Influence of estrogen levels on anticholinergic activity of tricyclic antidepressives

Prog. Neuro-Psychopharmocol. & Biol. Psychiai. Printed in Great Britain. All rights reserved.

1986,

Vol. 10, pp. 49-55 Copyright

INFLUENCE OF ESTROGEN LEVELS ON ANTICHOLINERGIC TRICYCLIC ANTIDEPRESSIVES

0

0276-5646166 $0.00 + .50 1986 Pergamon Press Ltd.

ACTIVITY

OF

EKIC C. KOK, BEVERLEY J. WILSON AND BEN POTGIETER School of Pharmaceutical Sciences, Rhodes University, Grahamstown, South Africa (Final form, August 1985) Abstract Kok, Eric C., Beverley J. Wilson and Ben Potgieter: Influence of estrogen levels on anticholinergic activity of tricyclic antidepressives. Prog. Neuro-Psychopharmacol.& Bio. Psychiat. 1986, -lo(l): 49-55. 1. Anticbolinergic activity of a number of tricyclic antidepressives and nomifensine was de~nstrated and their order of affinity for the cholinergic receptors of rat jejunum was determined. 2. The influence of ethinyl estradiol and a conjugated estrogen product, PremarintR1 on the binding of several tricyclic antidepressivesto the hepatic mixed function oxidase system was investigated. 3. The influence of these steroids on the metabolism of the antidepressives was evaluated and ethinyl estradiol was shown to have a marked influence on the metabolism of the antidepressives studied, while conjugated estrogens were shown to have little effect. hepatic mixed function oxidase, nomifensine,

Introduction The tricyclic antidepressives possess peripheral (Blackwell et al., 1978: Ho et al., 1966: Pinder et al., 1977: Shein and Smith. 19781 and central (Blackwell et al.. 1978: Bve et al., 1978; Ho et al., 1966; Pinder et al.,' 1977) antimuscarinic activity: The-side effects commonly encountered in man include dryness of the mouth, drowsiness or sedation, and constipation. Occassionally, extrapyramidal symptoms, blurred vision, postural hypotension, sweating and tachycardia are noted. Other related symptoms occur more rarely. It is of some interest to determine the cause of the side effects and to attempt to establish the cause of the higher incidence of these effects in women than in men. A step toward finding reasons for these effects was taken in a series of studies on the rat. Methods Animals. Female, Wistar rats of from 6 to 7 months of age and weighing 200 -+20 g USed.

Drugs: The foilowing antidepressives and steroids were used: Anwressives: 1. amitriptyline, Frosst-MSD 2. protriptyline, Frosst-MSD 3. trimipramine, Maybaker dothiepin, Boots Z: nortriptyline, Eli Lilly 67. clomiprpmine, Ciba Geigy . imipramine, Ciba Geigy

