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P157 interaction of verapamil and cimetidine in human liver microsomes
Posters / European Journal of Pharmaceutical Sciences 2 (1994) 117-194
158
P157 INTERACTION OF VERAPAMIL AND CIMETIDINE IN HUMAN LIVER MICROSOMES
U. Fischer1, R. Wacke1, F.-P.Nitschke2, B. Drewelow1 1 ClinicalPharmacology,Instituteof PharmacologyandToxicology,Universityof Restock,18055Restock,Germany 2 TransplantationCenter,Clinicof Urology,Universityof Restock,18055Rostesk,Germany Studies concerning the interaction of Verapamil - a high clearance calcium channel blocker undergoing extensive metabolism by Cytochrom P 450 and the H2-antargonist contradictory
results.
Cimetitidine
Attemps
to
and its clinical relevance give
explain the
extent
of Verapamil
metabolism by Cimetidine seen during in vitro and in vivo experiments using
K i- and IC50-values were not close agreement with
clinical
observations. The aim of our study was to find out wether the intrinsic clearance
P158 EFFECT OF TEMPERATURE ON THE DISTRIBUTION OF BARBITURATE IN ISOLATED PERFUSED RAT LIVER
C.-H.Chou,M. Rowland Departmentof Pharmacy,UniversityofManchester,ManchesterM139PL,U.K.
There is an increasing interest in the influence of hypothermia on drug disposition and cellular functions (z). In the present study, the distribution kinetics of 5-methyl-5-ethyl barbituric acid (C1) in the isolated perfused rat liver ( n = 4 ) at different temperatures (36, 20, 15 and 10°C) was examined using the multipleindicator dilution technique. The liver was perfused with ~rotein-free Krebs bicarbonate medium at 15ml/min in a single-pass mode. :4C-Sucrose (extracellular marker), all-water (cellular marker) and C1 were injected simultaneously into the portal vein, and the outflow profiles were observed. The profiles of sucrose and water, and hence the respective spaces were unaffected by temperature. However, temperature dramatically affected the profiles of C1 (Fig.l). The distribution kinetics of C1 shifts from a perfusion-rate limitation at 36°C toward a barrier-rate limitation at temperature below 20°C. 0.07
(Clin t = Vma x / Km) is more suitable to descdbe the above findings.
0.06-
Therefore microsomes derived from 8 preparations of 4 human livers (not
0.05-
accepted for transplantation) were incubated with Verapamil in presence of
0.04-
increasing concentrations of Cimetidine (5, 10, 25, 50 und 100 pM) in a
o.o3-
NADPH regenerated system. We quantified the formation of the verapamil
0.02-
metabolites Norverapamil, D702, D 703 and D 617 with and without
0.010~
Cimetidine and determined Vma x, K m and Clin t. It was observed a Cimetidine concentration dependent inhibition of verapamil metabolism. The decline of Clin t amounted to 10 - 20 % for 25 pM and 40 - 55 % for 100 pM Cimetidine respectivly. These findings are in accordance with in vivo observations in human regarding the degree of inhibition of verapamil metabolite formation by Cimetidine. This approach taking into account the intrinsic clearance model seems to be suitable for in vitro prediction of drug interactions at the level of the cytochrom P 450 mediated metabolism. P159 ESTIMATION OF VASCULAR, EXTRAVASCULAR AND INTRACELLULAR DISTRIBUTION SPACES IN THE DUAL PERFUSED RAT LIVER
S. Sahin,M. Rowland Departmentof Pharmacy,Universityof Manchester,ManchesterM139PL,U.K.
Most work involving the perfuscd liver have been concerned with single input via the portal vein (PV). Yet, physiologically the liver receives a dual blood supply, PV and the hepatic artery (HA), each with the potential for specific associated aqueous distribution spaces. The indicator dilution technique ~ was employed to determine the vascular, extravascular and intracellular distribution spaces of the liver perfused through both PV and HA. The in situ rat livers (n = 7 : I0) were perfused dually using protein-free Krebs-bicarbonate solution. The markers (red blood cells, t2Slalbumin, ~4C-sucrose, t"C-urea, 3H-water) were injected in a random order into either the HA or PV. The frequency outflow profiles were analyzed using statistical moment theory. 0. 025 ¢~
0.02
.. ~/~ 1'0 2'0 3'0 4'0 5'0 6'0 7'0 8'0 9t0 100
T(secI Fig.1
Temperature
dependence of hepatic efflux curves.
