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Effects of omeprazole on ethanol metabolism: an in vitro and in vivo rat and human study
EFFECTS OF OMEPRAZOLE ON ETHANOL METABOLISM: AN IN VITRO AND IN VZVO RAT AND HUMAN STUDY G. POZZATO, *G. BENEDETTI,
F. FRANZIN, M. MORETTI, *R. SABLICH, *M. MARIN, L. CAMPANACCI
“T. LACHIN, l-M. STEBEL
and
Institute of Putologia Medica, University School of Medicine, Trieste, Italy; “GastroenteroloKy Department, Pordenone General Hospital, Pordenone, Italy crnd ?Depurtment of Biochemistry, Uni\*ersity School of Medicine, Trieste, Italy
SUMMARY Since some H2-receptor antagonists, like cimetidine or ranitidine, affect ethanol metabolism by interference with gastric and/or hepatic alcohol dehydrogenase (ADH) it was investigated whether omeprazole has a similar effect and its effects were compared with those of cimetidine, an inhibitor of gastric ADH. The firstpass metabolism (FPM), quantified by measuring the difference between areas under the curve (AUC) of ethanol blood concentrations after oral intake or intravenous administration of the same amount (0.3 g kg-’ b.w.) of ethanol (EtOH), was studied before and after 1 week of omeprazole (20 mg daily) or cimetidine (800 mg daily) administration in 10 normal male volunteers. ADH activity was determined in gastric mucosal samples, collected during endoscopy, before and after 1 month of omeprazole treatment. The effect of the drugs on gastric and hepatic ADHs was studied in vitro in both rat and man. No significant effect of omeprazole was found on AUCs of the blood EtOH concentrations. The ADH activity in antral mucosa before and after omeprazole therapy did not show significant differences. In vitro, omeprazole reduced the activity of the low Km gastric ADH with a Ki of 5.6 mM in rat and the hepatic ADH activity with a Ki of 2.4 mM in man, whereas the drug did not show any effect on hepatic ADH in rat and gastric ADH in man. On the contrary, cimetidine increased the AUCs of EtOH blood concentrations after both gastric and intravenous route and, in the in vitro gastric and hepatic ADH in both man and rat. These results assay, inhibited indicate that omeprazole does not affect EtOH metabolism in man and seems to be safer than cimetidine in subjects unable to reduce ethanol intake during the therapy for peptic ulcer or other hypersecretory conditions. KEYWORDS: alcohol dehydrogenase, ethanol metabolism, omeprazole.
Correspondence to: Gabriele 447. 34149 Triestc. Italy. 1043-66
Po7zato.
I x/94/0 10047- I3/$08.00/0
lstituto di Patologia
Medica,
Ospedalc
01994
di Cattinara,
Strada di Fiume
The Italian Pharmacological
Society
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INTRODUCTION Recent studies in men [ 1, 21 and in rats [3] indicate that a significant fraction of ethanol (EtOH) is oxidized in the stomach by an alcohol dehydrogenase (ADH) placed in gastric mucosa [4]. This fraction can be calculated from the difference between areas under the curve (AUC) of blood EtOH concentrations when the same amount of alcohol is given intravenously (i.v.) or orally. This ‘first-pass metabolism’ (FPM) of ethanol seems to present gender difference [5] and can be reduced by pharmacological interferences. Common drugs like aspirin [6] or cimetidine [7] can reduce the FPM by inhibition of gastric ADH activity. In addition to cimetidine other H2-receptor antagonists had been studied with minor effect on ADH activity and FPM [S, 91. Until now several studies have been reported on the effects of omeprazole [lo, 1 l] on ethanol metabolism. This new drug presents a molecular structure very different from H2-receptor antagonists but an inhibitory action cannot in principle be ruled out. The aim of this study was: (1) to investigate, in an in vitro assay, the effects of omeprazole on gastric and hepatic ADH activity in rat and in man; (2) to assess in viva the effect of the drug on blood ethanol levels achieved after the ingestion of a moderate amount of alcohol; (3) to measure the ADH activity in gastric mucosal samples before and after 1 month of therapy with omeprazole; (4) to compare the effects of the omeprazole and the cimetidine, a well known inhibitor of ADH activity, on EtOH metabolism in man.
MATERIALS AND METHODS Chemicals NAD was purchased from Boehringer Mannheim (Italy); omeprazole powder was kindly provided by Hassle AB (Sweden) and cimetidine by BioResearch S.p.A. (Italy). All other chemicals were purchased from Carlo Erba (Italy) and were RPE-ACS grade. Rat study Adult male Wistar rats, 3 months old (Average weight 350 g), were housed under standard laboratory conditions prior to the experiments. Non-fasted rats were killed by decapitation, the liver and stomach immediately removed and washed with ice-cold sucrose 0.25 M-EDTA 1 mM pH 7.4 solution. The gastric mucosa was scraped with a lancet, weighed and homogenized with a Potter containing the above solution to obtain a 20% (w/v) homogenate. The liver was gently cut, weighed and placed in the Potter with the same solution to obtain a 5% (w/v) homogenate. Liver and gastric homogenates were centrifuged at 100 000 g at 4°C for 60 min to obtain cytosolic fractions. Protein concentration was determined by BCA method [ 121 using bovine serum albumin as standard. Five rats were used for each set of experiments. ADH activity was measured by addition of a small volume of the cytosolic fraction to the assay mixture, reaching a final protein concentration of 50-150
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pug mll’. Readings were taken using a Jasco UV/VIS spectrophotometer at 340 nm. Kinetics usually were followed for 3 min. In order to calculate kinetic parameters (Km and Ki) the data were plotted according to Lineweaver-Burk and Dixon. Kinetics were repeated thrice. The activity of the low Km gastric ADH, in presence and in absence of the drug, was measured in glycine/NaOH buffer 0.1 M, pH 9.5-NAD 2.5 mM in presence of increasing ethanol concentrations (50400 mM). The same procedure was followed for hepatic ADH except for ethanol concentration range (0.28-1.0 mM). The effect of the drugs on high Km gastric ADH was assessed in Tris/HCl buffer 0.1 M, pH 7.5-NAD 2.5 mM-4-methylpyrazole 1 mM, at ethanol concentration in the molar range (0.6-2.5 M). Omeprazole was dissolved in polyethylene glycol 400 (PEG 400) at the concentration of 17.2 mg ml-’ (50 mM) immediately before use. Preliminary experiments demonstrated that PEG 400 has no effects on gastric ADH activity whereas it is somehow metabolized by the hepatic ADH. This was not surprising, PEG being an alcohol, and prompted the dissolution of omeprazole in water when measuring the possible inhibitory effect on hepatic enzyme. Cimetidine was dissolved in sodium acetate/acetic acid buffer 0.1 mol l-‘, pH 6.0 up to a concentration of 100 mmol 1-l. The addition of different amounts of cimetidine did not change the pH of the system.
