HBV (EC50 = 3.75 nM)

The EC50 value of entecavir monohydrate (also known as BMS200475 monohydrate or SQ34676 monohydrate) against HBV is 3.75 nM. It is incorporated into the HBV protein primer and then prevents reverse transcriptase from starting. The antiviral activity of BMS -200475 against other RNA and DNA viruses is notably lower [1]. Compared to Iamivudine and other deoxyguanosine analogs (Penciclovir, Ganciclovir, Lobucavir, and Aciclovir), entecavir monohydrate is phosphorylated to its active metabolite more easily. Entecavir has a 15-hour intracellular half-life [2].

In chronically infected woodchucks, daily oral treatment of Entecavir (monohydrate) at a dose range of 0.02-0.5 mg/kg for 1-3 months can effectively lower the levels of viremia caused by the woodchuck hepatitis virus (WHV) [3].

Extracellular HBV DNA analysis.[1]
HepG2 2.2.15 cells were obtained from George Acs and were maintained in RPMI 1640 supplemented with 2 mM L-glutamine, 50 U of penicillin per ml, 50 mg of streptomycin per ml, 500 mg of G418 per ml, and 5% fetal bovine serum. Stock antiviral solutions were prepared in phosphate-buffered saline (PBS) and were stored at 220°C. Concentrations were determined by UV spectroscopy. Working dilutions of antiviral compounds were prepared in medium containing 2% fetal bovine serum and were stored at 4°C for the duration of the experiment. 2.2.15 cells were plated at a density of 5 3 105 cells per well on 12-well Biocoat collagen-coated plates and were maintained in a confluent state for 2 to 3 days before being overlaid with 1 ml of medium spiked with antiviral compound. Compoundcontaining medium was changed daily for 9 days. On day 10, the media were collected and clarified by pelleting the cellular debris with a 5-min microcentrifuge spin at 16,000 3 g. Supernatants were processed for HBV DNA by the alkaline lysis method of Korba and Milman with minor modifications. Released HBV DNA was immobilized on a Magnagraph nylon membrane by using a dot blot manifold, hybridized to a probe prepared from a full-length HBV genome template excised from plasmid pTHBV-1, and 32P-radiolabeled to a specific activity of 2 3 109 cpm/mg by using a random primer kit. Quantitation was performed on a Storm 860 phosphorim-ager. All drug concentrations were tested in duplicate or triplicate, with antiviral effects being scored as reductions in the amount of HBV DNA present in the medium relative to that in untreated controls after the 10-day treatment period.

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Intracellular HBV DNA analysis. [1]
2.2.15 cells were treated with antiviral agents as described above. Monolayers were washed with PBS and were then lysed at room temperature with 1 ml of lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 5 mM MgCl2, 0.2% Nonidet P-40). Removal of cellular debris was accomplished by a 5-min microcentrifuge spin at 16,000 3 g. Lysates were then subjected to an overnight immunoprecipitation at 4°C with a rabbit anti-HBV core protein antibody coupled to protein A–Sepharose CL-4 beads. The core particles were disrupted by a 2-h proteolytic digestion with 1 mg of proteinase K per ml at 56°C, followed by phenol-chloroform-isoamyl alcohol extraction. Nucleic acids were ethanol precipitated and separated on a 1% agarose–Tris-borateEDTA gel. DNA products were depurinated (0.25 N HCl) for 15 min, denatured (0.5 N NaOH) for 30 min, and neutralized (1 M Tris-HCl [pH 7.5], 1.5 M NaCl) for 30 min. DNA was transferred to the nylon membrane by the method of Southern, hybridized to a 32P-radiolabeled HBV genomic probe, and visualized by autoradiography as described above. Assay for inhibitory activity against other viruses. The antiviral activity of BMS-200475 against influenza virus strain A/WSN/33(HIN1) was determined in an MDBK cell protection assay as described previously. Anti-human immunodeficiency virus type 1 (anti-HIV-1) strain RF activity was determined in CEM-SS and MT-2 cells by an XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]- 2H-tetrazolium-5-carboxanilide inner salt) assay. Plaque reduction assays were used to determine the activity of BMS-200475 against varicella-zoster virus (VZV) strain Ellen and herpes simplex virus type 1 (HSV-1) strain Schooler in WI-38 cells and against human cytomegalovirus (HCMV) strain AD169 in human diploid foreskin fibroblast (HFF) cells. Cytotoxicity in these cell lines was assessed by either 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MDBK, WI-38, HFF) or XTT (CEM-SS, MT-2) dye uptake assays.

Cytotoxicity.[1]
Cytotoxicity was assessed in HepG2 cells (American Type Culture Collection), the stably transfected 2.2.15 derivative cell line, and HuH-7 cells by metabolic uptake of methyl-tritiated thymidine ([methyl3 H]dT) with a specific activity of 6.7 Ci/mmol. The cells were grown to approximately 80% confluence in 24-well Biocoat plates, exposed to various concentrations of BMS-200475 for 24 h, and then pulse-labeled for 3 h with 8 mCi of [methyl3 H]dT per well. After lysis of the cells (with 20 mM Tris-HCl [pH 7.6], 30 mM NaCl, 10 mM EDTA, 1% sodium dodecyl sulfate) and proteolytic digestion, the 3 H-labeled cellular DNA was recovered by precipitation with trichloroacetic acid and was quantified by scintillation counting.

