Abstract
L-754,394, a furanopyridine derivative, is an experimental HIV protease inhibitor. Previous studies from this laboratory have demonstrated that L-754,394 is cleared very rapidly in animals, and that this drug is a potent mechanism-based inactivator (suicide inhibitor) for CYP3A4 in human liver microsomes. Because L-754,394 is a high-clearance drug and an enzyme inactivator, it is expected that this drug will be subject to significant first-pass metabolism, and that the degree of enzyme inactivation will be dependent not only on the dose, but also on the route of administration. The purpose of this study is to examine the effects of dose and route of administration on the kinetics of L-754,394 using rats and dogs as animal models. In both rats and dogs, L-754,394 exhibited marked dose-dependent pharmacokinetics after i.v. and oral administration. Irrespective of i.v. or oral administration, the area under the plasma concentration-time curve from zero to infinity increased with dose in a greater than proportional manner. However, the magnitude of area under the plasma concentration-time curve from zero to infinity increase was much greater after oral dosing than after i.v. administration, indicating route-dependent pharmacokinetics. Data from in vitro and in vivo studies suggested that the dose- and route-dependent pharmacokinetics were due mainly to the inactivation (destruction) of the enzymes responsible for its own metabolism.
L-754,394, a furanopyridine derivative, is an experimental HIV protease inhibitor (Fig.1). Previous studies from this laboratory have shown that L-754,394 exhibits dose-dependent pharmacokinetics in animals (Lin et al., 1995). This dose dependence does not follow Michaelis-Menten kinetics. Although L-754,394 in plasma declines log-linearly with time, the plasma t1/2increases with dose. In vitro experiments with liver microsomal preparations from humans revealed that L-754,394 is a highly potent and selective mechanism-based inactivator (suicide inhibitor) for cytochrome P450 (Chiba et al., 1995). P450 3A4-mediated 6β-hydroxylation of testosterone was inhibited strongly by L-754,394 in a time-dependent manner. The enzyme inactivation is further supported by the evidence that L-754,394 undergoes cytochrome P450-dependent oxidation of the fused furan ring, leading to the formation of chemically reactive metabolites (Sahali-Sahly et al., 1996).
The liver is the major site of drug metabolism, and the entire blood supply of the upper gastrointestinal tract passes through the liver before reaching the systemic circulation. Thus, it is expected that L-754,394, a high-clearance drug and an enzyme inactivator, will be subject to significant first-pass metabolism, and that the degree of enzyme inactivation and first-pass metabolism will be dependent not only on the dose, but also on the route of administration. This study was undertaken to investigate the first-pass metabolism of L-754,394 and the effect of the route of administration on the kinetics of L-754,394 in rats and dogs.
Experimental Procedures
Materials.
L-754,394 and its analog, L-741,051, used as analytical internal standards (Fig. 1), were synthesized at Merck Research Laboratories. The radioactive L-754,394 was synthesized with14C at the 1-position of the pentanamide, and was at least 98% pure by HPLC. Testosterone, cortisone, glucose-6-phosphate (G6P)1, glucose-6-phosphate dehydrogenase (G6PDH), and NADPH were purchased from Sigma Chemical Co. (St. Louis, MO). 6β-Hydroxylated testosterone was obtained from Steraloids, Inc. (Wilton, NH). All other reagents were of analytical grade.
Fresh rat and dog livers were obtained from drug-free animals. Hepatic microsomes were prepared by standard procedures described in detail elsewhere (Fujita et al., 1990). Microsomes were washed, and final pellets were resuspended in 0.15 M Tris-HCl buffer (pH 7.4). Microsomal protein was measured by the method of Lowry et al. (1951) with BSA as the standard.
Animals.
Male Sprague-Dawley rats (Taconic Farms, Germantown, NY), weighing 300 to 400 g, and male pure-bred beagles (Marshall Research, North Rose, NY), weighing from 8 to 10 kg, were used for in vitro and in vivo studies. Animals were housed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and maintained under a 12-h light/dark cycle with free access to water, and were fed with standard diets from Purina (St. Louis, MO): Rodent Laboratory Chow for rats, and Lab Canine Diet for dogs.
