Pharmacological effects of diethylthiocarbamic acid methyl ester, the active metabolite of disulfiram?

European Journal of Pharmacology, 166 (1989) 419-425

419

Elsewer EJP 50849

Pharmacological effects of diethylthiocarbamic acid methyl ester, the active metabolite of disulfiram? Erling N. P e t e r s e n Department of Pharmacology, A / S Dumex, Prags Boulevard 37, DK 2300 Copenhagen S, Denmark Received 24 November 1988, revised MS received 15 March 1989, accepted 18 April 1989

A recently &scovered metabohte, diethyltluocarbamic acid methyl ester (Me-DTC), has been f o u n d in the plasma of m a n and rats m much tugher concentratxons than any other described metabolite after therapeutic doses of disulfiram M e - D T C , in contrast to other dlsulflram metabohtes, is a potent mlubltor of hver aldehyde dehydrogenase ( A L D H ) m wtro. Like &sulflram, M e - D T C had a p r o n o u n c e d h y p o t h e r n u c effect in rats. This hypotherrmc effect and the augmented blood pressure response to ethanol challenge in rats developed rapidly with M e - D T C but were somewhat delayed with &sulflram. The blood pressure response outlasted the presence of M e - D T C m plasma ( < 24 h); a significant effect was found 48 h after pretreatment but not 72 h after a single dose. N o effect was observed when ethanol was gwen 15 nun before M e - D T C or disulhram. These latter two observations are consistent with a f u n c t m n of M e - D T C as a s m o d e inhibitor of A L D H . Since M e - D T C has been reported to lnlubit A L D H m xatro, even u n d e r anaerobic con&tions, M e - D T C may be the active metabolite of dlsulfiram.

Dlsulfiram; Dlethyltluocarbanuc aod methyl ester (Me-DTC); Ethanol; Hypotherrma; Blood pressure; (Rat, Toxloty)

1. Introduction

Disulfiram (Antabuse, Esperal) has gained wide acceptance as a supportive drug m the treatment of alcoholism. When treated properly, the alcoholic will experience a disulfiram-ethanol reaction when ethanol is ingested. The clinical symptoms are highly unpleasant and fear of or experience of these symptoms should help the alcohohc to abstain from regular drinking or episodes of heavy drinking. Years of research into the pharmacokinetics of disulfiram have led to the conclusion that dlsulfiram is rapidly converted into several metabolites, such as diethyldithiocarbamate (DDC) and its methyl ester Me-DDC, and substantlal amounts of carbon&sulfide (CS2) (about 30%) are formed; simultaneous diethylamme is excreted into the urine (StriSmme, 1965; Sauter et al., 1977; C o b b y

et al., 1977; Yourick and Faiman, 1987; Johansson, 1986). When disulfiram and its ident/fied metabolites were tested for their ability to inhibit low K m aldehyde dehydrogenase ( A L D H ) in an in vitro bioassay without metabolic capacity, 1.e. human erythrocytes, only &sulfiram was active in the extremely high concentration of 500 /~M (Johansson, m press). Since disulfiram disappears within seconds after incubation in h u m a n blood, and plasma concentrations have not been detected consistently in patients and animals, it is htghly unlikely that this very weak inhibitory activity plays a role in the observed effects of dlsulfiram treatment. H u m a n erythrocyte A L D H is, however, highly sensitive to inhibition by disulfiram treatment in vivo in m a n (Johansson and Stankiewicz, in press). This suggests that disulfiram only serves as a prodrug for an active metabolite. The &s-

0014-2999/89/$03 50 © 1989 Elsevaer Science Pubhshers B V (Biomedical Dixqslon)

420 C2H 5 k N

S II - C -

S

-

/

S

-

S C2H 5 II / C - N \

C2H5

C2H 5

C2H 5

disulfiram DSF )

S

x

II N

- C

- SH

/ C2H 5

diethyldlthiocarbamic

C2H 5

acid ( DDC )

S

\

II N

-

C

- SCH 3

/

diethyldithiocarbamic acid methyl ester ( Me-DDC )

C2H 5

C2H 5 \ N

-

O )I C - SCH 3

/ diethylthiocarbamic acid methyl ester ( Me-DTC )