Human studies In vitro. To examine the kinetics of human gastric ADH activity a pool of 40 biopsies of gastric antral mucosa collected during diagnostic endoscopy was used. The specimens (weight range from 4.7 to 9.8 mg) were immediately frozen in liquid nitrogen and then kept frozen at -80°C until determination. Median age of patients was 40&12, none of them took any kind of drug before endoscopy. Within 2 weeks, the pooled mucosal samples underwent the same procedure previously described for rat study. Since two ADH isoenzymes have been discovered recently in human gastric mucosa, the effects of variable concentrations of omeprazole and cimetidine on ADH activities were measured at 50 mM ethanol pH 9.6 for the low Km and at 1 .O M ethanol at pH 7.5 in the presence of 1 .O mM 4-methylpyrazole for the high Km. Large human liver specimens were obtained through autopsy within 12 h of death from apparently healthy individuals who had succumbed to sudden, traumatic death. Liver samples were immediately washed and homogenized in icecold sucrose 0.25 M-EDTA 1 mM pH 7.4 with a Potter. Homogenates were centrifuged at 100 000 g at 4°C for 60 min to obtain cytosolic fractions. As multiple ADH isoenzymes [13] are present in the human liver, the activity was tested with wide ethanol concentration (from 0.5 to 200 mM). Twenty male volunteers, aged from 23 to 27 In vivo. Pharmacokinetic study. years (average weight 69.3+6.7 kg), with a daily ethanol consumption less than 30 g, participated in this study which was approved by the Ethical Committee of the Cattinara Hospital. All the subjects abstained from alcoholic drinks for at least 1 week preceding the test. None of them were smokers, drug-addicts and did not assume any kind of drug. The subjects received in a random order for 2
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consecutive days, 1 h after a standard meal of 550 KCalories (Bread, eggs, milk, jelly), 0.3 g kg-’ of body weight of ethanol p.o. or i.v. in 200 ml of 5% glucose. The solution was either drunk in 10 min or infused in 20 min. Subjects remained supine or sitting throughout the necessary time. After the start of ethanol administration, blood samples were collected, through an indwelling venous catheter, at 15min intervals for 165 min. The samples were collected in PCA 0.33 M; after centrifugation the supernatants were kept refrigerated at 4°C and assayed within 24 h in duplicate for alcoholemia by enzymatic method (Test Combination Alcoholemia, Boehringer-Mannheim Italia, Milan, Italy). After the basal study, 10 subjects received 20 mg of omeprazole as a single daily dose and 10 subjects, comparable as regards age and weight, received 400 mg of cimetidine twice daily for 7 days. After the treatment, the intragastric and i.v. administration of ethanol were repeated. On days 8 and 9 the doses of omeprazole and cimetidine were administered with the standard breakfast 1 h before ethanol administration. The AUC of ethanol blood concentrations was calculated using the trapezoidal method of integration from the beginning of the administration to the time when ethanol was no longer detectable. According to Julkunen et al. [l] the ‘first-pass metabolism’ of ethanol (expressed as mmol 1-l h-‘) was obtained from the difference between AUC after i.v. administration and AUC after oral intake. Ethanol pharmacokinetic parameters were calculated from blood EtOH concentrations after i.v. infusion. The rate of EtOH disappearance (expressed as mmoles d-’ h-‘) and the volume of EtOH distribution in the body (1 kg-‘) were respectively obtained from the slope and the y intercept of the regression line, calculated by the least squares method. The regression analysis was conducted following the distribution phase of EtOH (usually 30 min after the end of EtOH infusion). Ethanol elimination rate (expressed in mmol kg-’ body weight h-‘) was obtained by the volume of distribution and by the rate of EtOH disappearance [141.
Gastric Alcohol Dehydrogenase Activity. In a small group of patients the gastric ADH activity was evaluated before and after omeprazole therapy. Ten patients (nine men and one woman; ages from 25 to 39), referred to the Gastroenterology Department for recurrent abdominal pain and/or dyspeptic syndrome, underwent diagnostic endoscopy which revealed the presence of a duodenal ulcer. None of them assumed H2-receptor blockers or other drugs affecting alcohol metabolism or acid secretion. Besides diagnostic biopsy, additional biopsies were performed in antral mucosa Subsequently, the patients received apparently free from rough alterations. omeprazole (20 mg day-‘). One month later the patients underwent control endoscopy which revealed in all cases healing of the duodenal ulcer. At this point, second specimens of antral mucosa were collected. The samples were immediately frozen in liquid nitrogen after collection and Within 2 weeks the biopsies were thawed, kept at -80°C until determination. weighed (range 5.5-10.3 mg) and homogenized in sucrose 0.25 M-EDTA 1 mM pH 7.4. After centrifugation at 10 000 g at 4°C for 10 min, the supernatants were spun at 100 000 g at 4°C for 60 min to obtain cytosolic fraction. The ADH activity was measured in duplicate or triplicate in glycine buffer 0.1 M pH 9.5, with ethanol
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500mM and NAD 2.4 mM, and the reduction
of NAD at 22°C was registered a computerized spectrophotometer. Protein concentration was determined BCA [ 121 method using bovine serum albumine as standard.
using with
Statistics
The significance of the differences before and after treatment with the drugs was tested by Student’s t-test, which was applied to paired comparison. A P value under 0.05 was considered to indicate statistical significance.
RESULTS The data indicate the presence in the cytosolic fraction of rat gastric mucosa of two distinct ethanol-dependent NAD reductases with an apparent Km for ethanol of 220+10 and 1043f24 mM respectively. The latter value is arbitrary as neither substrate saturation nor inhibition have been found up to a concentration of 2.5 M ethanol. Omeprazole did not present any inhibitory effect on high Km ADH, whereas the Dixon plot evidenced an inhibition of low Km ADH (Fig. la). This result was further confirmed by a Lineweaver-Burk plot (Fig. lb). Both tests are consistent with a linear-mixed type of inhibition with a Ki of 5.85-0.9 mM. As shown in Fig. 2 cimetidine is a strong inhibitor of the gastric ADH activity. The Dixon plot (Fig. 2a) and the Lineweaver-Burk plot (Fig. 2b) indicated a noncompetitive inhibition of the low Km ADH with a Ki of 0.167+0.009 mmol 1-l.
1
mti
-4 pz Omeprazole
??&OH 50 mmol I 0 EtOH 150 mm011
0 (mm01
’ ’
Z
I ’1
-z
II
4 2 EtOH (mm01
1 ’)
0 Omeprazole 3.5 mm01 1~ ’ 0 Omeprazole 0.0 mm01 1 ’
Fig. 1. Effect of omeprazole on low Km rat gastric ADH according to Dixon (a) and Lineweaver-Burk (b): 20 ~1 of cytosol in 900 ~1 of mixture (Buffer glycine/NaOH 0.1 M/NAD 2.5 mM) in presence of ethanol ranging from 50 to 800 mM. Final protein concentration 82 ,ul ml-‘. Each point represents an average of 3 experimemts.