49

were

50

E. C. Kok et al.

8. desipramine, Ciba Geigy 9. doxepin 10. nomifensine, Hoechst Steroids: 1. ethinyl estradiol, Sigma Chemical 2. conjugated estrogens, Premarin, Ayerst Apparatus. Microsomes were obtained by centrifugation in an MSE 18 High Speed centifuge; UV/visible spectra were obtained on a Beckman Acta M VI spectrophotometer, separation of incubation mixtures was obtained on a Varian A 8500 liquid chromatograph equipped with a s-topflow septumless injector and fitted with a Cl8 column (39 mm x 30 cm) a Varichrom variable wavelength UV detector, and a Varian Aerograph CDS III recorder. Experimental Procedure. Studies on the anticholinergic effects of the antidepressives involved the use of the rat jejunum suspended in Tyrodes solution at 37°C in an aerated, 10 ml organ bath. Organs were attached to a Metrohm flat bed recorder via an electronic isotonic transducer. Cumulative log dose-response curves were drawn using carbachol as agonist. Once 2 similar curves had been obtained, the organ was washed 4 times with Tyrodes solution and was allowed to rest for 10 min. Log Affinity Values. A set concentration of agonist was added to 10 ml of fresh Tyrodes solution in the organ bath and the tissue allowed to equilibrate for 5 min, whereafter a dose-response curve was again obtained with carbachol. After a IO-min rest period in fresh Tyrodes the experiment was repeated with a concentration of agonist three times solution, higher than had previously been used. After every 2 exposures to antagonist, the tissue was washed 4-6 times with Tyrodes solution, allowed to rest for IO min, and a control log doseresponse curve was again drawn to determine whether any deviation from the original control values had resulted. If deviation was noted, further controls were carried out until two similar curves had been obtained. In any subsequent determinations of affinity values (expressed as pA2 values) the immediately preceding control agonist curves were used. The value is the negative log of that molar concentration of the antagonist in whose PA pr 5 sence the concentration of the agonist must be doubled to obtain the same effect as in the absence of the antagonist. Log affinity constants were calculated and expressed as the mean of 6 determinations using the formula expressed below. Rat Hepatocyte Studies. Studies on rat hepatocytes were carried out as described by Moldeus et al. (19/8) Viability of cells was determined by the trypan blue staining technique. Incubations 'were carried out at 37°C for 10 min in suspensions containin 1x 106 cells/ml, tricyclic antidepressives (1 x 10-4 M) and conjugated estrogens (1 x lo-B M). Reaction was stopped with 10% trichloroacetic acid. Microsomal Studies. Microsomes were prepared as by Cinti et al. (1972); animals were For both incubations and spectral studies microsomal sacrificed at 09haO in all cases. preparations of protein concentration of 1 mg/ml were used. Incubations were carried out as Protein determinations were performed by Cinti et al. (1972), for 10 min at 37'C. according to the method of Lowry et al. (1951) as modified by Miller (1959). The effect of conjugated estrogens or ethinyl estradiol on the binding of imipramine was measured spectrally in the presence of concentrations of the steroids which caused maximum Binding spectra are obtained by the addition of xenobiotics to the sample binding spectra. Such spectra are refered cuvette when both cuvettes contain suspensions of the microsomes. to as difference spectra and are designated Type 1 if the peak appears near 390 nm and the The amplitude of the peak and trough are represented as the change in trough near 420 nm. from the plot of the absorbance, AA (390 - 420). The binding constant, Ks, is obtained reciprocal of the concentration of the xenobiotic,l/[Sl, versus the reciprocal of the change in absorbance. Spectra were obtained between 350 and0 nm from suspensions of microsomes in phosphate buffer, 0,l M, ph 7,4. The effect of ethinyl estradiol on the metabolism of_4tricyclic antidepressives M antidepressive and from measured in the presence of concentrations of 1 x 10 IO-4 to 1 x IO-3 M steroid.

was

1 x

Estrogen

and anticholinergic

activity

51

of antidepressants

Analysis of Incubate. Analysis of the incubation mixture involved alkalinizing with NaOH. the supernatant extracted with hexanelisoamyl alcohol It was then centrifuged, decanted, the organic phase was extracted with a dilute solution of HCl. An and, then, (99/l), aliquot of the aqueous extract was then injected into the liquid chromatograph under a solvent system of methanol/acetonitrile/phosphate buffer, (41/15/44), 091 M, pH 7,6, containing pentane sulphonate and maintaining a flow rate of 1,5 ml/min (Proelss et al., Extent of conversion of substrate to product was calculated on the basis of the 1978). relative peak size of an internal standard. Mathematical determinations

Constants Treatment. using the equation

were

calculated

and

expressed

as

the

mean

of

6

EAB _= EM is the effect of the agonist A, produced in the presence of the antagonist, B; EM The intrinsic activity, 01, ximal effect obtainable by an agonist on the system. of carbachol is taken to be 1. K is the affinity constant of the antagonist, CBI is the KA is the affinity constant of the agonist and log molar concentration of the antigonist, [Alisthe log molar concentration of the agonist (in this instance, carbachol).

rFeF;eEP(I

Results All animals were sacrificed at the same time of day in order to avoid diurnal in factors which might influence response to the agents employed.

