Pharmacokinetics of Diazepam in the Rat: Influence of a Carbon Tetrachloride-Induced Hepatic Injury JUAN M. DiAZ-GARCIA'*, JESUSOLIVER-BOTANA', AND DANIELFOS-GALVE" Received Au ust 6, 1990, from 'Depaflamento Fannacia y Tecnologia FannacBuHca, Facultad de Fannacia, Univenidad de Navarra, *Present address: Department of Pharmacy, University of Manchester, Accepted for publication August 7, 1991. 31008 Pampkna, Spain. Manchester M13 9PL, U.K. Abotracl 0 The pharmacokineticsof diazepam in normal rats and in rats pretreated with carbon tetrachloride to induce hepatic Cirrhosis (Cirrhotic rats) was studied after intravenous and oral administration of the drug (4
mg/kg). Animals pretreated with this hepatotoxic agent showed a significant prolongation in the half-life of diazepam in plasma that is due more to an increase in volume of distribution rather than to a decrease in clearance. This study confirmed that diazepam was highly extracted by the rat liver and was not affected by the hepatotoxic agent, although
there probably was a saturation of the activity of the cytochrome, P enzyme when the drug was administered orally. Diazepam binds to plasma proteins to a high degree in both normal and cirrhotic rats: however, in the latter, a significant increase in the fraction of unbound drug in plasma was observed. Pretreatment of rats with carbon tetrachloridedid not produce any change either in the distribution of diazepam into erythrocytes or in the disposition of the metabolite desmethyldiazepam.
Diazepam is a benzodiazepinederivative that is extensively used clinically as a n anticonvulsant, anxiolytic, and muscle relaxant and in the treatment of acute alcohol withdrawal. Its metabolites, desmethyldiazepam, temazepam, and oxazepam, are pharmacologically active. The pharmacokinetics of diazepam in humans are known to be altered in liver disease states.' Absorption of the drug after oral administration is diminished when liver cirrhosis develops. In this case, the volume of distribution increases significantly primarily because of a decrease in binding to plasma proteins, and clearance from plasma is reduced to 50% of its normal value. The increase in half-life in plasma is more marked in patients with cirrhotic livers than in patients with acute viral hepatitis. There are many studies about the pharmacokinetic behavior of diazepam in normal rats. Values for many pharmacokinetic parameters have been reported,s7 but there are striking differences among some of them. The pharmacokinetics of diazepam in rats with an experimentally induced liver disease have not been studied extensively. Trennery and Warin@ reported a longer half-life in rats with thioacetamide-induced hepatic cirrhosis, possibly because of the shorter duration of the distribution phase; they also found a significant reduction in the clearance of diazepam from blood. The extraction ratio of diazepam across the liver (E), is a well-studied pharmacokinetic parameter. However, there is a wide discrepancy among the reported results.2.6.8.@This important parameter can predict the extent to which changes in binding to protein, hepatic blood flow, or hepatocellular enzyme capacity will affed the hepatic clearance of diazepam.10 Oral administration of the hepatotoxic agent carbon tetrachloride (CCl,) a t a dose of 0.02 mU100 g leads to a cirrhotic condition in the rat." Liver damage was therefore induced in rats with CCl, as a model to study factors affecting the pharmacokinetic behavior of diazepam in animals with liver disease. 768 I Journal of Phannaceutical Sciences Vol. 81, No. 8, August 1992