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For the high Km ADH, increasing cimetidine concentration, according to Dixon (Fig. 2c), showed a competitive inhibition with a Ki of 2.3f0.2 mmol-‘. The rat hepatic ADH, tested in the range 0.28-1.037 mM of ethanol showed an apparent Km of 0.54+0.06 mM. Evidence of inhibition for omeprazole, dissolved
60
Ki = 0.167 I
-0.5
0.0
I
0.5
Cimetidine
1.0
(
1.5
V EtOH
135 mmol
1
I
I
I
4
6
EtOH
0 Cimetidine ‘I Cimetidine
’
(mm01 1 -‘I
0.0 mmol 0.1 mmol 0.2 mmol
V Cimetidine
I
’1
2
1~’)
(mm01
0 EtOH 67 mmol I ml
mm011
1 1 1-l 1-l
I
I
Ki = 2.3
I
0’-2!I
0
Cimetidine
0 EtOH 1.5 mm011 0 EtOH
2.5 mmol 1
mmol 1~'
I
I
2
4
(mm01 1~ ’1
1
’
on low Km rat gastric ADH according to Dixon (a): the lines are Fig. 2. Effect of cimetidine parallel, therefore no Ki can be calculated. A Lineweaver-Burk (b) plot was drawn at two inhibitor levels resulting in the same Ki value for both (0.167 mM). In Fig. (c) the effect of increasing cimetidine concentration on high Km ADH is reported. Final protein concentration: 68 ,ug ml-‘.
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in water, range 0.06-0.59 mM, was not found. On the contrary, cimetidine was able to inhibit competitively hepatic ADH with an apparent Ki of 6.8f0.2 mmol 1-l. As recently reported [ 151, also in human gastric mucosa, two different ADH isoenzymes are present. The results of the present study are slightly different from previous reports: at low ethanol concentration the gastric ADH displayed an apparent Km of 2.1k1.3 mM (vs. 5.5f2.2), at high ethanol concentration the apparent Km was 1.05f0.8 M (vs. 2.09f0.09). The low Km gastric ADH showed high sensitivity to the inhibitory effect of 4-methylpyrazole with an apparent Ki of 2.83kO.8 mM, whereas the high Km ADH was refractory. Omeprazole had no significant effects on human gastric ADH, neither on the low nor on the high Km isoenzyme, while both were inhibited by cimetidine. Dixon and Lineweaver-Burk plots showed a competitive inhibition on low Km ADH (at pH 9.6) with an apparent Ki of 0.122+0.03 mrvr, and a non-competitive inhibition on the high Km (at pH 7.5 in the presence of 1 mM 4-methylpyrazole) with an apparent Ki of 1.26f0.4 mM. Human liver ADH presented an apparent Km of 3.04f1.9 mM, which is probably the result of multiple Class I ADH isoenzymes [13] present in the liver. Omeprazole presented an inhibitory effect on hepatic ADH: the Dixon and Lineweaver-Burk plots are consistent with a mixed-linear type of inhibition with an apparent Ki of 2.4f0.2 mM. Cimetidine produced a competitive inhibition of hepatic ADH with an apparent Ki of 6.0fl.O mM. Neither after i.v. nor after oral ingestion of alcohol did areas under the curve of blood ethanol concentrations show significant differences after omeprazole administration (see Table I and Fig. 3). However, cimetidine treatment greatly affected ethanol metabolism: when alcohol was given orally the AUC was significantly larger after cimetidine than before, and when ethanol was given i.v. the resulting AUC was greater as well. Therefore, the difference between the AUCs after iv. and intragastric administration was only slightly reduced. Also, the peak of ethanol concentration was significantly higher after cimetidine treatment by either route (see Table II and Fig. 4). Neither omeprazole nor cimetidine changed the volume of distribution of EtOH and the other pharmacokinetic parameters. The ADH activity in antral mucosa samples before and after omeprazole
Table I Ethanol pharmacokinetic parameters before and after omeprazole therapy (20 mg per die) in 10 male subjects AUC i.v. Before After
9.9f1.9 10.7k2.0
AUC p.0.
FPM
Peak
EtOH DR
EtOh ER
6.7k2.1 7.1+2.1
3.3f2.2 3.7f2.6
8.W2.2 8.5k2.8
0.3fO. 1 0.28f0.3
1.63f0.4 1.69f0.2
VD 0.62+0.1 0.64kO.l
Values are meansfso. AUC: area under the curve of blood EtOH concentrations (mmol 1-l h-l). FPM; gastric first-pass metabolism of EtOH (mmol 1-l h-l). Peak: peak value of ethanol blood concentration after i.v. administration (mmol 1-l). EtOH DR: rate of EtOH disappearance (mmol dl-’ h-‘). EtOH ER: rate of EtOH elimination (mmol kg-’ body weight h-‘). VD: volume of EtOH distribution in the body (1 kg-‘). All parameters considered showed no change after omeprazole administration.
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After omeprazole O--O Intravenous m Intragastric
0 Min
30 60 90 120150 180 Min
Fig. 3. Effect of omeprazole on blood alcohol levels after either oral or i.v. administration of ethanol (0.3 g kg-’ body wt) to 10 normal volunteers. The areas under the curve of ethanol
concentrations did not reveal any significant difference after either oral or i.v. administration.
Table II Ethanol pharmacokinetic parameters before and after cimetidine therapy (800 mg per die) in ten male subjects
Before After
AUC i.v.
AUCp.o.
FPM
Peak
EtOH DR
EtOh ER
VD
9.8f 1.5 15.1+2.4*
5.7k1.8 12.5+4.3*
4.111.3 2.6+3.5*
7.9f2.5 10.3&l .2*
0.29&O. I 0.3 l&O.3
1.69+0.17 1.79f0.3
0.64fO. 1 0.63&O. 1
Values are mean&xx AUC: area under the curve of blood EtOH concentrations (mmol 1-l h-‘). FPM: gastric first-pass metabolism of EtOH (mmol I-’ h-l). Peak: peak value of ethanol blood concentration after i.v. administration (mmol 1-l). EtOH DR: rate of EtOH disappearance (mmol dl-’ h-l). EtOH ER: rate of EtOH elimination (mmol kg-’ body weight h-l). VD: volume of EtOH distribution in the body (1 kg-‘). FPM, AUCs and peak value were significantly (*P
therapy showed minor changes with a slight were expressed as Ug-’ of tissue.
increase
(Table
III) when the results
DISCUSSION The in vitro gastric ADH been shown omeprazole administration.
results of this study indicate that omeprazole does not inhibit human activity, whereas only a minor effect on low Km gastric ADH has in rats. The pharmacokinetic studies did not reveal any effect of on AUC of blood EtOH concentration after either i.v. or oral On the contrary, cimetidine, a powerful and selective inhibitor of
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I
Before cimetidine
After cimetidine
i\
I \
1 J
I
II
\
O--O Intravenous W Intragastric
30 60 90 120150 180 210 Min
Min
Fig. 4. Effect of cimetidine on blood alcohol levels after either oral or i.v. administration of the same amount (0.3 g kg-’ body wt) of ethanol to 10 normal volunteers. Areas under the curve of ethanol concentrations after administration of alcohol by either route as well as peak values were affected by cimetidine.
