HIV-1

Amprenavir promotes the specific interactions between the nuclear receptor pregnane X receptor (PXR) and the coactivators SRC-1 and PBP. Amprenavir is docked into the high-resolution crystal structure of human PXR in complex with SR12813. Amprenavir occupies all four subpockets, and its hydroxyl group forms a hydrogen bond with Ser247, which is located in the connection region of PXR, to help to position the drug in the optimal orientation inside the receptor. Amprenavir forms direct contacts with one residue on αAF of the PXR activation function-2 (AF-2) surface, Phe429, which may stabilize the active AF-2 conformation of the receptor and contribute to the agonist activity of amprenavir on PXR. Amprenavir induces the expression of bona fide PXR target genes involved in phase I (CYP3A4), phase II (UGT1A1), and phase III (MDR1) metabolism in both HepaRG cells and LS180 cells.

---

Fosamprenavir is a phosphate ester prodrug that, upon dissolution, is cleaved to the poorly soluble yet readily absorbable parent drug amprenavir. In this study, a novel cell-free in vitro setup with quasi-continuous monitoring of the dynamic dissolution/bio-conversion/permeation of fosamprenavir was designed and tested. It consists of side-by-side diffusion cells, where the donor and acceptor compartments are separated by the biomimetic barrier PermeaPad®, and sampling from the donor compartment is accomplished via a microdialysis probe. Externally added bovine alkaline phosphatase induced bioconversion in the donor compartment. Microdialysis sampling allowed to follow the enzymatic conversion of fosamprenavir to amprenavir by the bovine alkaline phosphatase in an (almost) real-time manner eliminating the need to remove or inactivate the enzyme. Biomimetic conversion rates in the setup were established by adding appropriate amounts of the alkaline phosphatase. A substantial (6.5-fold) and persistent supersaturation of amprenavir was observed due to bioconversion at lower (500 µM) concentrations, resulting in a substantially increased flux across the biomimetic barrier, nicely reflecting the situation in vivo. At conditions with an almost 10-fold higher dose than the usual human dose, some replicates showed premature precipitation and collapse of supersaturation, while others did not. In conclusion, the proposed novel tool appears very promising in gaining an in-depth mechanistic understanding of the bioconversion/permeation interplay, including transient supersaturation of phosphate-ester prodrugs like fosamprenavir [2].

Amprenavir increases atherogenic LDL cholesterol fractions in WT mice, but not in PXR−/− mice. Amprenavir stimulates expression of known PXR target genes, including CYP3A11, glutathione transferase A1, and MDR1a, in the intestine of WT mice but not in PXR−/− mice. Amprenavir-mediated PXR activation stimulates the expression of both LipF and LipA in the intestine of WT mice, but not in PXR−/− mice, indicating a possible role of intestinal PXR in mediating dietary lipid breakdown and absorption in mammals.

Correlation between true concentration and concentration in the perfusate: extraction-efficiency correction factor [2]
Standards were prepared in beakers containing increasing concentrations of amprenavir (0.94, 2.44, 9.76, 24.4, and 48.8 µM) and fosamprenavir (5.6, 14.2, 56.9, 142, and 284 µM) together in HBSS. The beakers were stirred with a magnetic stirbar and heated to 37 °C on a heating plate. The microdialysis probes were consecutively placed in the respective beakers to determine the extraction-efficiency correction factor. The perfusate contained 0.2% (w/V) Vitamin E TPGS in HBSS and was pumped through the tubes and probes with a syringe pump at a constant rate of 10 µL/min. Samples were collected in time intervals of 15-20, 20-25, and 25-30 min and analyzed with UHPLC-UV.

---

Microdialysis extraction-efficiency correction factor in side-by-side cells for bioconversion/permeation studies [2]
Solutions containing 69.8 µM fosamprenavir and 53.9 µM amprenavir were filled into both the donor and acceptor compartments of side-by-side cells separated by PermeaPad®. The water jackets of the side-by-side cells were kept at 37 °C while the microdialysis was running for 30 min to equilibrate. Afterwards, the correction factor was determined by measuring inside the side-by-side cells as described in Section 2.5.1.

