partisil@ lo/25 ODS-2 (25 cm X 0.46 cm i.d., Whatman Inc., Clifton, NJ, U.S.A.). Samples (10 ~1) were eluted at room temperature with a linear gradient of methanol in 0.5% (w/v) ammonium dihydrogen phosphate buffer (pH 3.0), from 50-65s at 2%/min. This system is known to be suitable for separating oestrogens. The flow rate was 2 ml * min-’ . The radioactive content of each sample was determined by liquid scintillation spectrometry using a Packard Tri-Carb 4640 liquid scintillation counter. Analysis of OH-TOL in microsomal incubations A linear relationship between the amount of OH-TOL formed and the concentration of microsomal protein was observed between 0.5 and 4.0 mg of protein/ml incubate. In a standard incubation mixture containing 2 mg of microsomal protein the reaction was linear to 10 mm. The substrate concentration used in a standard assay (2 mM) was in excess of the Km of the enzyme (0.63 mM, unpublished observation). The following reaction mixture was used: TOL (2 mM), EDTA (1 mM). KC1 (1 mM), MgCl* (5 mM), AQ, CQ, MQ, PQ, QD and Q (0.5 mM), microsomes (1 ml of 2 mg * ml-‘) and NADPH (1 mM). Incubations were performed at 37°C for 9 min in an agitating water bath. Microsomal reactions were stopped by addition of 100 ~1 HCl (6 M), and then extracted twice with ether (2 X 3 ml.). OH-TOL present in the ether extracts was analysed by HPLC as described previously [14,15]. In brief, TOL (2 mM) was added to the incubation flask, and evaporated to dryness under nitrogen. Following incubation, the reaction mixture was transferred to 10 ml sovirel tubes containing internal standard (I.S. chlorpropamide, 25 ~1 of 0.2 mg * ml-l solution). The ether extracts of the samples were evaporated to dryness, and reconstituted in methanol (100 ~1). A Spectra-Physics SP 8700 solvent delivery pump was used connected to an SP 8500 dynamic mixer. Separations were-performed on a Partisil@ lo/25 ODS-2 column. Samples (20 ~1) were eluted at room temperature with a mobile phase of methanol: 0.05% phosphoric acid (50 : 50, v/v). The flow rate was 1.8 ml * min-’ . The ratio of the peak height of OH-TOL to I.S. was plotted against the concentration of OH-TOL to give standard curves. The ICsO values for MQ, PQ, QD and Q (0.05, 0.1, 0.5 and 1.0 mM) on TOL hydroxylation were determined. It was not possible to determine these values for AQ and CQ due to solubility problems at high concentrations. Statistical analysis Data were analysed by l-factor analysis of variance. When the overall F value was significant, differences between any treatment vs. control were only considered as significant if the critical value for Dunnett’s test was exceeded. Data are presented as mean f S.D.
