Stereoselective interaction between gossypol and rat plasma

CONTRACEPTION

STEREOSELECTIVE

INTERACTION

BETWEEN GOSSYPOL AND RAT PLASMA

D.F. Wu and M.M. Reidenberg Departments of Pharmacology and Medicine Cornell University Medical College 1300 York Avenue, New York, New York, U.S.A.

ABSTRACT Gossypol, a potential male oral contraceptive, is chiral and chemically reactive. The present study was done to learn more about the stereoselective activity of this drug. The isomers were equipotent in hemolyzing erythrocytes in protein-free buffer while (+) gossypol was a more potent hemolysin than (-) in Both isomers disappeared from buffer at the same rate plasma. while (-) disappeared from plasma much faster than (+). Treating plasma with aspirin or DNFB to react with the free amino groups on the protein, slowed the disappearance of (-) gossypol. We conclude that (-) gossypol binds to free amino groups on protein and this stereoselective protein binding may account for some of the pharmacokinetic or pharmacodynamic difference between the two isomers of gossypol.

Correspondence

and reprint requests:

Marcus M. Reidenberg, M.D. Department of Pharmacology Cornell University Medical College 1300 York Avenue New York, N.Y. 10021 U.S.A.

Submitted for publication October 23, 1989 Accepted for publication January 9, 1990

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INTRODUCTION

Gossypol has the structure shown in Fig.1. Because of the presence of both phenolic and carbonyl groups, . gossypol can react with either acids or bases. It reacts with several amines to form stable derivatives and aromatic amines to form Schiff's bases (1). The phenolic groups of this compound may contribute to the instability and the tendency of gossypol to be oxidized, a property utilized for its electrochemical detection (2).

CHO

Fig. 1

OH

Chemical structure of gossypol.

Gossypol has various pharmacologic effects. It is a systemic contraceptive for males (3) and has been used to control certain gynecologic disorders such as uterine myoma and endometriosis (4). Other reported pharmacological effects of gossypol include anti-viral (5, 6), anti-neoplastic (7,8) and anti-parasitic activities (9), and the inhibition of many enzymes (IO). The uncoupling of oxidative phosphorylation by gossypol has been proposed as a mechanism of the antifertility effect (11). Gossypol has chiral properties because of the hindered rotation about the internaphthyl bond. Racemic gossypol has been separated into (+) and (-) stereoiosmers by different laboratories (12, 13 ), and the antifertility (14, 15) and antitumor activity (16) reside mainly in (-) gossypol. Furthermore, the half-life of the (-) enantiomer was found to be much shorter than that of (+) gossypol in humans, dogs (17) and rats (18). The present investigation was done to study the interaction of gossypol stereoisomers with plasma proteins from rats to shed some light on the mechanism of phannacodynamic and pharmacokinetic differences between the (+) and (-) gossypol.

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MATERIALS AND METHODS

Male Sprague-Dawley rats weighing 200-350 Animals: Water and Purina rat chow were allowed ad libitum.

g were used.

Chemicals and solutions: Racemic gossypol acetic acid was purchased from Sigma Chemical Co., St. Louis, MO. Gossypol enantiomers and gossypol dimethyl ether (internal standard) were gifts from the Institute of Materia Medica, Chinese Academy of Medical Sciences, Beijing, China. 2,4-Dinitrofluorobenzene (DNFB), aspirin and Triton X-100 were also purchased from Sigma Chemical Co. Dimethyl sulfoxide was from Aldrich Chemical Co., Inc. (+)Gossypol, (-) gossypol or gossypol dimethyl ether was dissolved in ethanol to a concentration of 100 ug/ml and kept at -10 C for the stability study. The gossypol enantiomers were dissolved in dimethyl sulfoxide to achieve a high concentration for the hemolysis experiment immediately before use. For injection into rats, the gossypol material was dissolved in saline to a concentration of 1 mg/ml by adding dropwise 10 N NaOH until it was in solution and then the pH value was rapidly brought back to 7.4 by dropwise addition of 5 N HCl. Normal buffer (148 mM NaCl, 5 mM KCl, 17 mM Tris base, pH 7.4) and Drabkin's solution (200 mg/l of potassium ferricyanide, 50 mg/ml of potassium cyanide, 140 mg/ml of potassium dihydrogen phosphate, 0.1% Triton X-100, pH 7.4) were prepared according to a modified method (19). Preoaration of plasma: Rat blood was collected from the ophthalmic venous plexus using methoxyflurane anesthesia. DNFBtreated plasma was obtained by adding varying amounts of DNFB to plasma and incubating at 37 C overnight in the dark in a shaking water bath (20). Aspirin-treated plasma was prepared by adding aspirin to plasma, stirring at room temperature for 2 hours, and then bringing the pH to 7.4 by adding 1N NaOH. Plasma ultrafiltrate was obtained by putting the fresh plasma--into a CENTRIFREE tube (Amicon Co.,Danvers, MA 01923) and centrifuging at 1000 g at 4 C for 30 minutes. Recovery of qossvpol enantiomers from rat nlasma and the procedure of sossvool assay: (+) or (-) gossypol was added to different plasma samples or normal buffer to a concentration of 5 ug/ml. The samples were incubated at 37 C for varying periods, and the gossypol concentration was assayed by an HPLC method which has been established in this laboratory (2). Briefly, gossypol in plasma was extracted into acetonitrile and analyzed using a reversed-phase column and a coulometric detector in the redox mode. To separate the enantiomers, (R)-(-)-2-amino-lpropanol was added to the acetonitrile layer which was then

