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Antimicrobial Agents and Chemotherapy, September 2006, p. 2926-2931, Vol. 50, No. 9
0066-4804/06/$08.00+0     doi:10.1128/AAC.01566-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Metabolic Activation of Pradefovir by CYP3A4 and Its Potential as an Inhibitor or Inducer

Chin-chung Lin,^* Che Fang, Salete Benetton, Gui-fen Xu, and Li-Tain Yeh

Valeant Research & Development, Costa Mesa, California

Received 8 December 2005/ Returned for modification 14 March 2006/ Accepted 8 June 2006

Top Abstract Introduction Materials and Methods Results Discussion References

 
Metabolic activation of pradefovir to 9-(2-phosphonylmethoxyethyl)adenine (PMEA) was evaluated by using cDNA-expressed CYP isozymes in portal vein-cannulated rats following oral administration and in human liver microsomes. The enzyme induction potential of pradefovir was evaluated in rats following multiple oral dosing and in primary cultures of human hepatocytes. The results indicated that CYP3A4 is the only cDNA-expressed CYP isozyme catalyzing the conversion of pradefovir to PMEA. Pradefovir was converted to PMEA in human liver microsomes with a K_m of 60 µM, a maximum rate of metabolism of 228 pmol/min/mg protein, and an intrinsic clearance of about 359 ml/min. Addition of ketoconazole and monoclonal antibody 3A4 significantly inhibits the conversion of pradefovir to PMEA in human liver microsomes, suggesting the predominant role of CYP3A4 in the metabolic activation of pradefovir. Pradefovir at 0.2, 2, and 20 µM was neither a direct inhibitor nor a mechanism-based inhibitor of CYP3A4, CYP2D6, CYP2C9, CYP2C19, CYP2E1, and CYP1A2 in human liver microsomes. In rats, the liver was the site of metabolic activation of pradefovir, whereas the small intestine did not play a significant role in the metabolic conversion of pradefovir to PMEA. Daily oral dosing (300 mg/kg of body weight) to rats for 8 days showed that pradefovir was not an inducer of P450 enzymes in rats. Furthermore, pradefovir at 10 µg/ml was not an inducer of either CYP1A2 or CYP3A4/5 in primary cultures of human hepatocytes.

Top Abstract Introduction Materials and Methods Results Discussion References

 
9-(2-Phosphonylmethoxyethyl)adenine (PMEA) (Fig. 1) is an acyclic phosphonate analogue of adenine which has been shown to be effective against the hepatitis B virus (HBV) in stably transfected human hepatocellular carcinoma cell lines, primary duck hepatocytes infected with duck hepatitis B virus, and a duck model of hepatitis B (8, 9). PMEA is phosphorylated to PMEA diphosphate (PMEApp) by cellular kinases or 5-phosphoribosyl-1-pyrophosphate synthetase, which inhibits HBV DNA polymerase (reverse transcriptase) by competing with the natural substrate dATP (15) and by causing DNA chain termination after its incorporation into viral DNA (1). However, PMEA is poorly absorbed in a number of species, including rats (20), monkeys (4), and humans (3). The low oral bioavailability of PMEA appears to be a consequence, in part, of the limited intestinal permeation of phosphonate, which is ionized at physiological pH (18).

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FIG. 1. Structure and activation of pradefovir to PMEA.

Adefovir dipivoxil is an oral prodrug of PMEA. Marcellin et al. (14) reported that in patients with HBeAg-positive chronic hepatitis B, 48 weeks of treatment with 10 or 30 mg of adefovir dipivoxil per day resulted in histological liver improvement, reduced serum HBV DNA and alanine aminotransferase levels, and increased the rates of HBeAg seroconversion. The 10-mg dose has a favorable risk-benefit profile for long-term treatment. No adefovir-associated resistance mutations were identified in the HBV DNA polymerase gene. Hadziyannis et al. (7) reported that in patients with HBeAg-negative chronic hepatitis B, 48 weeks of treatment with 10 mg of adefovir dipivoxil treatment resulted in significant histological, virological, and biochemical improvement, with an adverse event profile similar to that of a placebo. There was no evidence of the emergence of adefovir-resistant HBV polymerase mutations. With the 30-mg dose, the incidence of increases in serum creatinine levels (0.3 to <0.5 mg/dl from those at the baseline) was significantly higher than that with the 10-mg dose (5). Therefore, treatment with a suboptimal dose of 10 mg was recommended.