For kinetic studies, all rats had an indwelling cannula (silicone rubber/polyethylene) implanted in the right jugular vein for blood sampling and i.v. drug administration. The surgery was performed under light pentobarbital (40 mg/kg, i.p.) 1 day before the experiment. During the experiments, all animals were housed individually in plastic metabolism cages.
In Vivo Kinetic Studies.
Four groups of rats each received a single i.v. dose of L-754,394 at 1, 2, 5, or 10 mg/kg as a solution (0.1 ml/kg) in dimethyl sulfoxide (DMSO). Blood samples were collected at appropriate time intervals, and plasma samples were separated immediately by centrifugation and kept frozen (−20°C) until assayed by HPLC.
After an overnight fast, another four groups of rats each received an oral dose of L-754–394 (free base) at 1, 2, 5, or 10 mg/kg as a solution (1 ml/kg) in 0.05 M citric acid. Blood samples were drawn periodically from the jugular vein at appropriate time points.
The effect of route of administration on the elimination kinetics of L-754,394 was studied further in rats by comparing the drug concentration in the systemic circulation during portal vein and femoral vein infusion of the drug. One group of rats (n= 3) received a portal vein infusion at 2 μg/min for 360 min. Ten days later, the same animals received a femoral vein infusion at the same infusion rate. Another group of rats (n = 4) received portal vein and femoral vein infusion at a higher infusion rate of 5 μg/min in a crossover fashion as described in the above study. Blood samples were collected at 15, 30, 60, 90, 120, 180, 240, and 360 min. Each animal had a cannula implanted in the right jugular vein for blood sampling. Cannulation of portal vein and femoral vein for drug infusion was performed 1 day before the experiment. The surgical procedures were carried out under pentobarbital anesthesia (35 mg/kg i.v.).
Four male beagles each received four i.v. doses of L-754,394 at 1, 2, 5, and 10 mg/kg in a crossover manner with a 2-week washout period between doses. The dosing solutions were prepared as described for the rat. Blood samples were collected periodically from the jugular vein at appropriate time points.
After a 4-week recovery period, the dogs used in the i.v. study were fasted overnight and received four oral doses at 1, 2, 5, and 10 mg/kg in a crossover fashion. The dosing solutions were prepared as described for rats. Blood samples were collected periodically from the jugular vein at predetermined time points.
In Vitro Studies.
Single-pass rat liver perfusion
Male Sprague-Dawley rats were used as liver donors. The surgical procedures and perfusion apparatus were identical with those described elsewhere (Morris et al., 1988). Liver was perfused in a single-pass fashion at a constant flow rate of 20 ml/min per liver at 37°C. Perfusion medium (pH 7.4) was protein-free and consisted of 4% dextran and 300 mg/dl glucose in Krebs-Henseleit bicarbonate solution. The perfusate was oxygenated (95% oxygen and 5% carbon dioxide) before being delivered to the liver. L-754,394 was added to the perfusate to yield a final concentration range of 0.005 to 0.05 μg/ml. Concentration of L-754,394 in the influent (Cin) and effluent (Cout) was measured by HPLC. The hepatic extraction ratio (EH) was calculated as:
Plasma protein binding of 14C-L-754,394.
Binding of 14C-L-754,394 to rat and dog plasma was determined by ultrafiltration. The pH of plasma was adjusted to 7.4 by the addition of a very small amount of HCl (less than 0.1% of plasma volume). 14C-L-754,394 was added to plasma to yield a final concentration range of 0.1 to 10 μg/ml. After incubation of the plasma samples at 37°C for 15 min, 1 ml of plasma was transferred immediately to a Centrifree tube (Amicon Co., Danvers, MA) and centrifuged at 1500g for 15 min at 37°C. Under these conditions, approximately 200 μl of filtrate was obtained. The unbound fraction of the drug was estimated directly from the ratio of drug concentration in the filtrate to that in the original plasma samples before centrifugation. Negligible binding of the drug to the ultrafiltration devices was observed.
