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]

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]

Metabolism / Metabolites
Hepatic glucuronidation (UGT1A3-mediated)

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Biological Half-Life
56.3 to 69.5 hours

[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 obtained by the formal condensation of 2,2-dimethylsuccinic acid with the 3-hydroxy group of betulinic acid. It is isolated from the Chinese herb Syzygium claviflorum. The first in the class of HIV-1 maturation inhibitors to be studied in humans, bevirimat was identified as a potent HIV drug candidate and several clinical trials were conducted, but development into a new drug was plagued by numerous resistance-related problems. It has a role as a metabolite and a HIV-1 maturation inhibitor. It is a pentacyclic triterpenoid, a dicarboxylic acid monoester and a monocarboxylic acid. It is functionally related to a betulinic acid.
Bevirimat, also known as PA-457 or YK-FH312, is investigated in clinical trials for treating HIV infection. Bevirimat is a solid. This compound belongs to the androgens and derivatives, which are hydroxylated C19 steroid hormones. They are known to favour the development of masculine characteristics. They also show profound effects on scalp and body hair in humans. Bevirimat targets the protein gag-pol polyprotein. Bevirimat is derived from a betulinic acid-like compound, first isolated from Syzygium claviflorum, a Chinese herb. It is not currently FDA-approved, but is undergoing clinical trials conducted by the pharmaceutical company Panacos.
Bevirimat is a drug derived from a betulinic acid-like compound, first isolated from the Chinese herb Syzygium claviflorum, with activity against human immunodeficiency virus (HIV). Bevirimat acts by binding to the Gag capsid precursor protein and blocking its conversion to mature capsid protein by protease cleavage. It potently inhibits replication in both wild-type and drug-resistant (reverse transciptase or protease) HIV-1 isolates.

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Drug Indication
Investigated for use/treatment in HIV infection.

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Mechanism of Action
Bevirimat has a novel mechanism of action, specifically inhibiting cleavage of spacer peptide 1 (SP1) from the C-terminus of capsid which results in defective core condensation. Specifically, bevirimat binds at the SP1/capsid junction, preventing cleavage by HIV protease.

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.)