HBV/hepatitis B virus

In this article, we describe a novel phosphonate nucleoside,  9-[1-(phosphonomethoxycyclopropyl)methyl]guanine (PMCG, 1), which exhibits a highly potent and selective anti-HBV activity (EC50 = 0.5 μM). Its orally available drug candidate, 9-[1-(phosphonomethoxycyclopropyl)methyl]-6-deoxyguanine Dipivoxil (Besifovir Dipivoxil (PMCDG Dipivoxil), 2) (Figure 1) is also described. Evaluation of anti-HBV activity of these synthetic compounds was conducted with HepG2 2.2.15 cells transfected with the HBV genome. As shown in Table 1, 1 and 16 are the most potent (EC50 = 0.5 μM). The other modified guanine base analogues also exhibit moderate to high anti-HBV activity (EC50 = 1.5−8.0 μM), while the thymine base analogue 11 and 6-ethylaminoguanine analogue 14 do not show anti-HBV activity at 30 μM. Among the modified guanine base analogues, the anti-HBV activity of 8, 15, and 16 are comparable to that of 1. Compounds 8, 15, and 16 would most likely be converted to 1 by oxidation or hydrolysis inside the cell. All novel phosphonate nucleoside analogues show an excellent cellular toxicity profile with CC50 > 1.0 mM and do not inhibit replication of HIV-1 and HSV-1 at 30 μM [6].

In conclusion, the introduction of a cyclopropyl moiety at the 2‘-position to restrict conformational mobility of an acyclic phosphonate nucleoside was shown to result in highly potent, specific, and selective anti-HBV activity. The orally available drug candidate Besifovir Dipivoxil (PMCDG Dipivoxil)(2) is a double prodrug of PMCG (1), which drastically reduced DNA titers of woodchuck hepatitis B virus (WHBV) in an in vivo study with woodchucks at 5 mg/kg per day.19 The drug candidate 2 has successfully completed phase I and is currently undergoing phase II clinical study for evaluation of efficacy in human.[6]

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Orally administered prodrug LB80380 (30, 60, 120, and 240 mg) is quickly absorbed and transforms into Besifovir (LB80331), the parent medication (LB80380 was not found in plasma). For LB80331, the range of the median Tmax was 1.0–2.0 hours after dosage. After that, the concentration of LB80331 in plasma dropped monoexponentially, with an average t1/2 of 2.5–3.3 hours.

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Background: LB80380 is potent antiviral agent against hepatitis B virus (HBV) in vitro and in the woodchuck model. It has an excellent preclinical safety profile including lower potential for renal toxicity than adefovir. It is effective against both wild-type and YMDD mutant HBV. LB80380 is converted to its parent drug, Besifovir (LB80331), after oral absorption, and further metabolized to its active form, LB80317.
Aims/methods: This randomized placebo-controlled Phase I/II clinical study of LB80380 was conducted to assess the safety, antiviral activity and pharmacokinetics of its parent drug Besifovir (LB80331) and its active form LB80317 in 29 Asian adults with chronic hepatitis B positive for hepatitis B e antigen in four escalating dose groups (30, 60, 120 and 240 mg once per day) for 4 weeks with a 12-week follow-up period.
Results: The mean maximum HBV DNA reduction was 3.05, 4.20, 3.67 and 3.68 log10 copies/ml for 30, 60, 120 and 240 mg per day, respectively. Viral dynamic analysis suggested a high degree of inhibition of HBV replication at doses of 60 mg or higher per day. LB80380 was well tolerated at all dose groups, and no dose-related clinical or laboratory adverse event was reported.
Conclusion: LB80380 is shown to be a potent and safe antiviral agent for HBV. Marked HBV DNA suppression was observed in all dose groups. The HBV DNA suppression was approximately constant at doses of 60 mg and higher over the 28-day dosing period. The dose response of LB80380 will be evaluated further in large clinical studies.[2]

