anti-HBV, HepG2 cell(EC50=3.75 nM)

Entecavir-triphosphate is a highly potent inhibitor of wild-type HBV Pol and is 100- to 300-fold more potent than lamivudine-triphosphate against 3TC-resistant HBV Pol. Entecavir inhibits the replication of 3TC-resistant HBV, but 20- to 30-fold higher concentrations are required. Entecavir results in an impressive reduction of serum viral DNA with covalently closed circular DNA and hepatitis B viral core antigen negativity in liver biopsy specimens. Entecavir has potent activity (EC50, 0.1 nM) against HIV in a unique single-cycle, single-cell-based pseudovirus assay (24) with CD4+ lymphocytes using a green fluorescent protein reporter fluorescence-activated cell sorter assay as the endpoint.

Entecavir causes a 4-log drop in serum DHBV DNA levels within 80 days and a slower 2- to 3-log drop in serum DHBV surface antigen (DHBsAg) levels within 120 days in ducks. Entecavir treatment reduces DHBV DNA replicative intermediates 70-fold in the liver, while the level of the stable, template form, covalently closed circular DNA decreases only 4-fold in ducks. Entecavir treatment reduces both the intensity of antigen staining and the percentage of antigen-positive hepatocytes in the liver, but the intensity of antigen staining in bile duct cells appeares not to be effected in ducks. Daily oral treatment with BMS-200475 at doses ranging from 0.02 to 0.5 mg/kg of body weight for 1 to 3 months effectively reduces the level of woodchuck hepatitis virus (WHV) viremia in chronically infected woodchucks

BMS-200475 has a EC50 of 3.75 nM against HBV. It is incorporated into the protein primer of HBV and subsequently inhibits the priming step of the reverse transcriptase. The antiviral activity of BMS-200475 is significantly less against the other RNA and DNA viruses. Entecavir is more readily phosphorylated to its active metabolites than other deoxyguanosine analogs (penciclovir, ganciclovir, lobucavir, and aciclovir) or lamivudine. The intracellular half-life of entecavir is 15 h.

BMS 200475 is prepared in phosphate-buffered saline (PBS) and diluted with appropriate medium containing 2% fetal bovine serum. HepG2 2.2.15 cells are plated at a density of 5×105 cells per well on 12-well Biocoat collagen-coated plates and are maintained in a confluent state for 2 to 3 days before being overlaid with 1 mL of medium spiked with BMS 200475. Quantification of HBV was performed on day 10.

This study was designed to test the efficacy of antiviral treatment with entecavir (ETV) in combination with DNA vaccines expressing duck hepatitis B virus (DHBV) antigens as a therapy for persistent DHBV infection in ducks. Ducks were inoculated with 10(9) DHBV genomes at 7 days of age, leading to widespread infection of the liver and viremia within 7 days, and were then treated orally with either ETV (0.1 mg/kg of body weight/day) or distilled water from 21 days posthatch for 244 days. Treatment with ETV caused a 4-log drop in serum DHBV DNA levels within 80 days and a slower 2- to 3-log drop in serum DHBV surface antigen (DHBsAg) levels within 120 days. Following withdrawal of ETV, levels of serum DHBV DNA and DHBsAg rebounded to match those in the water-treated animals within 40 days. Sequential liver biopsy samples collected throughout the study showed that ETV treatment reduced DHBV DNA replicative intermediates 70-fold in the liver, while the level of the stable, template form, covalently closed circular DNA decreased only 4-fold. ETV treatment reduced both the intensity of antigen staining and the percentage of antigen-positive hepatocytes in the liver, but the intensity of antigen staining in bile duct cells appeared not to be effected. Intramuscular administration of five doses of a DNA vaccine expressing the DHBV presurface, surface, precore, and core antigens, both alone and concurrently with ETV treatment, on days 50, 64, 78, 127, and 141 did not result in any significant effect on viral markers.[2]
Drug administration. Ducks from groups 1 and 2 were weighed three times a week, and ETV at a dose of 0.1 mg/kg/day was administered by gavage to these ducks each day for 244 days. Control ducks (groups 3, 4, and 5) were weighed weekly and given 2 ml of distilled water by gavage daily.[2]