Table Order of Affinity

1

for the Cholinergic Receptors for Various Antidepressives

Antidepressive

a

Each pA Methods.2

value

in the Rat Jejunum

PACT

Amitriptyline

8,0 + 0,l

Protriptyline

8,0 f 0,l

Trimipramine

7,9 + 0,2

Oothiepin

7,8 f 0.2

Nortriptyline

7,7 * 0,2

Clomipramine

7,6 * 0,2

Imipramine

7,6 + 0,2

Desipramine

6,8 + 0,2

Nomifensine

5,3 f 0,3

represents

variation

the mean and range of 6

determinations.

For

details

see

52

E. C. Kok et al.

The Magnitude of the Anticholinergic Effect. This is demonstrated in the study in which the DA, values for a number of the antideoressiveshas been determined (Table 1). The dibenzo/ycloheptane derivatives were found'to be more potent anticholinergics than the dibenzapine derivatives. Within the dibenzocycloheptaneseries, substitution of C-11 with a sulphur atom (dothiepin) reduced the affinity for the cholinergic receptors, while methylation of the terminal nitrogen increased the activity so that, for example, imipramine shows greater affinity than does desipramine. With regard to the propyl side chain, introduction of an unsaturation (amitriptyline)has little effect (compared with protriptyline). Nomifensine has less affinity for the receptors than does any of the tricyclic antidepressives. The Study Carried Out on _RatHepatocytes. It involved the interaction of a conjugated estrogen mixture, PremarinK, and imipramine. In this case, it was seen that the conjugated estrogens have only a small and insignificant effect on the metabolism of imipramine by the hepatocytes when added in equimolar concentrations, as measured in terms of the conversion of imipramine to its metabolites (not shown). Effect of Estrogens on Binding. When the effect of the estrogens on the binding of imipramine was investigated, it was seen that the conjugated estrogens were less inhibitor than was ethinyl estradiol (Figure 1). The KS for imipramine was increased from 0,12 x IO-g (compare with von Bahr and Orrenius, 1971; 0,22 x 10-6 M) to 0,t6x 10-6 M in the presence M in the presence of of saturating levels of conjugated estrogens and to I,33 x 10 ethinyl estradiol. The finding that conjugated estrogens have less effect is not surprising since conjugates are not normally substrates for the hepatic monooxygenase system.

Figure 1. Inhibition by conjugated estrogens and ethinyl estradiol of the Type 1 binding spectral changes6 of imipramine hydrochloride. Binding constant (K ) of imipramine (IS] 0,8 mg/ml (0) and M) was determined in the presence of 0,65 mg/ml $4, expressed as 10 1,0 mg/ml (0) of microsomal protein from rat liver (see Methods). Inhibition was measured in the presence of saturating concentrationsof conjugated estrogens (e) and of ethinyl estradiol (m).

Estrogen and anticholinergic

activity of antidepressants

53

Metabolic Effects. Ethinyl estradiol inhibited, in a dose-dependent fashion, the metabolism of all six of the antidee$essives studied (Figure 2). Most affected by the M (that is, equimolar with the antidepressive)was estrogen at a concentration of 1 x 10 doxepin hydrochloride, the metabolism of which to-its major metabolite was reduced to M ethinyl estradiol, the metabolism approximately 35%, while at a concentration of 3 x 10 of clomioramine hvdrochloride was -reduced to approximately 15%. All metabolism was eliminated in the presence of 1 x 10mJ M ethinyl estradiol.

