selective effect of liver disease on the activities of specific metabolizing enzymes: investigation...

PHARMACOI(INETICS AND DRUG DISPOSITION

Selective effect of liver disease on the activities of specific metabolizing enzymes: Investigation of cytochromes M50 2.Cl9 and 2D6

Backgroztnd and Objectives: Drug metabolism is influenced by liver disease because of the central role that the liver plays in metabolic activities in the body. However, it is still unclear how activities of specific drug- metabolizing enzymes are influenced by the presence and severity of liver disease. As a consequence, alteration in metabolism of specific drugs cannot be easily predicted or appropriate dosage adjustment recommendations made. Methods: The activities of cytochromes P450 (CYP) 2C19 and 2D6 were investigated in a group of patients with mild or moderate liver disease (n = 18) and a group of healthy control subjects (n = 10). The disposition of racemic mephenytoin for CYP2C19 and debrisoquin for CYP2D6 were characterized in plasma and urine samples collected over 192 hours. Results: The elimination of S-mephenytoin was severely reduced among patients with liver disease, result- ing in a 79% decrease in plasma clearance for all patients combined. This reduction was related to the severity of disease, patients with moderate disease being affected more severely than patients with mild disease. Similar differences were observed in the urinary excretion of 4’-hydroxymephenytoin metabolite. By contrast, there was no effect on the disposition of R-mephenytoin or debrisoquin. Conclusion: These results show selectivity in the effect of liver disease on activities of specific metabolizing enzymes, CYP2C19 being more sensitive than CYP2D6. They suggest that recommendations for moditi- cation in drug dosage in the presence of liver disease should be based on knowledge of the particular enzyme involved in metabolism of the drug. The results emphasize the need for further studies of each specific drug-metabolizing enzyme in the presence of liver disease. (Clin Pharmacol Ther 1998;64:8-17.)

Adedayo Adedoyin, PhD, Patricia A. Arm, MD, W. 0. Richards, MD, Grant R Wilkinson, PhD, and Robert A. Branch, MD Pittsburgh, Pa., and Nashville, Tenn.

From the Center for Clinical Pharmacology, University of Pittsburgh Medical Center, Department of Pharmaceutical Sciences, Univer- sity of Pittsburgh, Pittsburgh, and the Division of Clinical Phar- macology, Vanderbilt University School of Medicine, Nashville.

Supported in part by U.S. Public Health Service grant GM31304. Received for publication Nov. 11, 1997; accepted Feb. 21, 1998. Reprint requests: Robert A. Branch, MD, Center for Clinical Phar-

macology, 623 Scaife Hall, University of Pittsburgh Medical Cen- ter, 200 Lothrop St., Pittsburgh, PA 15213-2582.

Copyright 0 1998 by Mosby, Inc. 0009-9236/98/$5.00 + 0 13/l/89799

Most drugs are eliminated from the body by means of metabolism, which is mediated by enzymes. These enzymes are concentrated in the liver, and this makes it the main metabolizing organ in the body. It follows that any condition that affects the functional integrity of the liver will be expected to influence its metabolic function and consequently the disposition of drugs. Liver disease has been shown to affect these functions, including metabolism and elimination of several

8

CLINICAL PHABMACOLOGY & THERAPEUTICS VOLUME 64, NUMBER 1 Adedoyin et al. 9

Table I. Subject characteristics

Patient/subject No. Sex Pugh score Child class

Patients with mild liver disease 1 2 3 4 5 6 7* 8 9*

Patients with moderate liver disease 10 11 12 13 14 15 16* 17 18

Control subjects 1 2

4 5 6*

8 9t lot

61 Male 43 Female 50 Female 46 Female 72 Female 74 Male 59 Male 38 Female 52 Female

53 Female 47 Male 61 Male 33 Male 51 Male 58 Male 50 Male 68 Male 77 Male

45 Male 51 Male 57 Female 71 Female 61 Male 64 Female 39 Male 54 Female 32 Female 73 Male

7 7 8

7

9

TYP2D6 poor metabolizer. 7CYP2C19 poor metabolizer.

drugs.‘-5 However, the relative effect of liver disease debrisoquine)lgJa However, such studies tended to use on individual drug-metabolizing enzymes that mediate only single-drug probes, making it difficult to assess these reactions is not well characterized. These selectivity, because assessment of selectivity required enzymes include members of the cytochrome P450 comparison of results from different studies that had (CUP) superfamily of enzymes, which mediate most of different designs and study populations. A more appro- the oxidative metabolic reactions in the body. The priate approach to assess the selectivity of the effect on superfamily consists of individual members that are different enzymes is to use known selective probes for products of specific genes and mediate specific reac- different specific enzymes in the same study populations.6-8 tion.