Distribution of 14C-L-754,394 in blood.
The distribution of 14C-L-754,394 between blood cells and plasma in rats and dogs was determined in vitro by incubation of fresh blood with the drug at 37°C.14C-L-754,394 was added to preincubated blood to yield a final concentration of 0.5 to 20 μg/ml. Aliquots of blood (1 ml) were taken at 5, 10, 30, and 60 min, and centrifuged immediately for about 1 min to separate blood cells and plasma. Blood and plasma samples were combusted to14CO2 in a sample oxidizer for radioactivity measurement.
Antibody inhibition studies.
Goat polyclonal antiserum raised against rat CYP3A2 and control goat serum were obtained from Gentest (Woburn, MA). The cross-reactivity of anti-rat CYP3A2 antiserum on CYP3A-catalyzed activity was examined by comparing the inhibitory effect of the antiserum on CYP3A marker activity, testosterone 6β-hydroxylation, in rat and dog liver microsomes. Microsomes (0.05 mg of protein per 50-μl sample) were preincubated with various concentrations of the antiserum (2, 5, 10, and 20 μl per 0.05 mg of protein) for 20 min at room temperature. After antiserum incubation, samples were preincubated for 5 min at 37°C. Testosterone metabolism was then initiated by the addition of 50 μl of a substrate mixture containing an NADPH-generating system (20 mM G6P; 4 I.U./ml G6PDH; 20 mM MgCl2; 1 mM NADPH) and testosterone at a final concentration of 500 μM. Reactions were carried out at 37°C for 20 min and terminated by the addition of ice-cold ethyl acetate (1 ml). Cortisone (20 μl of 20 μM) was added as internal standard. The resultant mixture was vortexed, and the ethyl acetate layer was separated by centrifugation, followed by evaporation to dryness under nitrogen. The residue was reconstituted in 150 μl of 20% methanol in water and injected onto the HPLC for analysis. The formation of the 6β-hydroxylated metabolite of testosterone was measured.
The contribution of CYP3A in the metabolism of L-754,394 in dog and rat liver microsomes was examined using anti-rat CYP3A2 antiserum at a maximal inhibitory concentration of 20 μl/0.05 mg protein. Microsomes (0.05 mg per 50-μl sample) were incubated with either control serum or antiserum at the indicated concentration for 20 min at room temperature. After a 5-min preincubation at 37°C, an equal volume of a substrate mixture, containing an NADPH-generating system (20 mM G6P; 4 I.U./ml G6PDH; 20 mM MgCl2; 1 mM NADPH) and L-754,394 for an initial concentration of 1 μM, was added to initiate L-754,394 metabolism. Immediately after the addition of substrate, an aliquot (100 μl) for the zero time point was transferred to a microcentrifuge containing ice-cold acetonitrile (1 ml) to terminate the reaction. Subsequent samples were taken at various time points (5, 10, and 30 min). Terminated samples were vortexed, and the supernatant was separated by centrifugation. The supernatant was dried under nitrogen, and reconstituted in 30% acetonitrile in water, and the concentration of L-754,394 was analyzed by liquid chromatography/mass spectrometry.
The involvement of CYP3A in the metabolism of L-754,394 in rat and dog liver microsomes was additionally confirmed by a ketoconazole inhibition study. Metabolism of L-754,394 (1 μM) was compared in the presence and absence of ketoconazole (10 μM). The details of the ketoconazole inhibition study have been described elsewhere (Chiba et al., 1995).
Time-dependent inhibition by L-754,394.