Experimental Section Animals-Male Wistar rats were divided in nine groups. Groups I and 111 (10 rats each) were used as controls. Diazepam was administered intravenously (iv) to rats in group I and orally to those in group 111. In groups I1 and IV (10 rats each), CCl, was previously administered. Diazepam was administered iv to rats in group II and orally to those in group IV.Group V (15 control rats) was used for binding studies. Group VI included 15 rats with CC1,-induced liver damage and was used also for binding studies. Groups VII and VIII (6 rats each) were used to study the distribution of diazepam into erythrocytes. The 10 rats in group IX were used to characterize the hepatic damage caused by CCl, [activities of aspartate aminotransferaee (GOT) and alanine aminotransferaee (GF'T) in serum were measured 48 h after administration of CCl, (0.02 mu100 g); blood was collected by retroorbital plexus puncture]. Weights of animals in all g r o u p ranged between 246 and 259 g. Diazepam (4 mgkg) was administered iv through the right jugular vein of animals under light ether anesthesia. The drug was dissolved in a solution of propylene glyco1:benzyl alcoho1:polyethylene glycol 250 (2:20:78, wlw). Blood was collected from the left jugular vein of animals at 2,5, 15,30,60,90, 120, 180, and 240 min after diazepam adminiatration in control rats and, in addition, at 300 and 360 min after diazepam administration in injured rats. When the drug was administered orally (4 mgkg), diazepam was dissolved in the aforementioned solution and blood was collected from the left jugular vein of animals at 10,15,30,45,60,90,120,180, and 240 min after the administration of diazepam in control rats and, in addition, at 300 and 360 min in injured rats. Blood samples (-400 &) were immediately centrifuged at 3400 rpm for 20 min, and plasma was kept frozen a t -25 "C until analysis. In all cases, blood was replaced with a n equal volume of saline. Induction of Liver Damage-CCl, was orally administered to rats at a dose of 0.02 mU100 g, the cirrhotic condition being characterized by measurement of activities of GOT and GPT in serum with specific kits (Monotest GOT no. 124362 and Monotest GPT no. 124524, Boehringer Mannheim GmbH). Chemicals-All fine chemicals were obtained from Scharlau (Barcelona, Spain). Other chemicals were either generally available or purchased from Panreac (Barcelona, Spain). Diazepam and medazepam were supplied by Barcia (Barcelona, Spain) and Impex Qufmica (Barcelona, Spain), respectively. Desmethyldiazepam was generously provided by Roche (Madrid, Spain). AnalysigConcentrations of diazepam and desmethyldiazepam in blood and plasma were determined by electron-capture gas chromatography according to the method described by Laescher.12A HewlettPackard gas chromatograph (model 5890A) equipped with a s3Ni electron capture detector and silanized glass tubing (2 m x 4 mm id.) that was packed with 50:50 methylsi1icone:phenylsilicone (SP2250 on Supelcoport; 80/100 mesh) was used. Temperature settings were 290 "C for the oven and 320 "C for the injector and detector. Argonmethane was used as the carrier gas at a flow rate of 50 mumin, and the volume of injection was 4 &. Medazepam was used as internal standard, and benzene was used as the extraction solvent. Standard curves for diazepam were produced over the concentration range 31.25-1500 ng/mL of plasma or blood. The intraday coefficients of variation for diazepam at 31.25 ng/mL were 3.58 and 7.51% in plasma and blood, respectively. For diazepam at 1500 ng/mL, the coefficients of variation were 0.16 and 1.34% in plasma and blood, respectively. In addition, standard curves for desmethyl0022-3549/92/0800-0768$02.68$02.50/0 0 1992, American Pharmaceutical Association