From many studies of the effect of liver disease on metabolizing activities, it is unclear whether there is a differential effect on these specific metabolizing enzymes. This is because in most studies the drugs used are considered general metabolic probes, such as antipyrine (INN, phenazone) and hexobarbital,s-16 or those the metabolism of which is mediated by unknown enzymes. In a few instances, drugs that are probes for individual enzymes have been used, such as caffeine,17 theophylline, l”,18, dapsonelg and debrisoquin (INN,

This study investigated the effect of liver disease on two specific drug-metabolizing enzymes, CYP2C19 and CYP2D6, with the specific drug probes, S- mephenytoirGtJ2 and debrisoquin23324 respectively, in the same population of patients. CYP2C19 is a geneti- cally polymorphic enzyme the activity of which gener- ally divides the population into a predominant extensive and a minority poor metabolizer group, but also an additional group of intermediate metabolizers.2s,26 Mephenytoin is a racemic drug. Among extensive

10 Adedoyin et al. CLINICAL PHARh3A COLOGY & THERAPEUTICS

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1.0 =: E .

5

Y 8 * 0.1

5

2

’ I I I 1

20 40 60 60 100 TIME (hours)

Fig. 1. Plasma concentration-time profiles of mephenytoin enantiomers (squares, R-mephenytoin; circles, S-mephenytoin) among extensive metabolizer control subjects (n = 8; mean values f SD).

metabolizers, mephenytoin exhibits stereoselective disposition because the S-enantiomer is more rapidly eliminated than the R-enantiomer. In contrast, among per- sons with mutant alleles of the enzyme (poor metabolizers), the two enantiomers have similar elimination characteristics.25 This stereoselective disposition and the almost exclusive 4’-hydroxylation of S-mephenytoin have been used to establish the phenotypic classi- fication of individuals into extensive metabolizers and poor metabolizers*7,** and develop a quantitative measure for in vivo activity of this enzyme.

CYP2D6 is a drug-metabolizing enzyme that also exhibits genetic polymorphism. The presence of mutant alleles results in a poor metabolizer phenotype.*9,30 The extent of metabolism of debrisoquin to 4-hydroxydebrisoquin has been extensively used as an in vivo measure of the expression and activity of the enzyme.23v24 These selective probes were used in the same study patient population to evaluate the selectivity of the effect of liver disease on the activity of specific drug- metabolizing enzymes. The two probe drugs have been used simultaneously in other studies, and neither has been found to interfere with measurement of the enzyme activity of the other.31

METHODS The study was approved by the biomedical institu-

tional review board of Vanderbilt University. The 10 control subjects were five men and five women between the ages of 32 and 73 years. The other subjects were 18 patients with liver disease (11 men and 7 women)

between the ages of 33 and 77 years (Table I). Three control subjects (two for mephenytoin and one for debrisoquin) and three patients with liver disease (all for debrisoquin) found to be poor metabolizers were excluded from data analysis. The control subjects were defined as normal by physical examination, medical history, and laboratory tests of renal and hepatic function. The patients were found to have cirrhosis con- firmed by biopsy, and the disease was classified accord- ing to severity as mild or moderate on the basis of Pugh score and Child class (Table I).sJ*Js

The pharmacokinetic study was conducted after an overnight fast and after simultaneous oral administration of racemic mephenytoin (100 mg Mesantoin, 460 pmol) and debrisoquin (10 mg Declinax). Blood samples (5 ml) were obtained before and at %, 1, l%, 2, 2%, 3,4, 6, 8, 10, 12,24, 36,48,72,96, 120, 144, and 168 hours after administration, and plasma was harvested by means of centrifugation. Total voided urine was collected for the intervals from 0 to 8, 8 to 24, 24 to 36, 36 to 48, 48 to 72,72 to 96,96 to 120, 120 to 144, 144 to 168, and 168 to 192 hours after drug administration. Volume and pH were measured, and an aliquot with the plasma samples was stored frozen at -20” C until analysis.