304 RESULTS
MQ, PQ and Q significantly inhibited EE2 metabolism in vitro at all three concentrations studied, as shown by the increase in unmetabolised EE2 (Table I). QD also significantly inhibited EE2 metabolism at 0.2 mM and 0.5 mM, but not at 0.1 mM. AQ showed a significant increase in unmetabolised EE2 at all concentrations, but only a significant decrease in the main metabolite, 2-hydroxyethinyloestradiol (2-OHEEz) at 0.2 and 0.5 mM. CQ showed no inhibition of EE2 metabolism in vitro. PQ was the most potent inhibitor and this is most evident when comparing data at 0.1 mM. In the presence of PQ (0.1 mM) the percentage of unTABLE
I
THE EFFECT
OF VARIOUS
QUINOLINE
Results are mean + S.D. of 4 experiments from controls; ***P < 0.001, significantly Cont. (mM)
DRUGS ON EE, METABOLISM IN VITRO in each group. **P< O.Ol,significantlydifferent different from controls. % of total radioactivity
as
P-OHEE,
EE,
Controls
0.1 0.2 0.5
49.9 t 12.9 53.3 2 4.0 60.0 2 2.9
12.1 + 4.5 6.1 ? 1.7 12.7 2 2.5
AQ
0.1 0.2 0.5
44.2 A 4.4 41.7 2 1.6 46.9 + 3.4*
27.6 + 4.6** 25.8 2 3.9** 29.0 + 4.3**
CQ
0.1 0.2 0.5
60.4 2 53.8 t 60.0 2
4.9 1.7 4.0
8.1 ? 3.9 6.6 2 3.3 12.8 2 3.9
MB
O.lb 0.2 0.5b
44.8 2 45.5 2 5.1 +
3.1 9.3 3.1***
33.3 t 1.7*** 27.9 ? 8.9** 83.0 2 3.2***
PQ
0.1 0.2 0.5
15.9 + 5.0** 5.9 2 1.0*** 1.5 ? 0.4***
60.7 2 4.1*** 73.2 2 1.4*** 86.3 ? 4.6***
QD
0.1 0.2 0.5
48.5 2 12.5 33.0 ? 7.9** 30.4 2 5.2***
19.1 ? 7.0 39.5 ?r 6.0*** 54.8 + 6.1***
Q
0.1 0.2 0.5
37.6 t 15.5 20.0 + 9.0*** 18.1 2 4.5***
32.9 f 6.0*** 48.5 I! 9.0*** 64.6 + 3.8***
‘Control incubations contained DMSO. A separate control tion studied. bData previously published in Riviere and Back [ 51.
applies
to each drug concentra-
305 metabolised EE2 was increased from 12.1 + 4.5% to 60.7 + 4.1%; the corresponding value for MQ was an increase to 33.3 f 1.7% and for Q, 32.9 f 6.0%. MQ, PQ, QD and Q significantly inhibited TOL metabolism at the highest concentration studied (1.0 mM), as shown by the reduction in enzyme activity (Table II). PQ showed significant inhibition of TOL metabolism at all four concentrations studied, whereas MQ and Q only showed significant inhibition at 0.5 and 1.0 mM. Both AQ and CQ showed no inhibition of TOL metabolism in vitro. PQ was the most potent inhibitor and this can be seen when comparing data at 0.5 mM. In the presence of PQ (0.5 mM) TOL hydroxylase activity
TABLE II THE EFFECT
OF VARIOUS
QUINOLINE
Results are mean + S.D. of 4 experiments from controls; **P < 0.01, significantly cantly different from controls. Cont. (mW
DRUGS
ON TOL METABOLISM
in each group. *P < 0.05, significantly different different from controls; ***P < 0.001, signifi.Enzyme activity (nmol * mine’ * mg protein-‘)
ControP
IN VITRO
1.52 2 0.33
% Control
100
AQ
0.5
1.49 + 0.38
CQ
0.5
1.79 ? 0.35
MB
0.05 0.1 0.5 1.0
1.16 1.11 0.24 0.11
+ ? 2 +
0.14 0.22 0.17*** 0.13***
77.92 7.9 74.65 12.6 16.12 13.0 9.52 11.9
0.05 0.1 0.5 1 .o
0.91 0.65 0.07 0.03
?: 0.24** 2 0.40*** k 0.04*** 2 0.01***
61.3 + 13.5 44.52 7.0 5.12 3.0 1.92 1.2
QD
0.05 0.1 0.5 1 .o
1.58’2 1.31 ? 1.08 + 0.73 2
0.37 0.21 0.21 0.21*
Q
0.05 0.1 0.5 1.0
1.39 1.02 0.57 0.38
0.44 0.19 0.12** 0.11***
PQ
%ontrol
-
incubation
contained
DMSO.
? + tt-
99.7 2 0.92 118.52
5.0
106.1 !I 23.1 87.3f: 6.8 72.5 2 14.6 47.75 4.8 94.2 2 68.7 + 38.2% 25.22
33.7 12.0 4.7 5.0
306 TABLE
III
INHIBITION OF TOL LIVER MICROSOMES
HYDROXYLASE
ACTIVITY
QUINOLINE
DRUGS
IN
RAT
IC,, (clW
MQ PQ QD Q
BY
value -
.-_.
