active hepatitis and alcoholic cirrhosis

Transcription

1 Br J clin Pharmac 1994; 37: Liver blood flow, antipyrine clearance, and antipyrine metabolite formation clearance in patients with chronic active hepatitis and alcoholic cirrhosis LARRY A. BAUER, TERESA O'SULLIVAN, WILLIAM G. REISS*, JOHN R. HORN, KENT OPHEIM, D. EUGENE STRANDNESS & ROBERT L. CARITHERS Departments of Pharmacy, Laboratory Medicine, Surgery, and Medicine, University of Washington, Seattle, Washington, USA 1 Duplex scanning was used to measure liver blood flow (hepatic artery and main branches of the portal and hepatic veins) in six healthy subjects, five cirrhotic patients, and six hepatitis patients. Antipyrine clearance and formation clearances to its metabolites were also measured. 2 Compared with healthy control subjects, cirrhotic patients had a lower hepatic vein blood flow (-76%, P <.5). This was due primarily to a lower portal vein blood flow (-36%, NS). A statistically significant difference in liver blood flow between patients with hepatitis and normal subjects was not detected. 3 Antipyrine half-life, clearance, and the area under the serum drug concentration vs time curve were significantly different in cirrhotic patients compared with the healthy subjects (mean ± s.d.-healthy controls: t, 2 = 13.7 ± 3. h, CL = 3. ± 8.6 ml h-' kg-', AUC = 549 ± 139 mg 1-1 h; cirrhotic patients: ti,2 = 32.4 ± 1.7 h, CL = 12.3 ± 2.1 ml h-1 kg-', AUC = 161 ± 218 mg 1-1 h; P <.8). Antipyrine halflife, clearance, and the area under the serum drug concentration vs time curve were not significantly different in hepatitis patients compared with the healthy subjects (hepatitis patients: tb = 14.3 ± 3.7 h, CL = 29.3 ± 8.5 ml h-1 kg-i, AUC = 498 ± 142 mg I1- h). The volume of distribution of antipyrine was similar in all three groups of subjects. 4 Formation clearances to 3-OH-methylantipyrine, 4-OH-antipyrine, and norantipyrine were all lower (P <.9) in cirrhotic patients compared with normal subjects and hepatitis patients. However, only the formation clearance to norantipyrine was lower (P <.2) in hepatitis patients when compared with normals. 5 There were strong correlations between the logarithmic transformations of the formation clearances for all antipyrine metabolites (r = , P <.1). However, the only orthogonal regression line with a slope not significantly different from one was that for norantipyrine and 3-OH-methylantipyrine indicating that these two metabolites may be metabolized by a common enzyme(s). 6 Weak correlations were found between the logarithmic transformations of hepatic vein blood flow and antipyrine clearance and the logarithmic transformations of hepatic vein blood flow and formation clearances to antipyrine metabolites (r= , P <.5). 7 Cirrhotic patients may have difficulty metabolizing drugs with high hepatic extraction ratios or drugs that are eliminated by the same liver enzyme systems which produce the three major metabolites of antipyrine. Hepatitis patients may have difficulty metabolizing drugs that share the same hepatic enzyme system responsible for the formation of norantipyine from antipyrine. Keywords antipyrine formation clearance liver blood flow hepatitis cirrhosis Correspondence: Dr Larry A. Bauer, Department of Pharmacy SC-69, University of Washington, Seattle, Washington 98195, USA *Current address: School of Pharmacy, University of Maryland, Baltimore, MD, USA 375

2 376 L. A. Bauer et al. Introduction The measurement of liver blood flow and hepatic drug metabolism capacity in patients with liver disease has been of interest for many years. Liver blood flow in patients with cirrhosis or hepatitis has usually been measured using indocyanine green or bromsulphthalein clearance even though the hepatic extraction of these compounds may be decreased [1-4]. Duplex scanning is a relatively new noninvasive technique that can be used to measure liver blood flow through the hepatic artery and branches of the portal and hepatic veins [5-8]. Antipyrine clearance and the formation clearances to its metabolites have been used to measure P-45 hepatic drug metabolism capacity [9-12]. The purpose of this study was to measure liver blood flow in cirrhotic and hepatitis patients using a noninvasive, direct measurement technique and to determine if cirrhosis and hepatitis