Enantiomers of mephenytoin in plasma were determined by means of chiral gas chromatography as described previously.34 Briefly, internal standard (3- methyl-5-phenyl-5-isopropylhydantoin) and 1 ml of 0.01 mol/L acetic acid were added to an aliquot of plasma (1 ml), and the mixture was placed over solid- phase extraction columns (Chem Elut 1003; Ana- lytichem) After allowing to sit for 5 minutes, the sample was eluted with 6 ml methylene chloride into glass tubes and evaporated to dryness under a stream of nitrogen in a warm water bath. The residue was reconsti- tuted with 10 p.1 ethylacetate before an aliquot was injected onto the gas chromatograph (Varian model 2100). Separation of the enantiomers was achieved on a Chirasil-Val III chiral capillary column (25 m x 0.32 mm; Alltech) maintained at a temperature of 175” C. Detection was achieved with a nitrogen-phosphorus detector.

4’-Hydroxymephenytoin metabolite was determined in urine by means of HPLC.27 The internal standard (phensuximide) and 1 ml concentrated hydrochloric acid was added to an aliquot of urine (0.5 ml). The tubes were incubated at 100” C for 1 hour and cooled. The mixture was placed over prewashed and predried solid- phase extraction columns (Chem Elut 1003) and allowed to sit for 5 minutes. The sample was eluted with 6 ml methylene chloride into glass tubes and evaporated to dryness under a stream of nitrogen in a warm

CLINICAL PHAIWA COLOGY & THERAPEUTICS VOLUME 64, NUMBER 1 Adedoyin et al. 11

Table II. Pharmacokinetic parameters of mephenytoin enantiomers among control subjects and patients with liver disease

Subject S-Mephenytoin plasma clearance (mUmin) R-Mephenytoin plasm clearance (mWmin)

Control subjects (n = 8) Patients

All (n = 18) Mild liver disease (n = 9) Moderate liver disease (n = 9)

1986.9 f 1878.4 24.0 f 10.6

408.6 zt 974.9* 23.2 f 37.4 745.1 f 1324.8 21.2 f 33.7 72.0 f 97.4*t 25.2 f 42.6

Data are mean values f SD. *p < 0.005, significantly different from control subjects. tp < 0.05, significantly different from patients with mild liver disease.

water bath. The residue was dissolved in 100 ~140% methanol in water before injection of an aliquot onto the HPLC (model 6000; Waters Chromatography). Sep- aration was achieved on a reversed-phase Crs column (51.1, 4.6 x 250 mm; Alltech) through which a mobile phase of 0.05 mol/L potassium phosphate pH 6.5 and methanol (65:35) was pumped at a rate of 1 ml/min and the eluent monitored at 211 nm.

Debrisoquin and 4-hydroxydebrisoquin in plasma and urine were determined by means of modification of a previously reported gas chromatography-mass spectrometry method.35 An aliquot of plasma (500 pl) or urine (100 pl) was taken, and the volume was adjusted to 600 pl with water. To this were added internal standards (deuteriated debrisoquin and 4-hydroxydebrisoquin), 200 pl saturated sodium bicarbonate, 200 pl hexafluoroacetylacetate, and 1.5 ml toluene. The mixture was heated at 100” C for 1% hours and allowed to cool. Five milliliters of 3 mol/L sodium hydroxide solution was added. The tube was vortex mixed and centrifuged, and the toluene was transferred to glass tubes and evaporated to dryness under a stream of nitrogen in a warm water bath. The residue was dissolved in 10 pl dodecane, and an aliquot was injected onto the gas chromatograph. Separation was achieved with a DB 1701 capillary column (J & W Scientific) maintained at an initial temperature of 170” C for % minute and increased at the rate of 20” C per minute to a final temperature of 250” C. The compounds were monitored by means of selected ion-monitoring mass spectrometry. Concentrations of all compounds were determined by means of interpolation with standard curves constructed by means of spiking the respective biologic fluids with the respective compounds and tak- ing them through the processes described earlier.