240 60 930 330
was 0.07 f 0.04 nmol - min-’ * mg protein-’ as compared to control (1.52 f 0.33). The order of potency was PQ (0.07 + 0.04 nmol * min-’ * mg protein-’ ) 2 MQ (0.24 f 0.17 nmol - min-’ * mg protein-’ ) > Q (0.57 f 0.12 nmol * min-’ * mg protein-’ ) > QD (1.08 f 0.21 nmol * min-’ * mg protein-‘) > AQ (1.49 + 0.38 nmol * min-’ * mg protein-‘) B CQ (1.79 f 0.35 nmol - min-’ * mg protein-‘). ICSO values were determined for MQ, PQ, QD and Q on TOL hydroxylase activity (Table III). PQ was found to be four times as potent as MQ against TOL hydroxylase. Q was approximately three times more potent than its stereoisomer (QD) against TOL hydroxylase. l
DISCUSSION
The present work provides more evidence of the potential of some quinoline drugs to inhibit hepatic microsomal enzyme activity. With data from two substrates (EE2 and TOL) it is clear that PQ, MQ and to a lesser extent Q, produce the most marked inhibitory effects and that CQ (over the range of concentrations studied) does not inhibit these enzymes. QD and AQ are of intermediate potency. These results are substantially in agreement with the study by Murray [2] on the effect of five of the quinoline drugs (not MQ) on aminopyrine N-demethylase activity, which indicated an order of potency Q >, PQ > AQ >QD 2 CQ. It was suggested that PQ and Q interact with the haem iron of cytochrome P-450 via the side chain amino nitrogen and the quinuclidine nitrogen respectively, as both provide a lone pair of electrons that are sterically accessible to the haem (Fig. 1). In contrast, CQ has relatively hindered nitrogen atoms in its alkylaminoalkyl side chain and in the quinoline ring and the quinuclidine nitrogen in QD is sterically inaccessible. The inhibition produced by MQ is probably the result of the interaction with the haem iron via the sterically accessible piperidine nitrogen. The fact that a compound shows enzyme inhibition in vitro does not mean that it will automatically lead to clinically important pharmacokinetic interactions if co-administered with other drugs in man. Indeed, despite the findings of the present study, we have demonstrated that in normal thera-
307 Cl
AH ‘CH(CH,),N(CzHd, AH,
CH3
H,N
(CH,),~”
CF3
NH
(3)
CH,O CH =CH2
(4)
(5) Fig. 1. The structures of the quinoline derivatives used in the study: (1) amodiaquine; (2) chloroquine; (3) mefloquine; (4) primaquine; (5) quinine and quinidine. The asterisk represents the centre of asymmetry.
peutic doses, PQ but not MQ inhibits antipyrine metabolism in man [6,7]. Although this could represent a selectivity in inhibitory effect it is far more likely to be a reflection of the different pharmacokinetics and hepatic accumulation of the antimalarials. Since enzyme inhibition in vivo is a reflection not only of binding to the enzyme but also of actual drug concentrations present in the 1iver;particularly in the vicinity of the enzyme it is possible that hepatic concentrations of MQ are very much less than PQ. There is evidence of the avid binding of MQ to both plasma proteins and red blood cells [ 161 and hence hepatic extraction may be low. Further studies in animals and man, both in vitro and in vivo are in progress in an attempt to understand the clinical relevance of the inhibitory potential of the quinolines.