The temporal effect of preincubation of L-754,394 on CYP3A-mediated testosterone 6β-hydroxylation was examined in rat and dog liver microsomes. Microsomes (4 mg/ml protein) were preincubated with or without L-754,394 (2, 20, and 100 μM) for a designated time period (0, 5, 10, 20, and 30 min) in the presence of 1 mM NADPH. Aliquots (62.5 μl) were then transferred to a reaction mixture (187.5 μl) containing an NADPH-generating system (20 mM G6P; 4 I.U./ml G6PDH; 20 mM MgCl2; 1 mM NADPH) and testosterone at a final concentration of 500 μM in 250 μl, and preincubated at 37°C for 5 min. Reactions were conducted at 37°C for 20 min and terminated by the addition of ice-cold ethyl acetate (1.0 ml). An additional 1.5 ml of ethyl acetate was added to extract metabolites. Cortisone (50 μl of 20 μM) was added to each sample as internal standard. Samples were vortexed, and the ethyl acetate layer was separated by centrifugation, followed by evaporation to dryness under nitrogen gas. The residue was reconstituted in 250 μl of 20% methanol in water and injected onto the HPLC. The formation of the 6β-hydroxylated metabolite of testosterone was measured.
Analytical Procedures.
HPLC assay for testosterone
A Thermo Separation Products (Piscataway, NJ) HPLC system consisting of a TSP spectra system P4000 pump, an AS3000 autosampler, and a UV2000 UV detector set at 240 nm was used to measure 6β-hydroxytestosterone hydroxylations. Separation was performed on a Phenomenex (Torrance, CA) Jupiter C18 column (250 × 4.6 mm, 5 μm). The HPLC method involved isocratic elution for 10 min with a 75:25 (v/v) mixture of 30% methanol in water (mobile phase A) and 10% acetonitrile in methanol (mobile phase B) at a flow rate of 1.0 ml/min. A linear gradient was then initiated for 20 min, during which mobile phase A was decreased to 50%. Mobile phase A was returned to 75% at 25 min and was retained for an additional 5 min. Retention times for 6β-hydroxytestosterone, cortisone, and testosterone were 10, 19, and 26 min, respectively.
HPLC assay for L-754,394.
The concentration of L-754,394 in plasma was determined by HPLC. To 0.2 ml (rat) or 0.5 ml (dog) plasma was added 250 ng of internal standard (L-741,051) and 5 ml of dimethyl ether. After shaking for 15 min, the samples were centrifuged and the organic layer was transferred to a clean tube where it was evaporated to dryness under nitrogen gas. The residue was dissolved in 250 μl of mobile phase, and 200 μl was injected onto a Zorbax RX-C8 (4.6 mm × 25 cm) analytical column. The flow rate of the mobile phase, acetonitrile/phosphoric acid (15 mM) (30:70, v/v, pH 3.2), was 1.5 ml/min. The limit of detection was 50 nM for rat plasma samples and 20 nM for dog plasma samples.
Pharmacokinetic analysis.
Because of the time- and dose-dependent nature of pharmacokinetic behavior, the term of clearance used in the study reflected time-averaged clearance. The clearance (CL) of L-754,394 was calculated as the i.v. dose divided by the total area under the plasma concentration-time curve from zero to infinity (AUC), or as the infusion rate (Ro) divided by the steady-state plasma concentration (Css):
Results
After i.v. administration, L-754,394 exhibited marked dose-dependent pharmacokinetics in rats. The clearance decreased from 89 ml/min/kg at 1 mg/kg to 12 ml/min/kg at 10 mg/kg (Table1). At the low dose (1 mg/kg), L-754,354 had a high clearance in rats that approximated hepatic blood flow (80 ml/min/kg; Boxenbaum, 1980). Dose-dependent absorption kinetics also were observed in rats; both the Cmax and AUC increased with dose in a greater than proportional manner (Table1). After oral dosing, L-754,394 was absorbed rapidly, and the peak concentration (Cmax) occurred within 60 min. The magnitude of disproportionate AUC increase was much greater after oral dosing than after i.v. administration. A 10-fold increase in the oral dose resulted in an 825-fold increase in the AUC, whereas after i.v. administration over the same dose range there was only a 66-fold increase in the AUC (Table 1), suggesting route-dependent nonlinear pharmacokinetics.