Procedure To Evaluate Anti-HBV Activity and Cytotoxicity.Anti-HBV Activity. [6]
The compounds listed above were evaluated for anti-HBV activity, referring to the article.13 The HepG2 2.2.15 cells, hepatitis B virus (HBV) producing cell line, were cultured at 37 °C in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum, 1% Antibiotic-Antimycotics, and 400 μg/mL Geneticin in the presence of 5% CO2. The HepG2 2.2.15 cells were plated at a density of 1.5 × 104 cells/well on 96-well plates and incubated for 2 days. When confluence was about 80−90%, the media were replaced with fresh DMEM containing 2% FBS, 1% ABAM, and 400 μg/mL G 418. The synthetic compounds with concentrations of 100, 20, 4, 0.8, 0.16, and 0 μM were treated every other day for 9 days. On day 10 the culture media were collected, boiled, and diluted serially. The samples were analyzed by real-time PCR using a Rotor-Gene 2000. Amplification primers were HBV2005F (5‘-TCA GCT CTG TAT CGG GAA GCC TTA G-3‘) and HBV2122R (5‘-CAC CCA CCC AGG TAG CTA GAG TCA-3‘), and TaqMan probe was 5‘-6-FAM-CCT CAC CAT ACT GCA CTC AGG CAA-BHQ-1−3‘. PCR samples were denatured for 10 min at 94 °C, followed by 40 cycles of 94° C for 30 s, 55 °C for 30 s, and 86 °C for 30 s. Emitting fluorescence was detected at 86 °C and the data analyzed statistically using PRISM.

Cytotoxicity. [6]
The 50% cytotoxic concentration of each compound was determined in HepG2 2.2.15 cell line. After compounds treatments for the evaluation of anti-HBV activity, each well of the 96-well plate was treated with 0.5 mg/mL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazdium bromide (MTT) and incubated for 2 h at 37 °C. At the end of the incubation period, the medium was removed, and the converted dye was solubilized with acidic isopropyl alcohol (0.05% HCl in 95% isopropyl alcohol). The metabolized formazan reduction product was colorimetrically measured at 490 nm.

Methods: An open-label, single- dose, randomized-sequence, 2-treatment crossover study was conducted in 32 Korean male volunteers. Subjects received either a combination of 60 and 90 mg tablets of the free base LB80380 formulation or a 183 mg (150 mg as a free base) tablet of the maleate LB80380 formulation. Then, after a 14- day washout period, each subject received the other formulation. Plasma and urine concentrations of LB80331 and LB80317 (active metabolites of LB80380) were measured by validated liquid chromatographytandem mass spectrometry assays. A safety assessment, which included vital signs, adverse events, electrocardiograms and clinical laboratory tests, was performed for each subject. [1]
Results: A total of 32 healthy subjects was enrolled, and 26 subjects completed the study. Single oral administrations of LB80380 maleate tablets did not result in clinically significant differences in the safety profile compared to the LB80380 free base tablets. The 90% confidence intervals (CIs) for the geometric mean ratios of Cmax and AUClast for LB80331 of the two treatments (maleate versus free base formulation) were 0.986 - 1.1240 and 0.9848 - 1.0533, respectively. The 90% CIs for the geometric mean ratios of Cmax and AUClast for LB80317 were 0.8379 - 0.9696 and 0.7224 - 0.9196. [1]
Conclusions: In healthy male subjects, the 183 mg LB80380 maleate tablet was pharmacokinetically equivalent to the 60 and 90 mg LB80380 free base tablets.

Besifovir (LB80380) is a relatively new oral acyclic nucleotide phosphonate. This article reviews the pharmacokinetic characteristics of LB80380 and discusses its role in the treatment of chronic hepatitis B virus infection. The content includes: LB80380 is a prodrug of LB80331 and LB80317. It is rapidly absorbed after oral administration. Dosage escalation of besifovir results in a linear increase in plasma concentration. Doses above 60 mg effectively inhibit hepatitis B virus (HBV) in humans. For example, at 60 mg, the maximum plasma concentration of LB80331 is 397 ng/mL. The time to peak plasma concentration and elimination half-life are 2.0 hours and 3.0 hours, respectively. Besifovir and its metabolites are primarily excreted via the kidneys. Its antiviral efficacy is non-inferior to 0.5 mg entecavir (ETV) daily. Regarding renal and bone toxicity, besifovir is generally safe. The most common adverse reaction is carnitine depletion, which occurs in almost all patients taking bexifovir and requires carnitine supplementation. Expert opinion: Bexifovir exhibits predictable pharmacokinetic characteristics in humans. There are currently few clinical studies on bexifovir. More data are expected in the future, especially for specific populations. Adverse reactions should be monitored for long-term use. Large-scale head-to-head trials should be conducted to compare the efficacy of bexifovir with existing nucleoside (acid) analogues, especially tenofovir alafenamide. [3]

