HIV-1 viral particle maturation; release of mature capsid protein from CA-SP1
HIV-1 Gag polyprotein precursor (specifically targeting the capsid (CA)-SP1 junction) [2]
HIV-1 Gag polyprotein precursor (focusing on the CA-SP1 cleavage site) [3]

Determination of bevirimat plasma concentrations.
Bevirimat plasma concentrations were measured using reverse-phase high-performance liquid chromatography assays with tandem mass spectrometric detection over the calibration ranges of 20.0 to 16,000 ng/ml (75-mg dose) and 100 to 60,000 ng/ml (150- and 250-mg doses). For the lower concentration range, heparinized plasma samples (100 μl) were treated with 50 μl of internal standard (1.50-μg/ml solution of 2,2-dimethylsuccinyl-4-dihydrobetulinic acid ester [DSD] in acetonitrile with 0.1% acetic acid) followed by 450 μl of cold, acidified acetonitrile to precipitate plasma proteins. After digestion and vortexing, the samples were centrifuged at 4°C and 13,000 × g for 10 min. The supernatant (500 μl) was evaporated to dryness with nitrogen at 30°C and the residue reconstituted in 80% methanol in 0.1% acetic acid. Samples were maintained at 4°C in the autosampler, and a 30-μl aliquot was injected into the liquid chromatography-tandem mass spectrometry system. For the higher concentration range, plasma samples (100 μl) were treated in a similar manner except that 10 μl of supernatant was directly injected without evaporation and reconstitution.
For both concentration ranges, prepared samples were chromatographed over a Luna C18 (2) high-performance liquid chromatography column (2.0 mm by 50 mm; 3.0-μm particles; part 00B-4251-B0) maintained at 40°C using a mobile phase consisting of 83% methanol in 75 mM ammonium acetate buffer. The mobile-phase flow rate was 0.2 ml/min. The resulting retention times for bevirimat and DSD were approximately 5.0 and 6.5 min, respectively. Bevirimat and DSD were detected using a triple quadrupole mass spectrometer , with multiple reaction monitoring (bevirimat, 583.3 to 455.2 m/z; DSD, 585.3 to 457.3 m/z).
Linearity was observed over both calibration curve ranges. The overall accuracy (percent analytical recovery) and precision (percent coefficient of variation [CV]) of the assay were determined from the plasma quality control samples that were analyzed during the analysis of the study samples. For the 20.0- to 16,000-ng/ml calibration range, the accuracy and precision were calculated as 97.7% (92.2 to 102%) and 6.78% (4.87 to 7.81%), respectively. For the 100- to 60,000-ng/ml calibration range, the accuracy and precision were calculated as 98.1% (93.0 to 104%) and 5.34% (3.92 to 7.92%), respectively. The quantitation limit of the assays was 20.0 ng/ml and 100 ng/ml, respectively, for the lower and upper curve ranges. Bevirimat has demonstrated stability in heparinized human plasma for 95 days when stored at −70°C .[1]

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Bevirimat is distinguished from the currently available antiretrovirals by its unique target and mode of action. While the specific interactions responsible for activity have yet to be fully characterized, it is clear that the target for bevirimat is the Gag polyprotein precursor, the main structural protein responsible for assembly and budding of virion particles.[2]

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1. Inhibition of HIV-1 replication: Bevirimat potently inhibits the replication of both wild-type and drug-resistant HIV-1 isolates in vitro, with similar 50% inhibitory concentration (IC50) values for both types [3]
2. Disruption of viral maturation: The drug specifically blocks the final rate-limiting step in Gag processing, preventing the cleavage of the CA-SP1 precursor to mature CA protein. This results in the production of immature, non-infectious virion particles [2]
3. Induction of drug resistance mutations: Serial drug passage studies identified six single amino acid substitutions that independently confer Bevirimat resistance. These mutations localize at or near the CA-SP1 cleavage site, a region not associated with resistance to other antiretroviral drugs [3]

Bevirimat [3-O-(3',3'-dimethylsuccinyl)betulinic acid] is the first in a new class of anti-human immunodeficiency virus (HIV) drugs that inhibit viral maturation by specifically blocking cleavage of the Gag capsid (CA) precursor, CA-SP1, to mature CA protein, resulting in defective core condensation and release of immature noninfectious virions. The bevirimat pharmacokinetics was well described by an oral two-compartment linear model (r(2), 0.98), with a mean (percent coefficient of variation) half-life of 60.3 (13.6) h and apparent oral clearance of bevirimat from the plasma compartment of 0.17 (18) liters/h. HIV RNA was modeled as being produced in infected CD4 cells, with bevirimat inhibiting infection of new CD4 cells thru a Hill-type function (r(2), 0.87). Single oral doses of bevirimat were well tolerated and demonstrated a dose-dependent reduction in viral load. The average maximum reduction from baseline following the 150- and 250-mg doses was greater than 0.45 log(10), with individual patients having reductions of greater than 0.7 log(10). No bevirimat resistance mutations were detected during the course of the study.[1]