diazepam in plasma were produced over the same concentration range. The intraday coefficients of variation were 7.60% (31.25 ng/mL) and 1.06% (1500ng/mL). Recoveries for diazepam, desmethyldiazepam, and medazepam either from blood or plasma were >96%. In plasma and blood, the limit of detection was 28 ng/mL for diazepam and 20 ng/mL for desmethyldiazepam. Although the gas chromatographic asmy detected other metabolites of diazepam (temazepam and oxazepam), the levels of these metabolites in blood or plasma detected after diazepam administration were not quantifiable. Pharmacokinetic Analysis-Biexponential equations were fitted to data with both JANA13 and PCNONLIN" programs. Criteria for discriminating between alternative polyexponential models16 were the information provided by the PCNONLIN program, Akaike criterion, and F ratio test.18 Pharmacokinetic parameters were estimated by conventional methods. Binding to Plaema Protein-Experiments on the binding of diazepam to plasma protein were carried out in vitro by equilibrium dialysis with the DIANORM (Diachema AG, Zurich) system. Aliquots (1 mL) of different concentrations of diazepam in plasma (1300,1000, 500, 250, and 125 ng/mL) were dialyzed against 1 mL of 0.1 M phosphate buffer (pH 7.4). Plasma was obtained by centrifugation (4000 rpm for 20 min) of blood collected by direct heart puncture from rats in groups V and VI. The albumin concentration in each sample was determined by using the method of Doumas et al.17 Distribution of Diazepam into Erythrocytes-Two studies were performed to assess the distribution of diazepam into erythrocytes. In the in vivo assay, diazepam (4 mgkg) was administered iv to three rats in group VII or VIII, and blood was collected after 2,15,30,and 60 min. Then, 200 4 of blood was taken from each sample; the remaining blood was centrifuged, and 200 of plasma was taken. These aliquots of plasma and blood were analyzed for diazepam. In the in vitro m a y , blood was collected directly by heart puncture in three rats in groups VII and VIII. Four aliquots (1 mL) of blood were transferred into glass centrifuge tubes and preincubated for 5 min at 37 "C. Different amounts of diazepam (1000,500,250, and 125 ng) were added, and the aliquots were incubated at 37 "C for 20 min. Then, 200 4 of blood and 200 p L of plasma were analyzed for diazepam. Hematocrit was estimated in duplicate for each animal by using a microhematomit centrifuge (GRI-CELL; 12 000 rpm). The blood-to-plasma ratio (BPR) was taken to be the concentration of diazepam in blood divided by that in plasma. Estimation of EH and the AfRnity of Diazepam for Erythrocytes ( y W H was indirectly estimated with eq 1 for iv administration and eq 2 for oral administration of diazepam:10
In these equations, QH is the hepatic blood flow (QH= 15 mIJminlB), CLHis hepatic clearance, AUC,, and AUC,, are the areas under the curyes of the concentrations of diazepam in plasma versus time after oral and iv administration of the drug, respectively, and F is the oral bioavailability. (It is assumed that diazepam completely traverses the gastrointestinal wall and no other organ, except the liver, significantly contributes to the elimination of the drug from the body.) y was calculated with eq 3: y=
(BPR- 1 + H)/Hf,
(3)
In eq 3,H is the hematocrit in the rat andf,, is the fraction of unbound drug in plasma. Statistical Method-Data were statistically analyzed by the conventional two-tailed t test.
Table C O O T and GPT Actlvltlei In !%rum'
Rat 1 2 3 4 5 6 7 8 9 10
Induction of Hepatic Iqjury-The activities of GOT and GPT in serum prior to the administration of CC1, to rats in group IX are statistically significantly (p < 0.01)lower than those determined 48 h after administration of the hepatotoxic agent (Table I). Pharmacokinetic Profile of Diazepam in Control and
48h
Oh
48 h
62 55
325 328 330 357 302 292 329 332 299 376 327 (26)
22
122 119 143 138 117 106 124 120 128 110 123 (11)
53 63 58
60 53 69 72
50
23 18 18 23 23 22 28 30 25 22 (6)
Mean (SD)b 60 (7) 'Determined prior to and 48 h after administration of 0.05 mL of CCI, to rats in group IX. SD, Standard deviation.
