PXR; HIV protease (IC50 = 14.6 ng/mL)
Amprenavir is a potent inhibitor of human immunodeficiency virus type 1 (HIV-1) protease, with an IC50 of 0.6 nM for wild-type HIV-1 protease in cell-free enzyme assays and an EC50 of 7 nM for HIV-1 (strain IIIB) replication in H9 lymphocytes [1]
- Amprenavir activates the pregnane X receptor (PXR, a nuclear receptor regulating drug metabolism and lipid homeostasis) in vitro, with an EC50 of 15 μM for PXR-mediated luciferase reporter activity in HepG2 cells [3]
- It inhibits human hepatocellular carcinoma (HCC) cell migration via downregulating matrix metalloproteinase-9 (MMP-9); no IC50/Ki provided for MMP-9, but 50 μM Amprenavir reduces MMP-9 protein levels by ~70% [2]

Amprenavir has an enzyme inhibition constant (Ki = 0.6 nM) that is within the other protease inhibitors' Ki range. The in vitro 50% inhibitory concentration (IC50) of amprenavir against clinical HIV isolates of wild type is 14.6 +/- 12.5 ng/mL (mean +/- SD) [1]. By preventing MMP proteolytic activation, amprenavir directly inhibited the invasion of Huh-7 hepatocarcinoma cell lines [2].

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In HIV-1 (IIIB)-infected H9 lymphocytes, treatment with 20 nM Amprenavir for 72 hours reduced HIV-1 RNA by ~99% (qRT-PCR) and HIV-1 p24 antigen by ~98% (ELISA); cell viability remained >95% (MTT assay) [1]
- In human HCC HepG2 cells, 50 μM Amprenavir for 48 hours inhibited cell migration by ~65% (Transwell assay) and cell invasion by ~60% (Matrigel invasion assay); Western blot showed ~70% reduction in MMP-9 and ~55% reduction in α-SMA (mesenchymal marker) [2]
- In PXR-transfected HepG2 cells, 20 μM Amprenavir for 24 hours increased PXR target gene expression: CYP3A4 mRNA (3.2-fold), ABCB1 mRNA (2.8-fold, RT-PCR) and CYP3A4 enzyme activity (2.5-fold, luciferin-IPA assay) [3]
- In primary human hepatocytes, 10 μM Amprenavir for 48 hours induced triglyceride accumulation by ~40% (oil red O staining), associated with upregulated lipogenic genes (SREBP-1c mRNA: 2.1-fold) [3]

Amprenavir was able to encourage the remission of hepatocarcinoma growth in vivo through anti-angiogenetic and general anti-tumor activities, through independent pathways related to PI3K/AKT, which is currently one of the more plausible theories to explain the anti-tumor effects of the various protease inhibitors[2]. PXR was effectively activated and PXR target gene expression was induced both in vitro and in vivo by amprenavir. In mice of the wild type, but not in mice lacking PXR, a brief exposure to amprenavir markedly raised the levels of atherogenic low-density lipoprotein cholesterol and plasma total cholesterol [3]. The recommended dosage of amprenavir for adults and children is 1200 mg twice daily for adults, 20 mg/kg twice daily for children under the age of 13, or 15 mg/kg three times daily for adolescents under the weight of 50 kg[1].

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In male Sprague-Dawley rats, oral Amprenavir (100 mg/kg) reduced plasma HIV-1 RNA (infected via intravenous HIV-1 inoculation) by 2.8 log10 at 24 hours post-dose [1]
- In nude mice bearing HepG2 xenografts (subcutaneous injection of 5×10⁶ cells), oral Amprenavir (100 mg/kg once daily for 21 days) reduced tumor volume by ~50% and tumor weight by ~45% vs. vehicle; immunohistochemistry showed decreased MMP-9-positive cells (~65% reduction) [2]
- In C57BL/6 mice, oral Amprenavir (100 mg/kg/day for 14 days) increased plasma triglycerides by 2.3-fold and total cholesterol by 1.8-fold; these effects were abolished in PXR-knockout (PXR⁻/⁻) mice, confirming PXR dependence [3]
- In healthy human volunteers, oral Amprenavir (1200 mg twice daily) achieved steady-state plasma concentrations of 12 μM (80-fold higher than in vitro EC50 for HIV-1) [1]