affect liver blood flow, antipyrine pharmacokinetics, and the formation clearances of antipyrine to its metabolites in man. Additionally, correlations were investigated between liver blood flow and antipyrine clearance, liver blood flow and antipyrine metabolite formation clearances, and between the formation clearances to the various antipyrine metabolites. Methods Six healthy subjects were admitted to the study as a control group (Table 1). They had normal physical examinations, medical histories, and laboratory values (blood chemistry, blood cell counts, urinalysis). Six patients with chronic active hepatitis and five patients with alcoholic cirrhosis were entered into the investigation as separate groups. All had physical examinations, medical histories, and laboratory values consistent with their disease. Study subjects did not smoke tobacco-containing products Table 1 Subject and patient characteristics Age BSA Weight Number (years) (mn2) (kg) Sex Pugh score Medications Healthy subjects M NA None F NA None M NA None F NA None F NA None F NA None Mean M/4F s.d Patients with chronic active hepatitis M 5 Interferon A 2B F 5 Transdermal oestrogen M 5 Vitamin K, trazodone, piroxicam, sucralphate M 5 None F 5 None F 5 None Mean M/3F 5 s.d Patients with alcoholic cirrhosis M 5 Frusemide, spironolactone, neomycin F 6 Spironolactone, propranolol (mild ascites) M 7 Frusemide, spironolactone, (bilirubin = 29.1 gmol 1-') multivitamins, ranitidine albumin = 32 g 1-') F 8 Spironolactone, frusemide, (mild ascites, tranexamic acid, folic acid, bilirubin = 59.9.mol l-l) Vitamin K M 8 Frusemide, hydrochlorothiazide, (mild ascites, triamterene albumin = 25 g 1-') Mean M/2F 6.8 s.d NA = not applicable.

3 Antipyrine kinetics and liver blood flow in hepatic disease 377 (except for hepatitis patients 8 and 11, and cirrhotic patient 13), had negative urine screens for drugs of abuse and ethanol, had normal electrocardiograms and chest X-rays, and did not take medications other than those listed in Table 1. The diagnosis of cirrhosis was confirmed by liver biopsy. Hepatitis patients had positive hepatitis serum titres (1 hepatitis B, 5 hepatitis C). The severity of disease was scored by Pugh's modification [13] of the Child's classification. The study was approved by the Investigational Review Board and Human Subjects Committee at the University of Washington. All subjects gave informed consent before participating in the study. Liver blood flow was measured in all subjects before administration of antipyrine. To reduced abdominal gas, subjects consumed a bland diet for 24 h and began fasting at 22. h the evening before a study day. Studies began between 7.3 h and 9.3 h to reduce any influence of circardian rhythms. Before blood flow measurements, subjects were placed in the supine position with their heads elevated at 2- to 3-degrees for 1 h to allow liver blood flow to stabilize. After blood flow measurements, subjects were given 1 g antipyrine orally at approximately 1. h. Blood samples for the assay of serum antipyrine concentration were obtained at,.5, 2, 3, 4, 8, 12, 24, 36, and 48 h from the healthy subjects and at, 4, 8, 12, 24, 36, 48, 72, 96, 12, and 144 h from the patients. Blood flows in the proper hepatic artery (distal to the common hepatic artery) and main branches of the portal and hepatic veins were measured using a duplex scanner [4-7]. (Acuson Corp., Mountain View, CA). The B-mode of the duplex scanner was used to measure vessel cross-sectional area, and pulsed Doppler ultrasound was used to measure blood velocity. Blood flow was computed using vessel cross-sectional area and the mean of 3-5 velocity measurements over a 3-4 min observation period. Serum antipyrine concentrations were measured by h.p.l.c. with a coefficient of variation of 4.6% at.2 gmol ml-' [14]. The terminal elimination rate constant (X) was calculated from a semi-logarithmic plot of post-absorption concentrations using linear regression. AUC values were computed using the linear trapezoidal rule and the quotient of the last antipyrine concentration and Xz. Assuming complete oral bioavailability [15], volume of distribution (V ) and clearance (CLO) were computed using standard pharmacokinetic formulae [16]. Urine concentrations of antipyrine and its metabolites were also measured