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1. Efficacy in HIV-infected adults (Phase I/II clinical trial): Four cohorts of six HIV-infected adults (CD4 counts >200, plasma viral loads 5,000–250,000 transcripts/ml, not on antiretroviral therapy) were randomized to single oral doses of placebo, 75 mg, 150 mg, or 250 mg Bevirimat. The drug demonstrated a dose-dependent reduction in viral load: the average maximum reduction from baseline was >0.45 log₁₀ for 150 mg and 250 mg doses, with individual patients achieving reductions >0.7 log₁₀ [1]
2. Resistance profile in vivo: No Bevirimat resistance mutations were detected during the clinical trial period [1]

1. HIV-1 infection and viral maturation assay: HIV-1-susceptible cell lines are seeded in culture plates and infected with wild-type or drug-resistant HIV-1 isolates. Bevirimat is added to the culture system at gradient concentrations, with a vehicle control group set up. After incubation for a specified period, the culture supernatant is collected to isolate viral particles. The maturity of viral particles is analyzed by detecting the cleavage status of the Gag CA-SP1 precursor (e.g., via Western blot to identify mature CA protein and uncleaved CA-SP1). The infectivity of viral particles is evaluated by infecting indicator cells and measuring viral replication markers [2]
2. Drug resistance induction assay: HIV-1-infected cells are continuously passaged in the presence of suboptimal concentrations of Bevirimat. After multiple passages, viral isolates are collected, and the Gag gene (especially the CA-SP1 region) is sequenced to identify amino acid substitutions associated with drug resistance [3]

Metabolism/Metabolites
Hepatic glucuronidation (UGT1A3 mediated)
Biological half-life
56.3 to 69.5 hours
1.Absorption: Peak plasma concentration is reached approximately 1-3 hours after oral administration of Bevirimat[3]
2.Distribution: Oral two-compartment linear model can well describe its pharmacokinetics[1]
3.Metabolism and excretion: It is mainly eliminated through hepatic glucuronidation and hepatobiliary excretion. Renal clearance is extremely low, with less than 1% of the administered dose excreted in the urine [3]
4. Half-life: The mean plasma elimination half-life is 60.3 (13.6) hours (Phase I/II clinical trials) [1]
5. Clearance: The apparent oral plasma clearance is 0.17 (18) liters/hour [1]
6. Dosage frequency: The long half-life (58-80 hours) supports once-daily dosing [3]

1. Clinical tolerability: Single oral doses of Bevirimat (75 mg, 150 mg, 250 mg) were well tolerated in HIV-infected adults [1]. 2. Short-term safety: Short-term (≤14 days) administration of Bevirimat was well tolerated, even when used in combination with other antiretroviral drugs [3].

[1]. Phase I and II study of the safety, virologic effect, and pharmacokinetics/pharmacodynamics of single-dose 3-o-(3',3'-dimethylsuccinyl)betulinic acid (bevirimat) against human immunodeficiency virus infection. Antimicrob Agents Chemother. 2007 Oct;51(10):3574-81.

[2]. Maturation inhibitors: a new therapeutic class targets the virus structure. AIDS Rev. 2007 Jul-Sep;9(3):162-72.

[3]. Bevirimat: a novel maturation inhibitor for the treatment of HIV-1 infection. Antivir Chem Chemother. 2008;19(3):107-13.