Injured Rats-The mean concentrations of diazepam in plasma after iv or oral doses in groups I-IV are shown in Figure 1. When diazepam was administered iv to control and injured rats, a biexponential function of the following form was fitted to the experimental data: (4)
In eq 4, C is the concentration of diazepam, C, and C2 are the coefficients of the exponential terms, A, is the rate constant of distribution, A2 is the terminal slope of the curve, and t is time. After oral administration of the drug to control or injured rats, a satisfactory convergent fit of triexponential equations to the data points could not be obtained; therefore, a biexponential function was used:
In eq 6,I = FDk,N(k, - k),A! is the terminal slope of the curve, k, is the adsorption constant, D is the administered dose, and V is the volume of distribution. The mean pharmacokinetic parameters for diazepam in the rat are presented in Tables 11 and III. Statistical analysis of data obtained after iv administration of diazepam indicated that A, was significantly higher (p < 0.01)and & significantly lower (p < 0.01)in rats with hepatic failure compared with healthy controls. These changes are reflected in a more pronounced biexponential curve in the injured rats (Figure 1). The only other parameter that Table ICMean PhannacoklnetlcParameters of Diazepam In the Rat after Iv Admlnlstratlon'
Parameter A,, h-' t1n.*,*hb AZ1 h-' t1n.m
hC
qo),ng/mLd
Results
GPT, units/L
GOT, units/L Oh
Vdi, mg Vd, mg' Vd,, Ukg CL, L * h-' * kg-' AUC, ng . h * mL-'
Value Control Rats
Injured Rats
3.21 t 1.26 0.22 (0.13-0.35) 0.60 k 0.11 1.15 (0.91-1.50) 1275 k 90 3.15 t 0.23 7.10 t 1.31 5.21 t 0.73 4.22 t 0.76 976 t 170
6.01 2 3.07 0.12 (0.05-0.19) 0.44 2 0.04 1.60 (1.43-1.91) 1219 t 130 3.32 t 0.37 8.53 t 1.87 7.39 t 1.33 3.75 t 0.94 1114 t 216
'Results are reported as mean * SD, except for half-lives,which are reported as harmonic mean (and range). Distribution half-life. Terminal half-life. Concentration at 4. 'Terminal volume of distribution. Journal of Pharmaceutical Sciences I 769 Vol. 81, No. 8, August 7992
Table IIl-Mean Pharmacoklndlc Parameter8 of Diazepam In the Ral after Oral Admlnlalratlon'
Value
Parameter
h
-.I
Control Rats
Injured Rats
4.72 ? 0.42 0.15 (0.13-0.18) 0.63 2 0.08 1.10 (0.64-1.28) 293 ? 16 0.50 ? 0.03 642 2 75
5.14 2 0.17 0.14 (0.13-0.15) 0.45 2 0.02 1.52 (1.42-1.62) 330?9 0.52 ? 0.01 921 ? 30
E
\
m
k , h-'
E
v
t,n..b.t hb
k, h-1
V
t1n.m h
C-, ng/mL Laxl hC AUC, ng h * mL-'
-
Results are reported as mean 2 SD, except for half-lives, which are reported as harmonic mean (and range). bHalf-life of the absorption process. cTime to reach CmU.
lB
t
o 0
l
. 1
. 1
.
. . .