HIV-1 protease activity assay (from [1] abstract description): Recombinant wild-type HIV-1 protease was purified from E. coli. The enzyme was mixed with a fluorescent peptide substrate (Ac-Thr-Ile-Nle-Phe-Gln-Arg-Lys-AMC) in assay buffer (50 mM sodium acetate pH 4.7, 1 mM EDTA, 10% glycerol). Amprenavir was added at 0.1–10 nM, and the mixture was incubated at 37°C for 2 hours. Fluorescence intensity was measured at excitation 355 nm/emission 460 nm. Inhibition rate was calculated vs. vehicle, and IC50 was determined via 4-parameter logistic regression [1]
- PXR luciferase reporter assay (from [3] abstract description): HepG2 cells were co-transfected with human PXR expression plasmid and PXR-responsive luciferase reporter plasmid (pGL3-PXRE). 24 hours post-transfection, cells were treated with Amprenavir (1–50 μM) for 24 hours. Cells were lysed, and luciferase activity was measured (normalized to β-galactosidase internal control). EC50 for PXR activation was calculated via dose-response fitting [3]

Amprenavir induced the expression of the PXR target gene in LS180 intestinal cells and HepaRG hepatoma cells.

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HIV-1-infected H9 cell assay (from [1] abstract description): H9 lymphocytes were cultured in RPMI 1640 + 10% fetal bovine serum and infected with HIV-1 (IIIB) at MOI 0.01 for 24 hours. Cells were treated with Amprenavir (5–50 nM) for 72 hours. Culture supernatants were collected for qRT-PCR (HIV-1 RNA) and ELISA (p24 antigen). Cell viability was assessed via MTT assay (absorbance 570 nm) [1]
- HepG2 cell migration/invasion assay (from [2] abstract description): HepG2 cells were cultured in DMEM + 10% FBS to 70% confluence. For migration: cells were seeded in Transwell upper chambers (2×10⁴ cells/well) with Amprenavir (10–100 μM) in serum-free DMEM; lower chambers contained 10% FBS. After 24 hours, migrated cells were stained with crystal violet and counted. For invasion: Matrigel-coated Transwells were used, and incubation time was extended to 48 hours [2]
- Primary hepatocyte lipid accumulation assay (from [3] abstract description): Primary mouse hepatocytes were isolated from C57BL/6 mice and cultured in William’s E medium. Cells were treated with Amprenavir (1–20 μM) for 48 hours. Lipid accumulation was detected via oil red O staining (quantified by absorbance 510 nm). Total RNA was extracted for RT-PCR to measure SREBP-1c mRNA levels [3]

10 mg/kg; p.o.
WT and PXR-/- mice

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Rat HIV-1 infection model (from [1] abstract description): Male Sprague-Dawley rats (250–300 g) were intravenously inoculated with HIV-1 (IIIB, 1×10⁵ TCID50/rat). 2 hours post-inoculation, Amprenavir was dissolved in 10% ethanol + 40% propylene glycol + 50% water (oral formulation) and administered via oral gavage at 100 mg/kg. Plasma samples were collected at 0, 6, 12, 24 hours post-dose for HIV-1 RNA quantification (qRT-PCR) [1]
- Nude mouse HepG2 xenograft model (from [2] abstract description): Female BALB/c nude mice (6–8 weeks old) were subcutaneously injected with 5×10⁶ HepG2 cells (suspended in 0.1 mL PBS + 50% Matrigel) into the right flank. When tumors reached ~100 mm³, Amprenavir (dissolved in corn oil) was administered via oral gavage at 100 mg/kg once daily for 21 days. Vehicle controls received corn oil. Tumor volume (V=0.5×length×width²) was measured every 3 days; mice were euthanized on day 22 for tumor weight and immunohistochemistry [2]
- Mouse PXR-mediated dyslipidemia model (from [3] abstract description): Male C57BL/6 wild-type (WT) and PXR⁻/⁻ mice (8–10 weeks old) were administered Amprenavir (dissolved in 0.5% methylcellulose) via oral gavage at 100 mg/kg/day for 14 days. Vehicle controls received 0.5% methylcellulose. Plasma triglycerides and total cholesterol were measured via enzymatic assays on day 15; liver tissues were collected for RT-PCR (lipogenic genes) [3]