Table III Gastric ADH activity before and after one month of omeprazole (20 mg per die) administration Putient
Se.\-
Age I/x’
FE ML CC SL ID VG FC MF UL GP Mean
M M M M M M F M M M
26 2s 33 31 33 38 26 38 31 39 32f5
After
Before
7.032 2.201 1.601 6.117 0.907 2.719 2.097 0.0 I.814 3.437 2.79f2.21
UPul: ’ 3.38 6.00 2.20 4.57 4.48 3.60 1.41 0.0 3.12 3.40 3.2lfl.71
ux
’
8.768 2.130 4.143 6.374 6.484 4.159 2.863 0.016 0.0 4.012 4.06f2.6
UPUX’ 3.39 4.22 2.80 5.71 7.05 4.27 1.32 2.11
0.0 3.28 3.41f2.05
Activity was determined with a spectrophotometric method (see text for details) and expressed as nmol NADH min g-’ of tissue (U g-‘) or nmol NADH mini’ pg-’ cytosolic protein (U j@‘). Means are medium plus/minus SD. Student’s r-test did not reveal significant differences between ADH activity before and after omeprazole therapy either expressed as U g-’ or UP& ‘.
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gastric ADH, seems to markedly interfere with EtOH metabolism. The drug increase the magnitude and the duration of the rise in blood ethanol levels after alcohol ingestion, while such an effect was only partially observed when the same dose of EtOH was given i.v., indicating that the cimetidine exerts its main action outside of the systemic circulation in agreement with in vitro studies. Though both cimetidine and omeprazole showed in vitro a moderate inhibitory action on hepatic ADH, no modifications of pharmacokinetic parameters have been found after EtOH i.v. administration. This finding can be accounted for by a rather high Ki (6.0 mM and 2.4 mM respectively) and, moreover, by the ability of these drugs to concentrate in the stomach, where specific receptors are present, and not in the liver [1.5]. Besides the inhibition of gastric ADH, other activities of cimetidine have to be supposed. Otherwise the alteration of the AUC and of the peak of ethanol concentration after i.v. alcohol administration cannot be explained. It is well known that cimetidine binds to hepatic microsomal cytochrome P450 and impairs the clearance of several drugs by inhibiting the P4.50-dependent biotransformation system [16]. Since there is a microsomal ethanol oxidizing system (MEOS) [17] involving P450 [ 181, the raised blood ethanol levels after i.v. administration could be related to inhibition of MEOS. Contrary to cimetidine and other H2_antagonists, omeprazole does not affect the FPM of ethanol. Since omeprazole is a potent antisecretory drug which suppresses gastric acid output [19], the lack of its effect on FPM indicates that the action of Hz-receptor antagonists, like cimetidine or ranitidine, on EtOH metabolism is not due to inhibition of acid output, but rather to interference with gastric ADH activity. The absence of any inhibitory action on gastric ADH is pointed out also by the determination of antral ADH activity before and after therapy. In this group of patients, statistical analysis shows a slight increase in ADH activity when activity was expressed as U g-’ of tissue. This result could be accounted for by the reduction of oedema in antral mucosa, in fact no modification has been found when the results are expressed as U mg-’ of cytosolic protein. Duodenal ulcer is usually (80%) associated with antral gastritis [20, 211 due to the greater acid secretory capacity of these patients 1221, consequently, the inhibition of the acid secretion improved gastritis together with healing duodenal ulcer. In conclusion, this study reveals that ethanol metabolism is not affected by short-term therapy with omeprazole and this drug seems to be safer than cimetidine in those patients who are not able to reduce EtOH consumption while under therapy for peptic ulcer or other hypersecretory conditions [23, 241. Possible effects of omeprazole on ethanol metabolism in long-term therapy or at higher doses remain to be assessed.
ACKNOWLEDGEMENTS The authors wish to thank Prof. criticism. M.S. was the recipient Studi Fegato”, Italy.
Charles Lieber for his constructive advice and of a Career Development Award from “Fond0
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REFERENCES I. DiPadova C, Womer TM, Julkunen RJK, Lieber CS. Effects of fasting and chronic alcohol consumption on the first-pass metabolism of ethanol. Gastroenterology 1987; 92: 1169-73. 2. Julkunen RJK, Tannenbaum L, Baraona E, Lieber CS. First pass metabolism of ethanol: an important determinant of blood levels after alcohol consumption. Alcohol 1985; 3: 43741. RJK, DiPadova C, Lieber CS. First pass metabolism of ethanol-a 3. Julkunen gastrointestinal barrier against the systemic toxicity of ethanol. Life Sci 1985; 37: 567-73. localization 4. Pestalozzi DM, Buhler R, von Wartburg JP, Hess M. Immunohistochemical of alcohol dehydrogenase in the human gastrointestinal tract. Gastroenterolo,qy 1983; 85: 1011-16. 5. Frezza M, Di Padova C, Pozzato G, Terpin M, Baraona E, Lieber CS. High blood alcohol levels in women. The role of decreased gastric alcohol dehydrogenase activity and firstpass metabolism. N Engl J Med 1990; 322: 95-9. Roine R, Gentry RT, Hernandez-Munoz R, Baraona E, Lieber CS. Aspirin increases blood alcohol concentrations after ingestion of ethanol. JAMA 1990; 264: 2406-08. Caballeria J, Baraona E, Rodamilans M, Lieber CS. Effects on gastric alcohol dehydrogenase activity and blood ethanol levels. Gastroenterology 1989; 96: 388-92. Caballeria J, Baraona E, Deulofeu R, Hemandez-Munoz R, Rod& J, Lieber CS. Effects of H2-receptor antagonists on gastric alcohol dehydrogenase activity. Digest Dis Sci 1991. B. In vivo 9. Seitz HK, Veith S, Czygan P, Bosche J, Simon B, Gugler R, Kommerell interactions between H2-receptor antagonists and ethanol metabolism in man and in rats. Hepatology 1984; 4: 12314. R, Baraona E, Lieber CS. Effects of 10. Roine R, Di Padova C, Frezza M, Hemandez-Munoz omeprazole, cimetidine and ranitidine on blood ethanol concentration. Gastroenterology 1990; 98: 114 (A). 11. Guram R, Adam E, Altan CP. Effects of omeprazole on ethanol metabolism. Alcoholism: Clin Exper Res 1991; 15: 1084-5. of protein using bicinchoninic acid. 12. Smith PK, Krohn RI, Mallia AK et al. Measurement Anal Biochem 1985; 150: 76-85. P, Horjales E et al. Comparison of three classes of human liver 13. Eklund H, Muller-Willie alcohol dehydrogenase. Eur J Biochem 1990; 193: 303-10. 14. Vestal RE, McGuire EA, Tobin JD et al. Aging and ethanol metabolism. Clin Pharmacol Ther 1976; 21: 343-54. R, Caballeria J, Baraona E, Uppal R, Greenstein R, Lieber CS. 15. Hemandez-Munoz Human gastric alcohol dehydrogenase: its inhibition by H2-receptor antagonists, and its effects on the bioavailability of ethanol. Alcoholism: Clin Exper Res 1990; 14: 946-50. basis for H2-antagonists drug interaction: 16. Powell JR, Donn KH. The pharmacokinetic concepts and implications. J Clin Gastroenterol 1983; 5 (suppl. 1): 95-l 11. ethanol oxidizing system: in vitro 17. Lieber CS, DeCarli LM. Hepatic microsomal characteristics and adaptive properties in vivo. J Biol Chem 1970; 245: 2305-12. of the microsomal ethanol oxidizing system 18. Ohnishi K, Lieber CS. Reconstitution (MEOS): qualitative and quantitative, changes of cytochrome P450 after chronic ethanol consumption. J Biol Chem 1977; 252: 7 124-3 1. of gastric acid secretion by E, Junggren U et a/. Inhibition 19. Larsson H, Carlsson omeprazole in the dog and rat. Gastroenterology 1983; 85: 900-7. 20. Cheli R, Giacosa A. Duodenal ulcer and chronic gastritis. Endoscopy 1986; 18: 125-31. 21. Hui W-M, Lam S-K, Ho J et al. Chronic antral gastritis in duodenal ulcer. Natural history and treatment with prostaglandin El. Gastroenterology 1986; 91: 1095-2003. 22. Blair AJ, Feldman M, Bamett C, Walsh JH, Richardson T. Detailed comparison of basal and food-stimulated gastric acid secretion rates and serum gastrin concentration in duodenal ulcer patients and normal subjects. J Clin Invest 1987; 79: 582-7.