C2H 5

F~g 1. Metabohc route from dlsulflramto Me-DTC

ulfiram inhibition of low K m liver A L D H might be explaaned by a rapid conversion of disulfiram into its active metabolite(s) (fig. 1). Because of its evident ability to inhibit fiver A L D H in vivo and to provoke the ethanol reaction, and despite the lack of a fiver A L D H inhibitory effect in vitro, Me-DDC ,s suggested to be the active metabolite of disulfiram (Yourick and Falman, 1987). However, improved analytical techniques have now revealed the presence in rats and man of much higher concentrations of a recently discovered metabolite, Me-DTC, after therapeutic doses of disulfiram. Plasma levels of this metabolite, about ten fold those of Me-DDC have been reported ,n human volunteers and rats (Johansson et al., 1989; Johansson and Stankiewicz, in press), whereas lower levels have been found m alcoholics (Johansson and Stankiewlcz, in press).

Me-DDC appears to undergo oxidative desulphurylation to Me-DTC in hepatocytes in VlVO as well as in vitro (Johansson et al., 1989). Me-DTC is a potent inhibitor of low K m liver A L D H in vitro and does not seem to need further oxidative metabohsm for its effect (Johansson et al., 1989). The study describes some of the characteristic pharmacodynamlc effects of Me-DTC in rats.

2. Materials and methods 2.1 Blood pressure experiments

Male Sprague-Dawley rats (Mol:sprd) (300-400 g) were housed in standard cages and allowed free access to rat chow (Althromm) and tap water. The rats were pretreated lntraperitoneally (1.p.) with

421 disulfiram or Me-DTC at fixed txmes before challenge with ethanol 1 g / k g (10% W / V ) Lp. The rats were anaesthetized with urethane (1.5 g / k g i.p.) 30-45 min before the challenge. The hypothermic effect of anaesthesia was counterbalanced by a thermastor-controlled heating pad adjusted to a body temperature of 37.5°C. A polyethylene cannula filled with heparinized saline (20 units/ml) was then inserted into the carotid artery after ~psilateral vagotomy and the full blood pressure wave was recorded on a Watanabe Mark III pen recorder. The heart rate was calculated from the blood pressure pulse curve with a tachometer. The blood pressure (BP) and the heart rate (HR) were continuously recorded throughout the experiment. At the time of the challenge ethanol was rejected i.p. and the cardiovascular response was monitored for a further 30 rain, at which time the ethanol BP response had stabihzed. The mean systolic and dlastohc BP levels 5 min before and at the time of injection were taken as the basal levels to which the ethanol response was compared (% of basal level). Four to eight rats were used per dose. The results obtained 30 rain after ethanol were analyzed by Student's t-test. In some experiments the reverse order of treatment was used, i.e. ethanol (1 g / k g ) was administered 15 rain before injection of Me-DTC or &sulfiram. The BP was recorded for 30 min and 120 rain after Me-DTC and dlsulfiram, respectively. 2 2. Body temperature effects Male Sprague-Dawley rats (150-200 g) were fitted with rectal probes and placed in restraimng boxes for temperature experiments. The animals were allowed to adapt to the experamental room for 1 week before the experiment. The environmental temperature was set at 20 +_ 1 ° C. The rectal temperature was recorded for 15 rain before 1.p. dosing with Me-DTC or dxsulfiram and the automatic recording continued for 240 min (EL-LAB, Copenhagen). Six rats were used per dose. The drug-induced hypothermia was compared with the initial temperatures by means of Student's t-test.

2 3. Acute toxicity m mtce Male mace (NMRI-Bom) 20-25 g were injected i.p. with Me-DTC m the doses 50, 100, 200 and 400 m g / k g , and were observed for lethality during the next 48 h. Four mace were used per dose level. 2 4. Drugs Disulfiram (Dumex, Copenhagen) was used m a rmcrocrystalhne form (disulfiram hydrophile) and Me-DTC was synthesized by T. Koch in our laboratories. Disulfiram was suspended m soybean o11, and Me-DTC was miscible with soybean oil. The injection volume was 1 m l / k g to rats and 10 m l / k g to mice.