Pharmacokinetic parameters were determined with standard noncompartmental methods, as follows: elimination rate constant (k) as slope of log-linear regression of the terminal phase, half-life as 0.693/k, area under the curve (AUC) with the log-trapezoidal

method, clearance as ratio of dose to total AUC, volume of distribution at steady state with the ratio of (dose . AUMC) to (AUC)*, in which AUMC is the area under the first moment curve estimated with the log-trapezoidal method. Metabolite formation clearances were calculated as the product of the fraction excreted in urine as the metabolite and the total clearances. Statistical analysis was performed by comparison of control subjects and patients or the two patient groups by means of the nonparametric Mann-Whitney test, with the level of significance set at p I 0.05.

RESULTS Mephenytoin exhibited stereoselective disposition

among extensive metabolizer control subjects with wide interindividual variability. This was reflected in a substantial difference in first-pass effect. Peak S- mephenytoin concentration was about 39% of peak R- mephenytoin concentration. Interindividual variability also was reflected in a much more rapid decline in plasma concentration of the S- than the R-enantiomer after a racemic dose (Fig. 1). These differences were caused by the higher clearance of S-mephenytoin than R-enantiomer (about an eightyfold difference; Table II). The disposition among patients with liver disease also was stereoselective (Fig. 2), but the difference between the two enantiomers was less than that among control subjects. There was no difference in clearance of R- mephenytoin between the control subjects and the patients, but clearance of S-mephenytoin among the patients, though still higher than that of R-mephenytoin, was significantly reduced compared with control subjects (Table II). Thus the difference in enantiomeric clearance was about 18-fold among patients compared with about eightyfold among control subjects. Similar differences were shown with the peak concentrations and decline in plasma concentration (Fig. 2).

The reductions in S-mephenytoin clearance and first- pass loss and the decline in plasma concentration

12 Adedoyin et al. CLINICAL I’HAFZvIA COLOGY & THERAPEUTICS

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0.01 --

” E . s Y 8 0.1

f

3 0

0 4 a 12 20 40 60 a0 100 0 4 a 12 20 40 60 a0 loo TIME (hours1 TIME (hours)

Fig. 2. Plasma concentration-time profiles of mephenytoin enantiomers among patients with mild (left panel, n = 9) and moderate (right panel, n = 9) liver disease (mean values f SD). Squares, R-Mephenytoin; circles, S-mephenytoin.

Table III. Urinary recovery of 4’-hydroxymephenytoin among control subjects and patients with liver disease

4 *-Hydroxymephenytoin urinary recovery (~01)

Subject O-8 Hours O-24 Hours O-72 Hours (total)

Control subjects (n = 8) Patients

All (n = 18) Mild liver disease (n = 9) Moderate liver disease (n = 9)

96.0 f 45.6 134.9 + 34.1 252.5 f 96.8

48.3 f 52.0* 97.1 + 70.5 138.4 + 82.0* 79.2 f 57.8 136.9 + 68.7 182.0 f 86.6 17.4 f 16.2***t 57.2 +47.4**t 94.9 f 50.2***t

Data are mean values f SD. *p < 0.05, significantly different from control subjects. **p < 0.01, significantly different from control subjects. ***p < 0.005, significantly different from control subjects. tp < 0.05, significantly different from patients with mild liver disease.

among patients depended on severity of the disease. For example, when findings for all patients were combined, there was a significant and substantial (79%) decrease in S-mephenytoin clearance. This combined average reduction was a result of a smaller decrease (63%) among patients with mild liver disease and a larger decrease (96%) among patients with moderate liver disease (Table II). These differences were reflected in the changes in peak concentration and decline in plasma concentration (Fig. 2).

Urinary excretion of 4’-hydroxymephenytoin over 72 hours showed the same profile as plasma clearance of S-mephenytoin, from which it is almost exclusively formed. Excretion among control subjects (252 ymol) approximated the dose (50 mg, 230 pmol) of the S- enantiomer (Table III). Urinary excretion of this metabolite among patients with liver disease was less than that among the control group, and the reduction in

excretion depended on severity of disease. For all patients combined, the reduction was about 45%, which was a reflection of 28% and 62% decreases among patients with mild and patients with moderate liver disease, respectively (Table III). Partial recoveries of this metabolite over 8 and 24 hours reflected similar differences between patients and control subjects (Table III).

