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Inhibition of hepatic microsomal enzymes by gossypol in the rat
CONTRACEPTION
INHIBITION OF HEPATIC MICROSOMAL ENZYMES BY GOSSYPOL IN THE RAT
Ma Xiao-Nian and D.J. Back*
National Research Institute for Family Planning, Beijing, China and *Department of Pharmacology & Therapeutics, University of Liverpool, P.O. Box 147, Liverpool, U.K.
ABSTRACT The effect of the male contraceptive, gossypol, on rat liver microsomal enzymes has been studied in vitro and in vivo. In vitro, gossypol inhibited aminopyrine N-demethylase activity, the concentration causing 50% inhibition being approximately 0.03 mM; Also the metabolism of the inhibition was non-competitive. ethinylestradiol (a drug with a number of metabolic pathways) was inhibited with the main effect being reduced 2- and 16hydroxylation. In vivo, gossypol, after 4 weeks of daily administration (30 mg/kg/day) caused a significant reduction in microsomal protein, cytochrome P450 and aminopyrine N-demethylase However, despite reduced enzyme activity, the metabolism activity. of tolbutamide (a drug with a single pathway of metabolism and hence a model substrate) was not impaired by either acute or chronic gossypol administration. Submitted for publication February 21, 1984 Accepted for publication June 1, 1984
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1984 VOL. 30 NO. 1
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CONTRACEPTION INTRODUCTION The antifertility effect of the polyphenolic compound gossypol in animals and man has been well documented (1). A number of studies have also investigated more general interactions between gossypol and enzyme systems. It has been reported to reduce succinic dehydrogenase, cytochrome oxidase and xanthine oxidase activities in vitro at certain concentrations but to increase activity at othersThe same enzyme activities are reported to be unaffected by (2). gossypol pretreatment in vivo (3). In contrast, lipid peroxidation activity is inhibited by gossypol in viva and in vitro (4) and the inhibition in vitro can be overcome by the addition of Fe+". Recently, Tang -_ et al.(5) have reported that gossypol inhibits rat liver catechol-O-methyl transferase activity in vitro with an IC50 of 0.01 mM; the inhibition was non-competitive. They also found that blood serum or SSA could significantly reduce the inhibitory effect of Inactivation of lactate dehydrogenase isozymes and malate gossYpol. dehydrogenase and inhibition of glutathione-S-transferase has been reported in two studies (6,7). It is clear that high concentrations of gossypol and/or metabolites accumulate in the liver (8) and it is therefore of vital importance to investigate the extent and magnitude of effects on It is particularly important that studies are hepatic enzymes. carried out both in vitro and in vivo and this paper reports work designed to investigate the interaction of gossypol with hepatic drug metabolising enzymes.
METHODS
1. In vitro studies Rats were killed by cervical dislocation, the livers rapidly removed and homogenized in ice-cold M/15 phosphate buffer (pH 7.4) containing 0.15 M KC1 using a Teflon in glass homogeniser. The 25% homogenate was centrifuged at 13,000g for 20 min at 4t_ The resulting supernatant was decanted without disturbing the pellet and centrifuged at 105,000 g for 60 min at 4-C. The microsomal pellet was resuspended in 0.2 ml phosphate buffer. The N-demethylation of aminopyrine was carried out with the following reaction mixture: aminopyrine (2.5 mM), semicarbizide (9.37 mM), gossypol acetic acid (0.0001-0.1 mM in 5Oul dimethylsulfoxide, DMSO) or DMSO alone, microsomes (0.5 ml of 4mg/ml suspension) and Formaldehyde The final incubation volume was 5 ml. NADPH (0.6 mM). production was measured with Nash reagent and absorbance determined at In some studies the effect of two fixed concentrations of 415 nm. gossypol (0.001 and 0.01 mM) on the kinetics of aminopyrine NIn other demethylation (O-25-2.5 mM aminopyrine) was investigated. studies the potential interaction of ferric chloride (0.1 mM) and gossypol on the N-demethylation reaction was investigated. The in vitro metabolism of ethinylestradiol (EE2) was studied as follows: In duplicate incubation flasks, 3H-EE2 (1 nCi; 10 nM) was evaporated to dryness, and then microsomal suspension (1 ml of 4 mg/ml suspension), NADPH (0.25 ml; 0.6 nmoles), gossypol (0.1 mM and 1 mM in 50 j.11 DMSO) and buffer (1.7 ml) added to a final volume of 3 ml. The