Pradefovir is a cyclodiester prodrug of PMEA. It is one of the HepDirect prodrugs, which are designed to be efficiently and specifically activated through an oxidative reaction catalyzed by CYP3A4, which is located mainly in the liver. This activation results in the generation of a highly charged nucleotide intermediate, which is trapped inside the hepatocytes, where it is further converted to PMEApp (13). The conversion of pradefovir to PMEA has been demonstrated in rat liver microsomes (M. D. Erion et al., personal communication). It has recently been reported (12) that following oral dosing (30 mg/kg of body weight) of rats with [¹⁴C]pradefovir, pradefovir was extensively converted to PMEA and other metabolites (metabolites A, B, C, and D). The plasma PMEA area under the concentration-time curve (AUC) accounted for about 23% of the plasma radioactivity AUC, and the amount of PMEA excreted in urine from time zero to 24 h accounted for about 66% of the urinary radioactivity, suggesting a major role of PMEA formation in the metabolism of pradefovir in animals.

In this study, the metabolic activation of pradefovir to PMEA was evaluated in various in vitro and in vivo studies. The potential of pradefovir as a P450 inhibitor or inducer was also evaluated.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Reagents and standards. NADPH (ß-NADP, reduced form), phosphate monobasic, phosphate dibasic, testosterone, 6ß-hydroxytestosterone, and ketoconazole were purchased from Sigma-Aldrich (St. Louis, MO). We purchased pooled human liver microsomes from 10 subjects (20 mg/ml); cDNA-expressed CYPs (supersomes) 1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, and 3A5; an insect cell control; and a CYP3A4-inhibitory monoclonal antibody (MAb) from BD Biosciences (Bedford, MA). PMEA, [¹³C]PMEA, pradefovir, [¹⁴C]pradefovir mesylate, and [¹³C]pradefovir were obtained from Valeant Research & Development (Costa Mesa, CA).

Incubation conditions. To evaluate the activities of different CYP enzymes toward the formation of PMEA, pradefovir (1.63 µM) was incubated with a panel of cDNA-expressed CYPs (supersomes; 20 nM). Pradefovir at 1.63 µM was incubated with cDNA-expressed CYP3A4, with or without ketoconazole (0, 0.05, 0.1, 0.2, 0.5, 1, and 2 µM). Pradefovir (1.63, 4.08, 16.3, or 40.8 µM) or testosterone (50 µM) was also incubated with pooled human liver microsomes (0.4 mg/ml), with or without ketoconazole (various concentrations), or CYP3A4-inhibitory monoclonal antibody. All incubations were performed in 1.5-ml Eppendorf tubes containing potassium phosphate buffer (50 mM; pH 7.4). The mixtures were preheated at 37°C for 3 min.

The reactions were initiated by the addition of NADPH (1 mM) and proceeded for 10 to 60 min. Incubations were stopped by the addition of 250 µl acetonitrile. After centrifugation at 14,000 rpm for 10 min, 150 µl supernatant was dried by N₂ and redissolved in 150 µl of reconstitution solution (10 mM ammonium acetate/3% dimethylhexylamine/1.5% hydroxyacetate) and 10 µl of internal standard solution (dextrorphan for testosterone or [¹³C]PMEA for pradefovir). The supernatant was analyzed by liquid chromatography/tandem mass spectrometry (LC-MS/MS) for PMEA or 6ß-hydroxytestosterone.

LC-MS/MS assays of PMEA. A high-pressure liquid chromatography (HPLC) system consisting of a LEAP Technologies HTC PAL autosampler and an Agilent 1100 LC pump was used. An Agilent ZORBAX Eclipse XDB-C8 column (5 µm; 4.6 by 150 mm) was used for the analysis. Twenty microliters of (reconstituted) sample was injected into the LC. The flow rate was 0.9 ml/min, with a run time of 6 min. The mobile-phase solution consisted of 90% solvent A (0.1% acetic acid in water) and 10% solvent B (0.1% acetic acid in acetonitrile) for 0.5 min, was switched to 50% solvent A and 50% solvent B at 3.5 min and kept there for 1 min, and was then returned to 90% solvent A and 10% solvent B. The effluent from the LC system was connected directly to an API 4000 triple-quadrupole mass spectrometer. The analysis was performed by using negative electrospray to monitor the ion transitions of 272134 and 277139 for PMEA and [¹³C]PMEA, respectively.