3. Results The present experiments showed that Me-DTC, when given to rats, exerted the same effects as disulfiram with respect to hypothermia and sedation. Me-DTC produced significant hypothermla even with 3 m g / k g i.p., and with 30 m g / k g i.p. the hypothern'na was marked, down to 31°C (fig. 2). The hypothermlc effect of Me-DTC developed rapidly, with a fall in temperature of 2 ° C already 30 nun after both 10 and 30 m g / k g i.p. Hypothernua was followed by obvious signs of sedation while the rats were restrained and by a marked reduction m locomotor activity when they were released in an open field. Hypothermaa after disulfiram developed more gradually, with only a 1 ° C fall at 30 min. D1sulfiram, 30 m g / k g l.p., was needed to produce the same degree of hypothernua as that attained with Me-DTC, 3-10 m g / k g . The maximum effect of Me-DTC was reached after about 60 man (see result with 10 mg/kg), m contrast to the 2-4 h required for &sulfiram. Moreover, Me-DTC appeared to be at least three tames more potent than disulfiram in this paradigm (fig. 2). Based on the hypothermaa results, the interaction with ethanol was studied m a blood pressure (BP) model at the time when the full effects of the compounds could be expected. Dose-response curves were made at 1 h for Me-DTC and at 4 h

422

t ~ ~C

k :±~ 3 mg/kg

10 mg/kg

- - " " = 30 mg/kg

°C

100 mg/kg

40

38

36

36.

34

34.

32.

32.

I

0

I

I

60

I

I

120

I

~

40

38

30

i t e m 10 mg/kg

I

I

30

I

180

240

I

I

0

30 rng/kg

I

I

60

I

120

I

I

I

I

180

240

min. after i.p. injection

min. alter i.p. injection

Fig. 2 T h e u m e c o u r s e o f the h y p o t h e n r u c r e s p o n s e to l.p injection o f d l s u l f i r a m (A) o r M e - D T C (B) to rats, a p < 0 05, b p < 0 01, c p < 0.001 c o m p a r e d to u m e 0 d e t e r m i n e d w i t h S t u d e n t ' s t-test V a l u e s a r e m e a n s a n d s t a n d a r d d e v i a t i o n s f o r SLX rats.

for disulfiram. It was found that both compounds were active at 30 m g / k g i.p., and that an increase m the disulfiram dose to 100 m g / k g did not appear to have a greater effect. The diastolic blood pressure was more sensitive than the systolic blood pressure to the acetaldehyde accumulated because of the inhibition of A L D H by disulfiram or MeDTC. The heart rate was not influenced (data not shown). There was very little difference in the potency of disulfiram and Me-DTC with respect

to the ethanol reaction when these drugs were tested at the two different time points (table 1). When the order of ethanol challenge and administration of Me-DTC or dlsulfiram was reversed, no reaction on the blood pressure was observed during the following 2 h period. This contrasts sharply with the reaction described above and could provide information about the mode of actions of these compounds (see table 2 and discussion).

TABLE l T h e d o s e - r e s p o n s e r e l a u o n s h t p o f the e t h a n o l b l o o d p r e s s u r e r e s p o n s e r e d u c e d b y p r e t r e a t m e n t w i t h & s u l f i r a m a n d M e - D T C at selected time p o i n t s b p < 0 01 ; c p < 0.001 as c o m p a r e d to the effects o f s a h n e d e t e r r m n e d w i t h S t u d e n t ' s t-test Substance

Pretreatment t i m e (h)

mg/kg I p.

Sahne

1

-

Dlsulhram

4 4 4

Me-DTC

1 1 1

B P r e s p o n s e to e t h a n o l % of l m t t a l v a l u e Systohc

N

Dlastohc

94.05:4.0

90.65:59

11

10 30 10(3

9005:41 90.0-t- 5.9 84.1 + 2.9 "