100 100

90

90

* 30 20

20

10

10

0

0 ETHYINYL OESTRADIOL

ETHYINYL OESTRADIOL

1O-4

Figure 2. Effect of ethinyl estradiol on the metabolism of tricyclic antidepressives. Metabolism measured was of (A) imipramine hydrochloride (P), amitriptyline hydrochloride (V), and doxepin hydrochloride (0) and of (B) dothiepin hydrochloride (m), trimipramine maleate (V) and clombpramine hydrochloride (0). In each case the concentration of antidepressive was 10 M. Each point represents the mean and range of three determinations. Discussion The chemical structure of the tricyclic antidepressiveswould lead one to assume that these compounds would act as multipotent antagonists with anticholinergic action. The assumption is supported by our data, which clearly show that the tricyclic antidepressives do bind to cholinergic receptors in the rat jejunum. It can further be seen that the extent of binding is affected by changes in the structure of the tricyclic skeleton itself, the side-

E. C. Kok et al.

54

chain, and the substituents on the terminal amine. Recognition of these factors possible the design of antidepressives with fewer and less severe side-effects.

makes

In addition to factors related to the binding of these antidepressives to cholinergic receptors are the factors related to the concentration of these agents in the body. One such pharmacokinetic factor is the rate, of metabolism. and another, the pathway, Few compounds have been studied more extensively than has imipramine so that the location of attack within the molecule and other metabolic characteristics are well defined (Bickel, 1977). Tricyclic antidepressives in general give rise to type 1 spectra in the presence of hepatic microsomes and the hepatic cytochrome P-450 dependent monooxygenase is involved in most of the early metabolic steps. This same oxidising system is involved in the oxidation of the steroid hormones testosterone, and estradiol, compounds with low K progesterone, values (Kuntzman et al., an indication that these compounds are amongst the norma 'rl 1965), physiological substrates. Ethinyl estradiol is similarly metabolised (Breuer 1972) and 1974.) It is therefore to be binds to the same enzyme system (Freudenthal and Amerson, expected that there will be competition for binding to cytochrome P-450 between the antidepressives and estrogens such as ethinyl oestradiol, as has been demonstrated here. Such competition at the metabolic level has already been demonstrated in the case of a number of endogenous steroids (Tephly and Mannerino, 1968 and Pasleau et al., 1981) and in vitro (Juchau and Fouts, 1966) and in vivo (Jori et al., certain synthetic steroids, 1969: Freudenthal and Amersonrm with resoect to soecific xen?%i?j???s. Our results The-finding that ethinyl estradiol inhibits, are in accord with these earlier findings. in a dose-dependent fashion, the metabolism of a number of antidepressives may contribute to an understanding of the differences in the effect of the antidepressives in women as compared with men and of the enhanced anticholinergic effects noted when ethinyl estradiol rather was administered along with imipramine to a group of women (Prange et al., 1976), than being due to anticholinergic action of the estrogens as 3uggested by Prange et al. (1976). However, the interaction noted in women between Premarin and imipramine (Khurana, is not likely due to an interference with metabolism but may well be due to changes 1972), at the receptor level (Bengtsson and Marshall, 1983; Fludder et al., 1977; Kendall et al., estrogens may 1981 and 1982). Another explanation could be that, in vivo, the conjugated be hydrolysed to the corresponding estrogens so th~~estrogens then are available within the hepatocytes.

Conclusions It is evident from these results that many tricyclic antidepressives have anticholinergic activity and that non-conjugated estrogens can be expected to increase these effects by hence, increasing their blood levels decreasing the metabolism of the antidepressives and, and duration of action.

Acknowleagements Support

by the Medical

Research

Council

of South Africa

is acknowledged

by B J Wilson.