90
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At the mixture was incubated at 3l"Cfor 1 h in a shaking water bath. end of the incubation period, the mixture was extracted twice with After centrifugation for 10 min at diethyl ether (6 ml) for 30 min. 2000 g, the organic layer was separated in a dry ice/methanol bath, evaporated to dryness and redissolved in methanol (50 ~1) with the addition of EB2 (10 ul of 2 mg/ml solution in methanol) and 2-hydroxy EE2 (10 ul of 2 mg/ml solution in methanol). Methanol extracts were then subjected to high performance liquid chromatography (HPLC) substantially according to the method of Maggs et al. (9). In brief, a twin pump aerograph model 8700 chromatograph (Spectra-Physics) was used. Separation was performed at room temperature on a Partisil lo/25 CDS column (0.46 cm int. diameter x 25 cm), protected by an in-line guard column packed with Co:Pell ODS. Samples were eluted with a linear The flow rate was 2 ml/min. gradient of methanol in aq. NHhH2PO4 buffer solution (0.5 g/100 ml; pH Eluate fractions were collected at 3.0), from 50-65% at Z%/min. 30-set intervals and the radioactive content determined by liquid scintillation spectrometry. Unlabelled estrogen standards were Positive monitored at 280 nm with a model LC W-VIS detector. metabolite identification has previously been made (9). 2. In vivo studies a) Tolbutamide metabolism. Adult male Wistar rats weighing 200-300 g were used. Rats were anaesthetized with pentobarbitone sodium (60 mg/kg; 60 mg/ml). The trachea, jugular vein and carotid artery were cannulated using polyethylene tubing (PE50). Heparin (200 U) was injected intravenously (i.v.) to prevent clotting of blood samples. Gossypol acetic acid (30 mg/kg; 5Oul/lOO g.b.w. in DMSO) or DMSO were injected intraperitoneally (i.p.) and then 30 min later In some tolbutamide (50 mg/kg; 50 mg/ml) was adminstered i.v. experiments, gossypol was administered orally (p.0.) for 2 weeks (30 mg/kg/day) prior to the study on tolbutamide metabolism. Blood samples were collected at 0, 15, 30, 60, 90, 120, 180, 240 and 300 min. After each sample was taken, 0.3 ml saline was returned Blood samples were centrifuged for 3 min and to the blood system. the plasma stored deep frozen for analysis by HPLC. Plasma tolbutamide and hydroxytolbutamide were measured by HPLC according to the method previously published from our laboratories A model 1lOA pump (Altex) linked to a model 110-10 (10). spectrophotometer (Hitachi) monitoring at 230 nm was used. Separations were performed at room temperature on a Partisil lo/25 ODS The mobile phase was column protected by an in-line guard column. methanol: 0.025% phosphoric acid (48:52, v/v). The flow rate was 1.8 ml/min. For measurement of tolbutamide, plasma (100 ul), internal standard (chlorpropamide; 25 ul of 1 mg/ml solution) and methanol (375 ~1) were vortexed and centrifuged at 2000 g for 10 min. Supernatant (20 ~1) was removed for injection onto the column. For measurement of hydroxytolbutamide, plasma (50 ul), internal standard (25 ~1) and methanol (125 ~1) were treated as above. Standard curves were prepared for both tolbutamide and hydroxytolbutamide and quantitation of plasma concentrations obtained by means of the peak height ratio to the internal standard. Tolbutamide half-life (tf) was calculated from the elimination rate constant (k) obtained by least squares regression analysis of
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1984 VOL. 30 NO. I