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0.02 to 0.5 mg/kg; oral

Ducks and Wookchucks

Absorption, Distribution and Excretion
Absorption Following oral administration in healthy subjects, entecavir peak plasma concentrations occurred between 0.5 and 1.5 hours. In healthy subjects, the bioavailability of the tablet is 100% relative to the oral solution.
renal cl=383.2 +/- 101.8 mL/min [Unimpaired renal function]
renal cl=197.9 +/- 78.1 mL/min [Mild impaired renal function]
renal cl=135.6 +/- 31.6 mL/min [Moderate impaired renal function]
renal cl=40.3 +/- 10.1 mL/min [severe impaired renal function]
apparent oral cl=588.1 +/- 153.7 mL/min [Unimpaired renal function]
apparent oral cl=309.2 +/- 62.6 mL/min [Mild impaired renal function]
apparent oral cl=226.3 +/- 60.1 mL/min [Moderate impaired renal function]
apparent oral cl=100.6 +/- 29.1 mL/min [severe impaired renal function]
apparent oral cl=50.6 +/- 16.5 mL/min [severe impaired renal function amnaged with Hemodialysis]
apparent oral cl=35.7 +/- 19.6 mL/min [severe impaired renal function amnaged with CAPD]
The bioavailability of /entecavir/ tablets was /equivalent/ to the oral solution.
Oral administration of 0.5 mg of entecavir given with a standard high fat meal resulted in a delayed in absorption (1 to 1.5 hours fed vs. 0.75 hours fasted), a decrease in Cmax of 44% to 46%, and a decrease in AUC of 18% to 20%.
/The estimated apparent volume of distribution is in excess of total body water/, entecavir is extensively distributed into tissues.
/Binding of entecavir to human serum proteins in vitro is/ low (13%)
For more Absorption, Distribution and Excretion (Complete) data for ENTECAVIR (7 total), please visit the HSDB record page.

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Metabolism / Metabolites
Entecavir is not a substrate, inhibitor, or inducer of the cytochrome P450 (CYP450) enzyme system. Entecavir is efficiently phosphorylated to the active triphosphate form.
Following administration of 14C-entecavir, no oxidative or acetylated metabolites were observed. Minor amounts of phase II metabolites (glucuronide and sulfate conjugates) were observed. Entecavir is not a substrate, inhibitor, or inducer of the cytochrome P450 enzyme system.

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Biological Half-Life
After reaching peak concentration, entecavir plasma concentrations decreased in a bi-exponential manner with a terminal elimination half-life of approximately 128-149 hours. The phosphorylated metabolite has a half-life of 15 hours.
Elimination /half-life is/ approximately 128 to 149 hours.
Accumulation /half-life is/ 24 hours