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23. Klinkeberg-Knol EC, Jansen JMBJ, Festen HPM, Meuwissem SGM, Lamers CBHW. Doubleblind multicenter comparison of omeprazole and ranitidine in the treatment of reflux oesophagitis. Lancet 1987; 1: 349-5 1. 24. Sandmark S, Carlsson R, Fausa 0, Lundell L. Omeprazole or ranitidine in the treatment of reflux esophagitis. Results of the double-blind, randomized Scandinavian multicenter study. Stand J Gastroenterol 1988; 23: 625-32.
F. FRANZIN, M. MORETTI, *R. SABLICH, *M. MARIN, L. CAMPANACCI
“T. LACHIN, l-M. STEBEL
and
Institute of Putologia Medica, University School of Medicine, Trieste, Italy; “GastroenteroloKy Department, Pordenone General Hospital, Pordenone, Italy crnd ?Depurtment of Biochemistry, Uni\*ersity School of Medicine, Trieste, Italy
SUMMARY Since some H2-receptor antagonists, like cimetidine or ranitidine, affect ethanol metabolism by interference with gastric and/or hepatic alcohol dehydrogenase (ADH) it was investigated whether omeprazole has a similar effect and its effects were compared with those of cimetidine, an inhibitor of gastric ADH. The firstpass metabolism (FPM), quantified by measuring the difference between areas under the curve (AUC) of ethanol blood concentrations after oral intake or intravenous administration of the same amount (0.3 g kg-’ b.w.) of ethanol (EtOH), was studied before and after 1 week of omeprazole (20 mg daily) or cimetidine (800 mg daily) administration in 10 normal male volunteers. ADH activity was determined in gastric mucosal samples, collected during endoscopy, before and after 1 month of omeprazole treatment. The effect of the drugs on gastric and hepatic ADHs was studied in vitro in both rat and man. No significant effect of omeprazole was found on AUCs of the blood EtOH concentrations. The ADH activity in antral mucosa before and after omeprazole therapy did not show significant differences. In vitro, omeprazole reduced the activity of the low Km gastric ADH with a Ki of 5.6 mM in rat and the hepatic ADH activity with a Ki of 2.4 mM in man, whereas the drug did not show any effect on hepatic ADH in rat and gastric ADH in man. On the contrary, cimetidine increased the AUCs of EtOH blood concentrations after both gastric and intravenous route and, in the in vitro gastric and hepatic ADH in both man and rat. These results assay, inhibited indicate that omeprazole does not affect EtOH metabolism in man and seems to be safer than cimetidine in subjects unable to reduce ethanol intake during the therapy for peptic ulcer or other hypersecretory conditions. KEYWORDS: alcohol dehydrogenase, ethanol metabolism, omeprazole.
Correspondence to: Gabriele 447. 34149 Triestc. Italy. 1043-66
Po7zato.
I x/94/0 10047- I3/$08.00/0
lstituto di Patologia
Medica,
Ospedalc
01994
di Cattinara,
Strada di Fiume
The Italian Pharmacological
Society
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INTRODUCTION Recent studies in men [ 1, 21 and in rats [3] indicate that a significant fraction of ethanol (EtOH) is oxidized in the stomach by an alcohol dehydrogenase (ADH) placed in gastric mucosa [4]. This fraction can be calculated from the difference between areas under the curve (AUC) of blood EtOH concentrations when the same amount of alcohol is given intravenously (i.v.) or orally. This ‘first-pass metabolism’ (FPM) of ethanol seems to present gender difference [5] and can be reduced by pharmacological interferences. Common drugs like aspirin [6] or cimetidine [7] can reduce the FPM by inhibition of gastric ADH activity. In addition to cimetidine other H2-receptor antagonists had been studied with minor effect on ADH activity and FPM [S, 91. Until now several studies have been reported on the effects of omeprazole [lo, 1 l] on ethanol metabolism. This new drug presents a molecular structure very different from H2-receptor antagonists but an inhibitory action cannot in principle be ruled out. The aim of this study was: (1) to investigate, in an in vitro assay, the effects of omeprazole on gastric and hepatic ADH activity in rat and in man; (2) to assess in viva the effect of the drug on blood ethanol levels achieved after the ingestion of a moderate amount of alcohol; (3) to measure the ADH activity in gastric mucosal samples before and after 1 month of therapy with omeprazole; (4) to compare the effects of the omeprazole and the cimetidine, a well known inhibitor of ADH activity, on EtOH metabolism in man.