by h.p.l.c. [17, 18]. The formation clearance to each antipyrine metabolite was computed from the product of the fraction of the antipyrine dose eliminated as metabolite in the urine and antipyrine clearance [11, 12]. Body surface area was calculated using the method of DuBois & DuBois [19]. Statistical analysis was by analysis of variance. When a significant difference was found among mean values, the t-test was used to determine if statistical differences existed between two sets of values. Orthogonal regression was used to determine relationships between metabolite formation clearances and also betwen metabolite formation clearances and liver blood flows. Orthogonal regression was chosen because both x and y values are subject to measurement error [12]. Logarithmic transformations of metabolite formation clearances and liver blood flows were used in the orthogonal regressions because the metabolite formation clearances and liver blood flows had different variances [12]. Pearson correlation coefficients were also computed. A P <.5 was considered to be statistically significant. Results Table 2 summarizes the results for each patient group. Hepatic vein blood flow in the cirrhotic patients was 76% lower than in normal subjects (P <.5). This was due primarily to a 36% decrease in portal vein blood flow, although this change did not achieve statistical significance. Mean values of portal vein (-21%), hepatic artery (-31%), and hepatic vein (-56%) blood flows were lower in patients with hepatitis compared with healthy subjects, but the differences were not statistically significant. The pharmacokinetic parameters of antipyrine for normal subjects and hepatitis patients were similar except for the urinary recovery of norantipyrine (P <.3) and the formation clearance to norantipyrine (P <.9). Both of these values were significantly lower in the hepatitis patients. In cirrhotic patients the mean antipyrine half-life was 136% (P <.1) higher, mean clearance was 6% lower (P <.2) and mean AUC was 93% higher (P <.2) than in control subjects. The fractions of the antipyrine dose excreted as 3-OH-methylantipyrine and norantipyrine were smaller in patients with cirrhosis (P <.7). Conversely, the fraction excreted as unchanged antipyrine was larger in the cirrhotic patients (P <.6). Cirrhotic patients had decreased formation clearances to all three antipyrine metabolites (P <.9) compared with normals and hepatitis patients, but the renal clearance of unchanged antipyrine was similar to that in the two other groups. The volume of distribution of antipyrine, the percentage of urinary 3-OH-methylantipyrine excreted as the glucuronide conjugate, and the total percentage of the antipyrine dose recovered in the urine was similar in all three groups. There were strong correlations between the formation clearances to each of the metabolites (Figure 1; Table 3). However, the only plot with a slope that was not significantly different from one was that of the log of norantipyrine formation clearance vs that of 3-OH methylantipyrine. Weak, but statistically significant, correlations were found between antipyrine clearance and hepatic vein blood flow, and the formation clearances to each metabolite and hepatic vein blood flow (Table 3). There were no correlations between antipyrine or metabolite formation clearances and portal vein or hepatic artery blood flows.

4 378 L. A. Bauer et al. Table 2 Antipyrine parameter CL, (ml h-' kg-') V (l kg-') t,, (h) AUC (mg 1-' h) CLR (ml h-1 kg-') Summary of study findings (mean ± s.d.) Urinary excretion (% dose) HMA (%) OHA (%) NORA (%) AP (%) Total (%) Formation clearance HMA (ml h-' kg-1) OHA (ml h-' kg-1) NORA (ml h-1 kg-') Blood flow Hepatic vein (ml min-' kg-l) Portal vein (ml min- kg-') Hepatic artery (ml min-' kg-') Normal subjects Hepatitis patients Cirrhotic patients 3. ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 3.6* 3.6 ± ± ± ± ± 1.3* 1.8 ± ± ± ± 2.l*'.58 ± ± 1.7*' 161 ± 218*W 1.4 ± ± 2.3* 23.6 ± ± 2.6* 1.8 ± 3.6*' 5.6 ± ±.3*O 2.9 ± 1.