Hepatotoxicity
Elevations in serum ALT levels occur in 2% to 10% patients with chronic hepatitis B treated with entecavir. These elevations appear to be due to a transient flare in the underlying chronic hepatitis B and occur both during and after therapy. On treatment, ALT flares typically occur during the first 1 to 2 months of therapy and are mild, asymptomatic and self-limited, accompanying the rapid declines in HBV DNA levels. Withdrawal flares in ALT levels occur in 8% to 12% of patients who receive entecavir therapy of hepatitis B and are subsequently withdrawn from treatment. The withdrawal flares arise within 1 to 3 months of stopping treatment and are usually preceded by marked and sudden rises in HBV DNA levels towards pretreatment values. Withdrawal flares of hepatitis B can be symptomatic and severe, and several instances of acute liver failure have been described in patients withdrawn from therapy after 1 to 3 years of treatment. There have been few reports of withdrawal flares after stopping entecavir, because the majority of patients have continued therapy indefinitely. However, the rate of flares and their severity are likely to be similar after stopping entecavir as after withdrawal of other therapies of hepatitis B.
Several instances of lactic acidosis have been reported in patients with advanced hepatitis B who were treated with entecavir; however, lactate levels were reported to be normal in patients with cirrhosis started on entecavir and followed prospectively, and in large clinical trials lactic acidosis has not been reported. The cases of lactic acidosis attributed to entecavir occurred largely in patients with severe, advanced disease and may have been due to septicemia, hypotension and/or hepatic failure rather than an adverse reaction to entecavir. Therapy with entecavir has not been associated with development of the typical syndrome of lactic acidosis, hepatic steatosis and liver failure in patients without severe preexisting liver disease that has been described with stavudine, didanosine and zidovudine treatment. Thus, clinically apparent direct hepatotoxicity from entecavir must be rare, if it occurs at all.
Likelihood score: E* (suspected but unproven cause of clinically apparent liver rinjury).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Entecavir has not been studied in nursing mothers being treated for hepatitis B infection. An alternate drug may be preferred, especially while nursing a newborn or preterm infant.
No differences exist in infection rates between breastfed and formula-fed infants born to hepatitis B-infected women, as long as the infant receives hepatitis B immune globulin and hepatitis B vaccine at birth. Mothers with hepatitis B are encouraged to breastfeed their infants after their infants receive these preventative measures.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
Binding of entecavir to human serum proteins in vitro is approximately 13%.

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Interactions
Potential pharmacokinetic interactions with drugs that reduce renal function or that may compete with entecavir for active renal tubular secretion; increased serum concentrations of entecavir or the concomitantly used drug may occur. Although the effect of concomitant use of such drugs with entecavir has not been specifically studied, patients receiving entecavir in conjunction with other drugs that may affect renal function or are excreted renally should be monitored closely for adverse effects.
In vitro evidence indicates that concurrent use of nucleoside reverse transcriptase inhibitors (NRTIs) and entecavir is unlikely to reduce the antiviral efficacy of entecavir against HBV or the antiretroviral activity of NRTIs (e.g., abacavir, didanosine, lamivudine, stavudine, tenofovir disoproxil fumarate, or zidovudine) against HIV.
Possible pharmacokinetic interaction (increased entecavir serum concentrations because of altered renal function) with cyclosporine or tacrolimus. Monitor renal function prior to and during entecavir treatment in patients (e.g., transplant patients) receiving cyclosporine, tacrolimus, or other immunosuppressive agents that may affect renal function.

[1]. Antimicrob Agents Chemother.2002 Aug;46(8):2525-32.

[2]. Antimicrob Agents Chemother.2003 Aug;47(8):2624-35.

[3]. Antimicrob Agents Chemother. 1997 Jul;41(7):1444-9.

[4]. Curr Med Res Opin.?2005 Nov;21(11):1845-57.

[5]. Antimicrob Agents Chemother.?1998 Dec;42(12):3209-18.

