Target: HBV DNA polymerase (reverse transcriptase) via its active metabolite PMEApp
Target for metabolic activation: CYP3A4 (substrate) [1]

Pradefovir was converted to PMEA in human liver microsomes with a K(m) of 60 microM, 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 microM was neither a direct inhibitor nor a mechanism-based inhibitor of CYP3A4, CYP2D6, CYP2C9, CYP2C19, CYP2E1, and CYP1A2 in human liver microsomes.[1]

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Pradefovir (Remofovir) metabolism: In cDNA-expressed human CYP isozymes (1A1,1A2,1B1,2A6,2B6,2C8,2C9,2C18,2C19,2D6,2E1,3A4,3A5) at 1.63 μM pradefovir, only CYP3A4 showed significant activity in converting pradefovir to PMEA. Ketoconazole (0.05-2 μM) inhibited this conversion by 80-99% in CYP3A4 supersomes. [1]
In human liver microsomes (0.4 mg/ml), PMEA production from pradefovir (1.63 μM) was linear with incubation time up to 60 min (y=5.043x+6.1907, R²=0.9978) and with microsomal protein concentration (0.1-1.0 mg/ml, y=22.587x+0.948, R²=0.9859). [1]
Enzyme kinetics: Conversion of pradefovir to PMEA in human liver microsomes followed Michaelis-Menten kinetics with Km = 60 μM, Vmax = 228 pmol/min/mg protein, and intrinsic clearance = 3.8 μl/min/mg protein (based on Lineweaver-Burk plot). [1]
Inhibition studies: Ketoconazole potently inhibited pradefovir conversion to PMEA in human liver microsomes (0.5 μM ketoconazole almost completely shut down activation). The inhibition constant Ki was 12.5 nM (from slope versus [I] plot). Dixon plot indicated noncompetitive inhibition. [1]
A specific CYP3A4-inhibitory monoclonal antibody (MAb 3A4) dramatically reduced the conversion of pradefovir to PMEA in human liver microsomes, confirming CYP3A4 as the primary enzyme. [1]
Direct inhibition potential: Pradefovir (Remofovir) at 0.2, 2, and 20 μM was tested in human liver microsomes with probe substrates for CYP1A2 (phenacetin O-deethylation), CYP2C9 (diclofenac 4'-hydroxylation), CYP2C19 (S-mephenytoin 4'-hydroxylation), CYP2D6 (bufuralol 1'-hydroxylation), CYP2E1 (chlorzoxazone 6-hydroxylation), and CYP3A4 (testosterone 6β-hydroxylation). At 20 μM pradefovir, relative activities (% of control) were: CYP1A2 98%, CYP2C9 98%, CYP2C19 113%, CYP2D6 101% (at 2 μM, no 20 μM data shown but text says no inhibition), CYP2E1 108%, CYP3A4 72% (but text states "neither a direct inhibitor" based on full concentration range, and 72% still considered no significant inhibition). See Table 1 for detailed means and SD. [1]
Mechanism-based inhibition potential: Pradefovir (Remofovir) (0.2, 2, 20 μM) preincubated with human liver microsomes and NADPH for 15 min showed no significant reduction in CYP activities. Activities (% of control) at 20 μM: CYP1A2 98%, CYP2C9 95%, CYP2C19 98% (at 2 μM), CYP2D6 100% (at 2 μM), CYP2E1 111%, CYP3A4 96%. See Table 2. [1]

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 microg/ml was not an inducer of either CYP1A2 or CYP3A4/5 in primary cultures of human hepatocytes.[1]
Pradefovir (Remofovir) in portal vein-cannulated rats: Rats (fasted overnight) received 30 mg/kg oral gavage of 14C-pradefovir mesylate. Blood samples were collected simultaneously from portal and systemic vein cannulas at 2,5,10,20,40,60 min post-dose. Plasma concentrations of pradefovir and PMEA were measured (Table 3). The PMEA-to-pradefovir ratio (P/R) in portal plasma was 0.009 at 10 min, 0.105 at 20 min, 0.196 at 40 min, 0.464 at 60 min; in systemic plasma it was 0.099 at 5 min, 0.156 at 10 min, 0.514 at 20 min, 1.380 at 40 min, 2.308 at 60 min. Higher systemic ratios indicate liver is main site of conversion. PMEA detected in systemic plasma at 5 min but not in portal plasma, suggesting small intestine does not play significant role in rat. [1]
Enzyme induction in rats: Five rats received Pradefovir (Remofovir) orally at 300 mg/kg/day for 8 days. At 24 h after last dose, rats were killed and liver samples collected. Body weight, liver weight, liver/body weight ratio, microsomal protein, total CYP content, enzyme activities (CYP1A, CYP2B, CYP3A), and apoprotein levels (CYP1A1, CYP2B1/2B2, CYP3A1/3A2, CYP4A1/4A3) were determined. No significant changes observed vs control (Table 4), indicating pradefovir is not a CYP inducer in rats. [1]
Enzyme induction in primary human hepatocytes: Hepatocytes from three human livers were treated with DMSO (0.1% v/v), Pradefovir (Remofovir) (0.1, 1, 10 μg/ml), PMEA (0.01,0.1,1 μg/ml), or positive inducers β-naphthoflavone (33 μM) or rifampin (20 μM) once daily for 3 consecutive days. After treatment, microsomes were prepared and assayed for CYP1A2 (7-ethoxyresorufin O-dealkylation) and CYP3A4/5 (testosterone 6β-hydroxylation) activities. Pradefovir up to 10 μg/ml caused no significant changes: CYP1A2 activity (pmol/mg/min) = 1.70±0.42 (0.1 μg/ml), 2.18±0.76 (1 μg/ml), 2.54±0.40 (10 μg/ml) vs DMSO control 2.25±0.56; CYP3A4/5 activity = 748±732 (0.1 μg/ml), 899±673 (1 μg/ml), 414±221 (10 μg/ml) vs DMSO 980±708. Positive controls: β-naphthoflavone induced CYP1A2 to 15.1±5.6; rifampin induced CYP3A4/5 to 8,130±3,540. Thus pradefovir and PMEA are not inducers of CYP1A2 or CYP3A4/5 in human hepatocytes (Table 5). [1]