*' 1.1 ±.4*' 1. ±.8* 1.8 ± ± 3.8 *P <.5, compared with normal subject; tp <.5, compared with hepatitis patient. AP = antipyrine, NORA = norantipyrine, HMA = 3-OH-methylantipyrine, OHA = 4-OH-antipyrine. Discussion Liver blood flow, intrinsic clearance, and plasma binding are primary factors defining hepatic drug clearance [2, 21]. Determination of these variables in patients with liver disease may be important as a decreased hepatic clearance could necessitate a decreased drug dosage rate. Liver blood flow is usually determined using a test compound with a high hepatic extraction ratio. Indocyanine green (ICG) and bromsulphthalein (BSP) both have high hepatic extraction ratios in subjects with normal liver function, and their clearance rates have been used as surrogate measures of liver blood flow [1-4]. Intrinsic clearance associated with the activity of the cytochrome P-45 enzyme system has been assessed from antipyrine clearance [22-24] and, more recently, from the formation clearances to antipyrine metabolites [9-12]. Significant dependence of hepatic drug clearance on liver blood flow requires that the compound has an hepatic extraction ratio greater than 7%. This requirement is crucial for compounds used as indicators of liver blood flow where, ideally, the extraction ratio should equal 1%. However, in patients with severe liver disease the mean (± s.d.) extraction ratios of both ICG (31 ± 1% [2], 52 ± 18% [1], and % [4]) and BSP (31 ± 18%) [3] are well below 7%. Thus, studies using these indicators, without invasive measurement of the hepatic extraction ratio, tend to underestimate liver blood flow in patients with liver disease. Further complications arise from a decrease in the extraction ratio of ICG with time [2], and enterohepatic recirculation of BSP [25]. Duplex scanning affords direct measurements of blood flow through the hepatic artery and branches of the portal and hepatic veins. These measurements are reproducible within subjects [8], noninvasive, and do not suffer from the problems found with the administration of test compounds when hepatic extraction ratio changes with disease or time after injection. Since blood flows are measured in branches of the portal and hepatic veins, total liver blood flow is not estimated using this technique. Nevertheless, changes in liver blood flow can be quantified. Thus, normal controls are used to compare disease induced changes in the portal and hepatic veins and the hepatic artery [6] or baseline blood flows are used to measure pharmacologically-induced changes in blood flow [5, 7]. Our estimates of relative liver blood flow in normal subjects and cirrhotic patients were similar to those reported by others using ICG [1, 2] or BPS [3] hepatic extraction ratios, antipyrine kinetics [22-24] and antipyrine metabolite formation clearances [11, 12]. Cirrhosis was associated with a 76% decrease in hepatic vein flow indicating that total liver blood flow was impaired. This change was principally due to a 36% decrease in portal vein flow probably caused by portal hypertension. Antipyrine clearance was lower, its half-life was longer, and its volume of distribution was unchanged in the cirrhotic patients compared with healthy subjects. The formation clearances to all three antipyrine metabolites were lower

5 Antipyrine kinetics and liver blood flow in hepatic disease 379 I E Co I -J E cm Co cm z - CJ._ E -i C) Co -j I a b ; log NORA formation CL (ml h - 1 kg - 1) log OHA formation CL (ml h -1 kg-') c.9 r a log OHA formation CL (ml h- -1 kg-1) Figure 1 a) Relationship between norantipyrine (NORA) and 3-OH-methylantipyrine (HMA) formation clearances. A high correlation coefficient (r =.92) and a regression line slope that is not statistically different from unity suggests that these metabolites are formed by the same or closely linked enzyme systems. b) Relationship between 4- OH-antipyrine (OHA) and norantipyrine (NORA) formation clearances. A regression line slope that is statistically different from unity suggests that these metabolites are formed by different enzyme systems. c) Relationship between 4-OH-antipyrine (OHA) and 3-OH-methylantipyrine (HMA) formation clearances. A regression line slope that is statistically different from unity suggests that these metabolites are formed by different enzyme systems. in the cirrhotic patients compared with both healthy controls and hepatitis patients. However, formation clearance to norantipyrine was not depressed preferentially to a larger extent in cirrhosis as reported by others [11]. A statistically significant decrease in liver blood flow in