308 ACKNOWLEDGEMENT
J.H.R. was in receipt of an MRC Studentship. REFERENCES 1 D.J. Back, H.S. Purba, C. Staiger, M.L’E. Orme and A.M. Breckenridge, Inhibition of drug metabolism by the antimalarial drugs chloroquine and primaquine in the rat, Biochem. Pharmacol., 32 (1983) 257. 2 M. Murray, In vitro effects of quinoline derivatives on cytochrome P-450 and aminopyrine N-demethylase activity in rat hepatic microsomes, Biochem. Pharmacol., 33 (1984) 3277. 3 M.I. Thabrew and C. Ioannides. Inhibition of rat hepatic mixed function oxidases by antimalarial drugs: selectivity for cytochromes P-450 and P-448, Chem.-Biol. Interact., 51 (1984) 285. 4 G.W. Mihaly, S.A. Ward, D.D. Nicholl, G. Edwards and A.M. Breckenridge, The effects of primaquine stereoisomers and metabolites on drug metabolism in the isolated perfused rat liver and in vitro rat liver microsomes, Biochem. Pharmacol., 34 (1985) 331. 5 J.H. Riviere and D.J. Back, Effect of mefloquine on hepatic drug metabolism in the rat: comparative study with primaquine, Biochem. Pharmacol., 34 (1985) 567. 6 D.J. Back, H.S. Purba, B.K. Park, S.A. Ward and M.L’E. Orme, Effect of chloroquine and primaquine on antipyrine metabolism, Br. J. Clin. Pharmacol., 16 (1983) 497. 7 J.H. Riviere, D.J. Back, A.M. Breckenridge and R.E. Howells, The pharmacokinetics of mefloquine in man: lack of effect of mefloquine on antipyrine metabolism, Br. J. Clin. Pharmacol., 20 (1985) 469. 8 H.M. Bolt and H. Kassel, Effect of insecticide synergists on microsomal oxidation of estradiol and ethinyloestradiol and on microsomal drug metabolism, Xenobiotica, 6 (1976) 33. 9 D.D. Breimer, Interindividual variations in drug disposition. Clinical implications and methods of investigation, Clin. Pharmacokin., 8 (1983) 371. 10 P. Jenner, B. Testa and F.J. DiCarlo, Xenobiotic and endobiotic metabolisingenzymes: an overstretched discrimination? in: J.W. Lamble (Ed.), Drug Metabolism and Disposition, Elsevier Science Publishers, New York, 1983, pp. 12-21. 11 F.P. Guengerich, L.M. Distlerath, P.E.B. Reilly, T. Wolft, T. Shimada, D.R. Umbenhauer and M.V. Martin, Human-liver cytochromes P-450 involved in polymorphisms of drug oxidation, Xenobiotica, 16 (1986) 378. 12 O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265. 13 J.L. Maggs, P.S. Grabowski, M.E. Rose and B.K. Park, The biotransformation of 17-aethynyl [3H]estradiol in the rat. Irreversible binding and biliary metabolites, Xenobiotica, 12 (1982) 657. 14 R.L. Nation, G.W. Peng and W.L. Chiou, Simple, rapid and micro high-pressure liquid chromatographic method for the simultaneous determination of tolbutamide and carboxytolbutamide in plasma, J. Chromatogr., 146 (1978) 121. 15 D.J. Back, F. Sutcliffe and J.F. Tjia, Tolbutamide as a model drug for the study of enzyme induction and enzyme inhibition in the rat, Br. J. Pharmacol., 81 (1984) 557. 16 R.C. San George, R.L. Nagel and M.E. Fabry, On the mechanism for the red-cell accumulation of mefloquine, an antimalarial drug, Biochim. Biophys. Acta, 803 (1984) 174.
Interactions,
Scientific
INHIBITION METABOLISM
JUDITH
59 (1986)
Publishers
Ireland
OF ETHINYLOESTRADIOL AND BY QUINOLINE DERIVATIVES
H. RIVIERE
and
D.J.
Department of Pharmacology Liverpool L69 3BX (U.K.) (Received (Revision (Accepted
301
301-308 Ltd.
April 21st, 1986) received July 3rd, July 21st, 1986)
TOLBUTAMIDE IN VITRO
BACK*
& Thempeutics.