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plasma drug concentrations. The apparent volume of distribution (Vd) was calculated by dividing the dose by the plasma concentration at zero time (Cpo). Tolbutamide clearance was calculated from Clp = 0.693 x vd. The area under the curve (AUC~_~) was tl calculated by the trapezoidal rule using a Hewlett-Packard programmable calculator. b) Iiepaticenzyme activity. Rats were dosed daily with gossypol At the end of this period acetic acid (30 mg/kg; p.0.) for 4 weeks. they were killed, the livers removed and microsomes prepared as previously described. Microsomal protein and cytochrome P450 were determined respectively by the methods of Lowry -et al. (11) and Crnura Aminopyrine N-demethylase activity was also estimated. & Sat0 (12). Student's unpaired t-test was used to determine statistical significance. Data are presented as mean -+ S.D. RESULTS 1. In vitro studies Gossypol inhibited aminopyrine N-demethylase activity (Fig. 1); the concentration causing 50% inhibition (IC50) being approximately 0.03 mM. The mechanism of inhibition of the enzyme was investigated in kinetic studies. Lineweaver-Burk plots (Fig. 2) with 2 concentrations of gossypol gave evidence of non-competitive inhibition (no change in em; change in Vmax). Adding ferric chloride (0.1 mM) did not prevent the inhibition; in fact there was a slight enhancing of inhibition. Gossypol inhibited EE2 metabolism (Table I). At concentrations of 0.1 and 1 mM, the formation of 16-hydroxy-EE2 was significantly reduced as was the formation of the heterogenous peak (as yet unidentified). At the high concentration, gossypol inhibited 2hydroxylation of EE2. 2. In vivo studies There were no significant changes in the pharmacokinetics of tolbutamide (Table II) or the production of hydroxytolbutamide after a single dose of gossypol or after 2 weeks daily dosing. In contrast, after 4 weeks dosing with gossypol, microsomal protein, cytochrome P450 and aminopyrine N-demethylase activity were all significantly reduced (Table III). DISCUSSION The results of this study have confirmed that gossypol is a potent inhibitor of hepatic microsomal enzymes in vitro. The IC50 for aminopyrine N-demethylation was 0.03 mM which makes gossypol some 300 times more potent an inhibitor of this enzyme than the widely In reported inhibitor of microsomal enzymes, cimetidine (13). contrast to a previous report that Fe+++ overcame the inhibition of lipid peroxidation (4), we found no evidence that ferric chloride could reduce the inhibition of aminopyrine N-demethylase activity by gossypol. The inhibition of the metabolism of the synthetic estrogen EE2r is also quite striking with both 2-hydroxylation and 16-
92
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1984 VOL. 30 NO. 1
CONTRACEPTION
.1
SO.
‘\I
l
4:
60.
P E. z 6 %I d 40fi
+
~
. I
20 .OOl
.l
.Ol Gossypol
1
ITIM
Fig. 1. Effect of gossypol on aminopyrine N-demethylase The results are the means + S.D. of four experiments.
activity.
Fig. 2. Lineweaver-Burk plots for aminopyrine N-demethylation at Each point is the mean of 4 various fixed gossypol concentrations. experiments.
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93
-
-
-
2OHEE2
2MeOEE2
20HME
*P < 0.05;
-
160HEE2
Gossypol (1 mn)
Gossypol (0.1 mM)
Control
**
** 2.1 + 2.4
***
15.4 + 3.1
2-methoxy-ethinylestradiol
2-hydroxy-ethinylestradiol
**p < 0.01;
1.1 + 0.3
**
1.7 + 0.5
2.6 ,+ 0.8
'X'
Mean ,+ S.D. (n = 4)
89.9 i 4.7
0.5 2 0.4
0.5 f 0.2
** 66.8 -+8.1
***
1.3 + 1.2
2MeOEE2
37.0 + 8.3
EE2
**Jp < 0.001; significantly different from controls.
2-hydroxy-mestranol
20HEE2
11.8 f 0.4
16-hydroxy-ethinylestradiol
1.6 + 0.7
** 1.9 + 0.6
* 6.8 -+4.0
2.0 2 0.5
12.3 -+5.1
160HEE2
Percent of 3H in HPLC column effluent as:
Effect of gossypol on ethinylestradiol (EE2) metabolism -in vitro
16.2 + 4.1
Heterogenous peak
Table I.
0.7 i 0.6
0.6 -+0.3
1.4 -+0.9
ZOHME
CONTRACEPTION
Effect of acute (30 mg/kg; single dose) and chronic (30 mg/kg/day for 2 weeks) treatment of gossypol on tolbutamide pharmacokinetic parameters
Table II.
CFQ
Vd
tf
AUC (mg/ml.min)
(ml/kg)
(h)
( w/ml)
ClP (ml/min/kg)
Acute Control
254
-+ 20
3.8
f 0.5
198
+ 15
83
+ 11
0.61
-+ 0.10
Gossypol
266
2 9
4.2
+ 0.6
189
+ 6
96
f 13 0.53
t 0.10
Control
214
-+ 16
3.2
+ 0.3
235
f 19
61
-+
8
0.84
j: 0.11
Gossypol
211
+
14
3.5
+ 0.3
237
+
65
+- 10
0.79
+ 0.12
(n=
5)
Chronic
Mean
+ S.D.