8685:112 77.85:8 5 b 75 8 5 : 6 7 c

4 4 4

3 10 30

100.7 5:10 2 94.0-1- 3.7 8225:49 ¢

92.25:6.4 86 7 5 : 4 6 7175:8.2 c

6 6 6

423 TABLE 2 The dlastohc BP response to Me-DTC and dlsulflram m rats pretreated with ethanol 1 g/kg 15 rain before the drug (reverse order of compounds as compared to table 1). Substance

mg/kg ip

Me-DTC

30

Dlsulflram

N

6

100

4

DmstohcBP % of tmtlal value 15 nun

30 nun

98.3 :t: 13.2

114 7 + 23.3

60 nun

120 nun

96 3 _+4.8

100 3 _+8 9

The long-term effects of M e - D T C were studied m the BP model. E t h a n o l challenge was carried out at various time p o i n t s from 0.16-72 h after peroral a d m i n i s t r a t i o n of M e - D T C (50 m g / k g ) . T h e results presented in fig. 3 show that there was n o significant response to the ethanol challenge at 0.16 h (10 min), a significant effect at 0.5 h, a n d a highly significant effect from 2-24 h. Some decline in the effect was observed at 48 h, a n d at 72 h n o effect was found. Except for one time p o i n t (2 h), n o significant fall in the systohc BP was seen, [~

%

Diastolic

Systohc

10C

90

I

8(3

7(3

60

] [ J

50 0

0,160,50

2

4

I ~/ I L_

8

Pretreatment time

24

J , 48

72

(hours)

Fig 3 The time course of the ethanol blood pressure effect (% of basal level) after oral admlmstraUonof Me-DTC (50 mg/kg) to rats b p < 0.01, c p < 0.0t31 compared to controls (0) given ethanol only, Student's t-test was used, n = 11 for controls and 6 for Me-DTC-treated rats. Values are the means, the standard devaatmns (not shown) vaned between 5-15% of the mean value

which shows that the pulse pressure m fact mcreased in response to the e t h a n o l challenge. T h e acute tOXlOty of M e - D T C was studied in mice after i n t r a p e r l t o n e a l injection. The LDs0 appeared to be a b o u t 170 m g / k g .

4. Discussion This s t u d y shows that M e - D T C has the same pharmacological effects as d l s u l f i r a m in rats. MeD T C even m i m i c k e d the a p p a r e n t l y irreversible i n t u b i t i o n of the low K m A L D H e n z y m e i n d u c e d b y dlsulflram. M e - D T C has been f o u n d in s u b s t a n t i a l a m o u n t s in rats, in h u m a n volunteers, a n d in some alcohohc patients u n d e r g o i n g t r e a t m e n t with therapeutic doses of disulfiram ( J o h a n s s o n et al., 1989; J o h a n s s o n a n d Stankiewicz, in press). The high c o n c e n t r a t i o n s of M e - D T C i n p l a s m a , its p h a r m a c o d y n a m i c profile a n d its rapid c o n v e r s i o n from M e - D D C , formerly believed to be the active metabolite of disulflram, seems to provide sufficient evidence to suggest that M e - D T C ts a n imp o r t a n t active m e t a b o l i t e of disulflram. M e - D T C is formed rapidly from M e - D D C injected into rats a n d can be present m p l a s m a m a m o u n t s 10 times higher t h a n M e - D D C 3 h after a d n u n l s t r a t i o n ( J o h a n s s o n et al., 1989). M e - D T C is also the pred o m i n a n t m e t a b o l i t e after disulfiram a d m i n i s t r a tion to h u m a n volunteers, with p l a s m a levels up to 30 times those of M e - D D C ( J o h a n s s o n a n d Stankiewlcz, in press). M e - D D C appears to be o n l y active w h e n oxadation can take place t h r o u g h oxidative d e s u l p h u r y l a t i o n ( J o h a n s s o n et al., 1989). Smce M e - D T C itself is active m hver h o m o g e n a t e s u n d e r a n i t r o g e n atmosphere, further oxidative m e t a b o l i s m of M e - D T C to sulphoxides or sulphones can be excluded as the basis for the effect (Johansson et al., 1989). Evadence is a c c u m u l a t i n g from clinical studies that the f o r m a t i o n of M e - D T C from M e - D D C is a zruclal step for the m a n i f e s t a t i o n of the disulflram effect in the alcoholic p a t i e n t ( J o h a n s s o n a n d Stankiewlcz, in press). High c o n c e n t r a U o n s of MeD D C , without detectable c o n c e n t r a t i o n s of MeD T C , have b e e n f o u n d in a few alcoholics who were u n a b l e to control their alcohol intake with