University
of
Liverpool,
P.O. Box
147,
1986)
SUMMARY
The effects of the quinoline derivatives amodiaquine (AQ), chloroquine (CQ), mefloquine (MQ), primaquine (PQ), quinine (Q) and quinidine (QD) on in vitro hepatic metabolism has been studied using as substrates ethinyloestradiol (EE,) and tolbutamide (TOL). The 2-hydroxylation of EE2 and the hydroxylation of TOL were determined in the presence of variable concentrations of each compound. MQ, PQ, AQ and Q significantly inhibited EE, metabolism at each of the concentrations studied (0.1, 0.2 and 0.5 mM) as shown by an increase in the percentage of unmetabolised EE,. QD significantly inhibited metabolism at 0.2 and 0.5 mM but CQ was without effect. In terms of recovery of 2OHEE,, PQ was the most potent inhibitor. At an inhibitor concentration of 0.5 mM the order of potency was PQ>MQ> Q> &Da AQ> CQ. TOL hydroxylase activity in control microsomes was 1.52 f 0.33 nmol. min-’ - mg protein-‘. The order of potency of the inhibitors (0.5 mM) was PQ> MQ> Q> QD> AQ> CQ. These data provide further evidence of the inhibitory potential of some of the quinoline derivatives. PQ, MQ, and to a lesser extent Q produce the most marked inhibitory effects. QD and AQ are of intermediate potency and CQ is essentially non-inhibitory. -
Key words: Enzyme inhibition - Hepatic microsomal line derivatives - Ethinyloestradiol - Tolbutamide
*To whom correspondence should be sent. Abbreviations: AQ, amodiaquine; CQ, chloroquine, EE,, quine; 2-OHEE,, 2-hydroxyethinyloestradiol; OH-TOL, primaquine; Q, quinine; QD, quinidine; TOL, tolbutamide.
0009-2797/86/$03.50 o 1986 Printed
Elsevier Scientific Publishers and Published in Ireland
Ireland
Ltd.
metabolism
ethinyloestradiol; hydroxytolbutamide;
- Quino-
MQ,
mefloPQ,
302 INTRODUCTION
There is increasing evidence from both in vitro and in vivo studies in animals [l-5] and in vivo studies in man [6,7] of the potential of some quinoline drugs to inhibit hepatic microsomal drug oxidation. Using aminopyrine as substrate in vitro, Murray [2] found that PQ, AQ and Q were relatively potent inhibitors of the N-demethylase activity, but that QD and CQ were essentially non-inhibitory. These findings were in agreement with our previous data which indicated that PQ inhibited aminopyrine N-demethylation at very much lower concentrations that CQ [l]. More recently we have shown that MQ inhibits hepatic microsomal enzymes both in vitro and in vivo and produces comparable inhibition in vitro to that seen with PQ [51* The present study was designed to investigate the relative inhibitory potential of six quinolines (AQ, CQ, MQ, PQ, QD and Q, Fig. 1) on two substrates, EE2 and TOL in vitro in the rat. There is evidence that steroids are oxidised by enzymes distinct from those responsible for the oxidation of drugs [B-lo] and that the specific cytochrome P-450 responsible for tolbutamide metabolism is distinct from other isoenzymes [ 111, hence these substrates were chosen for study to give information on two distinct forms of cytochrome P-450. MATERIALS
AND
METHODS
Materials Male Wistar rats (200-250 g) were from Bantin and Kingman, Hull, U.K. PQ diphosphate, CQ diphosphate, QD hydrochloride, Q hydrochloride and chlorpropamide were obtained from Sigma. TOL and hydroxytolbutamide (OH-TOL) were obtained from Hoechst. AQ hydrochloride was from Parke, Davis and Co., and MQ hydrochloride was a gift from Hoffman-La-Roche, Basel, Switzerland. [6,7,13H] 17a-EE2 (spec. act., 45.8 Ci/mmol) was from the New England Nuclear Corp. (F.R.G.) and EE, was a gift from Schering A.G. (Berlin, F.R.G.). All other reagents were obtained from B.D.H. Ltd. Preparation of microsomes Microsomes were prepared from rat liver as described Microsomal protein content was determined by the method
previously of Lowry
[5]. et al.