Table III.
15
The effect of chronic (30 mg/kg/day for 4 weeks) gossypol administration on hepatic enzyme activity
Control
Gossypol
443 f 25
391
Liver wt (g)
14.9 f 1.8
13.7 + 2.1
Microsomal protein (mg/g liver)
22.8 -+3.5
17.3 + 5.2
Cytochrome ~450 (nmoles/g liver)
11.2
8.4 + 1.7
* Body
wt (g)
+
28
*
* k 2.3
Aminopyrine N-demethylase activity (nmoles/min/mg protein) 14.3 !: 5.3
* 8.5 + 1.3
Mean * S.D. (n = 4) *P < 0.05; significantly different from control.
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hydroxylation reactions affected. Since both the N-demethylation and the hydroxylation reactions are cytochrome P450-dependent, there is clear evidence that gossypol inhibits the microsomal haemoprotein(s). The reduction in microsomal protein, cytochrome P450 and amino pyrine N-demethylase activity after chronic gossypol administration is in agreement with the in vitro study. However, some caution needs to be exercised in interpreting these results, due to the decrease in body weight. The lack of effect of gossypol, either given as a single dose or administered chronically, on tolbutamide pharmacokinetics indicates that it is not always possible to extrapolate from in vitro studies to predict how the metabolism of another compound may be influenced -in vivo. Tolbutamide is metabolised by a single pathway in the rat to form hydroxytolbutamide and this is a P450-dependent step. It is therefore, an extremely good substrate to use when investigating the effects of other compounds (such as gossypol) on hepatic drug metabolism. Since gossypol clearly inhibits cytochrome P450, the reason for the lack of effect on tolbutamide metabolism may be either that tolbutamide is metabolised by a form of P450 (at least 7 have been identified) not inhibited by gossypol or that, even though the enzyme is decreased there is still a sufficient amount to metabolise the substrate. It is important to recognize that there are fundamental differences between in vitro and in vivo studies. In vitro, a known concentration of the potential inhibitor is present for a set period of time, whereas in vivo, the actual concentration in the liver cell is not known and may decrease with time. The enzyme, due to membrane disruption, will be more accessible in vitro than in vivo. We believe that the results of this and previous (5) studies indicate that gossypol administration may have important effects on It is essential to bear in mind that not only drugs hepatic enzymes. (such as tolbutamide) but endogenous compounds (eg. steroids) and environmental chemicals are metabolised by cytochrome(s) P450. Hence, although in vivo tolbutamide metabolism was unaffected, we do not know as yet if also applies to other substrates. ACKNOWLEDGEMENT Ma Xiao-Nian gratefully acknowledges receipt of a World Health Organization Training Fellowship.
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REFERENCES 1.
Prasad, M.R.N. and Diczfalusy, E. supp1. 5, 53-70, 1982.
2.
Myers, B.D. and Tnroneberry, G.O. Effect of gossypol on some oxidative respiratory enzymes. Plant Physiol. 41, 787-791,
Gossypol. Int. J. Androl.
1966. 3.
Meksangsee, L.A., Clawson, A.J. and Smith, F.H. The -in vivo effect of gossypol on cytochrome oxidase, succinoxidase and succinic dehydrogenase in animal tissue. J. Agr. Food Chem. 18, 917-920, 1970.
4.
Skutches, C.L. and Smith, F.H. Effect of phenobarbital on the level of gossypol in the liver and the effect of gossypol and phenobarbital on liver microsomal 0-demethylation and lipid peroxidation activities in the rat. J. Nutr. 104, 1567-1575, 1914.
5.
Tang, F., Tsang, A.Y.F., Lee, Ching-po and Wong, P.Y.D. Inhibition of catechol-O-methyltransferase by gossypol: the 1983. effect of plasma proteins. Contraception 26, 515-519,
6.
Lee, C-Y.G., Moon, Y.S., Yuan, J.H. and Chen, A.F. Enzyme inactivation and inhibition by gossypol. Mol. Cell. Biochem. 47, 65-70, 1982.
7.
Tso, W.W.and Lee, C.S. Lactate dehydrogenase-X: an isozyme particularly sensitive to gossypol inhibition. Int. J. Androl. 5, 205-209, 1982.