Bevirimat is a pentacyclic triterpenoid compound formed by the condensation of 2,2-dimethylsuccinic acid and the 3-hydroxyl group of betulinic acid. It was isolated from the traditional Chinese medicine Syzygium claviflorum. As the first HIV-1 maturation inhibitor studied in humans, bevirimat was considered a potential HIV drug candidate and underwent several clinical trials; however, its development has been hampered by numerous drug resistance issues. It is both a metabolite of HIV-1 and a maturation inhibitor. It is a pentacyclic triterpenoid compound, as well as a dicarboxylic acid monoester and monocarboxylic acid. Its function is related to betulinic acid. Bevirimat, also known as PA-457 or YK-FH312, is currently undergoing clinical trials for the treatment of HIV infection. Bevirimat is a solid. This compound belongs to the androgen and its derivatives, and is a hydroxylated C19 steroid hormone. They are known to promote the development of male characteristics. They also have significant effects on human scalp and body hair. Bevirimat targets the Gag-pol polyprotein. Bevirimat is derived from a betulinic acid-like compound originally isolated from the medicinal herb Syzygium claviflorum. It has not yet received FDA approval but is undergoing clinical trials by the pharmaceutical company Panacos. Bevirimat is a drug derived from a betulinic acid-like compound originally isolated from the medicinal herb Syzygium claviflorum, which is active against human immunodeficiency virus (HIV). Bevirimat's mechanism of action involves binding to the Gag capsid precursor protein and preventing its conversion to the mature capsid protein via protease cleavage. It effectively inhibits the replication of both wild-type and drug-resistant (reverse transcriptase or protease) HIV-1 isolates.

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Drug Indications
It has been investigated for the treatment of HIV infection.

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Mechanism of Action
Bevirimat possesses a novel mechanism of action, specifically inhibiting the cleavage of spacer peptide 1 (SP1) from the C-terminus of the capsid, thereby leading to core aggregation defects. Specifically, Bevirimat binds to the SP1/capsid junction, preventing cleavage by the HIV protease.

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1. Drug Classification and Mechanism: Bevirimat is the first HIV-1 maturation inhibitor, and its mechanism of action is distinctly different from existing antiretroviral drugs (reverse transcriptase inhibitors, protease inhibitors). It targets Gag polyprotein precursor, a structural protein responsible for viral assembly and budding[2]
2. Indications:Bevirimat is being developed for the treatment of HIV-1 infection, aiming to address unmet needs of current therapies in overcoming viral resistance and tolerability issues[3]
3. Clinical development status: Phase I and II trials have demonstrated its safety and efficacy, and studies are currently underway to evaluate long-term efficacy and tolerability[3]
4. Structural characteristics:Bevirimat is a derivative of betulinic acid, chemically named 3-O-(3',3'-dimethylsuccinoyl)betulinic acid[1]

C36H56O6

584.8263

584.407

C, 73.93; H, 9.65; O, 16.41

174022-42-5

457928

White to off-white solid powder

1.1±0.1 g/cm3

662.7±40.0 °C at 760 mmHg

197.7±20.8 °C

0.0±4.3 mmHg at 25°C

1.548

10.15

2

6

7

42

1170

10

O(C(C([H])([H])C(C(=O)O[H])(C([H])([H])[H])C([H])([H])[H])=O)[C@@]1([H])C([H])([H])C([H])([H])[C@@]2(C([H])([H])[H])[C@]([H])(C1(C([H])([H])[H])C([H])([H])[H])C([H])([H])C([H])([H])[C@]1(C([H])([H])[H])[C@]2([H])C([H])([H])C([H])([H])[C@]2([H])[C@@]3([H])[C@]([H])(C(=C([H])[H])C([H])([H])[H])C([H])([H])C([H])([H])[C@]3(C(=O)O[H])C([H])([H])C([H])([H])[C@@]12C([H])([H])[H]

YJEJKUQEXFSVCJ-WRFMNRASSA-N

InChI=1S/C36H56O6/c1-21(2)22-12-17-36(30(40)41)19-18-34(8)23(28(22)36)10-11-25-33(7)15-14-26(42-27(37)20-31(3,4)29(38)39)32(5,6)24(33)13-16-35(25,34)9/h22-26,28H,1,10-20H2,2-9H3,(H,38,39)(H,40,41)/t22-,23+,24-,25+,26-,28+,33-,34+,35+,36-/m0/s1

(1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-9-((3-carboxy-3-methylbutanoyl)oxy)-5a,5b,8,8,11a-pentamethyl-1-(prop-1-en-2-yl)icosahydro-3aH-cyclopenta[a]chrysene-3a-carboxylic acid

YK FH312; YK FH-312; YK FH 312; MPC4326; MPC 4326; MPC-4326; PA457; PA 457; PA-457; FH11327; FH-11327; FH 11327; YK FH312

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 : 50~100 mg/mL ( 85.49 ~170.98 mM)
Ethanol : ~20 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.27 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 25.0 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: 2.5 mg/mL (4.27 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with heating and sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 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: ≥ 2.5 mg/mL (4.27 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly..

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Solubility in Formulation 4: 10% DMSO+40% PEG300+5% Tween-80+45% Saline: ≥ 2.5 mg/mL (4.27 mM)

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