9
4
I
1
6
0
t (h)
changed significantly was the volume of distribution at steady state (Vd,; Table 11). ARer oral administration, the maximum concentration of diazepam (C,,,& the absorption constant (Ita), and AUC,, were all significantly greater in injured animals (p < 0.01; Table III). The half-life (tl,.J values for desmethyldiazepam observed after either oral or iv administration of diazepam to group I or 11are presented in Table IV.Desmethyldiazepam concentrations in plasma were much lower than those of diazepam. This metabolite was the only one detected in plasma in quantifiable amounts. It seems that the appearance of desmethyldiazepam in plasma is very rapid, because in all cases, no defined maximum concentrations were observed. Metabolite concentrations in plasma were higher when diazepam was administered orally to rate. Thue, 2 min after the iv administration of the parent drug to normal rats, the mean concentration of desmethyldiazepam in plasma was 119 ng/mL compared with a value of 116 ng/mL observed 10 min after the oral administration of diazepam. In rate with hepatic failure, desmethyldiazepam concentrations in plasma reached values of 114 and 167 ng/mL at 2 and 10 min after iv and oral administration of diazepam, respectively. Binding of Diazepam to Plasma Protei&ignificant differences have been found in the binding of diazepam to plasma proteins. The amount of drug bound to these proteins was 96.4 0.9%inconhlratsand90.1? 2.5%ininjuredrats(p
mg/kg). Animals pretreated with this hepatotoxic agent showed a significant prolongation in the half-life of diazepam in plasma that is due more to an increase in volume of distribution rather than to a decrease in clearance. This study confirmed that diazepam was highly extracted by the rat liver and was not affected by the hepatotoxic agent, although
there probably was a saturation of the activity of the cytochrome, P enzyme when the drug was administered orally. Diazepam binds to plasma proteins to a high degree in both normal and cirrhotic rats: however, in the latter, a significant increase in the fraction of unbound drug in plasma was observed. Pretreatment of rats with carbon tetrachloridedid not produce any change either in the distribution of diazepam into erythrocytes or in the disposition of the metabolite desmethyldiazepam.
Diazepam is a benzodiazepinederivative that is extensively used clinically as a n anticonvulsant, anxiolytic, and muscle relaxant and in the treatment of acute alcohol withdrawal. Its metabolites, desmethyldiazepam, temazepam, and oxazepam, are pharmacologically active. The pharmacokinetics of diazepam in humans are known to be altered in liver disease states.' Absorption of the drug after oral administration is diminished when liver cirrhosis develops. In this case, the volume of distribution increases significantly primarily because of a decrease in binding to plasma proteins, and clearance from plasma is reduced to 50% of its normal value. The increase in half-life in plasma is more marked in patients with cirrhotic livers than in patients with acute viral hepatitis. There are many studies about the pharmacokinetic behavior of diazepam in normal rats. Values for many pharmacokinetic parameters have been reported,s7 but there are striking differences among some of them. The pharmacokinetics of diazepam in rats with an experimentally induced liver disease have not been studied extensively. Trennery and Warin@ reported a longer half-life in rats with thioacetamide-induced hepatic cirrhosis, possibly because of the shorter duration of the distribution phase; they also found a significant reduction in the clearance of diazepam from blood. The extraction ratio of diazepam across the liver (E), is a well-studied pharmacokinetic parameter. However, there is a wide discrepancy among the reported results.2.6.8.@This important parameter can predict the extent to which changes in binding to protein, hepatic blood flow, or hepatocellular enzyme capacity will affed the hepatic clearance of diazepam.10 Oral administration of the hepatotoxic agent carbon tetrachloride (CCl,) a t a dose of 0.02 mU100 g leads to a cirrhotic condition in the rat." Liver damage was therefore induced in rats with CCl, as a model to study factors affecting the pharmacokinetic behavior of diazepam in animals with liver disease. 768 I Journal of Phannaceutical Sciences Vol. 81, No. 8, August 1992