the hepatitis patients relative to that in normals was not detected. The kinetics of antipyrine were similar in the hepatitis patients and the healthy controls except for a 45% lower formation clearance to norantipyrine in the former (Table 2). As expected for a compound with a low hepatic extraction ratio, there were only weak correlations between liver blood flow and the various clearances of antipyrine (Table 3). Decreased antipyrine clearance and metabolic formation clearances were mostly observed in patients with severe decreases in hepatic vein blood flow (<1 ml min-1 kg-'). A high correlation (orthogonal regression) between two formation clearances with a slope not significantly different from unity suggests that the two metabolites are formed by the same or closely linked enzyme systems [12, 26]. High correlations were found between the log transformations of the various formation clearances (Figure 1, Table 2) in our patients. However, the only slope not significantly different from unity was for the formation clearances of norantipyrine and 3-OH-methylantipyrine. Therefore, these metabolites are probably produced by a common isoform of cytochrome P-45 whereas the formation of 4-OH-antipyrine is mediated by a different isoform. This finding is in agreement with the results of a study in normal volunteers, where it was further concluded that theophylline and 4-OH-antipyrine are metabolized by the same isoform (presumably CYPIA2) but different from that responsible for the oxidation of hexobarbitone and the formation of 3-OH-methylantipyrine and norantipyrine (presumably CYP2C9) [26]. However, in a similar study conducted in a variety of liver disease patients, the same investigators found a different pattern of correlations between hexobarbitone, theophylline and antipyrine metabolite formation clearances [12]. Liver blood flow, antipyrine clearance, and formation clearances of antipyrine metabolites are important factors to measure to determine the effects of liver disease on drug disposition. Doppler ultrasound measurements of liver blood flow not only allow the determination of blood flow through the different components of the hepatic vascular system, but also do not suffer the limitations of liver blood flow estimates using ICG or BSP clearance techniques. Completely noninvasive studies are possible if Doppler ultrasound measurements of liver blood flow are coupled with the assessment of antipyrine kinetics and metabolite formation clearances using saliva and urine [27, 28]. Funding was provided by Ciba-Geigy Pharmaceuticals, the Scholars in Pharmacy Fellowship from the Merck Foundation, and NIH grant GM A portion of this investigation was conducted in the Clinical Research Center at the University of Washington Medical Center (NIH grant RR-37). We greatly appreciate the help of Karen Carlsen, R.N. and Debra Brateng, R.N. for performing duplex scanning measurements, Jennifer Binford for recruiting study subjects, and Professor D. D. Breimer for providing antipyrine metabolite assay standards.

6 38 L. A. Bauer et al. Table 3 Orthogonal regression lines and Pearson correlation coefficients for formation clearances and hepatic vein blood flow Formation clearances or Pearson correlation hepatic vein blood flow Orthogonal -egression lines coefficients log NORA vs log HMA y =.91218x r =.92' log OHA vs log NORA y = x * r =.88t log OHA vs log HMA y = x * r =.93t log AP vs log HVBF y =.51661x * r =.54t log NORA vs log HVBF y = x * r =.54t log OHA vs log HVBF y =.799lx * r- =.62t log HMA vs log HVBF y = x +.348* r =.59t AP = antipyrine, NORA = norantipyrine, HMA = 3-OH-methylantipyrine, OHA = 4-OH-antipyrine, HVBF = hepatic vein blood flow. *Slope significantly different from 1, tp <.1; +P <.5. References 1 Caesar J, Shaldon S, Chiandussi L, Guevara L, Sherlock S. The use of indocyanine green in the measurement of hepatic blood flow and as a test of hepatic function. Clin Sci 1961; 21: Leevy CM, Mendenhall CL, Lesko W, Howard MM. Estimation of hepatic blood flow with indocyanine green. J clin Invest 1961; 41: Bradley SE, Ingelfinger FJ, Bradley GP. Hepatic circulation in cirrhosis of the liver. Circulation 1952; 5: Pessayre D, Lebrec D, Descatoire V, Peignoux M, Benhamou J-P. Mechanism for reduced drug clearance in patients with cirrhosis. Gastroenterology 