Pradefovir is a PMEA prodrug that is cyclic diester. It is one of the HepDirect prodrugs made to be selectively and potently activated by oxidative processes mediated by the liver-based enzyme CYP3A4. With a Km of 60 μM, a maximum metabolic rate of 228 pmol/min/mg protein, and an intrinsic clearance rate of around 359 L/min, pradefovir is transformed into PMEA in human liver microsomes [1].

Pradefovir is a PMEA prodrug that is cyclic diester. It is one of the HepDirect prodrugs made to be selectively and potently activated by oxidative processes mediated by the liver-based enzyme CYP3A4. With a Km of 60 μM, a maximum metabolic rate of 228 pmol/min/mg protein, and an intrinsic clearance rate of around 359 L/min, pradefovir is transformed into PMEA in human liver microsomes [1].

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Pradefovir is metabolically activated to PMEA by CYP3A4 in human liver microsomes with a Km of 60 µM, a maximum rate of metabolism of 228 pmol/min/mg protein, and an intrinsic clearance of about 3.8 µl/min/mg protein.[1]
Pradefovir at concentrations of 0.2, 2, and 20 µM was neither a direct inhibitor nor a mechanism-based inhibitor of CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, or CYP3A4 in human liver microsomes.[1]
Ketoconazole (a CYP3A4 inhibitor) and a CYP3A4-inhibitory monoclonal antibody (MAb 3A4) significantly inhibited the conversion of pradefovir to PMEA in human liver microsomes, confirming the predominant role of CYP3A4 in its metabolic activation. Ketoconazole at 0.5 µM essentially shut down the activation.[1]
In primary cultures of human hepatocytes, pradefovir at concentrations up to 10 µg/ml did not induce CYP1A2 or CYP3A4/5 enzyme activities.[1]

For eight days, rats were given 300 mg/kg of prdefovir orally. The rats' body weight, liver weight, liver weight to body weight ratio, liver microsomal protein content, total CYP content, enzyme levels of CYP1A, CYP2B, and CYP3A activity, and apoprotein content of CYP1A1, CYP2B1/2, CYP3A1/2, and CYP4A1/3 were all unaffected, suggesting that prdefovir does not induce CYP in rats [1].

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In portal vein-cannulated rats administered pradefovir orally (30 mg/kg), PMEA was detected in systemic plasma but not in portal plasma at 5 minutes post-dose, indicating that the liver, not the small intestine, is the primary site of metabolic activation in rats.[1]
Daily oral administration of pradefovir (300 mg/kg/day for 8 days) to rats did not induce CYP1A, CYP2B, or CYP3A enzyme activities or their apoprotein levels, indicating that pradefovir is not a CYP inducer in rats.[1]

To identify the CYP isozyme responsible for pradefovir activation, pradefovir (1.63 µM) was incubated with a panel of cDNA-expressed human CYP supersomes (including 1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, and 3A5). Only CYP3A4 showed significant activity in converting pradefovir to PMEA. Ketoconazole was added at various concentrations (0, 0.05, 0.1, 0.2, 0.5, 1, and 2 µM) to confirm inhibition.[1]
Enzyme kinetics were studied by incubating pradefovir (1.63 to 40.8 µM) with pooled human liver microsomes (0.4 mg/ml) for 15 minutes. The reaction followed Michaelis-Menten kinetics, and kinetic parameters (Km, Vmax) were derived from Lineweaver-Burk plots.[1]
Inhibition studies were performed by co-incubating pradefovir with ketoconazole or a CYP3A4-inhibitory monoclonal antibody in human liver microsomes. The inhibition constant (Ki) for ketoconazole was determined to be 12.5 nM using a slope versus inhibitor concentration plot, and Dixon plot analysis indicated noncompetitive inhibition.[1]
To evaluate pradefovir as a direct or mechanism-based inhibitor of various CYP isozymes, pradefovir (0.2, 2, 20 µM) was incubated with human liver microsomes and CYP-specific substrates (phenacetin for CYP1A2, diclofenac for CYP2C9, S-mephenytoin for CYP2C19, buturalol for CYP2D6, chlorzoxazone for CYP2E1, testosterone for CYP3A4). For mechanism-based inhibition assessment, pradefovir was pre-incubated with microsomes and NADPH for 15 minutes before adding the substrate. Metabolite formation was measured by LC-MS/MS.[1]

Portal vein-cannulated rat study: Rats were fasted overnight and administered pradefovir mesylate (30 mg/kg) orally by gavage. Blood samples were collected simultaneously from portal and systemic vein cannulas at 2, 5, 10, 20, 40, and 60 minutes post-dose into heparinized tubes. Plasma was separated by centrifugation and analyzed for pradefovir and PMEA concentrations by a validated LC-MS/MS method.[1]
Enzyme induction study in rats: Rats received daily oral doses of pradefovir (300 mg/kg/day) for 8 days. Twenty-four hours after the last dose, rats were euthanized and livers were collected. Liver weight, body weight, liver microsomal protein content, total CYP content, and specific enzyme activities (CYP1A, CYP2B, CYP3A) and apoprotein levels (CYP1A1, CYP2B1/2, CYP3A1/2, CYP4A1/3) were determined.[1]