Similar to the observations in rats, L-754,394 showed route- and dose-dependent pharmacokinetics in dogs. When given i.v., the clearance decreased from 23 ml/min/kg at 1 mg/kg to 1.8 ml/min/kg at 10 mg/kg (Table 2). The clearance of L-754,394 was intermediate at the low dose (1 mg/kg), relative to dog hepatic blood flow (40 ml/min/kg; Boxenbaum, 1980). When given orally, the drug was absorbed rapidly with a tmax (time reaching peak concentration) of less than 60 min. Again, both theCmax and AUC increased disproportionally with the oral dose (Table 2). Route-dependent nonlinear pharmacokinetics also was apparent in dogs. The degree of AUC increase in dogs was much greater after oral dosing than after i.v. administration. A 10-fold increase in the dose led to a 655-fold increase in the AUC after oral dosing, and a 134-fold increase in the AUC after i.v. administration (Table 2).
To gain an insight into the mechanism responsible for the route-dependent effect of L-754,394 kinetics, the pharmacokinetics of L-754,394 were studied in rats during femoral and portal vein infusion in a crossover fashion at a low (2 μg/min) or high (5 μg/min) infusion rate. In all animals, the concentration of L-754,394 reached a plateau within 90 min and remained relatively constant thereafter up to 360 min during either route of administration. Because L-754,394 is a high-clearance drug, it was expected that the drug would be subject to significant hepatic first-pass metabolism, and that the drug concentration in systemic circulation after intraportal administration would be lower than after i.v. dosing. Surprisingly, higher steady-state drug concentrations and lower clearances were observed in all three individual animals, when L-754,394 was infused at a constant rate of 2 μg/min via the portal vein as compared with the femoral vein infusion. The mean clearance was 30 ml/min/kg during portal vein infusion and 70 ml/min/kg during femoral vein administration (Table3). When the infusion rate was increased to 5 μg/min, there were no significant differences in clearance and steady-state concentrations between these two routes. The mean clearance was 9.8 ± 2.97 and 12.5 ± 1.47 ml/min/kg, respectively, during portal vein and femoral vein infusion (Table4). Together, these results suggest route- and rate-dependent effects of L-754,394 on hepatic metabolism.
The rate-dependent effect on hepatic metabolism was investigated further in rats using the technique of isolated perfused liver. As shown in Fig. 2, in a range of infusion rates varying from 2 to 20 μg/min, L-754,394 was metabolized very rapidly by rat liver with a hepatic extraction ratio (EH) of approximately 95%. At the lowest rate of 2 μg/min, the EH remained constant up to 90 min after the commencement of infusion and declined slowly afterward (Fig.2). On the other hand, at the highest rate of 20 μg/min, the EH remained unchanged only for 20 min and then declined very rapidly to less than 10% at 120 min after the start of infusion (Fig. 2). Again, these results showed that the reduction of hepatic metabolism caused by L-754,394 was rate-dependent.
Consistent with in vivo data, L-754,394 was metabolized rapidly by rat and dog liver microsomes. In both liver microsomes, less than 10% of the dose remained after a 30-min incubation at 37°C (Fig.3). To determine which isoform of cytochrome P450 is responsible for the metabolism of L-754,394 in rats and dogs, the effects of isoform-specific antibodies and chemical inhibitors on catalytic activity were studied. As shown in Fig. 3, anti-rat CYP 3A2 antibody strongly inhibited the hepatic metabolism of L-754,394 in both rat and dog liver microsomes. Antibodies against CYP 2C and CYP 2D had little effect on L-754,394 metabolism in both species (data not shown). The cross-reactivity of CYP 3A2 antibody against 6β-hydroxylation of testosterone has been examined in rat and dog liver microsomes. Anti-CYP3A2 antibody inhibited 95% of testosterone 6β-hydroxylase activity in rats, and 85% in dogs (Fig.4). These data point to the possible involvement of CYP 3A enzymes in the metabolism of L-754,394 in both species. The involvement of CYP 3A enzymes was supported further by the observations that ketoconazole, a potent CYP 3A inhibitor, significantly inhibited the metabolism of L-754,394 in both rat and dog liver microsomes (Fig. 5).