8.
Xue, S.P. Studies on the antifertility effect of gossypol, a new contraceptive for males. Symposium on Recent Advances in Fertility Regulation. Beijing, 1980 (Chang Chai Fen, D. Griffin and A. Woolman, Eds.). W.H.O., Geneva, 1981, p 122-146.
9.
Maggs,
J.L., Grabowski, P.S. and Park, B.K. Drug-protein conjugates. II. An investigation of the irreversible binding and metabolism of 17a-ethinylestradiol -in vivo. Biochem. Pharmac. 32, 301-308, 1983.
10.
Back, D.J., Sutcliffe, F., and Tjia, J.F. Tolbutamide as a model drug for the study of enzyme induction and enzyme inhibition in the rat. Br. J. Pharmac. (in press).
11.
Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. Protein measurement with the folin reagent. J. Biol. Chem. 193, 265-275, 1951.
12.
Omura, T. and Sato, R. The carbon monoxide binding pigment of liver microsomes. II. Solubilization, purification and properties. J. Biol. Chem. 239, 2379-2385, 1964.
13.
Pelkonen, 0. and Puurunen, J. The effect of cimetidine on in vitro and -in vivo microsomal drug metabolism in the rat. Biochem. Pharmac. 29, 3075-3080, 1980.
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INHIBITION OF HEPATIC MICROSOMAL ENZYMES BY GOSSYPOL IN THE RAT
Ma Xiao-Nian and D.J. Back*
National Research Institute for Family Planning, Beijing, China and *Department of Pharmacology & Therapeutics, University of Liverpool, P.O. Box 147, Liverpool, U.K.
ABSTRACT The effect of the male contraceptive, gossypol, on rat liver microsomal enzymes has been studied in vitro and in vivo. In vitro, gossypol inhibited aminopyrine N-demethylase activity, the concentration causing 50% inhibition being approximately 0.03 mM; Also the metabolism of the inhibition was non-competitive. ethinylestradiol (a drug with a number of metabolic pathways) was inhibited with the main effect being reduced 2- and 16hydroxylation. In vivo, gossypol, after 4 weeks of daily administration (30 mg/kg/day) caused a significant reduction in microsomal protein, cytochrome P450 and aminopyrine N-demethylase However, despite reduced enzyme activity, the metabolism activity. of tolbutamide (a drug with a single pathway of metabolism and hence a model substrate) was not impaired by either acute or chronic gossypol administration. Submitted for publication February 21, 1984 Accepted for publication June 1, 1984
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1984 VOL. 30 NO. 1
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CONTRACEPTION INTRODUCTION The antifertility effect of the polyphenolic compound gossypol in animals and man has been well documented (1). A number of studies have also investigated more general interactions between gossypol and enzyme systems. It has been reported to reduce succinic dehydrogenase, cytochrome oxidase and xanthine oxidase activities in vitro at certain concentrations but to increase activity at othersThe same enzyme activities are reported to be unaffected by (2). gossypol pretreatment in vivo (3). In contrast, lipid peroxidation activity is inhibited by gossypol in viva and in vitro (4) and the inhibition in vitro can be overcome by the addition of Fe+". Recently, Tang -_ et al.(5) have reported that gossypol inhibits rat liver catechol-O-methyl transferase activity in vitro with an IC50 of 0.01 mM; the inhibition was non-competitive. They also found that blood serum or SSA could significantly reduce the inhibitory effect of Inactivation of lactate dehydrogenase isozymes and malate gossYpol. dehydrogenase and inhibition of glutathione-S-transferase has been reported in two studies (6,7). It is clear that high concentrations of gossypol and/or metabolites accumulate in the liver (8) and it is therefore of vital importance to investigate the extent and magnitude of effects on It is particularly important that studies are hepatic enzymes. carried out both in vitro and in vivo and this paper reports work designed to investigate the interaction of gossypol with hepatic drug metabolising enzymes.