1978; 74: Reiss WG, Bauer LA, Horn JR, Zierler JR, Easterling TR, Strandness DE. The effects of oral nifedipine on hepatic blood flow in man. Clin Pharmac Ther 1991; 5: O'Sullivan TA, Bauer LA, Horn JR, Zierler BK, Strandness DE, Williams-Warren J, Smith AL, Unadkat JD. Disposition of drugs in cystic fibrosis III-hepatic blood flow. Clin Pharmac Ther 1991; 5: Bauer LA, McDonnell N, Horn JR, Zierler B, Opheim K, Strandness DE. Single and multiple doses of oral cimetidine do not change liver blood flow in humans. Clin Pharmac Ther 199; 48; Horn JR, Zierler B, Bauer LA, Reiss W, Strandness DE. Estimation of hepatic blood flow in branches of the hepatic vessels utilizing a non-invasive, duplex Doppler method. J clin Pharmac 199; 3: Poulsen HE, Loft S. Antipyrine as a model drug to study hepatic drug-metabolizing capacity. J Hepatology 1988; 6: Park BK, Kitteringham NR. Assessment of enzyme induction and enzyme inhibition in humans-toxicological implications. Xenobiotica 199; 2: Teunissen MWE, Spoelstra P, Koch CW, Weeda B, can Duyn W, Janssens AR, Breimer DD. Antipyrine clearance and metabolite formation in patients with alcoholic cirrhosis. Br J clin Pharmac 1984; 18: Schellens JHM, Janssens AR, van der Wart JHF, van der Velde EA, Breimer DD. Relationship between the metabolism of antipyrine, hexobarbital, and theophylline in patients with liver disease as assessed by a 'cocktail' approach. Eur J clin Invest 1989; 19: Pugh RNH, Murray-Lyon IM, Dawson JL, Pietroni MC, Williams R. Transection of the oesophagus for bleeding oesophageal varices. Br J Surg 1973; 6: Weber A, Opheim K, Smith AL. Simplified high performance liquid chromatographic quantitation of antipyrine. J Chromatograph Sci 1984; 22: Danhof M, van Zuilen A, Boeijinga JK, Breimer DD. Studies of the different metabolic pathways of antipyrine in man-oral versus IV administration and the influence of urinary collection time. Eur J clin Pharmac 1982; 21: Wagner JG. Model-independent, linear pharmacokinetic equations allowing direct calculation of many needed pharmacokinetic parameters from the coefficients and exponents of simple polyexponential equations which have been fitted to the data. J Pharmacokin Biopharm 1976; 4: Teunissen MWE, Meerburg-van er Torren JE, Vermeulen NPE, Breimer DD. Automated HPLC-determination of antipyrine and its main metabolites in plasma, saliva, and urine, including 4-4'-dihydroxyantipyrine. J Chromatogr Biomed Appl 1983; 278: Danhof M, de Groot-van der Vis E, Breimer DD. Assay of antipyrine and its primary metabolites in plasma, saliva, and urine by high pressure liquid chromatography and preliminary results in man. Pharmacology 1978; 18: DuBois D, Du Bois EF. The measurement of the surface of man. Arch intern Med 1916; 15: Rowland M, Benet LZ, Graham GG. Clearance concepts in pharmacokinetics. J Pharmacokin Biopharm 1973; 1: Wilkinson GR, Shand DG. A physiological approach to hepatic drug clearance. Clin Pharmac Ther 1975; 18: Andreasen PB, Ranek L, Statland BE, Tygstrup N. Clearance of antipyrine-dependence of quantitative liver function. Eur J clin Invest 1974; 4: Branch RA, James JA, Read AE. The clearance of antipyrine and indocyanine green in normal subjects and in patients with chronic liver disease. Clin Pharmac Ther 1976; 2: Klotz U, Fischer C, Muller-Seydlitz P, Schulz J, Muller WA. Alterations in the disposition of differently cleared drugs in patients with cirrhosis. Clin Pharmac Ther 1979; 26: Lorber SH, Oppenheimer MJ, Shay H, Lynch P, Siplet

7 Antipyrine kinetics and liver bloodflow in hepatic disease 381 H. Enterohepatic circulation of bromsulphthaleinintraduodenal, intraportal and intravenous dye administration in dogs. Am J Physiol 1953; 173: Schellens JHM, Van Der Wart JHF, Danhof M, Van Der Velde EA, Breimer DD. Relationship between the metabolism of antipyrine, hexobarbitone, and theophylline in man as assessed by a 'cocktail' approach. Br J clin Pharmac 1988; 26: Danhof M, Breimer DD. Studies on the different metabolic pathways of antipyrine in man-i. Oral administration of 25, 5, and 1 mg to healthy volunteers. Br J clin Pharmac 1979; 8: Teunissen MWE, Srivastava AK, Breimer DD. Influence of sex and oral contraceptive steroids on antipyrine metabolite formation. Clin Pharmac Ther 1982; 32: (Received 13 May 1993, accepted 16 November 1993)