The involvement of CYP3A in L-754,394 metabolism was further confirmed by the inhibitory activity of L-754,394 on the 6β-hydroxylation of testosterone. As shown in Fig. 6, L-754,394 inhibited 6β-hydroxylation of testosterone in rat liver microsomes in a concentration- and time-dependent manner. Similarly, concentration- and time-dependent inhibition was observed for 6β-hydroxylation in dog liver microsomes (Fig. 6). Collectively, these results support the notion that L-754,394 caused a time-dependent loss of enzyme activity of CYP 3A enzymes in both species.
When 14C-L-754,394 was incubated with fresh rat and dog blood at 37°C, an equilibrium between blood cells and plasma occurred readily within 5 min. The ratios of drug concentration in blood to that in plasma were about 0.96 for the rat and 0.90 for the dog, and the ratios were concentration-independent over a range of 0.5 to 20 μg/ml. This indicates that L-754,394 was distributed roughly evenly between blood cells and plasma. Therefore, the blood clearance of L-754,394 was approximately equivalent to the plasma clearance in both species. In addition, L-754,394 was bound significantly to rat and dog plasma. The unbound fraction of the drug in plasma was 0.15 for both species, and was independent of concentration.
After i.v. administration of 14C-L-754,394 (1 mg/kg) to rats and dogs, more than 85% of the administered dose was found in the feces over a 48-h collection, whereas only a small fraction of the dose was recovered in the urine (5%). In a separate study with bile duct-cannulated rats, approximately 65% of the dose was excreted in the bile and 4% in the urine over a period of 24 h after an i.v. dose of 14C-L-754,394 (1 mg/kg), whereas 60 and 5% of the dose was excreted in the bile and urine, respectively, after oral administration of the same dose. The latter suggests that L-754,394 was absorbed very well in this species. Analysis of the bile and urine samples revealed that the majority of the radioactivity was attributed to drug-related metabolites, and parent drug accounted for only a very small fraction (<2%) of the radioactivity, indicating that biotransformation is the major pathway of the elimination of L-754,394.
Discussion
The pharmacokinetics of L-754,394 were dose- and route-dependent. In both rats and dogs, the clearance decreased with increasing dose and the AUC increased in a greater than dose-proportional manner (Tables 1and 2). At the same dose range, the magnitude of disproportionate AUC increase after oral dosing was much greater than that after i.v. administration in both rats and dogs, implying unusual nonlinear pharmacokinetics. The literature in nonlinear pharmacokinetics indicates that the nonlinearity has been associated almost exclusively with dose-dependent changes in pharmacokinetic parameters (Lin, 1994;Lin and Lu, 1997). Route-dependent nonlinear pharmacokinetics has been rarely reported. As will be discussed later, the dose- and route-dependent pharmacokinetics of L-754,394 were most likely due to the inactivation (destruction) of the enzyme responsible for its own metabolism.
L-754,394 has been shown to be a highly potent and selective inactivator of cytochrome P450 3A enzymes in animals and humans (Chiba et al., 1995; Lin et al., 1995). It is probably the most potent mechanism-based inactivator (suicide inhibitor) of P450 3A4 yet described. In human liver microsomes, P450 3A4-mediated 6β-hydroxylation testosterone is inhibited strongly by L-754,394 withKI and kinactvalues of 7.5 μM and 1.62 min−1, respectively (Chiba et al., 1995). Furthermore, in vitro studies have demonstrated that L-754,394 undergoes metabolism in liver microsomes to chemically-reactive intermediates which bind covalently to microsomal enzymes, resulting in enzyme inactivation (Sahali-Sahly et al., 1996).