MATERIALS AND METHODS Chemicals NAD was purchased from Boehringer Mannheim (Italy); omeprazole powder was kindly provided by Hassle AB (Sweden) and cimetidine by BioResearch S.p.A. (Italy). All other chemicals were purchased from Carlo Erba (Italy) and were RPE-ACS grade. Rat study Adult male Wistar rats, 3 months old (Average weight 350 g), were housed under standard laboratory conditions prior to the experiments. Non-fasted rats were killed by decapitation, the liver and stomach immediately removed and washed with ice-cold sucrose 0.25 M-EDTA 1 mM pH 7.4 solution. The gastric mucosa was scraped with a lancet, weighed and homogenized with a Potter containing the above solution to obtain a 20% (w/v) homogenate. The liver was gently cut, weighed and placed in the Potter with the same solution to obtain a 5% (w/v) homogenate. Liver and gastric homogenates were centrifuged at 100 000 g at 4°C for 60 min to obtain cytosolic fractions. Protein concentration was determined by BCA method [ 121 using bovine serum albumin as standard. Five rats were used for each set of experiments. ADH activity was measured by addition of a small volume of the cytosolic fraction to the assay mixture, reaching a final protein concentration of 50-150
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pug mll’. Readings were taken using a Jasco UV/VIS spectrophotometer at 340 nm. Kinetics usually were followed for 3 min. In order to calculate kinetic parameters (Km and Ki) the data were plotted according to Lineweaver-Burk and Dixon. Kinetics were repeated thrice. The activity of the low Km gastric ADH, in presence and in absence of the drug, was measured in glycine/NaOH buffer 0.1 M, pH 9.5-NAD 2.5 mM in presence of increasing ethanol concentrations (50400 mM). The same procedure was followed for hepatic ADH except for ethanol concentration range (0.28-1.0 mM). The effect of the drugs on high Km gastric ADH was assessed in Tris/HCl buffer 0.1 M, pH 7.5-NAD 2.5 mM-4-methylpyrazole 1 mM, at ethanol concentration in the molar range (0.6-2.5 M). Omeprazole was dissolved in polyethylene glycol 400 (PEG 400) at the concentration of 17.2 mg ml-’ (50 mM) immediately before use. Preliminary experiments demonstrated that PEG 400 has no effects on gastric ADH activity whereas it is somehow metabolized by the hepatic ADH. This was not surprising, PEG being an alcohol, and prompted the dissolution of omeprazole in water when measuring the possible inhibitory effect on hepatic enzyme. Cimetidine was dissolved in sodium acetate/acetic acid buffer 0.1 mol l-‘, pH 6.0 up to a concentration of 100 mmol 1-l. The addition of different amounts of cimetidine did not change the pH of the system.
Human studies In vitro. To examine the kinetics of human gastric ADH activity a pool of 40 biopsies of gastric antral mucosa collected during diagnostic endoscopy was used. The specimens (weight range from 4.7 to 9.8 mg) were immediately frozen in liquid nitrogen and then kept frozen at -80°C until determination. Median age of patients was 40&12, none of them took any kind of drug before endoscopy. Within 2 weeks, the pooled mucosal samples underwent the same procedure previously described for rat study. Since two ADH isoenzymes have been discovered recently in human gastric mucosa, the effects of variable concentrations of omeprazole and cimetidine on ADH activities were measured at 50 mM ethanol pH 9.6 for the low Km and at 1 .O M ethanol at pH 7.5 in the presence of 1 .O mM 4-methylpyrazole for the high Km. Large human liver specimens were obtained through autopsy within 12 h of death from apparently healthy individuals who had succumbed to sudden, traumatic death. Liver samples were immediately washed and homogenized in icecold sucrose 0.25 M-EDTA 1 mM pH 7.4 with a Potter. Homogenates were centrifuged at 100 000 g at 4°C for 60 min to obtain cytosolic fractions. As multiple ADH isoenzymes [13] are present in the human liver, the activity was tested with wide ethanol concentration (from 0.5 to 200 mM). Twenty male volunteers, aged from 23 to 27 In vivo. Pharmacokinetic study. years (average weight 69.3+6.7 kg), with a daily ethanol consumption less than 30 g, participated in this study which was approved by the Ethical Committee of the Cattinara Hospital. All the subjects abstained from alcoholic drinks for at least 1 week preceding the test. None of them were smokers, drug-addicts and did not assume any kind of drug. The subjects received in a random order for 2
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consecutive days, 1 h after a standard meal of 550 KCalories (Bread, eggs, milk, jelly), 0.3 g kg-’ of body weight of ethanol p.o. or i.v. in 200 ml of 5% glucose. The solution was either drunk in 10 min or infused in 20 min. Subjects remained supine or sitting throughout the necessary time. After the start of ethanol administration, blood samples were collected, through an indwelling venous catheter, at 15min intervals for 165 min. The samples were collected in PCA 0.33 M; after centrifugation the supernatants were kept refrigerated at 4°C and assayed within 24 h in duplicate for alcoholemia by enzymatic method (Test Combination Alcoholemia, Boehringer-Mannheim Italia, Milan, Italy). After the basal study, 10 subjects received 20 mg of omeprazole as a single daily dose and 10 subjects, comparable as regards age and weight, received 400 mg of cimetidine twice daily for 7 days. After the treatment, the intragastric and i.v. administration of ethanol were repeated. On days 8 and 9 the doses of omeprazole and cimetidine were administered with the standard breakfast 1 h before ethanol administration. The AUC of ethanol blood concentrations was calculated using the trapezoidal method of integration from the beginning of the administration to the time when ethanol was no longer detectable. According to Julkunen et al. [l] the ‘first-pass metabolism’ of ethanol (expressed as mmol 1-l h-‘) was obtained from the difference between AUC after i.v. administration and AUC after oral intake. Ethanol pharmacokinetic parameters were calculated from blood EtOH concentrations after i.v. infusion. The rate of EtOH disappearance (expressed as mmoles d-’ h-‘) and the volume of EtOH distribution in the body (1 kg-‘) were respectively obtained from the slope and the y intercept of the regression line, calculated by the least squares method. The regression analysis was conducted following the distribution phase of EtOH (usually 30 min after the end of EtOH infusion). Ethanol elimination rate (expressed in mmol kg-’ body weight h-‘) was obtained by the volume of distribution and by the rate of EtOH disappearance [141.
Gastric Alcohol Dehydrogenase Activity. In a small group of patients the gastric ADH activity was evaluated before and after omeprazole therapy. Ten patients (nine men and one woman; ages from 25 to 39), referred to the Gastroenterology Department for recurrent abdominal pain and/or dyspeptic syndrome, underwent diagnostic endoscopy which revealed the presence of a duodenal ulcer. None of them assumed H2-receptor blockers or other drugs affecting alcohol metabolism or acid secretion. Besides diagnostic biopsy, additional biopsies were performed in antral mucosa Subsequently, the patients received apparently free from rough alterations. omeprazole (20 mg day-‘). One month later the patients underwent control endoscopy which revealed in all cases healing of the duodenal ulcer. At this point, second specimens of antral mucosa were collected. The samples were immediately frozen in liquid nitrogen after collection and Within 2 weeks the biopsies were thawed, kept at -80°C until determination. weighed (range 5.5-10.3 mg) and homogenized in sucrose 0.25 M-EDTA 1 mM pH 7.4. After centrifugation at 10 000 g at 4°C for 10 min, the supernatants were spun at 100 000 g at 4°C for 60 min to obtain cytosolic fraction. The ADH activity was measured in duplicate or triplicate in glycine buffer 0.1 M pH 9.5, with ethanol
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500mM and NAD 2.4 mM, and the reduction
of NAD at 22°C was registered a computerized spectrophotometer. Protein concentration was determined BCA [ 121 method using bovine serum albumine as standard.
using with
Statistics
The significance of the differences before and after treatment with the drugs was tested by Student’s t-test, which was applied to paired comparison. A P value under 0.05 was considered to indicate statistical significance.