METHODS
1. In vitro studies Rats were killed by cervical dislocation, the livers rapidly removed and homogenized in ice-cold M/15 phosphate buffer (pH 7.4) containing 0.15 M KC1 using a Teflon in glass homogeniser. The 25% homogenate was centrifuged at 13,000g for 20 min at 4t_ The resulting supernatant was decanted without disturbing the pellet and centrifuged at 105,000 g for 60 min at 4-C. The microsomal pellet was resuspended in 0.2 ml phosphate buffer. The N-demethylation of aminopyrine was carried out with the following reaction mixture: aminopyrine (2.5 mM), semicarbizide (9.37 mM), gossypol acetic acid (0.0001-0.1 mM in 5Oul dimethylsulfoxide, DMSO) or DMSO alone, microsomes (0.5 ml of 4mg/ml suspension) and Formaldehyde The final incubation volume was 5 ml. NADPH (0.6 mM). production was measured with Nash reagent and absorbance determined at In some studies the effect of two fixed concentrations of 415 nm. gossypol (0.001 and 0.01 mM) on the kinetics of aminopyrine NIn other demethylation (O-25-2.5 mM aminopyrine) was investigated. studies the potential interaction of ferric chloride (0.1 mM) and gossypol on the N-demethylation reaction was investigated. The in vitro metabolism of ethinylestradiol (EE2) was studied as follows: In duplicate incubation flasks, 3H-EE2 (1 nCi; 10 nM) was evaporated to dryness, and then microsomal suspension (1 ml of 4 mg/ml suspension), NADPH (0.25 ml; 0.6 nmoles), gossypol (0.1 mM and 1 mM in 50 j.11 DMSO) and buffer (1.7 ml) added to a final volume of 3 ml. The
90
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At the mixture was incubated at 3l"Cfor 1 h in a shaking water bath. end of the incubation period, the mixture was extracted twice with After centrifugation for 10 min at diethyl ether (6 ml) for 30 min. 2000 g, the organic layer was separated in a dry ice/methanol bath, evaporated to dryness and redissolved in methanol (50 ~1) with the addition of EB2 (10 ul of 2 mg/ml solution in methanol) and 2-hydroxy EE2 (10 ul of 2 mg/ml solution in methanol). Methanol extracts were then subjected to high performance liquid chromatography (HPLC) substantially according to the method of Maggs et al. (9). In brief, a twin pump aerograph model 8700 chromatograph (Spectra-Physics) was used. Separation was performed at room temperature on a Partisil lo/25 CDS column (0.46 cm int. diameter x 25 cm), protected by an in-line guard column packed with Co:Pell ODS. Samples were eluted with a linear The flow rate was 2 ml/min. gradient of methanol in aq. NHhH2PO4 buffer solution (0.5 g/100 ml; pH Eluate fractions were collected at 3.0), from 50-65% at Z%/min. 30-set intervals and the radioactive content determined by liquid scintillation spectrometry. Unlabelled estrogen standards were Positive monitored at 280 nm with a model LC W-VIS detector. metabolite identification has previously been made (9). 2. In vivo studies a) Tolbutamide metabolism. Adult male Wistar rats weighing 200-300 g were used. Rats were anaesthetized with pentobarbitone sodium (60 mg/kg; 60 mg/ml). The trachea, jugular vein and carotid artery were cannulated using polyethylene tubing (PE50). Heparin (200 U) was injected intravenously (i.v.) to prevent clotting of blood samples. Gossypol acetic acid (30 mg/kg; 5Oul/lOO g.b.w. in DMSO) or DMSO were injected intraperitoneally (i.p.) and then 30 min later In some tolbutamide (50 mg/kg; 50 mg/ml) was adminstered i.v. experiments, gossypol was administered orally (p.0.) for 2 weeks (30 mg/kg/day) prior to the study on tolbutamide metabolism. Blood samples were collected at 0, 15, 30, 60, 90, 120, 180, 240 and 300 min. After each sample was taken, 0.3 ml saline was returned Blood samples were centrifuged for 3 min and to the blood system. the plasma stored deep frozen for analysis by HPLC. Plasma tolbutamide and hydroxytolbutamide were measured by HPLC according to the method previously published from our laboratories A model 1lOA pump (Altex) linked to a model 110-10 (10). spectrophotometer (Hitachi) monitoring at 230 nm was used. Separations were performed at room temperature on a Partisil lo/25 ODS The mobile phase was column protected by an in-line guard column. methanol: 0.025% phosphoric acid (48:52, v/v). The flow rate was 1.8 ml/min. For measurement of tolbutamide, plasma (100 ul), internal standard (chlorpropamide; 25 ul of 1 mg/ml solution) and methanol (375 ~1) were vortexed and centrifuged at 2000 g for 10 min. Supernatant (20 ~1) was removed for injection onto the column. For measurement of hydroxytolbutamide, plasma (50 ul), internal standard (25 ~1) and methanol (125 ~1) were treated as above. Standard curves were prepared for both tolbutamide and hydroxytolbutamide and quantitation of plasma concentrations obtained by means of the peak height ratio to the internal standard. Tolbutamide half-life (tf) was calculated from the elimination rate constant (k) obtained by least squares regression analysis of