---

Dynamic bioconversion/permeation studies [2]
Bioconversion/permeation studies [2]
The bioconversion/permeation studies were carried out in side-by-side cells attached to a circulating water heating system at 37 °C and with the biomimetic barrier PermeaPad® at a surface area of 1.77 cm2 separating donor and acceptor compartments. The acceptor compartments were filled with 7 mL of 0.2% Vitamin E TPGS in HBSS, while the donor volume was 5.0 mL (setup 1), 6.0 mL (setup 2) and 5.5 mL (setup 3); the slight variation between the three replicates was due to minor differences in the geometry of the side-by-side cells as the volume had to be enough to fully cover both the membrane and the microdialysis probe. A microdialysis probe attached to a syringe pump filled with 0.2% Vitamin E TPGS in HBSS was immersed into the donor compartment (Images of the full setup can be found in the supplementary material). Both compartments were stirred at 500 rpm by cross-type magnetic stir bars.

The donor compartments were filled with either a 500 µM amprenavir suspension, which had equilibrated overnight in a shaking water bath, or a 437 µM fosamprenavir solution. 20 min prior to all dissolution/bioconversion experiments, the syringe pump was started with a flowrate of 10 µL/min. In the case of fosamprenavir, the donor was filled at this point and the microdialysis probe was immersed into the fosamprenavir solution to equilibrate. After 20 min, bovine alkaline phosphatase was added to the donor to initiate the experiment at concentrations corresponding to 5.3 U/mL or 53 U/mL, respectively. In the case of amprenavir suspension, the microdialysis probe was immersed into the donor compartment immediately after adding the amprenavir suspension to start the experiments. Dialysate samples were collected in 10 min intervals for a total of 180 min. Afterwards, the dialysate concentrations were quantified with UHPLC-UV and converted to donor concentrations using the probe-specific extraction-efficiency correction factors described in Section 2.5.2. Conventional 200 µL acceptor samples were withdrawn after 30, 60, 90, 120, and 180 min and quantified with UHPLC-UV.

---

Dissolution/bioconversion/permeation study with a human dose [2]
The dissolution step was added to the dynamic bioconversion/permeation experiments by placing an amount of fosamprenavir in the donor chambers equal to a final concentration of 4500 µM. These experiments were carried out the same way as the bioconversion/permeation experiments in Section 2.7.1. However, like with the amprenavir suspension, the microdialysis was calibrated for 20 min with the probe outside the donor. The microdialysis probe was added immediately to the donor after adding HBSS and 53 U/mL enzyme at the start of the experiment.

Permeation experiments in MDCK-wt cells [2]
Marbine-Darby Canine kidney wild-type (MDCK-wt) cells were maintained in culture medium composed of Dulbecco's Modified Eagle Medium with high glucose supplemented with 10% foetal bovine serum and 500U/mL penicillin/streptomycin. Cells were seeded on 6 well transwell-plates (0.4 µm polycarbonate membrane) at the cell density of 1.8 × 106 cells/transwell and differentiated for three days in cell culture media. After the differentiation, the cell culture media was aspirated, and the cells were washed three times with HBSS. Then, the filter supports with MDCK-wt cells were transferred into 6-well plates, and HBSS was added to the wells’ apical (1.5 mL) and basolateral (2.6 mL) sides. Before the bioconversion/permeation experiments, the plates were pre-incubated in HBSS for 20 min at 37 °C.

The bioconversion/permeation experiments were initiated by replacing the HBSS on the apical side with either 385 µM fosamprenavir solutions or 80 µM amprenavir solutions. The cells were incubated in Heidolph Titramax incubator at 400 rpm and 37 °C. Samples of 500 µL were withdrawn from the basolateral side at 10, 20, 30, 45, 60, 90, 120, 150, and 180 min. After sampling, 500 µL HBSS was added to the basolateral side as a replacement for the sample. The drug concentrations were quantified from the samples with UHPLC-UV.

The ntegrity of the MDCK-wt cells was investigated in a similar experiment with 1:1000 diluted radiolabeled 3H mannitol (15.9 Ci/mmol) in HBSS under the same conditions as in the bioconversion/permeation experiments with MDCK-wt cells. The 3H mannitol was quantified by mixing a 100 µL acceptor sample and 400 µL scintillation medium and measured on a radioactivity counter.