The data were analyzed using the dispersion model (2). The one-compartment model adequately described the profiles at 36°C, whereas the two-compartment form, which incorporates a cellular barrier, provided a better description of the data at lower temperatures. The estimated influx rate constants for 10, 15, 20°C are 0.17-+ 0.01, 0.37±0.03, 0.59±0.18 see't; the corresponding efflux rate constants are 0.05-+ 0.01, 0.08 ± 0.Ol and 0.15-+ 0.04 sec;t. The distribution volume of C1 (0.76 ± 0.06 ml/g liver) are similar at these temperatures. These data are consistent with an activation energy of 67 KJ/mol for hepatocyte permeability of C1. (1) Buylaert W A et al, Clln Pha~macokin 17, 10-26, 1989. (2) Chou C-H et al, Drug Metab Disp 21, 933-938, 1993. P160 DISTRIBUTION KINETICS OF SALICYLIC ACID IN ISOLATED PERFUSED CIRRHOTIC RAT LIVER
K. Nasseri,M. Rowland Departmentof Pharmacy,Universityof Manchester,ManchesterM139PL,U.K. Department of Pharmacy.University of Manchester, Manchester, U.K. M 13 9PL Many factors influence hepatic clearance including disease. Although the effect of altered blood flow, protein binding and enzyme activity induced by disease on clearance has been well studied, relatively little is known about changes in membrane permeability. We have examined this factor with salicylic acid(SA), a relatively poorly permeable compound, in the in situ isolated perfused liver (IPRL) of an experimental model of cirrhosis. 20 male Sprague-Dawley rats were used as liver donors. Proctor method Ill was adopted for induction of cirrhosis in 10 animals. The second group served as weight matched controls. The single-pass IPRL was employed using Krebs buffer at a flow rate calculated according to liver weight. Single bolus tracer doses of C14-SA and radiolabeled references (RBC, albumin, sucrose, water and urea) were injected into the portal vein and hepatic output was collected for up to 3 min. Hepatic cirrhosis was confirmed by histology in 70% of treated rats. Liver viability was assessed by gross apoarance, perfusion flow and pH, bile recovery, and oxygen consumption. AUC, %recovery, mean transit time (MTT) and volume of distribution (V) of each solute was calculated using statistical moment analysis[2]. Recovery of SA was higher than 90% with no difference between cirrhotic (CR) and control (C) livers. Figure shows that SA
0.015
o.o1
2.5
0.005 0 0
20
40
60
80
1' L~ 100 120
'~ 140
, 160
180
200
T(sec) Piq.1 Hepatic outflow prof£1es after HA and PV injections. Figure 1 shows a typical outflow profile of urea following HA and PV injections in the same liver preparation. The mean transit time and hence the volume of distribution, were larger after HA injection than after PV injection. These data provided the following estimates for vascular, interstitial (extravascular space - vascular space) and cellular (total water space extravascular space) spaces, respectively: HA -0.228,0.094,0.577ml/g liver; PV - 0.157,0.069,0.420 ml/g liver, respectively. These data indicate that HA and PV flows do not share completely a common space within the liver. Therefore, the route of delivery of drugs to the liver may affect their disposition in the organ.
Ipang. K.S. ¢t al, J . P h a ~ c o k i m a i o p h a ~ . , [ 6 , 5 9 5 -
603.]988.
o~
2
(meanvalue:~SD) MTI"(s) CV2 V (mUgliver)
48±10 2500"2:800 1.2~0.5
100~30 4800f.2000 2.5~0.7
d if_
°"o
0 50 100 150 r~(,) 200 elutes much faster from CR than C livers.This was associated with a much shorter MTF. While in part this is explained by a smaller V in CR livers, the permeability of CR livers was also much lower, as reflected by a smaller CV2, a measure of spreading. This decrease in permeability is consistant with an increase in fibrosis and collagen deposits in the Disse space associated with cirrhosis[3]. 1. Proctor,E. ; Chatamra,K. Gastroenterology 83:1183-90 (1982) 2. Hussain,Z.; McLachlan,A.; Rowland,M. Pbarm Res.(In press) 3. Varin,E; Huet,P.M.J.Clin. Invest. 76:1904-1912(1985)
158
P157 INTERACTION OF VERAPAMIL AND CIMETIDINE IN HUMAN LIVER MICROSOMES
U. Fischer1, R. Wacke1, F.-P.Nitschke2, B. Drewelow1 1 ClinicalPharmacology,Instituteof PharmacologyandToxicology,Universityof Restock,18055Restock,Germany 2 TransplantationCenter,Clinicof Urology,Universityof Restock,18055Rostesk,Germany Studies concerning the interaction of Verapamil - a high clearance calcium channel blocker undergoing extensive metabolism by Cytochrom P 450 and the H2-antargonist contradictory
results.
