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

Abstract

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 M, 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 M 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 g/ml was not an inducer of either CYP1A2 or CYP3A4/5 in primary cultures of human hepatocytes.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).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 ...

Overview of Biologically Active Nucleoside Phosphonates

“…A remarkably pronounced enhancement of plasma stability compared to 19 was observed with the selectively liver-activated cyclic 1-aryl-1,3-propanyl ester prodrug of PMEA ( 21 , Figure 2 ), also known as pradefovir (MB-06866) (Lin et al, 2006 ). The design of 21 was based on the HepDirect™ prodrug technology, which relied on the oxidative conversion of 21 to PMEA by the action of the hepatic cytochrome P450 isozyme CYP3A4, consequently allowing for decreased non-hepatic activation.…”

Cytochrome P450-Activated Prodrugs

“…Efforts to circumvent this limitation led to the development of the prodrug pradefovir (Figure 17) [104]. The prodrug is activated by CYP3A4 hydroxylation of the ring in a manner similar to that for cyclophosphamide (Figure 3) [105]. The monophosphate thus released is phosphorylated in situ by PRPP synthase, producing adefovir.…”

“…The monophosphate thus released is phosphorylated in situ by PRPP synthase, producing adefovir. The prodrug preferentially delivers adefovir to the liver, as indicated by a 12-fold higher liver/kidney ratio of adefovir with the prodrug versus the parent agent [105]. …”

Tenofovir and Adefovir as Antiviral Agents

“…For the treatment of hepatitis B, a novel adefovir prodrug with a cyclic phosphonate promoiety (pradefovir; Figure 24.5), which is selectively activated by CYP3A4 in the liver, is being developed with the goal of reducing the systemic exposure of adefovir [186]. This liver-targeting strategy would potentially allow higher and more efficacious doses of adefovir to be delivered, without the risk of nephrotoxicity.…”