Experimental Section Animals-Male Wistar rats were divided in nine groups. Groups I and 111 (10 rats each) were used as controls. Diazepam was administered intravenously (iv) to rats in group I and orally to those in group 111. In groups I1 and IV (10 rats each), CCl, was previously administered. Diazepam was administered iv to rats in group II and orally to those in group IV.Group V (15 control rats) was used for binding studies. Group VI included 15 rats with CC1,-induced liver damage and was used also for binding studies. Groups VII and VIII (6 rats each) were used to study the distribution of diazepam into erythrocytes. The 10 rats in group IX were used to characterize the hepatic damage caused by CCl, [activities of aspartate aminotransferaee (GOT) and alanine aminotransferaee (GF'T) in serum were measured 48 h after administration of CCl, (0.02 mu100 g); blood was collected by retroorbital plexus puncture]. Weights of animals in all g r o u p ranged between 246 and 259 g. Diazepam (4 mgkg) was administered iv through the right jugular vein of animals under light ether anesthesia. The drug was dissolved in a solution of propylene glyco1:benzyl alcoho1:polyethylene glycol 250 (2:20:78, wlw). Blood was collected from the left jugular vein of animals at 2,5, 15,30,60,90, 120, 180, and 240 min after diazepam adminiatration in control rats and, in addition, at 300 and 360 min after diazepam administration in injured rats. When the drug was administered orally (4 mgkg), diazepam was dissolved in the aforementioned solution and blood was collected from the left jugular vein of animals at 10,15,30,45,60,90,120,180, and 240 min after the administration of diazepam in control rats and, in addition, at 300 and 360 min in injured rats. Blood samples (-400 &) were immediately centrifuged at 3400 rpm for 20 min, and plasma was kept frozen a t -25 "C until analysis. In all cases, blood was replaced with a n equal volume of saline. Induction of Liver Damage-CCl, was orally administered to rats at a dose of 0.02 mU100 g, the cirrhotic condition being characterized by measurement of activities of GOT and GPT in serum with specific kits (Monotest GOT no. 124362 and Monotest GPT no. 124524, Boehringer Mannheim GmbH). Chemicals-All fine chemicals were obtained from Scharlau (Barcelona, Spain). Other chemicals were either generally available or purchased from Panreac (Barcelona, Spain). Diazepam and medazepam were supplied by Barcia (Barcelona, Spain) and Impex Qufmica (Barcelona, Spain), respectively. Desmethyldiazepam was generously provided by Roche (Madrid, Spain). AnalysigConcentrations of diazepam and desmethyldiazepam in blood and plasma were determined by electron-capture gas chromatography according to the method described by Laescher.12A HewlettPackard gas chromatograph (model 5890A) equipped with a s3Ni electron capture detector and silanized glass tubing (2 m x 4 mm id.) that was packed with 50:50 methylsi1icone:phenylsilicone (SP2250 on Supelcoport; 80/100 mesh) was used. Temperature settings were 290 "C for the oven and 320 "C for the injector and detector. Argonmethane was used as the carrier gas at a flow rate of 50 mumin, and the volume of injection was 4 &. Medazepam was used as internal standard, and benzene was used as the extraction solvent. Standard curves for diazepam were produced over the concentration range 31.25-1500 ng/mL of plasma or blood. The intraday coefficients of variation for diazepam at 31.25 ng/mL were 3.58 and 7.51% in plasma and blood, respectively. For diazepam at 1500 ng/mL, the coefficients of variation were 0.16 and 1.34% in plasma and blood, respectively. In addition, standard curves for desmethyl0022-3549/92/0800-0768$02.68$02.50/0 0 1992, American Pharmaceutical Association