RESULTS The data indicate the presence in the cytosolic fraction of rat gastric mucosa of two distinct ethanol-dependent NAD reductases with an apparent Km for ethanol of 220+10 and 1043f24 mM respectively. The latter value is arbitrary as neither substrate saturation nor inhibition have been found up to a concentration of 2.5 M ethanol. Omeprazole did not present any inhibitory effect on high Km ADH, whereas the Dixon plot evidenced an inhibition of low Km ADH (Fig. la). This result was further confirmed by a Lineweaver-Burk plot (Fig. lb). Both tests are consistent with a linear-mixed type of inhibition with a Ki of 5.85-0.9 mM. As shown in Fig. 2 cimetidine is a strong inhibitor of the gastric ADH activity. The Dixon plot (Fig. 2a) and the Lineweaver-Burk plot (Fig. 2b) indicated a noncompetitive inhibition of the low Km ADH with a Ki of 0.167+0.009 mmol 1-l.
1
mti
-4 pz Omeprazole
??&OH 50 mmol I 0 EtOH 150 mm011
0 (mm01
’ ’
Z
I ’1
-z
II
4 2 EtOH (mm01
1 ’)
0 Omeprazole 3.5 mm01 1~ ’ 0 Omeprazole 0.0 mm01 1 ’
Fig. 1. Effect of omeprazole on low Km rat gastric ADH according to Dixon (a) and Lineweaver-Burk (b): 20 ~1 of cytosol in 900 ~1 of mixture (Buffer glycine/NaOH 0.1 M/NAD 2.5 mM) in presence of ethanol ranging from 50 to 800 mM. Final protein concentration 82 ,ul ml-‘. Each point represents an average of 3 experimemts.
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For the high Km ADH, increasing cimetidine concentration, according to Dixon (Fig. 2c), showed a competitive inhibition with a Ki of 2.3f0.2 mmol-‘. The rat hepatic ADH, tested in the range 0.28-1.037 mM of ethanol showed an apparent Km of 0.54+0.06 mM. Evidence of inhibition for omeprazole, dissolved
60
Ki = 0.167 I
-0.5
0.0
I
0.5
Cimetidine
1.0
(
1.5
V EtOH
135 mmol
1
I
I
I
4
6
EtOH
0 Cimetidine ‘I Cimetidine
’
(mm01 1 -‘I
0.0 mmol 0.1 mmol 0.2 mmol
V Cimetidine
I
’1
2
1~’)
(mm01
0 EtOH 67 mmol I ml
mm011
1 1 1-l 1-l
I
I
Ki = 2.3
I
0’-2!I
0
Cimetidine
0 EtOH 1.5 mm011 0 EtOH
2.5 mmol 1
mmol 1~'
I
I
2
4
(mm01 1~ ’1
1
’
on low Km rat gastric ADH according to Dixon (a): the lines are Fig. 2. Effect of cimetidine parallel, therefore no Ki can be calculated. A Lineweaver-Burk (b) plot was drawn at two inhibitor levels resulting in the same Ki value for both (0.167 mM). In Fig. (c) the effect of increasing cimetidine concentration on high Km ADH is reported. Final protein concentration: 68 ,ug ml-‘.
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in water, range 0.06-0.59 mM, was not found. On the contrary, cimetidine was able to inhibit competitively hepatic ADH with an apparent Ki of 6.8f0.2 mmol 1-l. As recently reported [ 151, also in human gastric mucosa, two different ADH isoenzymes are present. The results of the present study are slightly different from previous reports: at low ethanol concentration the gastric ADH displayed an apparent Km of 2.1k1.3 mM (vs. 5.5f2.2), at high ethanol concentration the apparent Km was 1.05f0.8 M (vs. 2.09f0.09). The low Km gastric ADH showed high sensitivity to the inhibitory effect of 4-methylpyrazole with an apparent Ki of 2.83kO.8 mM, whereas the high Km ADH was refractory. Omeprazole had no significant effects on human gastric ADH, neither on the low nor on the high Km isoenzyme, while both were inhibited by cimetidine. Dixon and Lineweaver-Burk plots showed a competitive inhibition on low Km ADH (at pH 9.6) with an apparent Ki of 0.122+0.03 mrvr, and a non-competitive inhibition on the high Km (at pH 7.5 in the presence of 1 mM 4-methylpyrazole) with an apparent Ki of 1.26f0.4 mM. Human liver ADH presented an apparent Km of 3.04f1.9 mM, which is probably the result of multiple Class I ADH isoenzymes [13] present in the liver. Omeprazole presented an inhibitory effect on hepatic ADH: the Dixon and Lineweaver-Burk plots are consistent with a mixed-linear type of inhibition with an apparent Ki of 2.4f0.2 mM. Cimetidine produced a competitive inhibition of hepatic ADH with an apparent Ki of 6.0fl.O mM. Neither after i.v. nor after oral ingestion of alcohol did areas under the curve of blood ethanol concentrations show significant differences after omeprazole administration (see Table I and Fig. 3). However, cimetidine treatment greatly affected ethanol metabolism: when alcohol was given orally the AUC was significantly larger after cimetidine than before, and when ethanol was given i.v. the resulting AUC was greater as well. Therefore, the difference between the AUCs after iv. and intragastric administration was only slightly reduced. Also, the peak of ethanol concentration was significantly higher after cimetidine treatment by either route (see Table II and Fig. 4). Neither omeprazole nor cimetidine changed the volume of distribution of EtOH and the other pharmacokinetic parameters. The ADH activity in antral mucosa samples before and after omeprazole
Table I Ethanol pharmacokinetic parameters before and after omeprazole therapy (20 mg per die) in 10 male subjects AUC i.v. Before After
9.9f1.9 10.7k2.0
AUC p.0.
FPM
Peak
EtOH DR
EtOh ER
6.7k2.1 7.1+2.1
3.3f2.2 3.7f2.6
8.W2.2 8.5k2.8
0.3fO. 1 0.28f0.3
1.63f0.4 1.69f0.2
VD 0.62+0.1 0.64kO.l
Values are meansfso. AUC: area under the curve of blood EtOH concentrations (mmol 1-l h-l). FPM; gastric first-pass metabolism of EtOH (mmol 1-l h-l). Peak: peak value of ethanol blood concentration after i.v. administration (mmol 1-l). EtOH DR: rate of EtOH disappearance (mmol dl-’ h-‘). EtOH ER: rate of EtOH elimination (mmol kg-’ body weight h-‘). VD: volume of EtOH distribution in the body (1 kg-‘). All parameters considered showed no change after omeprazole administration.