Therapeutic Uses
Antiviral Agents
Entecavir has been evaluated for the treatment of chronic HBV infection in a phase III, randomized, double-blind, active-controlled study (AI463038) in HIV-infected patients receiving highly active antiretroviral therapy (HAART) that included lamivudine. These patients had recurrent HBV viremia (99% were HBeAg-positive), active HBV replication (median baseline serum HBV DNA levels 9.13 log10 copies/mL), and persistent elevations in serum ALT concentrations (mean serum ALT of 71.5 IU/L). Patients continued HAART (including lamivudine 300 mg daily) and were randomized to receive concurrent therapy with entecavir (1 mg daily) or placebo for 24 weeks followed by an additional 24-week open-label period during which all patients received entecavir (1 mg daily). Analysis of limited data at 24 weeks indicated that 6% of patients who received entecavir in conjunction with lamivudine-containing HAART had undetectable levels of serum HBV DNA (defined as less than 300 copies/mL by PCR assay) compared with 0% of those who received placebo and lamivudine-containing HAART. Thirty-four percent of patients receiving the regimen that included entecavir had normal serum ALT concentrations (i.e., biochemical response) at week 24 compared with 8% of those who did not receive entecavir. In addition, the mean decrease in serum HBV DNA levels from baseline was 3.65 log10 copies/mL at week 24 in those receiving the regimen that included entecavir compared with an increase of 0.11 log10 copies/mL in those receiving a lamivudine-containing HAART regimen alone. Median serum HIV-1 RNA levels remained stable at approximately 2 log10/mL during the 24-week blinded study period.
Entecavir has been evaluated for the treatment of lamivudine-refractory chronic HBV infection in a phase III, randomized, double-blind, active-controlled study (AI463026) in adults with active HBV replication (median baseline serum HBV DNA levels 9.36 log10 copies/mL), persistent elevations in serum ALT concentrations (mean serum ALT of 128 IU/L), and histologic evidence of active liver disease (mean Knodell necroinflammatory score of 6.5). 27 Seventy-six percent of patients in the study were male, 37% were Asian, 62% were Caucasian, and 52% had prior treatment with interferon alfa. Patients in the study had previously received lamivudine therapy for a mean duration of 2.7 years and lamivudine-resistant mutations were identified at baseline in 85% of patients. Patients were randomized to switch (without a washout or an overlap period) from lamivudine to entecavir (1 mg daily) or to continue lamivudine (100 mg daily) for 52 weeks. Data analysis at 48 weeks indicated that 55% of patients switched to entecavir had histologic improvement (defined as a reduction of at least 2 points in the Knodell necroinflammatory score with no concurrent worsening of the Knodell fibrosis score) compared with 28% of those who continued to receive lamivudine. Sixty-one percent of patients receiving entecavir had normal serum ALT concentrations (i.e., biochemical response) at week 48 compared with 15% of those receiving lamivudine. In addition, the mean decrease in serum HBV DNA levels from baseline was 5.11 log10 copies/mL at week 48 in those receiving entecavir compared with a decrease of 0.48 log10 copies/mL in those receiving lamivudine. Nineteen percent of patients who received entecavir had undetectable levels of serum HBV DNA (defined as less than 300 copies/mL by PCR assay) at week 48 compared with 1% of those who received lamivudine. Seroconversion to anti-HBe occurred in 8 or 3% of patients who received entecavir or lamivudine, respectively. Approximately 55% of patients treated with entecavir continued treatment for up to 96 weeks; 40% of these patients had undetectable levels of serum HBV DNA, 81% had normal ALT concentrations, and 10% achieved seroconversion.
Efficacy of entecavir for the management of HBeAg-negative, anti-HBe- and HBV-DNA-positive chronic HBV infection was evaluated in a phase III, randomized, double-blind, active-controlled study (AI463027) in nucleoside-naive adults with active HBV replication (median baseline serum HBV DNA levels 7.58 log10 copies/mL by a PCR-based assay), persistent elevations in serum ALT concentrations (mean serum ALT of 142 IU/L), and histologic evidence of active liver disease (mean Knodell necroinflammatory score of 7.8). Seventy-six percent of patients in the study were male, 58% were Caucasian, 39% were Asian, and 13% had prior treatment with interferon alfa. Data analysis at 48 weeks indicated that 70% of patients who received entecavir (0.5 mg daily) had histologic improvement (defined as a reduction of at least 2 points in the Knodell necroinflammatory score with no concurrent worsening of the Knodell fibrosis score) compared with 61% of those who received lamivudine (100 mg daily). Serum ALT concentrations also normalized at week 48 in 78% of patients who received entecavir compared with 71% of those who received lamivudine daily. In addition, the mean decrease in serum HBV DNA levels from baseline was 5.04 log10 copies/mL at week 48 in patients receiving entecavir 0.5 mg daily compared with a mean decrease of 4.53 log10 copies/mL in those receiving lamivudine 100 mg daily. Ninety percent of patients who received entecavir had undetectable levels of serum HBV DNA (defined as less than 300 copies/mL by PCR assay) at week 48 compared with 72% of those who received lamivudine. Eighty-five percent of patients treated with entecavir met the response criteria (determined at 48 weeks based on HBV virologic suppression [less than 0.7 MEq/mL by bDNA assay] and loss of HBeAg) and discontinued entecavir at 52 weeks per protocol; very few of these patients had undetectable levels of serum HBV DNA and 46% of patients maintained normal ALT concentrations during an additional 24 weeks of follow-up.
For more Therapeutic Uses (Complete) data for ENTECAVIR (6 total), please visit the HSDB record page.