The disposition of debrisoquin and 4-hydroxydebrisoquin were comparable between control subjects and patients with liver disease whether grouped together or evaluated separately as groups with mild or moderate disease (Fig. 3 and Table IV). There was no difference in any of the estimated pharmacokinetic parameters except for the AUC of 4-hydroxydebrisoquin when all patients were grouped together. Even though the mean AUC values for the metabolite were similar between mild, moderate, and all liver disease grouped together, it reached statistical significance

CLINICAL P- COLOGY & THERAPEUTICS VOLUME 64, NUMBER 1 Adedoyin et al. 13

C E

. 9 10.0

Y

8

0 25 50 75 100 125 150 175

TIME (hours)

I I - I * I - I r I * I ’ 8 ‘1

0 25 50 75 100 125 150 175 TIME (hours)

Fig. 3. Plasma concentration-time profiles of debrisoquin (circles) and 4-hydroxydebrisoquin (triangles)

among control subjects (left panel, n = 9) and patients with liver disease (right panel, n = 15). All patients with liver disease were combined because there was no difference between the patients with mild and the patients with moderate disease (mean values + SD).

compared with control subjects only when all patients were combined (Table IV). The urinary excretions of debrisoquin and 4-hydroxydebrisoquin showed similar patterns and were not different between patients and control subjects (Table V).

The difference in the effect of liver disease on S- mephenytoin and debrisoquin elimination is further illustrated by their relation to Pugh score among these subjects. Oral clearance of S-mephenytoin exhibited an inverse relation to Pugh score; patients with the highest Pugh scores had the lowest clearances (Fig. 4). This is consistent with the finding that patients with moderate liver disease had lower clearances and excreted less 4’-hydroxymephenytoin than patients with mild liver disease (Tables II and III). On the contrary, within this small sample, there was no apparent relation between oral clearance of debrisoquin and Pugh score but instead, there was significant overlap in the range of values among all subject groups (Fig. 4).

DISCUSSION This study showed the selective effect of liver disease

on the activity of specific cytochrome P450 enzymes by means of an investigation of the effect of cirrhosis on the activities of CYP2C19 and CYP2D6 in which the metabolism of S-mephenytoin and debrisoquin, respectively, was used as a probe. The differences in elimination of these drugs--S-mephenytoin clearance was reduced and R-mephenytoin and debrisoquin clearances were unaffected-showed that the responses of specific metabolizing enzymes to the effect of liver disease on

hepatic metabolizing activity are selective. This finding is consistent with and may explain apparent discrepan- cies in previous reports that showed decreased elimina- tiont3,36-39 or no effect39-42 of liver disease on the disposition of different drugs. This situation is further com- plicated by the dependence of the effect on the severity of liver disease for drugs whose eliminations are affected. Thus interpretation of reports of the effect of liver disease on metabolizing activities must consider the drug probe used and the condition of the patients.

The reduced elimination of mephenytoin in liver disease was stereoselective, as reflected by reduction in the clearance of S-mephenytoin but not of R-mephenytoin (Table II). In spite of the difference in effect on clearances of mephenytoin enantiomers, the disposition of mephenytoin was still stereoselective among patients with liver disease with higher clearance of S- than R-

mephenytoin. The reduction in S-mephenytoin clearance resulted in a significant reduction in urinary excretion of 4’-hydroxymephenytoin, which is almost exclusively formed from S-mephenytoin (Table III). The urinary recovery of 4’-hydroxymephenytoin among these patients was still much more than would be expected from poor metabolizers of CYP2C19, which is usually less than 25 pmo125,28 (data not shown). These findings provide unequivocal demonstration of selectivity in the effect of liver disease on activity of specific metabolizing enzymes because the two enantiomers are metabo- lized by different pathways catalyzed by different enzymes.43-45 S-mephenytoin is mostly 4’-hydroxylated among extensive metabolizers because of almost exclu-

14 Adedoyin et al. CLINICAL PHARMA COLOGY & THERAPEUTICS

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Table IV. Pharmacokinetic parameters of debrisoquin (INN, debrisoquine) and 4-hydroxydebrisoquin among control subjects and patients with liver disease

Patients with liver disease

Parameter

Debrisoquin Oral CL (ml/m@ tx (hr) v,, (L) Renal CL (ml/min) Formation CL (mEnin) CL (others) (ml/min)

I-Hydroxydebrisoquin tX (hr) AUC (ng . hr/ml)

Control (n = 9) All (n = 15)