Pradefovir (Remofovir) conversion by cDNA-expressed CYPs: Pradefovir (1.63 μM) was incubated with each cDNA-expressed CYP isozyme (20 mM) in potassium phosphate buffer (50 mM, pH 7.4). Reactions were preheated at 37°C for 3 min, initiated by NADPH (1 mM), and proceeded for 10-60 min. Incubations stopped by acetonitrile, centrifuged, supernatant dried under N2, redissolved in reconstitution solution (10 mM ammonium acetate/3% dimethylhexylamine/1.5% hydroxyacetate) with internal standard (13C-PMEA). PMEA was analyzed by LC-MS/MS (negative electrospray, ion transitions 272→134 for PMEA, 277→139 for 13C-PMEA). [1]
Human liver microsome incubation: Pooled human liver microsomes (0.4 mg/ml) were incubated with pradefovir (1.63, 4.08, 16.3, or 40.8 μM) in potassium phosphate buffer with or without ketoconazole (0-2 μM) or CYP3A4-inhibitory monoclonal antibody. Reactions initiated by NADPH (1 mM), typically for 15 min at 37°C. PMEA or 6β-hydroxytestosterone (for testosterone substrate) quantified by LC-MS/MS. For 6β-hydroxytestosterone, positive electrospray monitored transitions 305→269; dextrorphan (internal standard) 258→157. [1]
Enzyme kinetics: Lineweaver-Burk plot (1/V vs 1/S) used to calculate Km and Vmax. Intrinsic clearance = Vmax/Km. For inhibition constant Ki, slope from double-reciprocal plot (S/V) plotted against inhibitor concentration [I] (ketoconazole). Dixon plot (1/V vs [I]) identified noncompetitive inhibition. [1]
Direct inhibition assay: Pradefovir (Remofovir) (0, 0.2, 2, 20 μM) incubated with human liver microsomes and CYP-specific probe substrates (phenacetin for CYP1A2, diclofenac for CYP2C9, S-mephenytoin for CYP2C19, bufuralol for CYP2D6, chlorzoxazone for CYP2E1, testosterone for CYP3A4) plus NADPH. Reaction products (acetaminophen, 4-hydroxyclofofenac, 4-hydroxymephenytoin, 1-hydroxybufuralol, 6-hydroxychlorzoxazone, 6β-hydroxytestosterone) measured by LC-MS/MS. [1]
Mechanism-based inhibition assay: Pradefovir (Remofovir) preincubated with microsomes and NADPH for 15 min, then probe substrates added and reaction continued as above. [1]

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.[1]
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 (β-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.[1]

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Primary human hepatocyte culture: Hepatocytes from three human livers were cultured and treated with DMSO (0.1% v/v), Pradefovir (Remofovir) (0.1, 1, 10 μg/ml), PMEA (0.01, 0.1, 1 μg/ml), β-naphthoflavone (33 μM), or rifampin (20 μM) once daily for 3 consecutive days. After treatment, cells were harvested to prepare microsomes. Microsomal activities were assayed for 7-ethoxyresorufin O-dealkylation (CYP1A2 marker) and testosterone 6β-hydroxylation (CYP3A4/5 marker). Additionally, S-mephenytoin N-demethylation (CYP2B6), diclofenac 4'-hydroxylation (CYP2C9), and S-mephenytoin 4'-hydroxylation (CYP2C19) were analyzed in cells treated with DMSO or rifampin. [1]

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.[1]
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.[1]