LC-MS/MS assays of pradefovir and PMEA in rat plasma. The supernatant obtained from protein precipitation was analyzed by a high-performance LC-MS/MS method to determine the concentrations of pradefovir and PMEA in rat plasma. The calibration curves ranged from 1 to 100 ng/ml for pradefovir and 10 to 1,000 ng/ml for PMEA. The limits of quantification were 1 ng/ml for pradefovir and 10 ng/ml for PMEA. All analytical results were within acceptance criteria.

LC-MS/MS assays of 6ß-hydroxytestosterone. An HPLC system consisting of a LEAP Technologies HTC PAL autosampler and an Agilent 1100 LC pump was used. A Phenomenex Luna-Phenyl/Hexyl column (5 µm; 4.6 by 50 mm) was used for the analysis. Fifteen microliters of (reconstituted) sample was injected into the LC. The flow rate was 0.9 ml/min, with a run time of 6 min. The mobile-phase solution consisted of 60% solvent A (0.1% formic acid in water) and 40% solvent B (0.1% formic acid in methanol) for 0.5 min; was switched to 35% solvent A and 65% solvent B in 0.5 min, to 20% solvent A and 80% solvent B in 0.5 min, and to 5% solvent A and 95% solvent B in 1 min and was kept there for 1.7 min; and was then returned to 60% solvent A and 40% solvent B. The effluent from the LC system was connected directly to an API 4000 triple-quadrupole mass spectrometer. The analysis was performed by using positive electrospray to monitor the ion transitions of 305269 and 258157 for 6ß-hydroxytestosterone and dextrorphan, respectively.

Conversion of pradefovir to PMEA in portal vein-cannulated rats. Rats were fasted overnight and given 30 mg/kg of [¹⁴C]pradefovir mesylate by oral gavage. Blood samples were simultaneously collected from the portal vein and systemic vein cannulas at 2, 5, 10, 20, 40, and 60 min after dosing and placed in heparinized tubes; and whole blood was centrifuged to harvest the plasma. The plasma concentrations of pradefovir and PMEA were determined by a validated LC-MS/MS method.

Enzyme inhibition studies with human liver microsomes. To evaluate the potential of pradefovir as a direct inhibitor, pradefovir was incubated with liver microsomes containing typical substrates (phenacetin for CYP1A2, diclofenac for CYP2C9, S-mephenytoin for CYP2C19, bufuralol for CYP2D6, chlorzoxazone for CYP2E1, and testosterone for CYP3A4). The reaction was initiated by the addition of NADPH and was terminated by the addition of acetonitrile. The activity of each CYP isozyme was determined by measurement of the reaction products by LC-MS/MS (i.e., acetaminophen for CYP1A2, 4-hydroxydiclofenac for CYP2C9, 4-hydroxymephenytoin for CYP2C19, 1-hydroxybufuralol for CYP2D6, 6-hydroxychlorzoxazone for CYP2E1, and 6-ß-hydroxytestosterone for CYP3A4). To evaluate the potential of pradefovir as a mechanism-based inhibitor, pradefovir was preincubated with microsomes and NADPH for 15 min. The subsequent procedures after the addition of the respective substrate were as described previously.

Enzyme induction study with rats. Five rats received pradefovir (300 mg/kg/day) orally for 8 days. At 24 h after administration of the last dose, the rats were killed and liver samples were collected. Body weight, liver weight, liver protein content, and liver microsomal P450 contents were determined. The apoprotein levels for CYP1A1, CYP2B1/2B2, CYP3A1/3A2, and CYP4A1/4A3 and the enzyme activities for CYP1A, CYP2B, and CYP3A were also determined by Western blot and LC analysis, respectively.