424

disulfiram. This lends further support to the concept that Me-DTC is an important metabohte of dlsulfiram, but it rmght be premature to call it the active metabolite since other metabolltes maght add to the effect on both low K m enzymes and h i g h K m enzymes. It may, however, turn out to be the most important metabolite of disulfiram in man and in animals. One characteristic of dlsulfiram is the slow development of its effect, which Is then prolonged for up to 8 days after the last dose in man and rats. This has been ascribed to irreversible inhibition of the A L D H enzyme. Consequently, the return to normal enzyme activity depends on the synthesis of new enzyme. This takes days m man and animals. Oral admimstration of Me-DTC (50 m g / k g ) yields high levels of Me-DTC very quickly, as 6000 and 2500 n g / m l have been measured at 10 mln and 20 min, respectively, with concentrations declimng to below the detection hmit of 50 n g / m l at 24 h (PflgSrd Andersen, unpublished observations). The ethanol reaction resulting from MeDTC administration does not, however, follow the plasma concentration curve at all, as no slgmficant effect was found at 10 rmn, whereas an effect was found from 30 min to 24 h after the single administration. No effect was found at 72 h. This indicates that Me-DTC mimics disulfiram in this respect also (Koda et al., 1984). Me-DTC and ethanol administered in the reverse order (ethanol first) in the ethanol experiment led to a loss of the effect of Me-DTC. Contrary to expectations, Me-DTC or disulfiram did not cause hypotenslon, and two animals even experienced a hypertensive response after MeDTC. This result might be interpreted as evidence for an increased amount of the substrate, acetaldehyde, competing with Me-DTC for the active site on the A L D H enzyme, with the consequence that Me-DTC has less chance of binding irreversibly to the enzyme. This points to a competitive type of action of Me-DTC; under certain conditions Me-DTC may become covalently bound, whereby it functions as a so-called suicide inhibitor. In vitro studies with purified bovine low K m A L D H enzymes have shown that Me-DTC

requires 30-60 min to bind covalently with the enzyme, thus supporting the notion of a time dependency of tbas binding (Johansson, 1989). Irreversible covalent binding to A L D H is one of the prerequisites for a successful outcome of treatment with an alcohol-deterrent drug like disulflram. In this way it is less hkely that the patient can drink through the ethanol reaction, i.e. ingest excessive amounts of ethanol, so that acetaldehyde effectively washes away the inhibitor Moreover, irregularity in taking the prescribed dose, which is to be expected from an alcoholic patient, may not be so critical, as he can experience the ethanol reaction up to 8 days after the last dose. Both disulfiram and Me-DTC produced hypothermia in rats. The effect of Me-DTC was rapid in onset and much more potent and extensive (down to 31°C) than that of disulflram. Both drugs produced marked sedation, partly because of the hypothermac action, which may effect many bodily and central nervous system functions. This study was performed to establish the times at which dose-response curves should be made for the ethanol response of both compounds on the cardiovascular system. Owing to the different time points of the comparative curves for the ethanol-blood pressure effect (table 1), any direct calculation of the comparative potencies of dlsulfiram and Me-DTC is inadvisable. Moreover, whereas Me-DTC was admlmstered in soybean oil, from which it is rapidly released, disulfiram was adrmnistered as a suspension of the microcrystalline form in the quality called dlsulfiram hydrophile. Particle size as well as the wetting properties of the crystals are critical factors for disulfiram availability, factors which are often overlooked in most experimental studies. Disulfiram m suspension must first dissolve, then be absorbed into the blood-stream, reduced to diethyldithiocarbamic acid (DDC), methylated to Me-DDC, and finally oxidatlvely desulphurated to Me-DTC. If Me-DTC is responsible for most of the effects of dlsulfiram, the reason for the difficulty in estimating the relative potencies of the prodrug, dlsulfiram and its active metabolite, Me-DTC, is evident. The marked hypothermic effect of Me-DTC may add to any toxic effect of the compound and