Cimetitidine
Attemps
to
and its clinical relevance give
explain the
extent
of Verapamil
metabolism by Cimetidine seen during in vitro and in vivo experiments using
K i- and IC50-values were not close agreement with
clinical
observations. The aim of our study was to find out wether the intrinsic clearance
P158 EFFECT OF TEMPERATURE ON THE DISTRIBUTION OF BARBITURATE IN ISOLATED PERFUSED RAT LIVER
C.-H.Chou,M. Rowland Departmentof Pharmacy,UniversityofManchester,ManchesterM139PL,U.K.
There is an increasing interest in the influence of hypothermia on drug disposition and cellular functions (z). In the present study, the distribution kinetics of 5-methyl-5-ethyl barbituric acid (C1) in the isolated perfused rat liver ( n = 4 ) at different temperatures (36, 20, 15 and 10°C) was examined using the multipleindicator dilution technique. The liver was perfused with ~rotein-free Krebs bicarbonate medium at 15ml/min in a single-pass mode. :4C-Sucrose (extracellular marker), all-water (cellular marker) and C1 were injected simultaneously into the portal vein, and the outflow profiles were observed. The profiles of sucrose and water, and hence the respective spaces were unaffected by temperature. However, temperature dramatically affected the profiles of C1 (Fig.l). The distribution kinetics of C1 shifts from a perfusion-rate limitation at 36°C toward a barrier-rate limitation at temperature below 20°C. 0.07
(Clin t = Vma x / Km) is more suitable to descdbe the above findings.
0.06-
Therefore microsomes derived from 8 preparations of 4 human livers (not
0.05-
accepted for transplantation) were incubated with Verapamil in presence of
0.04-
increasing concentrations of Cimetidine (5, 10, 25, 50 und 100 pM) in a
o.o3-
NADPH regenerated system. We quantified the formation of the verapamil
0.02-
metabolites Norverapamil, D702, D 703 and D 617 with and without
0.010~
Cimetidine and determined Vma x, K m and Clin t. It was observed a Cimetidine concentration dependent inhibition of verapamil metabolism. The decline of Clin t amounted to 10 - 20 % for 25 pM and 40 - 55 % for 100 pM Cimetidine respectivly. These findings are in accordance with in vivo observations in human regarding the degree of inhibition of verapamil metabolite formation by Cimetidine. This approach taking into account the intrinsic clearance model seems to be suitable for in vitro prediction of drug interactions at the level of the cytochrom P 450 mediated metabolism. P159 ESTIMATION OF VASCULAR, EXTRAVASCULAR AND INTRACELLULAR DISTRIBUTION SPACES IN THE DUAL PERFUSED RAT LIVER
S. Sahin,M. Rowland Departmentof Pharmacy,Universityof Manchester,ManchesterM139PL,U.K.
Most work involving the perfuscd liver have been concerned with single input via the portal vein (PV). Yet, physiologically the liver receives a dual blood supply, PV and the hepatic artery (HA), each with the potential for specific associated aqueous distribution spaces. The indicator dilution technique ~ was employed to determine the vascular, extravascular and intracellular distribution spaces of the liver perfused through both PV and HA. The in situ rat livers (n = 7 : I0) were perfused dually using protein-free Krebs-bicarbonate solution. The markers (red blood cells, t2Slalbumin, ~4C-sucrose, t"C-urea, 3H-water) were injected in a random order into either the HA or PV. The frequency outflow profiles were analyzed using statistical moment theory. 0. 025 ¢~
0.02
.. ~/~ 1'0 2'0 3'0 4'0 5'0 6'0 7'0 8'0 9t0 100
T(secI Fig.1
Temperature
dependence of hepatic efflux curves.