Numerous underlying mechanisms have been proposed for dose-dependent pharmacokinetics (Lin, 1994). Among these, saturation of enzyme activity (saturable Michaelis-Menten kinetics) is probably the most common cause. For L-754,394, there are several lines of evidence that suggest that the dose-dependent pharmacokinetics are the result of dose-dependent autocatalytic destruction of enzymes, rather than saturable Michaelis-Menten kinetics. In the case of saturable Michaelis-Menten kinetics, the decline of drug concentration in plasma after high doses parallels that after low doses when the plasma concentrations are in a similar range. This means that the plasmat1/2 after high doses should be similar to that after low doses. Previous data from our laboratory showed that L-754,394 in plasma of rats and dogs declined log-linearly with time, and the decline of plasma concentration after a high i.v. dose (10 mg/kg) did not parallel the plasma decline over a similar concentration range at low i.v. doses (0.5 and 1 mg/kg). The plasma terminalt1/2 values of L-754,394 in rats were 20, 25, and 120 min after an i.v. dose of 0.5, 1, and 10 mg/kg, respectively, whereas the corresponding values were 53, 56, and 980 min in dogs (Lin et al., 1995).
The phenomenon of route-dependent pharmacokinetics of L-754,394 can be explained by the following theoretical considerations and experimental observations. As shown in eq. 4, a decrease in the CLint caused by enzyme inactivation will result in an almost proportional increase in the AUCpo. However, a similar degree of CLint decrease would have less effect on the AUCi.v., because the hepatic intrinsic clearance (CLint) is limited by hepatic blood flow (Qh), as indicated in eq. 3. This explains the greater increase in AUC after oral administration as compared with that after i.v. administration, even though the magnitude of changes in the CLint was similar after the two different routes.
Furthermore, it is known that the degree of enzyme inactivation depends on the concentration of inactivator, and on the duration of exposure (Silverman, 1988; Ito et al., 1998). The “initial drug concentration” entering the liver after oral administration may be many times greater than after an equivalent i.v. dose if the rate of absorption of a drug is rapid and complete. This is because all of the drug absorbed from the intestinal lumen enters the liver through the portal vein after oral dosing, whereas the drug distributes into the systemic circulation before reaching the portal vein after i.v. injection. Because L-754,394 is well and rapidly absorbed, the drug concentrations entering the liver after oral dosing would be much higher than after i.v. administration. This also can explain why the magnitude of AUC increase after oral dosing was greater than that after i.v. administration at the same dose range of L-754,394.
The initial drug concentration hypothesis was also supported by the data in Table 3. Although the duration of exposure and dose of L-754,394 was identical by either portal or femoral vein infusion, the concentration entering the liver was much higher during portal vein administration, suggesting that the degree of enzyme inactivation was greater during portal vein infusion than femoral vein infusion, as indicated by the clearance (30 versus 70 ml/min/kg). However, when the infusion rate was increased to 5 μg/min, the differences in the CL (9.8 versus 12.5 ml/min/kg) between portal vein and femoral vein infusion was much less pronounced as compared with the differences at 2 μg/min, reflecting saturation of enzyme inactivation. Consistent with the initial drug concentration hypothesis, the degree of enzyme inactivation was less significant when the same dose of L-754,394 was given i.v. at a slow infusion rate (2 μg/min) as compared with that given as a single i.v. dose. The clearance was 70 ml/min/kg after a 360-min infusion (Table 3) and only 34 ml/min/kg after a single dose (Table 1), even though in both cases, the same dose of L-754,394 was given i.v.
In summary, L-754,394 exhibited dose- and route-dependent pharmacokinetics in rats and dogs. Both in vitro and in vivo data suggest that dose- and route-dependent pharmacokinetics were due mainly to the autocatalytic destruction of CYP 3A enzymes.
Footnotes
-
Send reprint requests to: Jiunn H. Lin, Ph.D., Drug Metabolism, Merck Research Laboratories, WP75A-203, West Point, PA 19486. E-mail: jiunn_lin{at}merck.com
- Abbreviations used are::
- G6P
- glucose-6-phosphate
- G6PDH
- glucose-6-phosphate dehydrogenase
- AUC
- area under the plasma concentration-time curve from zero to infinity
- Received July 20, 1999.
- Accepted January 4, 2000.
- The American Society for Pharmacology and Experimental Therapeutics