Throughout the study, there was no significant difference in the overall incidence of adverse events between the two groups (Table 3). Similarly, there was no significant difference in the number of newly reported adverse events between weeks 48 and 96 in the BSV-BSV group (53.5%) and the TDF-BSV group (57 patients, 50.0%). Adverse events with an incidence ≥5% in the BSV-BSV group included nasopharyngitis, dyspepsia, nausea, back pain, headache, dizziness, fatigue, and elevated ALT; adverse events with an incidence ≥5% in the TDF-BSV group included nasopharyngitis, dyspepsia, gastritis, diarrhea, pruritus, and urticaria. No dyspepsia was reported in the BSV group, while 7 patients (8.33%) in the TDF group experienced 8 episodes of dyspepsia during the first 48 weeks of treatment. Although no serious adverse events leading to death occurred during the study, one patient with hepatocellular carcinoma (HCC) in the BSV group and two patients with HCC or elevated creatine phosphokinase in the TDF group discontinued treatment with the study drug during the first 48 weeks of treatment. In addition, one patient in the BSV-BSV group developed tuberculous colitis, and another patient in the TDF-BSV group developed essential thrombocytosis. Both patients discontinued study drug treatment during the extended treatment period. https://pubmed.ncbi.nlm.nih.gov/30448598/

[1]. Pharmacokinetic comparison of the maleate and free base formulations of LB80380, a novel nucleotide analog, in healthy male volunteers. Int J Clin Pharmacol Ther. 2012 Sep;50(9):657-64.

[2]. A randomized placebo-controlled, dose-finding study of oral LB80380 in HBeAg-positive patients with chronic hepatitis B. Antivir Ther. 2006;11(8):977-83.

[3]. Pharmacokinetic evaluation of besifovir for the treatment of HBV infection. Expert Opin Drug Metab Toxicol. 2018 Jan;14(1):101-106.

[4]. Efficacy and Safety of Besifovir Dipivoxil Maleate Compared With Tenofovir Disoproxil Fumarate in Treatment of Chronic Hepatitis B Virus Infection. Clin Gastroenterol Hepatol.2018 Nov 15. pii: S1542-3565(18)31244-8;
[5]. Hepatitis B virus: new therapeutic perspectives. Liver Int.2016 Jan;36 Suppl 1:85-92.

[6]. A novel class of phosphonate nucleosides. 9-[(1-phosphonomethoxycyclopropyl)methyl]guanine as a potent and selective anti-HBV agent. J Med Chem. 2004 May 20;47(11):2864-9.

Besifovir dipivoxil is an oral, small-molecule hepatitis B virus (HBV) polymerase inhibitor. HBV polymerase is an enzyme that catalyzes the synthesis of new RNA from existing RNA strands. Besifovir dipivoxil is thought to inhibit viral proliferation by interfering with the virus's replication mechanism.
See also: Besifovir (note moved here).

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Drug Indications
Studied for the treatment/treatment of hepatitis B (viral hepatitis).

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Mechanism of Action
Besifovir dipivoxil is an HBV polymerase inhibitor. It is thought to inhibit viral proliferation by interfering with the virus's replication mechanism.