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1984 VOL. 30 NO. I
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plasma drug concentrations. The apparent volume of distribution (Vd) was calculated by dividing the dose by the plasma concentration at zero time (Cpo). Tolbutamide clearance was calculated from Clp = 0.693 x vd. The area under the curve (AUC~_~) was tl calculated by the trapezoidal rule using a Hewlett-Packard programmable calculator. b) Iiepaticenzyme activity. Rats were dosed daily with gossypol At the end of this period acetic acid (30 mg/kg; p.0.) for 4 weeks. they were killed, the livers removed and microsomes prepared as previously described. Microsomal protein and cytochrome P450 were determined respectively by the methods of Lowry -et al. (11) and Crnura Aminopyrine N-demethylase activity was also estimated. & Sat0 (12). Student's unpaired t-test was used to determine statistical significance. Data are presented as mean -+ S.D. RESULTS 1. In vitro studies Gossypol inhibited aminopyrine N-demethylase activity (Fig. 1); the concentration causing 50% inhibition (IC50) being approximately 0.03 mM. The mechanism of inhibition of the enzyme was investigated in kinetic studies. Lineweaver-Burk plots (Fig. 2) with 2 concentrations of gossypol gave evidence of non-competitive inhibition (no change in em; change in Vmax). Adding ferric chloride (0.1 mM) did not prevent the inhibition; in fact there was a slight enhancing of inhibition. Gossypol inhibited EE2 metabolism (Table I). At concentrations of 0.1 and 1 mM, the formation of 16-hydroxy-EE2 was significantly reduced as was the formation of the heterogenous peak (as yet unidentified). At the high concentration, gossypol inhibited 2hydroxylation of EE2. 2. In vivo studies There were no significant changes in the pharmacokinetics of tolbutamide (Table II) or the production of hydroxytolbutamide after a single dose of gossypol or after 2 weeks daily dosing. In contrast, after 4 weeks dosing with gossypol, microsomal protein, cytochrome P450 and aminopyrine N-demethylase activity were all significantly reduced (Table III). DISCUSSION The results of this study have confirmed that gossypol is a potent inhibitor of hepatic microsomal enzymes in vitro. The IC50 for aminopyrine N-demethylation was 0.03 mM which makes gossypol some 300 times more potent an inhibitor of this enzyme than the widely In reported inhibitor of microsomal enzymes, cimetidine (13). contrast to a previous report that Fe+++ overcame the inhibition of lipid peroxidation (4), we found no evidence that ferric chloride could reduce the inhibition of aminopyrine N-demethylase activity by gossypol. The inhibition of the metabolism of the synthetic estrogen EE2r is also quite striking with both 2-hydroxylation and 16-
92
JULY
1984 VOL. 30 NO. 1
CONTRACEPTION
.1
SO.
‘\I
l
4:
60.
P E. z 6 %I d 40fi
+
~
. I
20 .OOl
.l
.Ol Gossypol
1
ITIM
Fig. 1. Effect of gossypol on aminopyrine N-demethylase The results are the means + S.D. of four experiments.
activity.
Fig. 2. Lineweaver-Burk plots for aminopyrine N-demethylation at Each point is the mean of 4 various fixed gossypol concentrations. experiments.
JULY 1984VOL. 30 NO. 1
93
-
-
-
2OHEE2
2MeOEE2
20HME
*P < 0.05;
-
160HEE2
Gossypol (1 mn)
Gossypol (0.1 mM)
Control
**
** 2.1 + 2.4
***
15.4 + 3.1
2-methoxy-ethinylestradiol
2-hydroxy-ethinylestradiol
**p < 0.01;
1.1 + 0.3
**
1.7 + 0.5
2.6 ,+ 0.8
'X'
Mean ,+ S.D. (n = 4)
89.9 i 4.7
0.5 2 0.4
0.5 f 0.2
** 66.8 -+8.1
***
1.3 + 1.2
2MeOEE2
37.0 + 8.3
EE2
**Jp < 0.001; significantly different from controls.
2-hydroxy-mestranol
20HEE2
11.8 f 0.4
16-hydroxy-ethinylestradiol
1.6 + 0.7
** 1.9 + 0.6
* 6.8 -+4.0
2.0 2 0.5
12.3 -+5.1
160HEE2
Percent of 3H in HPLC column effluent as:
Effect of gossypol on ethinylestradiol (EE2) metabolism -in vitro
16.2 + 4.1
Heterogenous peak
Table I.