170.8 f 114.2 180.5 f 110.7 42.5 + 15.2 35.1 + 16.2 5642612 457 +296

24.7 f 10.3 44.0 f 28.0 38.0 f 33.5 29.2 f 24.7

108.2 f 83.8 107.2 f 79.7

37.4 f 13.5 33.0 f 21.4 318.4 f 96.2 208.0 f 129.1*

Mild (n = 7) Moderate (n = 8)

196.3 f 150.0 166.5 2 68.8 31.8 f 4.2 38.0 f 22.1 498k356 4202252

37.8 f 20.8 49.5 * 33.5 35.5 f 33.8 23.5 + 12.5

123.0 f 103.2 93.5 + 55.7

29.1 f 12.3 36.5 + 27.5 199.2 k 143.0 215.5 + 125.3

Data are mean values f SD. CL, Clearance; tq half-life; V,,, steady-state volume of distribution; AUC, area under the plasma concentration-time curve. *p < 0.05, significantly different from control subjects.

Table V. Urinary excretion of debrisoquin and 4-hydroxydebrisoquin among control subjects and patients with liver disease

Patients with liver disease

Compound Control subjects (n = 9) All (n = 15) Mild (n = 7) Moderate (n = 8)

Debrisoquin (%) 21.0 f 13.1 28.5 + 16.3 26.2 f 14.7 30.6 + 18.2 4-Hydroxydebrisoquin (%) 20.6 + 8.0 15.8 f 6.2 16.5 f 5.1 15.1 f 7.4 Total (%) 41.6 f 13.1 44.3 + 15.0 42.7 f 12.4 45.7 f 17.8

Data are mean values f SD.

sive metabolism by CYP2C19.22 In contrast, R- mephenytoin is mostly demethylated by an as-yet unidentified enzyme. That the metabolism of only the S-enantiomer was affected suggests that liver disease was influencing the enzyme involved in its metabolism but not that of the R-enantiomer. Thus it can be con- cluded that liver disease substantially reduced the activity of this specific enzyme (CYP2C19) but did not affect the enzyme involved in R-mephenytoin metabolism.

The magnitude of the decrease in CYP2C19 activity caused by liver disease depended on the severity of the disease. There was a gradual decrease in clearance of S- mephenytoin and urinary excretion of 4’-hydroxymephenytoin from control subjects to patients with mild liver disease to patients with moderate liver disease, even though only the patients with moderate liver disease showed a statistically significant difference from control subjects (Tables II and III). The lack of statistical significance in patients with mild liver disease was probably caused by the very wide interindividual variability in the measures or insufficient power to detect the difference because of the relatively small number of patients

involved. This dependence of the decrease in activity on severity of disease is clearly shown by the inverse relation between S-mephenytoin clearance and Pugh score (Fig. 4) in which patients with the highest Pugh scores also had the lowest clearances. Interestingly, the two groups of patients with liver disease were significantly different from each other. This result is consistent with our earlier observation of a significant relation between phenotypic indices of mephenytoin disposition (WS enantiomeric ratio and cumulative 4’-hydroxymephenytoin excretion in &hour urine collection) and an independent measure of hepatic function (serum cholylglycine levels).46 In this observation, the higher the serum cholylglycine level, an indicator of more severe disease, the lower was the measure of mephenytoin elimination.

In contrast to the effect on S-mephenytoin elimination, the disposition of debrisoquin appears to be unaffected by liver disease, even with cirrhosis of moderate severity. None of the estimated pharmacokinetic parameters reached statistical significance except for the AUC of 4-hydroxydebrisoquin when all patients were combined (Tables IV and V). This difference in AUC

CLINICAL PHARMA COLOGY & THERAPEUTICS VOLUME 64, NUMBER 1 Adedoyin et al. 15

3 6000

E 1 g 5500

e f 4 3500 w

% A

0 5 6 7 6 9 0 5 6 7 6 9 PUGH SCORE PUGH SCORE

Fig. 4. Relation between S-mephenytoin clearance (left panel) and debrisoquine clearance (right panel) and Pugh score. Squares, Control subjects; circles, patients with mild liver disease; triangles, patients with moderate liver disease.