Enzyme induction study with primary cultures of human hepatocytes. The preparations of hepatocytes cultured from three separate human livers were treated with dimethyl sulfoxide (0.1 vol/vol), pradefovir (0.1, 1, or 10 µg/ml), PMEA (0.01, 0.1, or 1 µg/ml), and two known human P450 inducers (ß-naphthoflavone [33 µM] or rifampin [20 µM]) once daily for three consecutive days. The cultured hepatocytes were assessed daily by light microscopy to assess them for morphological normalcy, with confluence adequate for treatments. In general, the cultured human hepatocytes for all treatments exhibited a normal hepatocyte morphology. After treatment, the cells were harvested to prepare microsomes for the analyses of 7-ethoxyresorufin O-dealkylation (a marker for CYP1A2), and testosterone 6ß-hydroxylation (a marker for CYP3A4/5). Analyses of S-mephenytoin N-demethylation (a marker for CYP2B6), diclofenac 4'-hydroxylation (a marker for CYP2C9), and S-mephenytoin 4'-hydroxylation (a marker for CYP2C19) were also performed with microsomes from cells treated with dimethyl sulfoxide or rifampin).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Conversion of pradefovir to PMEA by cDNA-expressed CYPs. cDNA-expressed major human CYPs (supersomes) were used to further investigate which P450 enzyme was catalyzing the conversion of pradefovir to PMEA at a concentration of 1.63 µM pradefovir. The CYPs used included 1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, and 3A5. Among these, only CYP3A4 showed significant activity in the conversion of pradefovir to PMEA. Addition of ketoconazole to cDNA-expressed CYP3A4 greatly inhibited the conversion of pradefovir to PMEA; the extents of inhibition were 80, 90, 96, 98, and 99% for ketoconazole concentrations of 0.05, 0.1, 0.2, 0.5, and 2 µM, respectively. This confirms the role of CYP3A4 in the conversion of pradefovir to PMEA.

Conversion of pradefovir to PMEA in human liver microsomes. Pradefovir (1.63 µM) was incubated with human liver microsomes (0.4 mg/ml) for an extended period of time, ranging from 10 to 60 min. The production of PMEA increased linearly with the length of incubation time (y = 5.043x + 6.1907; R² = 0.9978). On the basis of these data, an incubation time of 15 min was selected for subsequent studies. A linear range of PMEA formation in relation to microsome concentration (from 0.1 to 1.0 mg/ml) was observed after a 15-min incubation (y = 22.587x + 0.948; R² = 0.9859). Therefore, a microsomal concentration of 0.4 mg/ml was selected for subsequent analyses.

Enzyme kinetics of pradefovir activation in human liver microsomes. The rate of conversion of pradefovir to PMEA in human liver microsomes depends on the substrate concentration. At low pradefovir concentrations (0 to 50 µM), the initial conversion velocity was nearly proportional to the pradefovir concentration, and this conversion was approximately a first-order reaction. As the pradefovir concentration increased, the initial rate reached saturation at concentrations greater than about 160 µM. The activation of pradefovir follows the Michaelis-Menten equation, and enzyme kinetic parameters were obtained from the Lineweaver-Burk (double-reciprocal) plot (y² = 0.365x – 0.0021; R² = 0.9972), with a K_m of 60 µM, a maximum rate of metabolism of 228 pmol/min/mg protein, and an intrinsic clearance of 3.8 µl/min/mg.

Inhibition of pradefovir conversion to PMEA in human liver microsomes by ketoconazole. The addition of ketoconazole to the incubation of pradefovir with human liver microsomes greatly reduced the activation of pradefovir (Fig. 2). This activation is basically shut down by ketoconazole at 0.5 µM, which is comparable to the 6ß-hydroxylation of testosterone, a CYP3A marker substrate. The reaction rate decreased with the increased amount of ketoconazole (0 to 0.1 µM) (Fig. 2). For each ketoconazole concentration, a double-reciprocal plot was drawn, and the slope values were obtained (see Fig. 6). The inhibition constant K_i was calculated to be 12.5 nM (Fig. 3) from the linear plot (y = 0.0108x + 0.1349; R² = 0.9682) of the slope versus inhibitor (ketoconazole) concentration ([I]). A Dixon plot was used to identify the type of inhibition for ketoconazole (1/V versus [I]) (Fig. 4). The intersection of the plots at the x axis indicated a noncompetitive inhibition.

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FIG. 2. Ketoconazole inhibition of conversion of pradefovir to PMEA in human liver microsomes as a percentage of the control activity (mean; n = 2) versus the concentration of pradefovir.