---

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

These two Phase I, open-label, single-dose, randomized, crossoverstudies in 40 healthymale subjects investigated the pharmacokinetic and safety profiles of various formulations of the amprenavir prodrug GW433908 in the presence and absence of food compared with amprenavir capsules. GW433908 is a phosphate ester prodrug of the antiretroviral protease inhibitor amprenavir, with improved solubility over the parent molecule and a potential for reduced pill burden on current dosing regimens. The calcium salt of the prodrug, GW433908G, was selected for further investigation, as it appeared to offer the greatest potential for the development of new drug formulations. In the fasting state, (1) GW433908G tablet and suspension were bioequivalent in terms of both AUC and Cmax, and (2) GW433908G tablet and suspension were bioequivalent to amprenavir capsules for AUC; however, Cmax was lower with GW433908G. After a high-fat meal compared with fasting, (1) the bioavailability of GW433908G suspension was decreased by 20% and Cmax by 41%, and (2) for GW433908G tablets, there was no influence on AUC(12% lower Cmax). After a low-fat meal compared with fasting, (1) there was bioequivalence for GW433908G tablets, but (2) bioavailability was decreased by 23% for amprenavir capsules (Cmax was also lower, by 46%). Overall, for GW433908G and amprenavir capsules, food had a negligible influence on plasma concentration at 12 hours postdose (C12). Whether administered as tablets or suspension, GW433908G pharmacokinetics was only slightly affected by food. GW433908G tablets were well tolerated and delivered plasma amprenavir concentrations equivalent to the recommended therapeutic amprenavir dose but with fewer tablets. The possibility of a lower pill burden offered by GW433908 may be of clinical benefit in the treatment of HIV infection.[1]

---

10 mg/kg; p.o.

WT and PXR-/- mice

Absorption, Distribution and Excretion
The absolute oral bioavailability of Fosamprenavir in humans has not been determined. Following a single fasting dose of 1400 mg Fosamprenavir, the AUC of ampravir in the Fosamprenavir oral suspension (50 mg/mL) and Fosamprenavir tablets (700 mg) was similar, but the Cmax of ampravir in the suspension was 14.5% higher than that in the tablets. The excretion of unchanged ampravir in urine and feces is minimal. Renal clearance of unchanged ampravir is approximately 1% of the administered dose; therefore, renal impairment is not expected to significantly affect ampravir clearance. Ampravir is the active metabolite of Fosamprenavir, metabolized in the liver by the cytochrome P450 enzyme system. Fosamprenavir is a prodrug that, after absorption, is rapidly hydrolyzed to ampravir by intestinal epithelial cell enzymes. In HIV-1 infected patients, the time to peak concentration (Tmax) of ampranasvir after a single dose of Fosamprenavir ranged from 1.5 to 4 hours (median 2.5 hours). The absolute oral bioavailability of ampranasvir after administration of Fosamprenavir in humans has not been determined. Compared to the fasting state, there were no significant changes in Cmax, Tmax, or AUC of ampranasvir after a single 1400 mg dose administered after a meal. The following are the area under the curve (AUC) values for ampravir based on different dosing regimens: Fosamprenavir 1400 mg, twice daily: 27.6 to 39.2 μg/hr/mL (median 33 μg/hr/mL); Fosamprenavir 1400 mg, once daily, plus ritonavir 200 mg, once daily: 59.7 to 80.8 μg/hr/mL (median 69.4 μg/hr/mL); Fosamprenavir 700 mg, twice daily, plus ritonavir 100 mg, twice daily: 69 to 90.6 μg/h/mL (median 79.2 μg/h/mL). Fosamprenavir can be taken with or without food.
Peak plasma concentration (Cmax): The Cmax values for ampranavir were as follows, depending on the treatment regimen used: Fosamprenavir 1400 mg once daily, ritonavir 200 mg once daily: 6.32 to 8.28 μg/mL (median 7.24 μg/mL); Fosamprenavir 1400 mg twice daily: 4.06 to 5.72 μg/mL (median 4.82 μg/mL); Fosamprenavir 700 mg twice daily, ritonavir 100 mg twice daily: 5.38 to 6.86 μg/mL (median 6.08 μg/mL)...
For more data on absorption, distribution, and excretion of Fosamprenavir (FOSAMPRENAVIR) (complete data for 10 cases), please visit the HSDB records page.

---

Metabolism/Metabolites
Fosamprenavir is rapidly and almost completely hydrolyzed in the intestinal epithelial cells during absorption to ampravir and inorganic phosphate before entering systemic circulation. Ampravir is metabolized in the liver via the cytochrome P450 3A4 (CYP3A4) enzyme system.
After oral administration, Fosamprenavir is rapidly and almost completely hydrolyzed to ampravir and inorganic phosphate before entering systemic circulation. This process occurs within the intestinal epithelial cells during absorption. Ampravir is metabolized in the liver via the cytochrome P450 3A4 (CYP3A4) enzyme system. The two major metabolites are the oxidation products of tetrahydrofuran and aniline. Small amounts of glucuronide conjugates of the oxidation products have been detected in urine and feces.

---

Biological Half-Life
The plasma elimination half-life of ampravir (the active metabolite) is approximately 7.7 hours. The plasma elimination half-life of ampoulenavir is approximately 7.7 hours.