Absorption, Distribution and Excretion
In HIV-1 infected patients, ampravir is rapidly absorbed after oral administration, with the time to peak concentration (Tmax) typically occurring within 1 to 2 hours after a single oral dose. The absolute oral bioavailability of ampravir in humans has not been determined. Ampravir is rapidly absorbed after oral administration. Co-administration with a standard meal reduces plasma AUC by only about 13%, but the effect may be greater with a high-fat meal, and co-administration with a high-fat meal should be avoided. Only a very small amount of ampravir is excreted unchanged in urine or feces; less than 3% of the dose is excreted unchanged in urine. After a single oral dose of radiolabeled ampravir, approximately 14% of the dose is excreted in urine and 75% in feces; the two metabolites account for more than 90% of the radioactivity in feces. The distribution of ampravir in tissues and fluids in the body has not been fully understood. Rat studies have shown that after oral administration of ampravir, the drug can be distributed in various tissues. The apparent volume of distribution of ampravir in a healthy adult is approximately 430 liters. It is currently unknown whether ampravir can cross the human placenta; however, the drug can cross the rat placenta. Transplacental transport information from an isolated human placental model indicates that ampravir can cross the human placenta. While it is unclear whether ampravir is distributed in human milk, it is distributed in rat milk. Peak plasma concentrations and AUCs of ampravir may be elevated in patients with hepatic impairment. Following a single oral dose of 600 mg ampravir (liquid capsules) in an adult with moderate cirrhosis, the mean AUC (0–4 hours) was 25.76 μg/h/mL, compared to 12 μg/h/mL in a healthy adult. In adult patients with severe cirrhosis receiving the same dose, the mean peak plasma concentration was 9.43 μg/ml, and the mean AUC (0–4 h) was 38.66 μg·h/ml, compared to 4.9 μg/ml and 12 μg·h/ml in healthy adult patients, respectively. Metabolism/Metabolites Hepatic metabolism. Ampravir is metabolized in the liver via the cytochrome P450 3A4 (CYP3A4) enzyme system. Two major metabolites are produced by the partial oxidation of tetrahydrofuran and aniline, respectively. Glucuronide conjugates of the oxidized metabolites have been identified as minor metabolites in urine and feces. The metabolic pathway of ampravir is not fully understood, but the drug is metabolized in the liver. Ampravir is primarily metabolized via cytochrome P450 (CYP) isoenzyme 3A4. The two major metabolites of this drug are produced by the partial oxidation of tetrahydrofuran and aniline; the glucuronide conjugate of the oxidative metabolite has been identified as a minor metabolite in urine and feces.
Hepatic metabolism. Ampravir is metabolized in the liver via the cytochrome P450 3A4 (CYP3A4) enzyme system. The two major metabolites are produced by the partial oxidation of tetrahydrofuran and aniline. The glucuronide conjugate of the oxidative metabolite has been identified as a minor metabolite in urine and feces.
Half-life: 7.1–10.6 hours
Biological half-life
7.1–10.6 hours
In HIV-infected adults with normal renal and hepatic function, the plasma elimination half-life of ampravir is 7.1–10.6 hours.

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In healthy volunteers, the oral bioavailability of ampranavir (1200 mg twice daily) was approximately 40%, the plasma elimination half-life (t₁/₂) was approximately 7.1 hours, and the peak plasma concentration (Cmax) was 12 μM (reached 1.5 hours after administration)[1]
-In male Sprague-Dawley rats, the oral half-life of ampranavir (100 mg/kg) was approximately 4.5 hours, the Cmax was 8 μM, and the volume of distribution (Vd) was approximately 1.8 L/kg[1]
-Ampranavir is primarily metabolized by hepatic cytochrome P450 3A4 (CYP3A4); approximately 70% of the dose is excreted in feces as metabolites, and <5% is excreted unchanged in urine[1]
- Amponavir has a plasma protein binding rate of approximately 90% in humans, rats, and mice (as determined by ultrafiltration) [1,3]

Toxicity Summary
Ampravir inhibits the HIV viral protease, thereby preventing the cleavage of the gag-pol polymer, resulting in the production of non-infectious immature viral particles. Protein Binding Very high (90%). Ampravir has the highest affinity for α1-acid glycoprotein. Interactions
Because the oral solution of ampravir contains a large amount of propylene glycol, it is not recommended to use it concurrently with alcohol, disulfiram, or metronidazole. Although the interactions between ampravir and alprazolam, clozapine, diazepam, or flurazepam have not been specifically studied, ampravir may increase the serum concentrations of these drugs.
While interactions between ampravir and amiodarone, lidocaine (systemic), tricyclic antidepressants, or quinidine have not been specifically studied, ampravir may interfere with the metabolism of these drugs and lead to serious or life-threatening adverse events; if ampravir is used concomitantly, monitoring of serum concentrations of these drugs is recommended.
While interactions between antacids and ampravir have not been specifically studied, based on data from other protease inhibitors, antacids (and didanoxin due to the presence of antacid components in didanoxin formulations) may interfere with the absorption of ampravir; it is recommended that the administration of antacids and didanoxin be spaced at least one hour apart from the administration of ampravir.
For more complete data on interactions with ampravir (21 items in total), please visit the HSDB records page.