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Current antiviral therapies have significantly improved the long-term prognosis of patients with chronic hepatitis B virus (HBV) infection. Interferon (IFN) and nucleoside (acid) analogue (NA) therapy have both been shown to slow the progression of liver disease in patients with chronic hepatitis B (CHB). However, persistent covalently closed circular DNA (cccDNA) may lead to viral relapse after discontinuation of antiviral therapy. Based on extensive research into the hepatitis B virus (HBV) lifecycle and virus-host interactions, a variety of novel drugs targeting both the virus and the host are currently being developed with the aim of curing HBV. Novel polymerase inhibitors, such as tenofovir alafenamide and bexifovir, offer effective and safer treatment options for CHB patients. Drugs targeting cccDNA, such as genetically engineered site-specific nucleases and RNA interference therapies, hold promise for eliminating cccDNA or inhibiting its transcription. HBV nucleocapsid assembly inhibitors can inhibit capsid formation and prevent HBV DNA synthesis. The HBV entry inhibitor Myrcludex-B has been shown to effectively inhibit cccDNA amplification and the spread of intrahepatic infection. Drugs targeting host factors that enhance innate and adaptive immune responses, including lymphotoxin-β receptor agonists, Toll-like receptor agonists, immune checkpoint inhibitors, and adenovirus-based therapeutic vaccines, may play a crucial role in clearing HBV-infected cells. With all these promising approaches, we hope to achieve the ultimate goal of curing HBV in the near future. 9-[1-(phosphonomethoxycyclopropyl)methyl]guanine (PMCG, 1) is a representative of a novel class of phosphonate nucleosides that inhibits HBV replication with extremely high potency (EC50 = 0.5 μM) in primary HepG2 2.2.15 cell culture. It did not exhibit significant cytotoxicity in various human cell lines at concentrations up to 1.0 mM. At a concentration of 30 μM, this compound did not inhibit the replication of human immunodeficiency virus (HIV-1) or herpes simplex virus (HSV-1). Many purine base analogs of compound 1 also exhibit inhibitory activity against hepatitis B virus (HBV), but pyrimidine analogs did not show inhibitory activity at a concentration of 30 μM. The potency of compound 1 is 4 times that of 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA), used as a positive control (EC50 = 2.0 μM). The characteristic cyclopropyl moiety at the 2' position of compound 1 was prepared via a titanium-mediated Kulinkovich cyclopropanation reaction. Compound 1 was modified to yield the orally effective candidate drug bexifovir dipivoxil (PMCDG Dipivoxil) (2). In a study of marmots infected with hepatitis B virus (WHBV), compound 2 demonstrated excellent efficacy at a dose of 5 mg/kg/day. Candidate drug 2 has successfully completed a Phase I clinical trial and is currently undergoing a Phase II clinical trial to evaluate its efficacy.

C10H14N5O4P

299.22

299.078

441785-25-7

Besifovir Dipivoxil maleate;441785-26-8; 441785-25-7; 1039623-01-2

5270766

Light yellow to yellow solid powder

1.9±0.1 g/cm3

668.4±65.0 °C at 760 mmHg

358.0±34.3 °C

0.0±2.1 mmHg at 25°C

1.826

-2.56

3

8

5

20

415

0

C1CC1(CN2C=NC3=CN=C(N=C32)N)OCP(=O)(O)O

KDNSSKPZBDNJDF-UHFFFAOYSA-N

InChI=1S/C10H14N5O4P/c11-9-12-3-7-8(14-9)15(5-13-7)4-10(1-2-10)19-6-20(16,17)18/h3,5H,1-2,4,6H2,(H2,11,12,14)(H2,16,17,18)

[1-[(2-aminopurin-9-yl)methyl]cyclopropyl]oxymethylphosphonic acid

Besifovir; 441785-25-7; Besifovir [INN]; ((1-((2-Amino-9H-purin-9-yl)methyl)cyclopropoxy)methyl)phosphonic acid; UNII-4PLG22CQUU; 4PLG22CQUU; PHOSPHONIC ACID, [[[1-[(2-AMINO-9H-PURIN-9-YL)METHYL]CYCLOPROPYL]OXY]METHYL]- (9CI); [1-[(2-aminopurin-9-yl)methyl]cyclopropyl]oxymethylphosphonic acid;

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)

DMSO: 250 mg/mL (835.51 mM)
H2O: ≥ 100 mg/mL (334.20 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (6.95 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 20.8 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: ≥ 2.08 mg/mL (6.95 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 20.8 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: ≥ 2.08 mg/mL (6.95 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 20.8 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.)