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Drug Warnings
The optimal duration of treatment with entecavir in patients with chronic hepatitis B infection and the relationship between treatment and long-term outcomes such as cirrhosis and hepatocellular carcinoma are unknown.
Severe acute exacerbations of hepatitis have occurred following discontinuance of hepatitis B virus (HBV) therapy, including entecavir therapy. In studies that evaluated safety of entecavir, exacerbations of hepatitis or ALT flare was defined as ALT elevations greater than 10 times the upper limit of normal (ULN) and greater than 2 times baseline serum concentrations. In clinical studies (AI463022, AI463027, AI463026), ALT flare occurred in 2, 8, or 12% of nucleoside-naive HBeAg-positive, nucleoside-naive HBeAg-negative, or lamivudine-refractory patients, respectively, following discontinuance of entecavir. The median time to exacerbations of hepatitis was 23 weeks. Rates of post-treatment ALT flare may be higher if entecavir therapy is discontinued without regard to previous response to therapy.
FDA Pregnancy Category C; RISK CANNOT BE RULED OUT. Adequate, well controlled human studies are lacking, and animal studies have shown risk to the fetus or are lacking as well. There is a chance of fetal harm if the drug is given during pregnancy; but the potential benefits may outweigh the potential risk.
Not known whether entecavir is distributed into milk. Discontinue nursing or the drug, taking into account the importance of the drug to the woman.
For more Drug Warnings (Complete) data for ENTECAVIR (15 total), please visit the HSDB record page.

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Pharmacodynamics
Entecavir is a guanosine nucleoside analogue with selective activity against hepatitis B virus (HBV). It is designed to selectively inhibit the Hepatitis B virus, blocking all three steps in the replication process. Entecavir is more efficient than an older Hepatitis B drug, lamivudine.

C12H15N5O3

277.28

277.117

C, 51.98; H, 5.45; N, 25.26; O, 17.31

142217-69-4

Entecavir monohydrate;209216-23-9;Entecavir-13C2,15N;(1R,3S,4R)-ent-Entecavir;188399-46-4;Entecavir-d2

135398508

White to off-white solid powder

1.8±0.1 g/cm3

734.2ºC at 760 mmHg

249-252ºC

397.9ºC

1.837

-0.96

4

5

2

20

480

3

O([H])[C@@]1([H])C([H])([H])[C@@]([H])(C(=C([H])[H])[C@]1([H])C([H])([H])O[H])N1C([H])=NC2C(N([H])C(N([H])[H])=NC1=2)=O

QDGZDCVAUDNJFG-FXQIFTODSA-N

InChI=1S/C12H15N5O3/c1-5-6(3-18)8(19)2-7(5)17-4-14-9-10(17)15-12(13)16-11(9)20/h4,6-8,18-19H,1-3H2,(H3,13,15,16,20)/t6-,7-,8-/m0/s1

2-amino-9-[(1S,3R,4S)-4-hydroxy-3-(hydroxymethyl)-2-methylidenecyclopentyl]-3H-purin-6-one

Entecavir; Baraclude; BMS-200475; BMS-200475; BMS-200475; FT-0083013; FT0083013; FT0083013; D07896; SQ 34676; SQ-34676; SQ34676;

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 (e.g. under nitrogen), 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)

DMSO : ≥ 44~55 mg/mL (158.68~198.35 mM)

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.)