Effects During Pregnancy and Lactation
◉ Overview of Use During Lactation
There is currently no publicly available information regarding the use of Fosamprenavir during lactation. Use of Fosamprenavir during lactation is not recommended. Achieving and maintaining viral suppression through antiretroviral therapy can reduce the risk of transmission through breastfeeding to below 1%, but not zero. For HIV-infected individuals receiving antiretroviral therapy with a persistently low viral load below the detection limit, their decision to breastfeed should be supported. If viral load is not suppressed, pasteurized donor breast milk or formula is recommended.
◉ Effects on Breastfed Infants
As of the revision date, no relevant published information was found.
◉ Effects on Lactation and Breast Milk
Gynecomastia has been reported in men receiving highly active antiretroviral therapy. Gynecomastia is initially unilateral, but approximately half of cases develop into bilateral gynecomastia. No changes in serum prolactin levels have been observed, and it usually resolves spontaneously within one year even with continued medication. Some case reports and in vitro studies suggest that protease inhibitors may cause hyperprolactinemia and galactorrhea in some male patients, but this conclusion remains controversial. The implications of these findings for lactating women are unclear. Prolactin levels in established lactating mothers may not affect their ability to breastfeed. Protein Binding: Very high (approximately 90%); primarily binds to α1-acid glycoprotein. The amount of free ampravir increases with increasing serum ampravir concentration. Interactions: Ampravir inhibits and induces cytochrome P-450 (CYP) isoenzyme 3A4; potential pharmacokinetic interactions (altering the metabolism of other drugs). Caution should be exercised when using Fosamprenavir concomitantly with substrates, inhibitors, or inducers of CYP3A4. Concomitant use with drugs with a narrow therapeutic index that are CYP3A4 substrates is not recommended. Ampravir does not inhibit CYP2D6, CYP1A2, CYP2C9, CYP2C19, or CYP2E1. Pharmacokinetic interactions with antacids (reducing peak concentration and AUC of ampravir). ...This pharmacokinetic interaction is not clinically significant, and there are no restrictions on the co-administration of Fosamprenavir with antacids. A pharmacokinetic interaction occurs when Fosamprenavir-enhanced ritonavir is used in combination with flecainide or propafenone (leading to increased plasma concentrations of the antiarrhythmic drug). Serious and/or life-threatening adverse reactions (e.g., arrhythmias) may occur. Concomitant use of Fosamprenavir-enhanced ritonavir with flecainide or propafenone is contraindicated. Pharmacokinetic interactions occur when Fosamprenavir is used in combination with amiodarone, benprimidil (discontinued in the US), systemic lidocaine, or quinidine (leading to increased concentrations of the antiarrhythmic drug). Serious and/or life-threatening adverse reactions may occur. Use with caution when using concurrently and monitor plasma concentrations of antiarrhythmic drugs as much as possible. For more complete interaction data on fosaprenavir (37 in total), please visit the HSDB record page.

[1]. Pharmacokinetics of GW433908, a prodrug of amprenavir, in healthy male volunteers. Mol Pharmacol.2013 Jun;83(6):1190-9.

[2]. In-vitro dynamic dissolution/bioconversion/permeation of fosamprenavir using a novel tool with an artificial biomimetic permeation barrier and microdialysis-sampling. Eur J Pharm Sci. 2023 Feb 1:181:106366.