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heated at 60 C for 100 min for derivatization. The mobile phase used to resolve the derivatised enantiomers was 0.2 M phosphate buffer(pH 3.5)-acetonitrile (38:62, v/v). At a flow rate of 1.5 ml/min, the retention times for derivatised (+) and (-) gossypol were 4.0 and 7.8 min, respectively. protocol of sossvool treatment: Three groups of 6 rats each were given either (+/-), (+I, 01: t-1 wssywl, 6 mg/kg/d for 9 days by subcutaneous injection. Blood was taken for concentrations of drug in plasma 24 hours after the last injection. Glutathione was added to the plasma and then the gossypol levels were measured as described (2). preparation of rat ervthrocvtes and hemolvsis assavs: This was done bv the method of Chenq et al. (21). Briefly, after beino separated from whole blood; the erythrocytes were-washed free of plasma and buffy coat by centrifugation in normal buffer and resuspended in the buffer. After incubation with gossypol and centrifugation, 50 ul of the supernatants was diluted with 1.5 ml of Drabkin's solution. Statistical methods: All gossypol recovery and hemolysis experiments were done in duplicate and the mean results reported. Statistical comparisons were by student's t test, with the Bonferroni correction when multiple comparisons were made. RESULTS Stabilitv of sossvool enantiomers in oH 7.4 buffer 5 ug/ml of (+) or (-) gossypol in the normal buffer (pH 7.4) was prepared and kept at 37 C in a shaking bath from 0 to 3 hours. The (+) and (-) enantiomers degraded at a similar rate in the buffer, with half-lives of 66 minutes and 62 minutes, respectively. mverv

of qossvpol enantiomers from rat ulasma

5 ug/ml of (+) or (-) gossypol in rat plasma was prepared and incubated at 37 C for different times. The concentration of (-) gossypol decreased at a rate similar to that in buffer (half -life of 55 min) while (+) gossypol concentration decreased much slower with about 90% recovery after 3 hours of incubation (Fig. 2). The difference in concentration between the two isomers was statistically significant at each time point after 0.

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10.000 A

A-A-A

A

Y % 0

I.000 -

'1 lcl

0.100

1

0.000

2 .Q#O

1.000

3.000

INCUBATION TIME (hrs) Fig. 2

Concentrations of (+I gassypol (A---A) and ) in rat plasma after different t-i gossypol r_ incubation time.

Stability of I-1 crossvpol ultrafiltrate.

in diluted

Plasma

and olasma

When a small amount of plasma (5%) or ultrafiltrate of plasma (5%) was added to buffer, the (-1 gossypol disappeared at Increasing the amount a slower rate than from the buffer alone. of plasma in the plasma/buffer mixture accelerated the rate of disappearance of the gossypol,suggesting the (-) gossypol irreversibly bound to the plasma protein during the incubation (data not shown). Recoverv

of

I-) aassvpol

in DNFB

or

aspirin

treated

Dlasma

It is believed that DNFB arylates amino groups of protein (22) and aspirin acetylates the same residues. (-]Gossypol was added to DNFB-treated and to aspirin-treated plasma to learn if these amino groups are involved in the disappearance of (-) gossypol from plasma. Both DNFB and aspirin pretreatment slowed the disappearance of (-) gossypol in plasma (Fig. 3). The halflives of (-) gossypol in DNFB and in aspirin-treated samples were 212 minutes and 161 minutes,respectively, compared to 60 minutes for the untreated plasma samples (p < 0.05 between the values at the various time points.)

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0.100

1

I

0.000

1 .ooo

2.000

3.000

INCUBATION TIME (hrs) Fig. 3. C-j Gossypol concentrations in DNFB (O_ 0, aspirin (A A) treated plasma and normal plasma (@--_a 1 after incubation.

concentration

of qossvnol

or

and ratio of isomers in rats qiven the

druq Table I lists total concentration of gossypol and the ratio of (+) to (-) isomer in the three groups of rats. Most of the gossypol which can be detected is (+) enantiomer. The concentration in (+) gossypol-treated group is about 2 times that in the racemate-treated group: no gossypol can be detected in (-) gossypol-treated group after 9 days of treatment.