[=I. Analysis of [3H] EE2 metabolites in microsomal incubations The following reaction mixture was used: [3H]EEz (1 PCi), EE2 (0.01 mM), ascorbic acid (1 mM), AQ, CQ, MQ, PQ, QD and Q (0.1, 0.2 and 0.5 mM), microsomes (1 ml of 6 mg - ml-’ ) and NADPH (0.6 mM). Incubations were performed at 37°C for 30 min in an agitating water bath. Microsomal reactions were stopped by extracting twice with ether (4 ml and 3 ml). Radiolabelled metabolites present in the ether extracts were analysed by
303 HPLC substantially according to the method described by Maggs et al. [ 131. A Spectra-Physics SP 8700 solvent delivery pump was used connected to an LKB 2112 Redirac fraction collector. A stainless-steel column was used packed with Partisil@ lo/25 ODS-2 (25 cm X 0.46 cm i.d., Whatman Inc., Clifton, NJ, U.S.A.). Samples (10 ~1) were eluted at room temperature with a linear gradient of methanol in 0.5% (w/v) ammonium dihydrogen phosphate buffer (pH 3.0), from 50-65s at 2%/min. This system is known to be suitable for separating oestrogens. The flow rate was 2 ml * min-’ . The radioactive content of each sample was determined by liquid scintillation spectrometry using a Packard Tri-Carb 4640 liquid scintillation counter. Analysis of OH-TOL in microsomal incubations A linear relationship between the amount of OH-TOL formed and the concentration of microsomal protein was observed between 0.5 and 4.0 mg of protein/ml incubate. In a standard incubation mixture containing 2 mg of microsomal protein the reaction was linear to 10 mm. The substrate concentration used in a standard assay (2 mM) was in excess of the Km of the enzyme (0.63 mM, unpublished observation). The following reaction mixture was used: TOL (2 mM), EDTA (1 mM). KC1 (1 mM), MgCl* (5 mM), AQ, CQ, MQ, PQ, QD and Q (0.5 mM), microsomes (1 ml of 2 mg * ml-‘) and NADPH (1 mM). Incubations were performed at 37°C for 9 min in an agitating water bath. Microsomal reactions were stopped by addition of 100 ~1 HCl (6 M), and then extracted twice with ether (2 X 3 ml.). OH-TOL present in the ether extracts was analysed by HPLC as described previously [14,15]. In brief, TOL (2 mM) was added to the incubation flask, and evaporated to dryness under nitrogen. Following incubation, the reaction mixture was transferred to 10 ml sovirel tubes containing internal standard (I.S. chlorpropamide, 25 ~1 of 0.2 mg * ml-l solution). The ether extracts of the samples were evaporated to dryness, and reconstituted in methanol (100 ~1). A Spectra-Physics SP 8700 solvent delivery pump was used connected to an SP 8500 dynamic mixer. Separations were-performed on a Partisil@ lo/25 ODS-2 column. Samples (20 ~1) were eluted at room temperature with a mobile phase of methanol: 0.05% phosphoric acid (50 : 50, v/v). The flow rate was 1.8 ml * min-’ . The ratio of the peak height of OH-TOL to I.S. was plotted against the concentration of OH-TOL to give standard curves. The ICsO values for MQ, PQ, QD and Q (0.05, 0.1, 0.5 and 1.0 mM) on TOL hydroxylation were determined. It was not possible to determine these values for AQ and CQ due to solubility problems at high concentrations. Statistical analysis Data were analysed by l-factor analysis of variance. When the overall F value was significant, differences between any treatment vs. control were only considered as significant if the critical value for Dunnett’s test was exceeded. Data are presented as mean f S.D.