0.7 i 0.6
0.6 -+0.3
1.4 -+0.9
ZOHME
CONTRACEPTION
Effect of acute (30 mg/kg; single dose) and chronic (30 mg/kg/day for 2 weeks) treatment of gossypol on tolbutamide pharmacokinetic parameters
Table II.
CFQ
Vd
tf
AUC (mg/ml.min)
(ml/kg)
(h)
( w/ml)
ClP (ml/min/kg)
Acute Control
254
-+ 20
3.8
f 0.5
198
+ 15
83
+ 11
0.61
-+ 0.10
Gossypol
266
2 9
4.2
+ 0.6
189
+ 6
96
f 13 0.53
t 0.10
Control
214
-+ 16
3.2
+ 0.3
235
f 19
61
-+
8
0.84
j: 0.11
Gossypol
211
+
14
3.5
+ 0.3
237
+
65
+- 10
0.79
+ 0.12
(n=
5)
Chronic
Mean
+ S.D.
Table III.
15
The effect of chronic (30 mg/kg/day for 4 weeks) gossypol administration on hepatic enzyme activity
Control
Gossypol
443 f 25
391
Liver wt (g)
14.9 f 1.8
13.7 + 2.1
Microsomal protein (mg/g liver)
22.8 -+3.5
17.3 + 5.2
Cytochrome ~450 (nmoles/g liver)
11.2
8.4 + 1.7
* Body
wt (g)
+
28
*
* k 2.3
Aminopyrine N-demethylase activity (nmoles/min/mg protein) 14.3 !: 5.3
* 8.5 + 1.3
Mean * S.D. (n = 4) *P < 0.05; significantly different from control.
JULY 1984 VOL. 30 NO. 1
95
CONTRACEPTION
hydroxylation reactions affected. Since both the N-demethylation and the hydroxylation reactions are cytochrome P450-dependent, there is clear evidence that gossypol inhibits the microsomal haemoprotein(s). The reduction in microsomal protein, cytochrome P450 and amino pyrine N-demethylase activity after chronic gossypol administration is in agreement with the in vitro study. However, some caution needs to be exercised in interpreting these results, due to the decrease in body weight. The lack of effect of gossypol, either given as a single dose or administered chronically, on tolbutamide pharmacokinetics indicates that it is not always possible to extrapolate from in vitro studies to predict how the metabolism of another compound may be influenced -in vivo. Tolbutamide is metabolised by a single pathway in the rat to form hydroxytolbutamide and this is a P450-dependent step. It is therefore, an extremely good substrate to use when investigating the effects of other compounds (such as gossypol) on hepatic drug metabolism. Since gossypol clearly inhibits cytochrome P450, the reason for the lack of effect on tolbutamide metabolism may be either that tolbutamide is metabolised by a form of P450 (at least 7 have been identified) not inhibited by gossypol or that, even though the enzyme is decreased there is still a sufficient amount to metabolise the substrate. It is important to recognize that there are fundamental differences between in vitro and in vivo studies. In vitro, a known concentration of the potential inhibitor is present for a set period of time, whereas in vivo, the actual concentration in the liver cell is not known and may decrease with time. The enzyme, due to membrane disruption, will be more accessible in vitro than in vivo. We believe that the results of this and previous (5) studies indicate that gossypol administration may have important effects on It is essential to bear in mind that not only drugs hepatic enzymes. (such as tolbutamide) but endogenous compounds (eg. steroids) and environmental chemicals are metabolised by cytochrome(s) P450. Hence, although in vivo tolbutamide metabolism was unaffected, we do not know as yet if also applies to other substrates. ACKNOWLEDGEMENT Ma Xiao-Nian gratefully acknowledges receipt of a World Health Organization Training Fellowship.
JULY
1984 VOL. 30 NO. 1
CONTRACEPTION
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4.
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1984 VOL. 30 NO. 1
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