Pradefovir is mainly metabolized and activated by hepatic CYP3A4 to its active form PMEA. [1]
The intrinsic clearance of pradefovir to PMEA in human liver microsomes is about 359 ml/min, which is lower than hepatic blood flow, indicating that not all pradefovir can be converted to PMEA in humans. [1]
In rats, after oral administration, pradefovir is mainly converted to PMEA in the liver, and the contribution of intestinal metabolism is minimal based on portal concentration comparison. [1]
The active metabolite PMEA is poorly absorbed orally due to the ionization of its phosphonate group at physiological pH, resulting in low oral bioavailability in various animals. [1]
In rats that have been given radiolabeled pradefovir, the plasma AUC of PMEA accounts for about 23% of the total plasma radioactivity AUC, and the AUC of PMEA in urine is also low. PMEA excretion accounts for about 66% of urinary radioactivity, indicating that PMEA formation is the main metabolic pathway. [1]

Pradefovir (at concentrations up to 20 µM) did not show direct or mechanistic inhibition of major CYP enzymes (CYP1A2, 2C9, 2C19, 2D6, 2E1, 3A4) in human liver microsomes, suggesting a low likelihood of metabolic drug interactions via CYP inhibition. [1] Pradefovir (at concentrations up to 10 µg/ml) did not induce CYP1A2 or CYP3A4/5 in primary cultures of human hepatocytes. [1] Pradefovir (300 mg/kg/day for 8 consecutive days) did not induce CYP enzymes in rats, with no changes in liver weight, body weight, microsomal protein, total CYP content, specific enzyme activity, or apolipoprotein levels. [1] The prodrug design of Pradefovir aimed to enhance liver-targeted delivery and reduce systemic exposure to PMEA, thereby improving safety by limiting off-target effects. [1]

[1]. Metabolic activation of Pradefovir by CYP3A4 and its potential as an inhibitor or inducer. Antimicrob Agents Chemother. 2006 Sep;50(9):2926-31.

Pradefovir mesylate (formerly known as MB-06886, Hepavir B, and ramofovir mesylate) is an orally administered small molecule compound belonging to the novel phosphate and phosphonate prodrug family of adefovir. Adefovir (Hepsera) is a noncyclic phosphonate analog of adenine used to treat hepatitis B virus infection. Due to the poor absorption and high nephrotoxicity of adefovir, Pradefovir mesylate is designed to specifically target the liver, reducing the risk to external tissues (especially the kidneys) while improving the efficacy of adefovir. Pradefovir is activated by cytochrome P-450 (CYP) 3A4-mediated oxidation, which is primarily expressed in the liver. This novel prodrug exhibits high stability in both plasma and tissues and has demonstrated potent anti-hepatitis B virus activity in both preclinical and clinical studies. Pradefovir is currently in a Phase II clinical trial for the treatment of chronic hepatitis B.
Pradifovir mesylate is the mesylate form of pradesfovir, a cyclic diester antiviral prodrug with specific activity against hepatitis B virus (HBV). Pradesfovir is primarily metabolized in the liver by hepatic enzymes (mainly CYP450 3A4) to adefovir. Adefovir is then phosphorylated by cellular kinases to its active form, adefovir diphosphate. Adefovir diphosphate is incorporated into viral DNA by competing with the natural substrate dATP, inhibiting RNA-dependent DNA polymerase. This leads to DNA chain termination, ultimately inhibiting HBV replication.
See also: Pradesfovir (note moved to).

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Drug Indications
It has been studied as a prodrug of Hepsera for the treatment of hepatitis B (viral hepatitis).

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Mechanism of Action
Pradifovir is activated by cytochrome P-450 (CYP) 3A4-mediated oxidation, which is primarily expressed in the liver. In this way, it can selectively increase the concentration of Hepsera in the liver.

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Pharmacodynamics
Pradifovir is activated by cytochrome P-450 (CYP) 3A4-mediated oxidation, which is mainly expressed in the liver. Therefore, pradifovir can concentrate Hepsera in the liver while maintaining a low concentration in other tissues. This novel prodrug is an orally administered small molecule compound belonging to the novel phosphate and phosphonate drug family. It is highly stable in both plasma and tissues.

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Pradifovir mesylate (Remofovir) is a cyclic diester prodrug of PMEA, belonging to the HepDirect class of prodrugs designed to be selectively activated in the liver via CYP3A4. [1]
Its activation mechanism involves CYP3A4-mediated oxidation, generating a charged intermediate that is retained in hepatocytes and further converted into the active diphosphate metabolite PMEApp, which inhibits HBV DNA polymerase. [1]
PMEA (active form) has shown anti-HBV activity in stably transfected human hepatocellular carcinoma cell lines, primary duck hepatocytes infected with duck hepatitis B virus, and duck hepatitis B models. [1]
The effects of CYP3A4 inhibitors (e.g., ketoconazole) or inducers (e.g., rifampin) on the pharmacokinetics of pradefovir have been noted, and related studies are ongoing. [1]
The prodrug strategy was adopted to overcome the problem of low oral bioavailability of the parent drug PMEA. [1]

C13H23CLN5O7PS

519.90

519.074

625095-61-6

9604653

White to off-white solid powder

4.573

2

11

6

33

668

2

CS(=O)(=O)O.C1CO[P@@](=O)(O[C@@H]1C2=CC(=CC=C2)Cl)COCCN3C=NC4=C(N=CN=C43)N

JXQUAHHUSMJUFV-HZPZRMRQSA-N

InChI=1S/C17H19ClN5O4P.CH4O3S/c18-13-3-1-2-12(8-13)14-4-6-26-28(24,27-14)11-25-7-5-23-10-22-15-16(19)20-9-21-17(15)23;1-5(2,3)4/h1-3,8-10,14H,4-7,11H2,(H2,19,20,21);1H3,(H,2,3,4)/t14-,28+;/m0./s1

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: 100 mg/mL (192.34 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)