304 RESULTS
MQ, PQ and Q significantly inhibited EE2 metabolism in vitro at all three concentrations studied, as shown by the increase in unmetabolised EE2 (Table I). QD also significantly inhibited EE2 metabolism at 0.2 mM and 0.5 mM, but not at 0.1 mM. AQ showed a significant increase in unmetabolised EE2 at all concentrations, but only a significant decrease in the main metabolite, 2-hydroxyethinyloestradiol (2-OHEEz) at 0.2 and 0.5 mM. CQ showed no inhibition of EE2 metabolism in vitro. PQ was the most potent inhibitor and this is most evident when comparing data at 0.1 mM. In the presence of PQ (0.1 mM) the percentage of unTABLE
I
THE EFFECT
OF VARIOUS
QUINOLINE
Results are mean + S.D. of 4 experiments from controls; ***P < 0.001, significantly Cont. (mM)
DRUGS ON EE, METABOLISM IN VITRO in each group. **P< O.Ol,significantlydifferent different from controls. % of total radioactivity
as
P-OHEE,
EE,
Controls
0.1 0.2 0.5
49.9 t 12.9 53.3 2 4.0 60.0 2 2.9
12.1 + 4.5 6.1 ? 1.7 12.7 2 2.5
AQ
0.1 0.2 0.5
44.2 A 4.4 41.7 2 1.6 46.9 + 3.4*
27.6 + 4.6** 25.8 2 3.9** 29.0 + 4.3**
CQ
0.1 0.2 0.5
60.4 2 53.8 t 60.0 2
4.9 1.7 4.0
8.1 ? 3.9 6.6 2 3.3 12.8 2 3.9
MB
O.lb 0.2 0.5b
44.8 2 45.5 2 5.1 +
3.1 9.3 3.1***
33.3 t 1.7*** 27.9 ? 8.9** 83.0 2 3.2***
PQ
0.1 0.2 0.5
15.9 + 5.0** 5.9 2 1.0*** 1.5 ? 0.4***
60.7 2 4.1*** 73.2 2 1.4*** 86.3 ? 4.6***
QD
0.1 0.2 0.5
48.5 2 12.5 33.0 ? 7.9** 30.4 2 5.2***
19.1 ? 7.0 39.5 ?r 6.0*** 54.8 + 6.1***
Q
0.1 0.2 0.5
37.6 t 15.5 20.0 + 9.0*** 18.1 2 4.5***
32.9 f 6.0*** 48.5 I! 9.0*** 64.6 + 3.8***
‘Control incubations contained DMSO. A separate control tion studied. bData previously published in Riviere and Back [ 51.
applies
to each drug concentra-
305 metabolised EE2 was increased from 12.1 + 4.5% to 60.7 + 4.1%; the corresponding value for MQ was an increase to 33.3 f 1.7% and for Q, 32.9 f 6.0%. MQ, PQ, QD and Q significantly inhibited TOL metabolism at the highest concentration studied (1.0 mM), as shown by the reduction in enzyme activity (Table II). PQ showed significant inhibition of TOL metabolism at all four concentrations studied, whereas MQ and Q only showed significant inhibition at 0.5 and 1.0 mM. Both AQ and CQ showed no inhibition of TOL metabolism in vitro. PQ was the most potent inhibitor and this can be seen when comparing data at 0.5 mM. In the presence of PQ (0.5 mM) TOL hydroxylase activity
TABLE II THE EFFECT
OF VARIOUS
QUINOLINE
Results are mean + S.D. of 4 experiments from controls; **P < 0.01, significantly cantly different from controls. Cont. (mW
DRUGS
ON TOL METABOLISM
in each group. *P < 0.05, significantly different different from controls; ***P < 0.001, signifi.Enzyme activity (nmol * mine’ * mg protein-‘)
ControP
IN VITRO
1.52 2 0.33
% Control
100
AQ
0.5
1.49 + 0.38
CQ
0.5
1.79 ? 0.35
MB
0.05 0.1 0.5 1.0
1.16 1.11 0.24 0.11
+ ? 2 +
0.14 0.22 0.17*** 0.13***
77.92 7.9 74.65 12.6 16.12 13.0 9.52 11.9
0.05 0.1 0.5 1 .o
0.91 0.65 0.07 0.03
?: 0.24** 2 0.40*** k 0.04*** 2 0.01***
61.3 + 13.5 44.52 7.0 5.12 3.0 1.92 1.2
QD
0.05 0.1 0.5 1 .o
1.58’2 1.31 ? 1.08 + 0.73 2
0.37 0.21 0.21 0.21*
Q
0.05 0.1 0.5 1.0
1.39 1.02 0.57 0.38
0.44 0.19 0.12** 0.11***
PQ
%ontrol
-
incubation
contained
DMSO.