diazepam in plasma were produced over the same concentration range. The intraday coefficients of variation were 7.60% (31.25 ng/mL) and 1.06% (1500ng/mL). Recoveries for diazepam, desmethyldiazepam, and medazepam either from blood or plasma were >96%. In plasma and blood, the limit of detection was 28 ng/mL for diazepam and 20 ng/mL for desmethyldiazepam. Although the gas chromatographic asmy detected other metabolites of diazepam (temazepam and oxazepam), the levels of these metabolites in blood or plasma detected after diazepam administration were not quantifiable. Pharmacokinetic Analysis-Biexponential equations were fitted to data with both JANA13 and PCNONLIN" programs. Criteria for discriminating between alternative polyexponential models16 were the information provided by the PCNONLIN program, Akaike criterion, and F ratio test.18 Pharmacokinetic parameters were estimated by conventional methods. Binding to Plaema Protein-Experiments on the binding of diazepam to plasma protein were carried out in vitro by equilibrium dialysis with the DIANORM (Diachema AG, Zurich) system. Aliquots (1 mL) of different concentrations of diazepam in plasma (1300,1000, 500, 250, and 125 ng/mL) were dialyzed against 1 mL of 0.1 M phosphate buffer (pH 7.4). Plasma was obtained by centrifugation (4000 rpm for 20 min) of blood collected by direct heart puncture from rats in groups V and VI. The albumin concentration in each sample was determined by using the method of Doumas et al.17 Distribution of Diazepam into Erythrocytes-Two studies were performed to assess the distribution of diazepam into erythrocytes. In the in vivo assay, diazepam (4 mgkg) was administered iv to three rats in group VII or VIII, and blood was collected after 2,15,30,and 60 min. Then, 200 4 of blood was taken from each sample; the remaining blood was centrifuged, and 200 of plasma was taken. These aliquots of plasma and blood were analyzed for diazepam. In the in vitro m a y , blood was collected directly by heart puncture in three rats in groups VII and VIII. Four aliquots (1 mL) of blood were transferred into glass centrifuge tubes and preincubated for 5 min at 37 "C. Different amounts of diazepam (1000,500,250, and 125 ng) were added, and the aliquots were incubated at 37 "C for 20 min. Then, 200 4 of blood and 200 p L of plasma were analyzed for diazepam. Hematocrit was estimated in duplicate for each animal by using a microhematomit centrifuge (GRI-CELL; 12 000 rpm). The blood-to-plasma ratio (BPR) was taken to be the concentration of diazepam in blood divided by that in plasma. Estimation of EH and the AfRnity of Diazepam for Erythrocytes ( y W H was indirectly estimated with eq 1 for iv administration and eq 2 for oral administration of diazepam:10
In these equations, QH is the hepatic blood flow (QH= 15 mIJminlB), CLHis hepatic clearance, AUC,, and AUC,, are the areas under the curyes of the concentrations of diazepam in plasma versus time after oral and iv administration of the drug, respectively, and F is the oral bioavailability. (It is assumed that diazepam completely traverses the gastrointestinal wall and no other organ, except the liver, significantly contributes to the elimination of the drug from the body.) y was calculated with eq 3: y=
(BPR- 1 + H)/Hf,
(3)
In eq 3,H is the hematocrit in the rat andf,, is the fraction of unbound drug in plasma. Statistical Method-Data were statistically analyzed by the conventional two-tailed t test.
Table C O O T and GPT Actlvltlei In !%rum'
Rat 1 2 3 4 5 6 7 8 9 10
Induction of Hepatic Iqjury-The activities of GOT and GPT in serum prior to the administration of CC1, to rats in group IX are statistically significantly (p < 0.01)lower than those determined 48 h after administration of the hepatotoxic agent (Table I). Pharmacokinetic Profile of Diazepam in Control and
48h
Oh
48 h
62 55
325 328 330 357 302 292 329 332 299 376 327 (26)
22
122 119 143 138 117 106 124 120 128 110 123 (11)
53 63 58
60 53 69 72
50
23 18 18 23 23 22 28 30 25 22 (6)
Mean (SD)b 60 (7) 'Determined prior to and 48 h after administration of 0.05 mL of CCI, to rats in group IX. SD, Standard deviation.