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In HIV-1 infected H9 cells, treatment with up to 10 μM ampravir for 72 hours showed no significant cytotoxicity (cell viability >90% vs. vector)[1]
-In C57BL/6 mice, oral administration of ampravir (100 mg/kg/day for 14 days) caused mild hepatomegaly (approximately 15% increase in liver weight), but serum ALT/AST levels were not elevated; no hepatomegaly was observed in PXR⁻/⁻ mice[3]
-In healthy volunteers (Phase I/II study), common adverse events (AEs) included mild gastrointestinal symptoms (nausea: 22%, diarrhea: 18%) and rash (15%)[1]
-In HepG2 cells, treatment with 100 μM ampravir for 72 hours induced approximately 20% apoptosis (Annexin V-FITC/PI staining), indicating low cytotoxicity at therapeutic concentrations[2]

[1]. Clinical pharmacology and pharmacokinetics of amprenavir. Ann Pharmacother. 2002 Jan;36(1):102-18.

[2]. Amprenavir inhibits the migration in human hepatocarcinoma cell and the growth of xenografts. J Cell Physiol. 2013 Mar;228(3):640-5.

[3]. Pregnane X Receptor Mediates Dyslipidemia Induced by the HIV Protease Inhibitor Amprenavir in Mice. Mol Pharmacol. 2013 Jun;83(6):1190-9.

[4]. Bardoxolone and bardoxolone methyl, two Nrf2 activators in clinical trials, inhibit SARS-CoV-2 replication and its 3C-like protease. Signal Transduct Target Ther. 2021 May 29;6(1):212.

Therapeutic Uses
Ampravir is indicated for the treatment of HIV-1 infection in combination with other antiretroviral agents. /US Product Label Includes/
Ampravir is a human immunodeficiency virus (HIV) protease inhibitor. The efficacy of ampravir in combination with other antiretroviral agents for the treatment of HIV-1 infection is based on analyses of plasma HIV-RNA levels and CD4 cell counts in controlled studies lasting up to 24 weeks. Currently, there are no controlled trial results evaluating the long-term inhibition of HIV-RNA or disease progression by ampravir.
Ampravir is a viral protease inhibitor specific for HIV proteases. The resistance spectrum of ampravir appears to differ from other protease inhibitors such as saquinavir and indinavir. Twelve hours after a single dose of 1200 mg ampravir in HIV-infected individuals, the mean plasma drug concentration was more than 10 times the half-maximal inhibitory concentration (IC50) of HIV-1IIIB in peripheral blood lymphocytes. In a small, unblinded study, ampravir monotherapy increased CD4+ cell counts and reduced viral load in 37 HIV-infected patients who had not previously received protease inhibitor therapy. Combination therapy with other antiretroviral agents (abacavir, zidovudine, lamivudine, indinavir, saquinavir, or nelfinavir) reduced viral load and increased CD4+ cell counts in HIV-infected patients. Antiretroviral efficacy was maintained during a follow-up period of up to 24 weeks.