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FIG. 6. Ketoconazole inhibition of conversion of pradefovir to PMEA (mean; n = 2) in human liver microsomes as the rate of conversion versus the concentration of pradefovir at different concentrations of ketoconazole.

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FIG. 3. Determination of Ki (slope versus [I] plot) for ketoconazole on conversion of pradefovir to PMEA (mean; n = 2).

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FIG. 4. Determination of type of inhibition (Dixon plot) for ketoconazole (Keto) on conversion of pradefovir to PMEA (mean; n = 2).

Inhibition of pradefovir conversion to PMEA in human liver microsomes by MAb 3A. The addition of a specific CYP3A-inhibitory monoclonal antibody (MAb 3A) dramatically lowered the activation of pradefovir with the increased amount of MAb 3A used (Fig. 5). This finding supports the results of the cDNA experiment and indicates that CYP3A is responsible for the activation of pradefovir.

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FIG. 5. MAb 3A4 inhibition of conversion of pradefovir to PMEA (mean; n = 2) in human liver microsomes.

Pradefovir inhibition of various P450 isozymes in human liver microsomes. Pradefovir was neither a direct inhibitor nor a mechanism-based inhibitor in human liver microsomes of CYP3A4 (6ß-hydroxylation of testosterone at 100 µM), CYP2D6 (1-hydroxylation of bufuralol at 25 µM), CYP2C9 (4-hydroxylation of diclofenac at 100 µM), CYP2C19 (4-hydroxylation of mephenytoin at 100 µM), CYP2E1 (6-hydroxylation of chlorzoxazone at 200 µM), or CYP1A2 (O-deethylation of phenacetin at 200 µM) (Tables 1 and 2).

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TABLE 1. Potential of pradefovir as a direct inhibitor for various CYP isozymes

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TABLE 2. Potential of pradefovir as a mechanism-based inhibitor for various CYP isozymes

Conversion of pradefovir to PMEA in portal vein-cannulated rats. The plasma levels of PMEA and pradefovir (Table 3) after oral dosing with pradefovir were used to calculate the ratio of the PMEA concentration to the pradefovir concentration (P/R) in both portal plasma and systemic plasma. The P/R ratios in portal plasma were 0 at 2 and 5 min, 0.009 at 10 min, 0.105 at 20 min, 0.196 at 40 min, and 0.464 at 60 min. The ratios in systemic plasma were 0 at 2 min, 0.099 at 5 min, 0.156 at 10 min, 0.514 at 20 min, 1.380 at 40 min, and 2.308 at 60 min. The higher P/R ratio in the systemic plasma clearly suggests that in the rat, the liver is the main site for the conversion of pradefovir to PMEA. Interestingly, 5 min after dosing with pradefovir, PMEA was detected in systemic plasma but not in portal plasma, indicating that the small intestine did not play a significant role in the metabolic conversion of pradefovir to PMEA in the rat. However, the data for the rats do not necessarily imply that human gastrointestinal CYP3A4 may not provide a greater amount of metabolism.

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TABLE 3. Mean plasma concentrations of pradefovir and PMEA in portal and systemic plasma of portal vein-cannulated ratsa

Enzyme induction of pradefovir in rats. Daily oral dosing of pradefovir (300 mg/kg) to rats for 8 days did not affect body weight; liver weight; liver weight-body weight ratio; liver microsomal protein content; total CYP content; enzyme activities for CYP1A, CYP2B, and CYP3A; and apoprotein contents for CYP1A1, CYP2B1/2, CYP3A1/2, and CYP4A1/3 (Table 4), indicating that pradefovir is not a CYP inducer in rats.

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TABLE 4. Effect of prolonged administration of pradefovir (300 mg/kg/day for 8 days) on CYP induction

Enzyme induction of pradefovir in primary cultures of human hepatocytes. Under the conditions in which the positive controls (ß-naphthoflavone and rifampin) caused appropriate, increased activity, pradefovir (up to 10 µg/ml) and PMEA (up to 1 µg/ml) caused no significant changes in 7-ethoxyresorufin O-dealkylase or testosterone 6ß-hydroxylase activity (Table 5). These results suggest that both pradefovir and PMEA are not inducers of either CYP1A2 or CYP3A4/5 in primary cultures of human hepatocytes.