suggests that there may be some minor effect of liver disease on formation of 4-hydroxydebrisoquin. The significance achieved when all patients were combined but not seen when they were divided into groups on the basis of mild or moderate disease could relate to sample size and sufficient power to detect the difference. A comparison of formation clearance also shows that among patients with moderate liver disease, the mean value was about 38% lower than for control subjects (a difference that was not statistically significant) whereas the value for patients with mild liver disease was not different from that of control subjects (Table IV). This trend sup- ports the suggestion that with more severe disease or a larger sample size, a statistically significant difference might be shown. In spite of the apparent trend toward reduced metabolism of debrisoquin among patients with moderate liver disease, it is clear that liver disease did not have an effect on debrisoquin metabolism comparable with the effect on S-mephenytoin metabolism. This reaction has been shown to be mediated by CYP2D6. Thus it can be inferred that liver disease (of mild or moderate severity) has little effect on the activity of this specific enzyme. Further studies with larger numbers and with patients with disease of different degrees of severity are needed to fully elucidate this effect.

Though this study showed a differential effect of liver disease on the activities of specific drug-metabolizing enzymes, the reasons for this difference are unclear. Dif- ferences in expression of specific enzymes in diseased liver has been reported.47 Thus the difference in activity may be a result of differential regulation of specific intrahepatic enzymes that respond differently to liver

disease to alter expression of the enzymes. Studies that simultaneously measure transcriptional and translational control of enzyme expression and activity are needed to elucidate this hypothesis.

Alternatively, whole-body measure of activity may also be reflecting the contributions of extrahepatic tis- sues to the metabolism of these drugs. Therefore the modest to no effect on debrisoquin metabolism might be caused by significant contributions from extrahepatic enzymes. The presence of CYP2D6 has been shown in the brain48 bladder mucosa,49 and peripheral blood mononuclear leukocytes.50 Interestingly, the expression of CYP2D6 messenger ribonucleic acid in bladder mucosa and peripheral blood mononuclear leukocytes has been shown to correlate significantly with whole- body measurements of CYP2D6 activity.49,5e This may

suggest coregulation of expression of the enzyme at these sites with that in the liver or considerable metabolism at these sites. This requires further investigation. Fenyves et a1.51 showed that factors other than decreased enzyme activity may contribute to differences seen in the presence of liver disease. S-Mephenytoin is a high- clearance drug, whereas R-mephenytoin and debrisoquin are both low-clearance drugs. The physiologic determinants of clearance of both types of drugs, blood flow for high-clearance drugs, and intrinsic activity and fraction of unbound drug for low-clearance drugstJ2 may respond differently in the presence of liver disease. Consequently the alteration in clearance of such drugs induced by liver disease will be different.

An important finding in this study, especially with respect to drug treatment of patients with liver disease,

16 Adedoyin et al. CLINICAL PHARMACOLOGY &THERAPEUTICS

JULY 19%

is the significant change in peak concentration of S- mephenytoin among patients with liver disease. It showed an increase in peak concentration from 0.32 f 0.17 pg/ml among control subjects to 0.96 f 0.58 pg/mI among patients with liver disease (0.98 + 0.68 pg/ml with mild disease and 0.94 + 0.49 pg/ml with moderate liver disease). S-Mephenytoin is a high-clearance drug with substantial first-pass loss after oral administration, and the increase in peak concentration is a reflection of reduction in first-pass loss. This change represents a 200% increase in peak concentration, which can have serious consequences if it involves a drug with narrow therapeutic index. Therefore such a change in the absorption and disposition of a drug should be taken into consideration in dosage adjustment recommendations in the care of patients with liver disease.

In conclusion, this study showed that liver disease affects the metabolic function of the liver but that the extent of effect varies with specific drug-metabolizing enzymes. CYP2C19 activity, as measured by means of S-mephenytoin hydroxylation, was affected markedly, and the effect depended on the severity of the disease. Patients with moderate liver disease were affected more than patients with mild liver disease. In contrast, CYP2D6 activity, in the same cohort of patients with liver disease, was comparable with that for control subjects. Thus CYP2C19 appears to be more sensitive in this regard than CYP2D6. The implication of this finding is that dosage adjustment for patients with liver disease has to depend on which particular enzyme mediates the metabolism of the drug involved and the severity of the disease itself. The study also emphasized the need for more studies investigating the effect of liver disease and the effect of different degrees of severity on the activity of specific metabolizing enzymes to achieve an under- standing of the pathophysiologic processes involved and to develop specific guidelines for dosage adjustment.

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