Injured Rats-The mean concentrations of diazepam in plasma after iv or oral doses in groups I-IV are shown in Figure 1. When diazepam was administered iv to control and injured rats, a biexponential function of the following form was fitted to the experimental data: (4)
In eq 4, C is the concentration of diazepam, C, and C2 are the coefficients of the exponential terms, A, is the rate constant of distribution, A2 is the terminal slope of the curve, and t is time. After oral administration of the drug to control or injured rats, a satisfactory convergent fit of triexponential equations to the data points could not be obtained; therefore, a biexponential function was used:
In eq 6,I = FDk,N(k, - k),A! is the terminal slope of the curve, k, is the adsorption constant, D is the administered dose, and V is the volume of distribution. The mean pharmacokinetic parameters for diazepam in the rat are presented in Tables 11 and III. Statistical analysis of data obtained after iv administration of diazepam indicated that A, was significantly higher (p < 0.01)and & significantly lower (p < 0.01)in rats with hepatic failure compared with healthy controls. These changes are reflected in a more pronounced biexponential curve in the injured rats (Figure 1). The only other parameter that Table ICMean PhannacoklnetlcParameters of Diazepam In the Rat after Iv Admlnlstratlon'
Parameter A,, h-' t1n.*,*hb AZ1 h-' t1n.m
hC
qo),ng/mLd
Results
GPT, units/L
GOT, units/L Oh
Vdi, mg Vd, mg' Vd,, Ukg CL, L * h-' * kg-' AUC, ng . h * mL-'
Value Control Rats
Injured Rats
3.21 t 1.26 0.22 (0.13-0.35) 0.60 k 0.11 1.15 (0.91-1.50) 1275 k 90 3.15 t 0.23 7.10 t 1.31 5.21 t 0.73 4.22 t 0.76 976 t 170
6.01 2 3.07 0.12 (0.05-0.19) 0.44 2 0.04 1.60 (1.43-1.91) 1219 t 130 3.32 t 0.37 8.53 t 1.87 7.39 t 1.33 3.75 t 0.94 1114 t 216
'Results are reported as mean * SD, except for half-lives,which are reported as harmonic mean (and range). Distribution half-life. Terminal half-life. Concentration at 4. 'Terminal volume of distribution. Journal of Pharmaceutical Sciences I 769 Vol. 81, No. 8, August 7992
Table IIl-Mean Pharmacoklndlc Parameter8 of Diazepam In the Ral after Oral Admlnlalratlon'
Value
Parameter
h
-.I
Control Rats
Injured Rats
4.72 ? 0.42 0.15 (0.13-0.18) 0.63 2 0.08 1.10 (0.64-1.28) 293 ? 16 0.50 ? 0.03 642 2 75
5.14 2 0.17 0.14 (0.13-0.15) 0.45 2 0.02 1.52 (1.42-1.62) 330?9 0.52 ? 0.01 921 ? 30
E
\
m
k , h-'
E
v
t,n..b.t hb
k, h-1
V
t1n.m h
C-, ng/mL Laxl hC AUC, ng h * mL-'
-
Results are reported as mean 2 SD, except for half-lives, which are reported as harmonic mean (and range). bHalf-life of the absorption process. cTime to reach CmU.
lB
t
o 0
l
. 1
. 1
.
. . .
9
4
I
1
6
0
t (h)
changed significantly was the volume of distribution at steady state (Vd,; Table 11). ARer oral administration, the maximum concentration of diazepam (C,,,& the absorption constant (Ita), and AUC,, were all significantly greater in injured animals (p < 0.01; Table III). The half-life (tl,.J values for desmethyldiazepam observed after either oral or iv administration of diazepam to group I or 11are presented in Table IV.Desmethyldiazepam concentrations in plasma were much lower than those of diazepam. This metabolite was the only one detected in plasma in quantifiable amounts. It seems that the appearance of desmethyldiazepam in plasma is very rapid, because in all cases, no defined maximum concentrations were observed. Metabolite concentrations in plasma were higher when diazepam was administered orally to rate. Thue, 2 min after the iv administration of the parent drug to normal rats, the mean concentration of desmethyldiazepam in plasma was 119 ng/mL compared with a value of 116 ng/mL observed 10 min after the oral administration of diazepam. In rate with hepatic failure, desmethyldiazepam concentrations in plasma reached values of 114 and 167 ng/mL at 2 and 10 min after iv and oral administration of diazepam, respectively. Binding of Diazepam to Plasma Protei&ignificant differences have been found in the binding of diazepam to plasma proteins. The amount of drug bound to these proteins was 96.4 0.9%inconhlratsand90.1? 2.5%ininjuredrats(p