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TABLE 5. Effects of pradefovir, PMEA, and prototypical inducers on the expression of P450 enzymes in primary cultures of human hepatocytes

Top Abstract Introduction Materials and Methods Results Discussion References

 
It is well known that CYP3A4 is the most abundant form of P450 isozyme and accounts for 25 to 30% of total P450 in human liver microsomes (17). The finding that CYP3A4 is the only P450 isozyme capable of converting pradefovir to PMEA seems to suggest that coadministration of ketoconazole, a potent P450 inhibitor (2), or rifampin, a potent P450 inducer (2), would significantly affect the pharmacokinetics of pradefovir in animals and humans. Several drug interaction studies with humans are in progress to evaluate the drug interaction potential of pradefovir and other drugs.

On the basis of a K_m of 60 µM, a maximum rate of metabolism of 228 pmol/min/mg protein, a liver weight of 25.7 g, and a microsomal protein content of 52.5 mg/g liver (10), an intrinsic clearance of about 359 ml/min was calculated, which is much less than the liver blood flow of 1,480 ml/min. This is in good agreement with findings that show that less than 60% of pradefovir is converted to PMEA in humans (11).

Testosterone is a typical substrate for CYP3A4 (17). As expected, addition of MAb 3A4 decreased the 6ß-hydroxylation of testosterone (Fig. 5). Similarly, addition of MAb 3A4 also greatly decreased the conversion of pradefovir to PMEA (Fig. 5), confirming that the conversion of pradefovir to PMEA is metabolized primarily by CYP3A4. Similarly, addition of ketoconazole also rapidly decreased the conversion of pradefovir to PMEA (Fig. 6), further affirming that the conversion of pradefovir to PMEA is catalyzed primarily by CYP3A4.

Ketoconazole inhibition of the conversion of pradefovir to PMEA was expressed as the plot of the rate of conversion (V) versus the substrate concentration (S). It was also expressed as 1/V versus 1/S to determine K_i. However, the intercept at the x axis was too small (Fig. 6) and was deemed to be inaccurate. As a consequence, a slope (S/V) was calculated and a plot of S/V versus [I] was used to determine K_i. A small K_i of 12.5 nM was thus calculated (Fig. 3), indicating that ketoconazole is a very potent inhibitor of the conversion of pradefovir to PMEA.

Our results (Fig. 6) indicate that ketoconazole is a noncompetitive inhibitor of the conversion of pradefovir to PMEA. This is in good agreement with the findings in the literature, which state that ketoconazole is also a noncompetitive inhibitor for midazolam (6), cyclosporine A (16), and estradiol (19).

It has been well established that P450 in the liver is responsible for the metabolism of most drugs. However, it is known that P450 is also present in the gastrointestinal tract (12). In order to elucidate whether pradefovir may already be converted to PMEA by gastrointestinal P450 prior to entry into the liver, a study was carried out with portal vein-cannulated rats. As shown in Table 3, the ratios of PMEA to pradefovir were much higher in systemic plasma than portal plasma; and PMEA was detected at the earliest time point (5 min) in systemic plasma but not in portal plasma, clearly indicating that gastrointestinal P450 did not play a significant role in the conversion of pradefovir to PMEA in rats. However, it should be noted that the findings for rats do not necessarily imply that human gastrointestinal CYP3A4 may not provide a greater amount of metabolism.

In a recent study, it has been shown that pradefovir was not an inducer of P450 enzymes in rats following daily oral dosing (300 mg/kg) for 8 days. In addition, pradefovir at concentrations up to 10 µg/ml was not an inducer of either CYP1A2 or CYP3A4/5 in primary cultures of human hepatocytes. However, further drug interaction studies are needed to confirm the lack of enzyme induction potential for pradefovir in humans.

Our data clearly demonstrate that pradefovir is a good substrate for liver CYP3A4. It is neither an inhibitor nor an inducer of P450. These results suggest that the coadministration of pradefovir would not affect the pharmacokinetics of other drugs.

 
* Corresponding author. Mailing address: Drug Development, Valeant Research & Development, Costa Mesa, CA 92626. Phone: (714) 545-1011. Fax: (714) 641-7201. E-mail: cclin{at}valeant.com.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Antimicrobial Agents and Chemotherapy, September 2006, p. 2926-2931, Vol. 50, No. 9
0066-4804/06/$08.00+0     doi:10.1128/AAC.01566-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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