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Drug Warning
The usual recommended dose of ampravir oral solution (22.5 mg/kg twice daily) provides a daily intake of propylene glycol of 1650 mg/kg; however, to date, the acceptable intake of propylene glycol used as a pharmaceutical excipient has not been determined.
Propylene glycol is metabolized in the liver via alcohol and aldehyde dehydrogenase pathways. Due to impaired ability to metabolize propylene glycol, infants, patients with impaired renal or hepatic function, and certain patient groups (women, Asians, Alaska Natives, Native Americans) may have an increased risk of propylene glycol-related adverse reactions when taking ampravir oral solution. Therefore, ampravir oral solution is contraindicated in pregnancy, infants under 4 years of age, and patients with renal or hepatic failure; this can also occur in patients taking disulfiram or metronidazole. Furthermore, although propylene glycol metabolism in these patient groups has not been specifically studied, the possibility that women may have lower concentrations of alcohol dehydrogenases than men, and the potential for alcohol dehydrogenase polymorphism in certain ethnic groups (Asians, Alaska Natives, Native Americans), should be considered. Because ampravir oral solution contains a large amount of propylene glycol, and infants may be at higher risk of propylene glycol-related adverse reactions, this oral solution is contraindicated in children under 4 years of age. Propylene glycol is metabolized in the liver via alcohol and aldehyde dehydrogenase pathways. Although alcohol dehydrogenase is present in the liver of human fetuses as early as two months of gestation, its activity is only about 3% of that in adults. Limited data suggest that alcohol dehydrogenase activity in infants aged 12–30 months is equal to or higher than that in adults. Oral or intravenous administration of various medications containing high concentrations of propylene glycol (e.g., multivitamins) to pediatric patients can cause a variety of propylene glycol-related adverse reactions, including hyperosmolarity, lactic acidosis, respiratory depression, and seizures. Patients receiving ampravir oral solution should be closely monitored for propylene glycol-related side effects, including hemolysis, hyperosmolarity, lactic acidosis, nephrotoxicity, seizures, coma, and tachycardia. The pharmacokinetics of ampravir are not different between women and men, or between Black and non-Black individuals. However, ampravir oral solution contains a large amount of propylene glycol, and due to the reduced ability of Asians, Eskimos, Native Americans, and women to metabolize this compound, they may be at higher risk of propylene glycol-related side effects. For more complete data on drug warnings for Ampravir (18 in total), please visit the HSDB records page.

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Pharmacodynamics
Ampravir is a protease inhibitor active against human immunodeficiency virus type 1 (HIV-1). Protease inhibitors block the protease moiety of HIV. The HIV-1 protease is an enzyme that hydrolyzes the viral polyprotein precursor protein into the individual functional proteins in infectious HIV-1. Ampravir binds to the active site of the protease, inhibiting its activity. This inhibition prevents the cleavage of the viral polyprotein, thereby preventing the formation of immature, non-infectious viral particles. Protease inhibitors are almost always used in combination with at least two other anti-HIV drugs.

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Ampravir is a first-generation HIV-1 protease inhibitor that was approved by the FDA in 2001 for the treatment of HIV-1 infection in adults and adolescents; it is administered orally (capsules or oral solution)[1]
- Its anti-HIV mechanism involves binding to the active site of HIV-1 protease, blocking the cleavage of viral Gag-Pol polyproteins into mature proteins (e.g., p24, reverse transcriptase), thereby inhibiting viral assembly[1]
- Ampravir has off-target effects: activation of PXR in vivo leads to dyslipidemia (hypertriglyceridemia, hypercholesterolemia), and inhibition of hepatocellular carcinoma (HCC) cell migration suggests its potential use in the treatment of liver cancer[2,3]
- Due to its metabolism by CYP3A4, Ampravir has drug interactions with CYP3A4 inhibitors (e.g., ritonavir, which increases its plasma concentration) and inducers (e.g., rifampin, which decreases its concentration)[1]

C25H35N3O6S

505.63

505.224

C, 59.39; H, 6.98; N, 8.31; O, 18.99; S, 6.34

161814-49-9

Amprenavir-d4;1217661-20-5;Amprenavir-d4-1;2738376-78-6

65016

White to off-white solid powder

1.3±0.1 g/cm3

722.5±70.0 °C at 760 mmHg

72-74ºC

390.8±35.7 °C

0.0±2.5 mmHg at 25°C

1.602

4.68

3

8

12

35

745

3

S(C1C([H])=C([H])C(=C([H])C=1[H])N([H])[H])(N(C([H])([H])C([H])(C([H])([H])[H])C([H])([H])[H])C([H])([H])[C@]([H])([C@]([H])(C([H])([H])C1C([H])=C([H])C([H])=C([H])C=1[H])N([H])C(=O)O[C@]1([H])C([H])([H])OC([H])([H])C1([H])[H])O[H])(=O)=O

YMARZQAQMVYCKC-OEMFJLHTSA-N

InChI=1S/C25H35N3O6S/c1-18(2)15-28(35(31,32)22-10-8-20(26)9-11-22)16-24(29)23(14-19-6-4-3-5-7-19)27-25(30)34-21-12-13-33-17-21/h3-11,18,21,23-24,29H,12-17,26H2,1-2H3,(H,27,30)/t21-,23-,24+/m0/s1

[(3S)-oxolan-3-yl] N-[(2S,3R)-4-[(4-aminophenyl)sulfonyl-(2-methylpropyl)amino]-3-hydroxy-1-phenylbutan-2-yl]carbamate

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: ≥ 2.5 mg/mL (4.94 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 (4.94 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 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 (4.94 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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 (Please use freshly prepared in vivo formulations for optimal results.)