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After omeprazole O--O Intravenous m Intragastric
0 Min
30 60 90 120150 180 Min
Fig. 3. Effect of omeprazole on blood alcohol levels after either oral or i.v. administration of ethanol (0.3 g kg-’ body wt) to 10 normal volunteers. The areas under the curve of ethanol
concentrations did not reveal any significant difference after either oral or i.v. administration.
Table II Ethanol pharmacokinetic parameters before and after cimetidine therapy (800 mg per die) in ten male subjects
Before After
AUC i.v.
AUCp.o.
FPM
Peak
EtOH DR
EtOh ER
VD
9.8f 1.5 15.1+2.4*
5.7k1.8 12.5+4.3*
4.111.3 2.6+3.5*
7.9f2.5 10.3&l .2*
0.29&O. I 0.3 l&O.3
1.69+0.17 1.79f0.3
0.64fO. 1 0.63&O. 1
Values are mean&xx AUC: area under the curve of blood EtOH concentrations (mmol 1-l h-‘). FPM: gastric first-pass metabolism of EtOH (mmol I-’ h-l). Peak: peak value of ethanol blood concentration after i.v. administration (mmol 1-l). EtOH DR: rate of EtOH disappearance (mmol dl-’ h-l). EtOH ER: rate of EtOH elimination (mmol kg-’ body weight h-l). VD: volume of EtOH distribution in the body (1 kg-‘). FPM, AUCs and peak value were significantly (*P
therapy showed minor changes with a slight were expressed as Ug-’ of tissue.
increase
(Table
III) when the results
DISCUSSION The in vitro gastric ADH been shown omeprazole administration.
results of this study indicate that omeprazole does not inhibit human activity, whereas only a minor effect on low Km gastric ADH has in rats. The pharmacokinetic studies did not reveal any effect of on AUC of blood EtOH concentration after either i.v. or oral On the contrary, cimetidine, a powerful and selective inhibitor of
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I
Before cimetidine
After cimetidine
i\
I \
1 J
I
II
\
O--O Intravenous W Intragastric
30 60 90 120150 180 210 Min
Min
Fig. 4. Effect of cimetidine on blood alcohol levels after either oral or i.v. administration of the same amount (0.3 g kg-’ body wt) of ethanol to 10 normal volunteers. Areas under the curve of ethanol concentrations after administration of alcohol by either route as well as peak values were affected by cimetidine.
Table III Gastric ADH activity before and after one month of omeprazole (20 mg per die) administration Putient
Se.\-
Age I/x’
FE ML CC SL ID VG FC MF UL GP Mean
M M M M M M F M M M
26 2s 33 31 33 38 26 38 31 39 32f5
After
Before
7.032 2.201 1.601 6.117 0.907 2.719 2.097 0.0 I.814 3.437 2.79f2.21
UPul: ’ 3.38 6.00 2.20 4.57 4.48 3.60 1.41 0.0 3.12 3.40 3.2lfl.71
ux
’
8.768 2.130 4.143 6.374 6.484 4.159 2.863 0.016 0.0 4.012 4.06f2.6
UPUX’ 3.39 4.22 2.80 5.71 7.05 4.27 1.32 2.11
0.0 3.28 3.41f2.05
Activity was determined with a spectrophotometric method (see text for details) and expressed as nmol NADH min g-’ of tissue (U g-‘) or nmol NADH mini’ pg-’ cytosolic protein (U j@‘). Means are medium plus/minus SD. Student’s r-test did not reveal significant differences between ADH activity before and after omeprazole therapy either expressed as U g-’ or UP& ‘.
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gastric ADH, seems to markedly interfere with EtOH metabolism. The drug increase the magnitude and the duration of the rise in blood ethanol levels after alcohol ingestion, while such an effect was only partially observed when the same dose of EtOH was given i.v., indicating that the cimetidine exerts its main action outside of the systemic circulation in agreement with in vitro studies. Though both cimetidine and omeprazole showed in vitro a moderate inhibitory action on hepatic ADH, no modifications of pharmacokinetic parameters have been found after EtOH i.v. administration. This finding can be accounted for by a rather high Ki (6.0 mM and 2.4 mM respectively) and, moreover, by the ability of these drugs to concentrate in the stomach, where specific receptors are present, and not in the liver [1.5]. Besides the inhibition of gastric ADH, other activities of cimetidine have to be supposed. Otherwise the alteration of the AUC and of the peak of ethanol concentration after i.v. alcohol administration cannot be explained. It is well known that cimetidine binds to hepatic microsomal cytochrome P450 and impairs the clearance of several drugs by inhibiting the P4.50-dependent biotransformation system [16]. Since there is a microsomal ethanol oxidizing system (MEOS) [17] involving P450 [ 181, the raised blood ethanol levels after i.v. administration could be related to inhibition of MEOS. Contrary to cimetidine and other H2_antagonists, omeprazole does not affect the FPM of ethanol. Since omeprazole is a potent antisecretory drug which suppresses gastric acid output [19], the lack of its effect on FPM indicates that the action of Hz-receptor antagonists, like cimetidine or ranitidine, on EtOH metabolism is not due to inhibition of acid output, but rather to interference with gastric ADH activity. The absence of any inhibitory action on gastric ADH is pointed out also by the determination of antral ADH activity before and after therapy. In this group of patients, statistical analysis shows a slight increase in ADH activity when activity was expressed as U g-’ of tissue. This result could be accounted for by the reduction of oedema in antral mucosa, in fact no modification has been found when the results are expressed as U mg-’ of cytosolic protein. Duodenal ulcer is usually (80%) associated with antral gastritis [20, 211 due to the greater acid secretory capacity of these patients 1221, consequently, the inhibition of the acid secretion improved gastritis together with healing duodenal ulcer. In conclusion, this study reveals that ethanol metabolism is not affected by short-term therapy with omeprazole and this drug seems to be safer than cimetidine in those patients who are not able to reduce EtOH consumption while under therapy for peptic ulcer or other hypersecretory conditions [23, 241. Possible effects of omeprazole on ethanol metabolism in long-term therapy or at higher doses remain to be assessed.
ACKNOWLEDGEMENTS The authors wish to thank Prof. criticism. M.S. was the recipient Studi Fegato”, Italy.
Charles Lieber for his constructive advice and of a Career Development Award from “Fond0
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23. Klinkeberg-Knol EC, Jansen JMBJ, Festen HPM, Meuwissem SGM, Lamers CBHW. Doubleblind multicenter comparison of omeprazole and ranitidine in the treatment of reflux oesophagitis. Lancet 1987; 1: 349-5 1. 24. Sandmark S, Carlsson R, Fausa 0, Lundell L. Omeprazole or ranitidine in the treatment of reflux esophagitis. Results of the double-blind, randomized Scandinavian multicenter study. Stand J Gastroenterol 1988; 23: 625-32.