425 m a k e it d i f f i c u l t f o r t h e m i c e t o survive. A c u t e toxicity studies using standard techniques showed t h e LDso in v a l u e i n m i c e t o b e 170 m g / k g , w h i c h c o r r e s p o n d s t o t h a t o f M e - D D C ( F a i m a n e t al., 1983). T h e h a l f - l i f e o f M e - D T C in r a t s a p p e a r e d t o b e r a t h e r s h o r t , as j u d g e d

from preliminary experi-

m e n t s , a n d the s a m e a p p e a r s to be the case in man,

as j u d g e d

from the plasma concentration

curve after &sulfiram admamstration (Johansson a n d Stankiewicz, in press). Thus, M e - D T C likely e x e r t s a ' h i t - a n d - r u n '

most

effect t h r o u g h irre-

v e r s i b l e i n h i b i t i o n o f t h e liver a l d e h y d e d e h y d r o genase. This m a y p e r m i t a d o s i n g schedule in clinical practice of only two or three doses of either disulfiram or M e - D T C a week. A successful outcome of dlsulfiram treatment s e e m s t o d e p e n d o n h o w well t h e a l c o h o l i c p a t i e n t can form Me-DTC Me-DTC, &sulfiram,

as

f r o m its p r o d r u g , d i s u l f i r a m .

the putatave

provides

a

active metabolite

very

valuable

tool

of for

improving the supportive disulfiram therapy for alcoholism.

Acknowledgement I thank Mrs A Wlllumsen for skalful techmcal help and Mrs R Sorensen and Mrs M Malboll for help in the preparation of the manuscript

References Cobby, J., M Mayersohn and S Selhah, 1977, Methyl diethyldxthlocarbamate, a metabohte of dlsulflram in man, Life Scl. 21, 937.

Falman, M D , L Artman and T Mazaasz, 1983, Dlethyl-dlthlocarbarmc acid-methyl ester &stnbutlon, ellnunation, and LDs0 in the rat after intraperltoneal admamstratlon, Alcohol Chn Exp. Res 7, 307 Johansson, B, 1986, Rapid and sensitive on-hne precolumn purification and lugh-performance hquld chromatograpluc assay for dlsulfiram and its metabohtes, J Chromatogr. 378, 419. Johansson, B, Cell membrane effects on distribution of disulflram and its cluef metabohtes and their inactivation of erythrocyte aldehyde dehydrogenase activity, Pharmacol Toxacol. (in press) Johansson, B, 1989, Diethyltluocarbarmc acid methyl ester A Smclde lnlubltor of hver aldehyde dehydrogenases, Pharmacol Toxacol. 64, 471 Johansson, B, H Angelo, J K Chnstensen, I W. Moller, P Ronsted and E. Arnold, Dose/effect relationslup of disulflram in human volunteers (II), Pharmacol Toxlcol (in press). Johansson, B, E N Petersen and E Arnold, 1989, Diethyltluocarbanuc acid methyl ester A potent inlubltor of aldehyde dehydrogenase found in rats treated with dlsulflram or dlethyldltluocarban'nc acid methyl ester, Biochem Pharmacol 38, 1053. Johansson, B and Z Stanklewlcz, Inlubltion of erythrocyte aldehyde dehydrogenase actlvaty and ehrmnation kinetics of dlethyldRluocarbamamc acid methyl ester and its monotluo analogue after single and repeated doses of dlsulfiram to humans, European J Chn Pharmacol (m press). Koda, L Y., S.G Madamba and F E Bloom, 1984. Hypotenslve response of ethanol in rats pretreated with dlsulflram or mtrefazole, Life Sci. 35, 1659 Sauter, A M , D Boss and J - P Von Wartburg, 1977, Reevaluation of the dlsulfiram-alcohol reaction m man, J. Stud. Alc 38, 1680 Stromme, J.H, 1965, Metabohsm of dlsulfiram and dlethyldlttuocarbamate m rats wah demonstration of an m vavo ethanol-reduced inlubition of the glucuromc acid conJugation of the tluol, Blochem Pharmacol 14, 393 Yourack, J J and M D Faiman, 1987, Diethyldltluocarbamac aod-methyl ester A metabolite of dxsulfiram and its alcohol sensmzlng properties m the disulflram-ethanol reaction, Alcohohsm 4, 463.