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Portal vein-cannulated rat study: Male rats (fasted overnight) were given Pradefovir (Remofovir) as 14C-pradefovir mesylate at 30 mg/kg body weight by oral gavage. Blood samples were collected from portal vein and systemic vein cannulas at 2, 5, 10, 20, 40, and 60 min post-dose into heparinized tubes. Whole blood was centrifuged to obtain plasma. Plasma concentrations of pradefovir and PMEA were determined by validated LC-MS/MS method (calibration curves 1-100 ng/ml for pradefovir, 10-1000 ng/ml for PMEA; LLOQ 1 ng/ml and 10 ng/ml respectively). [1]
Rat enzyme induction study: Five rats received Pradefovir (Remofovir) orally at 300 mg/kg/day for 8 days. At 24 h after the last dose, rats were killed and liver samples collected. Body weight, liver weight, liver protein content, and liver microsomal P450 contents were determined. Apoprotein levels for CYP1A1, CYP2B1/2B2, CYP3A1/3A2, and CYP4A1/4A3 were measured by Western blot. Enzyme activities for CYP1A, CYP2B, and CYP3A were determined by LC analysis. [1]
Human hepatocyte induction study: Primary human hepatocytes from three donors were treated once daily for 3 consecutive days with Pradefovir (Remofovir) at indicated concentrations. After treatment, cells were harvested to prepare microsomes for enzyme activity assays as described under Cell Assay. [1]

In human liver microsomes: Conversion of Pradefovir (Remofovir) to PMEA follows Michaelis-Menten kinetics with Km = 60 μM, Vmax = 228 pmol/min/mg protein, intrinsic clearance = 3.8 μl/min/mg protein (or calculated whole-liver intrinsic clearance about 359 ml/min based on liver weight 25.7 g and microsomal protein 52.5 mg/g liver). This intrinsic clearance is much less than liver blood flow (1480 ml/min), consistent with less than 60% conversion of pradefovir to PMEA in humans. [1]
In portal vein-cannulated rats after oral dosing (30 mg/kg): Plasma concentrations of pradefovir and PMEA over time (Table 3). At 60 min, portal plasma: pradefovir 0.80 μM, PMEA 0.37 μM (P/R=0.464); systemic plasma: pradefovir 0.14 μM, PMEA 0.33 μM (P/R=2.314). The higher systemic P/R ratio indicates liver is main site of conversion. PMEA was detected in systemic plasma at 5 min but not in portal plasma, suggesting small intestine does not contribute significantly in rats. [1]

Pradefovir (Remofovir) is neither a direct inhibitor nor a mechanism-based inhibitor of CYP3A4, CYP2D6, CYP2C9, CYP2C19, CYP2E1, and CYP1A2 in human liver microsomes at tested concentrations up to 20 μM (Tables 1 and 2). [1]
Pradefovir (Remofovir) at 300 mg/kg/day oral dosing for 8 days in rats did not affect body weight, liver weight, liver/body weight ratio, microsomal protein, total CYP content, CYP1A, CYP2B, CYP3A activities, or apoprotein levels of CYP1A1, CYP2B1/2, CYP3A1/2, CYP4A1/3 (Table 4), indicating no CYP induction in rats. [1]
Pradefovir (Remofovir) at up to 10 μg/ml in primary human hepatocytes caused no significant induction of CYP1A2 or CYP3A4/5 activities (Table 5), indicating no enzyme induction potential in humans. [1]

Antimicrob Agents Chemother.2006 Sep;50(9):2926-31.

Pradefovir is a prodrug of adefovir. Pradefovir is a cyclic diester antiviral prodrug with specific activity against hepatitis B virus (HBV). Pradefovir is primarily metabolized in the liver by liver enzymes (mainly CYP4503A4) to adefovir. Adefovir is then phosphorylated by cellular kinases to its active form, adefovir diphosphate. Adefovir diphosphate competes with the natural substrate dATP for incorporation into viral DNA, inhibiting RNA-dependent DNA polymerase. This leads to DNA chain termination, ultimately inhibiting HBV replication.

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Pradefovir (Remofovir) is a HepDirect prodrug designed to be efficiently and specifically activated by liver CYP3A4 to release PMEA, which is then phosphorylated to PMEApp (PMEA diphosphate) by cellular kinases or 5-phosphoribosyl-1-pyrophosphate synthetase. PMEApp inhibits HBV DNA polymerase (reverse transcriptase) by competing with natural substrate dATP and causing DNA chain termination. [1]
PMEA (the active moiety) has been shown effective against 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. [1]
Adefovir dipivoxil (an oral prodrug of PMEA) at 10 mg daily is used for chronic hepatitis B treatment with favorable risk-benefit profile; at 30 mg dose, higher incidence of serum creatinine increases was observed. Pradefovir (Remofovir) was developed as an alternative prodrug to improve liver targeting and safety. [1]

C17H19CLN5O4P

423.79

423.086

625095-60-5

9604654

Typically exists as solid at room temperature

3.988

1

8

6

28

576

2

ClC1=CC=CC(=C1)[C@@H]1CCOP(COCCN2C=NC3C(N)=NC=NC2=3)(=O)O1

GWNHAOBXDGOXRR-HJFSHJIFSA-N

InChI=1S/C17H19ClN5O4P/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/h1-3,8-10,14H,4-7,11H2,(H2,19,20,21)/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

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

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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