Table

I. Gossypol concentrations and ratio of isomers in plasma from rats given 6mg/kg/d racemic gossypol or the pure enantiomers for 10 days ______-__--~--~~-_-------~~--~~-_______------~----~----~_---_---Total gossypol cone Ratio of (+)/(-) Treatment (ng/ml)* (+/-)

gossYPo1 (n=6)

224.7+/-142.8

0.92/0.08

(f)

gossYPo1

518.6+/-152.9

l.OO/O.OO

(n=6) (-) gossYPo1

ND

ND

(n=6) ____-------~--------~~---~--~~~-~~~~~~~~~---------__________ * Mean +/- SD: ND: None detected. The two isomers were in a 1:l ratio in the racemic 392

gossypol.

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CONTRACEPTION

jiemolvtic effects of aossvool enantiomers in vitro The hemolytic effect of gossypol isomers on rat erythrocytes was studied to evaluate the biologic consequences of these differences between the two enantiomers. (+) Gossypol at 100-200 uM concentration caused more hemolysis than (-) gossypol when they were incubated in whole blood for one hour (Fig. 4)( p < 0.01). When the enantiomers were incubated with washed erythrocytes, no difference in hemolysis was found between the two gossypol isomers (Fig. 5). When the plasma was incubated with (+) or (-) gossypol for one hour, and then with red blood cells added and incubated at 37 C for one hour, the hemolytic effect of (-) gossypol at higher concentrations was less that of (+) gossypol (Fig. 6) (p = 0.05 for paired t test over concentration range of 100-400 uM).

100

0 0

Fig.

4

CO-

50

100

150

GOSSYPOL

(uM)

Hemolytic effects of (+) gossypol CA----A) O) on rat red blood cells when incubated

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200

250

and (-1 gossypol with whole blood.

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80

-

250

200

100 GOSSYPOL Fig. 5 Gossypol enantiomers-induced incubated with rat red blood cells plasma proteins. A = (+) gossypol,

(uM) hemolysis when in the absence of c)= (-1 gossypol.

80

60

40

20

0 0

100

200

300

400

GOSSYf’OL (uM) Fig. 6 Gossypol enantiomers-induced incubation of the drugs with plasma A= (+) gossyPol,O= (-1 g0ssyp01.

384

hemolysis after for one hour at 37 C.

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DISCUSSION The enantiomers of gossypol have been found to have different pharmacokinetics and different pharmacologic effects in several species including man (2, 14, 15,). Some in vitro

studies show different effects of the different enantiomers such as the anticancer activity of gossypol (16), ATP production (23) and the inhibition of lactate dehydrogenase isoenzyme LDH-C4(24) whichare effects of the (-) and not the (+) enantiomer. Other investigations have found similar in vitro effects for the two isomers such as the inhibition of the release of testosterone from cultured Leydig cells (25), and the spermicidal activities (26). Our data suggest that rat plasma protein binding of gossypol enantiomers is stereoselective for the (-) gossypol. A consequence of this is that (+) gossypol induced more hemolysis than (-) gossypol in the presence of plasma but that both enantiomers caused equal hemolysis of washed red blood cells suspended in a protein-free solution. This indicates that presence of plasma protein can contribute to some of the different effects of gossypol enantiomers. Conkerton and Frampton reported that gossypol reacted with free amino groups of a number of proteins decreasing the reaction of DNBF with proteins (27). Chemically, the Schiff bases formed by the reaction of gossypol with protein is slowly reversible, but this conjugated gossypol is not extracted by organic solvents in our assay. Our data suggest that (-) gossypol reacts with amino groups to form Schiff bases in rat plasma much more rapidly than (+) gossypol because only (-) gossypol disappeared quickly in the plasma, and DNFB and aspirin slowed this disappearance. This stereoselective disappearance of (-) gossypol in plasma complicates the interpretation of measurements of gossypol concentration. Interestingly, Xu, Wang and Qian reported (28) that large doses of aspirin (240-300mg/kg/d by mouth) antagonized the antifertility activity of racemic gossypol in rats. While these authors postulated that the mechanism of aspirin's effect was due to inhibition of prostaglandin synthesis, another possibility is that the aspirin acetylated the amino groups on a putative gossypol receptor thereby preventing the interaction of (-) gossypol with its receptor. Acknowledqments The authors are grateful to the staff of the Research Animal Resource Center at Cornell University Medical College and Dr. M.B. Jennings for her administration of gossypol to rats. This work was supported in part by a grant from the Rockefeller Foundation and D.F. Wu, on his leave, is a fellowship awardee from the Rockefeller Foundation.

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Den Boer PJ, Grootegoed JA. Mechanism of action of (-) gossypol on ATP production in isolated hamster spermatids. J Reprod Fert 1988; 83: 693-700

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Grootegoed JA. Differential effects of (+) and Den Boer PJ, (-) gossypol enantiomers on LDH-C4 activity of hamster spermatogenic epithelium. J Reprod Fert 1988; 83:701-g

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