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mtDNA content.[1]
To determine the effects of antiviral treatment on mitochondrial DNA (mtDNA) content, subconfluent HepG2 cells were treated with drug for 4 or 8 days, scraped off the plates into PBS, and transferred to microcentrifuge tubes. The cells were lysed by three freeze-thaw cycles, and mtDNA was extracted by the method of Tamura and Aotsuka. The mtDNA content was quantified by dot blot hybridization with a 32P-radiolabeled probe complementary to the mitochondrial cytochrome c oxidase subunit I gene excised from plasmid pHHCJ10.

Animals. [3]
All experimental procedures involving woodchucks were reviewed and approved by the Animal Care and Use Committee of the Wallingford, Conn., site of the Pharmaceutical Research Institute of Bristol-Myers Squibb Co. Woodchucks chronically infected with WHV were obtained from Marmotech Inc. At weekly intervals, animals were weighed, anesthetized by intramuscular injection of ketamine (100 mg/kg) and xylazine (10 mg/kg), and bled from the femoral vein for serum samples which were stored at −20°C. Liver biopsies were performed on animals anesthetized with ketamine-xylazine supplemented with isofluorane inhalation as needed. Liver slices of 0.5 to 1.5 cm were aseptically removed via a ventrolateral incision over the liver, snap frozen in liquid nitrogen, and stored at −70°C prior to analysis for WHV DNA.

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Compounds. [3]
BMS-200475 was prepared as a 1-mg-per-ml stock solution in pyrogen-free sterile water. 3TC was purchased from Glaxo-Wellcome and was prepared similarly. For oral delivery, each drug solution was mixed with a liquid diet formulation so as to achieve the desired dose (based on individual-animal body weights) in a final volume of 5 ml. Placebo-treated animals received liquid diet alone.

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0.02 to 0.5 mg/kg; oral

Ducks and Wookchucks

[1]. Innaimo SF, et al. Identification of?BMS-200475?as a potent and selective inhibitor of hepatitis B virus. Antimicrob Agents Chemother. 1997 Jul;41(7):1444-8.
[2]. Rivkin A, et al. A review of entecavir in the treatment of chronic hepatitis B infection. Curr Med Res Opin.?2005 Nov;21(11):1845-56.
[3]. Genovesi EV, et al. Efficacy of the carbocyclic 2'-deoxyguanosine nucleoside?BMS-200475?in the woodchuck model of hepatitis B virus infection. Antimicrob Agents Chemother.?1998 Dec;42(12):3209-17

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.
Antiviral Agents: Agents used in the prophylaxis or therapy of VIRUS DISEASES. Some of the ways they may act include preventing viral replication by inhibiting viral DNA polymerase; binding to specific cell-surface receptors and inhibiting viral penetration or uncoating; inhibiting viral protein synthesis; or blocking late stages of virus assembly.

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Absorption: 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.
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.
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.

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By competing with the natural substrate deoxyguanosine triphosphate, entecavir functionally inhibits all three activities of the HBV polymerase (reverse transcriptase, rt): (1) base priming, (2) reverse transcription of the negative strand from the pregenomic messenger RNA, and (3) synthesis of the positive strand of HBV DNA. Upon activation by kinases, the drug can be incorporated into the DNA which has the ultimate effect of inhibiting the HBV polymerase activity.
Entecavir is a nucleoside analogue with activity against HBV polymerase. It is efficiently phosphorylated to the active triphosphate form, which competes with the natural substrate deoxyguanosine triphosphate and inhibits all three activities of the HBV polymerase (reverse transcriptase): 1) base priming, 2) reverse transcription of the negative strand from the pregenomic messenger RNA and 3) synthesis of the positive strand of HBV DNA. Entecavir triphosphate is a weak inhibitor of cellular DNA polymerases alpha, beta, and delta and mitochondrial DNA polymerase gamma with Ki values ranging from 18 to > 160 microM.

C12H15N5O3.H2O

295.29

295.12805

C, 48.81; H, 5.80; N, 23.72; O, 21.67

209216-23-9

Entecavir;142217-69-4; 209216-23-9 (hydrate); 142217-69-4 (free); 911138-73-3; 188399-46-4 (enantiomer)

135526609

White to off-white solid powder

1.81

661.4ºC at 760 mmHg

259 °C

2.15E-18mmHg at 25°C

139.28

O=C1NC(N)=NC2=C1N=CN2[C@@H]3C([ C@H](CO)[C@@H](O)C3)=C.[H]O[H]

YXPVEXCTPGULBZ-WQYNNSOESA-N

InChI=1S/C12H15N5O3.H2O/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);1H2/t6-,7-,8-;/m0./s1

6H-Purin-6-one, 2-amino-1,9-dihydro-9-((1S,3R,4S)-4-hydroxy-3-(hydroxymethyl)-2-methylenecyclopentyl)- monohydrate

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)

Solubility in Formulation 1: ≥ 3 mg/mL (10.16 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 30.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 3 mg/mL (10.16 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 30.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 3 mg/mL (10.16 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 30.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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