The data were analyzed using the dispersion model (2). The one-compartment model adequately described the profiles at 36°C, whereas the two-compartment form, which incorporates a cellular barrier, provided a better description of the data at lower temperatures. The estimated influx rate constants for 10, 15, 20°C are 0.17-+ 0.01, 0.37±0.03, 0.59±0.18 see't; the corresponding efflux rate constants are 0.05-+ 0.01, 0.08 ± 0.Ol and 0.15-+ 0.04 sec;t. The distribution volume of C1 (0.76 ± 0.06 ml/g liver) are similar at these temperatures. These data are consistent with an activation energy of 67 KJ/mol for hepatocyte permeability of C1. (1) Buylaert W A et al, Clln Pha~macokin 17, 10-26, 1989. (2) Chou C-H et al, Drug Metab Disp 21, 933-938, 1993. P160 DISTRIBUTION KINETICS OF SALICYLIC ACID IN ISOLATED PERFUSED CIRRHOTIC RAT LIVER
K. Nasseri,M. Rowland Departmentof Pharmacy,Universityof Manchester,ManchesterM139PL,U.K. Department of Pharmacy.University of Manchester, Manchester, U.K. M 13 9PL Many factors influence hepatic clearance including disease. Although the effect of altered blood flow, protein binding and enzyme activity induced by disease on clearance has been well studied, relatively little is known about changes in membrane permeability. We have examined this factor with salicylic acid(SA), a relatively poorly permeable compound, in the in situ isolated perfused liver (IPRL) of an experimental model of cirrhosis. 20 male Sprague-Dawley rats were used as liver donors. Proctor method Ill was adopted for induction of cirrhosis in 10 animals. The second group served as weight matched controls. The single-pass IPRL was employed using Krebs buffer at a flow rate calculated according to liver weight. Single bolus tracer doses of C14-SA and radiolabeled references (RBC, albumin, sucrose, water and urea) were injected into the portal vein and hepatic output was collected for up to 3 min. Hepatic cirrhosis was confirmed by histology in 70% of treated rats. Liver viability was assessed by gross apoarance, perfusion flow and pH, bile recovery, and oxygen consumption. AUC, %recovery, mean transit time (MTT) and volume of distribution (V) of each solute was calculated using statistical moment analysis[2]. Recovery of SA was higher than 90% with no difference between cirrhotic (CR) and control (C) livers. Figure shows that SA
0.015
o.o1
2.5
0.005 0 0
20
40
60
80
1' L~ 100 120
'~ 140
, 160
180
200
T(sec) Piq.1 Hepatic outflow prof£1es after HA and PV injections. Figure 1 shows a typical outflow profile of urea following HA and PV injections in the same liver preparation. The mean transit time and hence the volume of distribution, were larger after HA injection than after PV injection. These data provided the following estimates for vascular, interstitial (extravascular space - vascular space) and cellular (total water space extravascular space) spaces, respectively: HA -0.228,0.094,0.577ml/g liver; PV - 0.157,0.069,0.420 ml/g liver, respectively. These data indicate that HA and PV flows do not share completely a common space within the liver. Therefore, the route of delivery of drugs to the liver may affect their disposition in the organ.
Ipang. K.S. ¢t al, J . P h a ~ c o k i m a i o p h a ~ . , [ 6 , 5 9 5 -
603.]988.
o~
2
(meanvalue:~SD) MTI"(s) CV2 V (mUgliver)
48±10 2500"2:800 1.2~0.5
100~30 4800f.2000 2.5~0.7
d if_
°"o
0 50 100 150 r~(,) 200 elutes much faster from CR than C livers.This was associated with a much shorter MTF. While in part this is explained by a smaller V in CR livers, the permeability of CR livers was also much lower, as reflected by a smaller CV2, a measure of spreading. This decrease in permeability is consistant with an increase in fibrosis and collagen deposits in the Disse space associated with cirrhosis[3]. 1. Proctor,E. ; Chatamra,K. Gastroenterology 83:1183-90 (1982) 2. Hussain,Z.; McLachlan,A.; Rowland,M. Pbarm Res.(In press) 3. Varin,E; Huet,P.M.J.Clin. Invest. 76:1904-1912(1985)