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BYE, C., CLUBLEY, M. AND PECK, A.W. (1978) Drowsiness, impaired performance and tricyclic antidepressant drugs. Br. J. Clin. Pharmacol. 6: 155-161. CINTI, D.L., MO#EUS, P. and SCHENKMAN. J.B. (1972) Kinetic parameters of drug-metabolizing enzymes in Ca -sedimented microsomes from rat liver. Biochem. Pharmacol. 212: 3249-3256. FLUDDER, J.M., TONGE, S.R. AND LEONARD, B.E. (1977) Modification by ethinyl oestradiol and imipramine on 5-hydroxytryptamine metabolism in discrete progesterone of the effects of areas of rat brain. Br. J. Pharmacol. 60: 309P. FREUDENTHAL, R.I. and AMERSON, E. (mjl4) Effect of synthetic estrogens and estrogenprogestin combinations on the hepatic microsomal enzyme system. Biochem. Pharmacol. -23: 2651-2656. FREEMAN, S.E., FREEMAN, W.P. and LLOYD, H.J. (1966) Action of tricyclic HO, A.K.S., Pharmacol. Biochem. antidepressant drugs on central processes involving acetylcholine. 15: 817-824. BIANCHETTI, A. and PRESTINI, P.E. (1969) Effect of contraceptive agents on drug Jon, A., metabolism. Eur. J. Pharmacol. 7: 1796-200. J.R. (7966) Effects of norethynodrel and progesterone on hepatic JUCHAU, M.R. and FOUTS, drug-metabolizing enzyme systems. Biochem. Pharmacol. 15: 891-898. KENDALL, D.A., STANCEL, G.M. and ENNA, S.J. (1981) ImiEamine: effect of ovarian hormones modification in serotonin receptor binding. Science 211: 1183-1185. KE:;ALL D A (1982) Theinfluence of sex hormones on STANCEL, G.M. and ENNA, S.J. J. Neurosci. -2: antidepressa&.-induced alterations in neurotransmitter receptor binding. 354-360. KHURANA, R.C. (1972) Estrogen-imipramine interaction. Letter to the editor, J. Amer. Med. Assn. 222: 702-703. KUNTZMAN;-- R., LAWRENCE, D. and CONNEY, A.H. (1965) Michaelis constants for the hydroxylation of steroid hormones and drugs by rat liver microsomes. Mol. Pharmacol. -1: 163-167. LOWRY, O.H., ROSEBROUGH, N.J., FARR, A.L. and RANDALL, R.J. (1951) Protein measurement with the Folin-phenol reagent. J. Biol. Chem. 193: 265-275. MILLER, G.L. (1959) Protein determinationxr large numbers of samples. Anal. Chem. -31: 964. MOLDEUS, P., HOGBERG, J. and ORRENIUS, S. (1978) Isolation and use of liver cells. In: Method in Enzymology LII, Part C, S. Fleischer and I. Packer, (eds), pp.60-71. Academic Press, New York, San Francisco and London. PASLEAU, F., KREMERS, P. and GEILEN, J.E. (1981) Competition between benzo(a)pyrene and various steroids for cytochrome P-450 dependent rat liver monooxygenases. Chem. Biol. Interact. 34: 279-286. PINDER, R.MT; BROGDEN, R.N., SPREIGHT, T.M. and AVERY, G.S. (1977) Doxepin up-to-date: a review of its pharmacological properties and therapeutic efficacy with particular reference Drugs 13: 161-218. to depression. PRANGE, A.J., G.R. and LIPTON, M.A. 1976. Hormonal Alteration Jr, WILSON, I.C., BRESE, of Imipramine Response: A Review. In: Hormones, Behaviour and Psychopathology, E.J. Sachar, (eds), pp. 41-67 Raven Press, New York. PROELSS, H.F., LOHMANN, H.J., MILES, D.G. (1978) High-performance liquid- chromatographic simultaneous determination of commonly used tricyclic antidepressants. Clin. Chem. -24: 1948-1953. SHEIN, K. and SMITH, S.E. 1978, Structure-activity relationships for the anticholinoceptor action of tricyclic antidepressants. Br. J. Pharmacol. 62: 567-571. TEPHLY, T.R. and MANNERING, G.J. (1968) Inhibition of %%g metabolism. V. Inhibition of drug metabolism by steroids. Mol. Pharmacol. 4: 10-14. VON BAHR, C. and ORRENIUS, S. (1971) SpectraT studies on the interaction of imipramine and some of its oxidized metabolites with rat liver microsomes. Xenobiotica -1: 69-78. Inquiries

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