Fosamprenavir is a sulfonamide drug with a structure based on sulfonamides, wherein the nitrogen atom of the sulfonamide is replaced by (2R,3S)-4-phenyl-2-(phosphonooxy)-3-({[(3S)-tetrahydrofuran-3-yloxy]carbonyl}amino)butyl. It is a prodrug of the HIV protease inhibitor and the antiretroviral drug ampravir. It functions as a prodrug. Its function is related to that of sulfonamides.
Fosamprenavir is a prodrug of ampravir, a human immunodeficiency virus (HIV) protease inhibitor.
Fosamprenavir is a protease inhibitor. The mechanism of action of Fosamprenavir is as an HIV protease inhibitor.
Fosamprenavir is a prodrug form of ampravir. In vivo, Fosamprenavir is metabolized to ampravir, a synthetic derivative of hydroxyethylamine sulfonamide, which selectively binds to and inhibits the activity of human immunodeficiency virus (HIV) proteases.
See also: Fosamprenavir calcium (note moved here). Drug Indications This drug is indicated for use in combination with other antiretroviral agents to treat human immunodeficiency virus (HIV-1) infection and for post-exposure prophylaxis in individuals at significant risk of HIV transmission who have been exposed to potentially infectious bodily fluids of a known HIV-infected person due to occupational or non-occupational reasons. Guidelines for its use are under revision due to its potential association with myocardial infarction and dyslipidemia in HIV-infected adults. FDA Label Mechanism of Action Fosamprenavir is a prodrug that is rapidly hydrolyzed into ampravir by phosphatases in intestinal epithelial cells during absorption. Ampravir is an inhibitor of HIV-1 proteases. During HIV replication, HIV proteases cleave the viral polypeptide products of the Gag and Gag-Pol genes to form the structural proteins of the viral core and essential viral enzymes. Ampravir interferes with this process by binding to the active site of the HIV-1 protease, thereby preventing the processing of viral Gag and Gag-Pol polyprotein precursors, ultimately leading to the formation of immature, non-infectious viral particles. Fosamprenavir is a prodrug of ampravir, an HIV protease inhibitor. Fosamprenavir is rapidly converted to ampravir in vivo by cellular phosphatases. Ampravir is an HIV-1 protease inhibitor. Ampravir binds to the active site of the HIV-1 protease, thereby preventing the processing of viral Gag and Gag-Pol polyprotein precursors, leading to the formation of immature, non-infectious viral particles.

---

Therapeutic Use
Fosamprenavir is indicated for use in combination with other antiretroviral drugs to treat HIV infection in adults.
/US Product Label Contains/

---

Drug Warnings
In clinical studies, approximately 19% of patients treated with Fosamprenavir reported a rash (usually maculopapular, mild to moderate, with or without pruritus); rash symptoms typically appeared about 11 days after starting Fosamprenavir, with a median duration of 13 days. In clinical studies, less than 1% of patients treated with Fosamprenavir reported serious or life-threatening skin reactions, including Stevens-Johnson syndrome.
Fosamprenavir contains a sulfonamide group, which may cause allergic reactions (e.g., rash) in some susceptible individuals. Cross-sensitivity between drugs containing sulfonamide groups and Fosamprenavir is not known. Patients with known allergies to sulfonamide-containing drugs should use Fosamprenavir with caution.
Patients with chronic hepatitis B or C virus infection, and patients with significantly elevated AST or ALT levels prior to Fosamprenavir treatment, may be at risk of further increases in liver enzyme levels. Liver function tests should be performed before starting Fosamprenavir treatment, and patients should be closely monitored during treatment. In clinical studies, approximately 4-8% of patients treated with Fosamprenavir reported elevated serum AST (SGOT) and/or ALT (SGPT) levels (more than 5 times the upper limit of normal). Ampravir resistance may occur. The potential impact of Fosamprenavir treatment on subsequent use of other HIV protease inhibitors is unclear. For more complete data on Fosamprenavir (14 in total), please visit the HSDB records page.

---

Pharmacodynamics
Fosamprenavir is hydrolyzed by cellular phosphatases to the antiretroviral protease inhibitor ampravir. This hydrolysis allows for the slow release of ampravir, thus reducing the number of tablets patients need to take.

C25H36N3O9PS

585.61

585.19

C, 47.70; H, 5.44; N, 6.67; Na, 7.30; O, 22.87; P, 4.92; S, 5.09

226700-79-4

Fosamprenavir Calcium Salt;226700-81-8;Fosamprenavir-d4;1133702-41-6; Fosamprenavir;226700-79-4; 226700-80-7

131536

Light yellow to yellow solid powder

1.4±0.1 g/cm3

1.612

3.36

4

11

14

39

912

3

CC(C)CN(C[C@H]([C@H](CC1=CC=CC=C1)NC(=O)O[C@H]2CCOC2)OP(=O)(O)O)S(=O)(=O)C3=CC=C(C=C3)N

MLBVMOWEQCZNCC-OEMFJLHTSA-N

InChI=1S/C25H36N3O9PS/c1-18(2)15-28(39(33,34)22-10-8-20(26)9-11-22)16-24(37-38(30,31)32)23(14-19-6-4-3-5-7-19)27-25(29)36-21-12-13-35-17-21/h3-11,18,21,23-24H,12-17,26H2,1-2H3,(H,27,29)(H2,30,31,32)/t21-,23-,24+/m0/s1

[(2R,3S)-1-[(4-aminophenyl)sulfonyl-(2-methylpropyl)amino]-3-[[(3S)-oxolan-3-yl]oxycarbonylamino]-4-phenylbutan-2-yl] phosphate

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 : ≥ 100 mg/mL (~170.76 mM)

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.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (4.27 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.

---

View More

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.

---

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

---

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