? + tt-
99.7 2 0.92 118.52
5.0
106.1 !I 23.1 87.3f: 6.8 72.5 2 14.6 47.75 4.8 94.2 2 68.7 + 38.2% 25.22
33.7 12.0 4.7 5.0
306 TABLE
III
INHIBITION OF TOL LIVER MICROSOMES
HYDROXYLASE
ACTIVITY
QUINOLINE
DRUGS
IN
RAT
IC,, (clW
MQ PQ QD Q
BY
value -
.-_.
240 60 930 330
was 0.07 f 0.04 nmol - min-’ * mg protein-’ as compared to control (1.52 f 0.33). The order of potency was PQ (0.07 + 0.04 nmol * min-’ * mg protein-’ ) 2 MQ (0.24 f 0.17 nmol - min-’ * mg protein-’ ) > Q (0.57 f 0.12 nmol * min-’ * mg protein-’ ) > QD (1.08 f 0.21 nmol * min-’ * mg protein-‘) > AQ (1.49 + 0.38 nmol * min-’ * mg protein-‘) B CQ (1.79 f 0.35 nmol - min-’ * mg protein-‘). ICSO values were determined for MQ, PQ, QD and Q on TOL hydroxylase activity (Table III). PQ was found to be four times as potent as MQ against TOL hydroxylase. Q was approximately three times more potent than its stereoisomer (QD) against TOL hydroxylase. l
DISCUSSION
The present work provides more evidence of the potential of some quinoline drugs to inhibit hepatic microsomal enzyme activity. With data from two substrates (EE2 and TOL) it is clear that PQ, MQ and to a lesser extent Q, produce the most marked inhibitory effects and that CQ (over the range of concentrations studied) does not inhibit these enzymes. QD and AQ are of intermediate potency. These results are substantially in agreement with the study by Murray [2] on the effect of five of the quinoline drugs (not MQ) on aminopyrine N-demethylase activity, which indicated an order of potency Q >, PQ > AQ >QD 2 CQ. It was suggested that PQ and Q interact with the haem iron of cytochrome P-450 via the side chain amino nitrogen and the quinuclidine nitrogen respectively, as both provide a lone pair of electrons that are sterically accessible to the haem (Fig. 1). In contrast, CQ has relatively hindered nitrogen atoms in its alkylaminoalkyl side chain and in the quinoline ring and the quinuclidine nitrogen in QD is sterically inaccessible. The inhibition produced by MQ is probably the result of the interaction with the haem iron via the sterically accessible piperidine nitrogen. The fact that a compound shows enzyme inhibition in vitro does not mean that it will automatically lead to clinically important pharmacokinetic interactions if co-administered with other drugs in man. Indeed, despite the findings of the present study, we have demonstrated that in normal thera-
307 Cl
AH ‘CH(CH,),N(CzHd, AH,
CH3
H,N
(CH,),~”
CF3
NH
(3)
CH,O CH =CH2
(4)
(5) Fig. 1. The structures of the quinoline derivatives used in the study: (1) amodiaquine; (2) chloroquine; (3) mefloquine; (4) primaquine; (5) quinine and quinidine. The asterisk represents the centre of asymmetry.
peutic doses, PQ but not MQ inhibits antipyrine metabolism in man [6,7]. Although this could represent a selectivity in inhibitory effect it is far more likely to be a reflection of the different pharmacokinetics and hepatic accumulation of the antimalarials. Since enzyme inhibition in vivo is a reflection not only of binding to the enzyme but also of actual drug concentrations present in the 1iver;particularly in the vicinity of the enzyme it is possible that hepatic concentrations of MQ are very much less than PQ. There is evidence of the avid binding of MQ to both plasma proteins and red blood cells [ 161 and hence hepatic extraction may be low. Further studies in animals and man, both in vitro and in vivo are in progress in an attempt to understand the clinical relevance of the inhibitory potential of the quinolines.
308 ACKNOWLEDGEMENT
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