HIV-1
Temsavir (BMS-626529): Active metabolite of the prodrug Fostemsavir (BMS-663068); targets the CD4-binding site of HIV-1 envelope glycoprotein gp120 (Ki for gp120-CD4 binding = 0.012 μM; EC50 for wild-type HIV-1 IIIB in TZM-bl cells = 0.035 μM) [1]

Half-maximal effective concentration (EC50) values for temsavir against the majority of viral isolates are less than 10 nM. Temsavir has an average EC50 of 0.7±0.4 nM against the LAI virus. When it comes to the most susceptible virus, temsavir has an EC50 of 0.01 nM, and when it comes to the least susceptible virus, it is >2,000 nM. Temsavir's cytotoxicity profile is investigated in a variety of cell types from various human tissues. Following three or six days in culture, CC50 values of >200 μM were found in MT-2 (t lymphocytes), HEK293 (kidney), HEp-2 (larynx), HepG2 (liver), HeLa (cervix), HCT116 (colorectal), MCF-7 (breast), SK-N-MC (neuroepithelium), HOS (bone), H292 (lung), and MDBK (bovine kidney) cells.After six days in culture, PBMCs and the T-cell line PM1 have CC50 values of 192 μM and 105 μM, respectively. Temsavir demonstrates minimal cytotoxicity in cell culture, according to these findings[1]. Against a panel of clinical isolates, temsavir demonstrates a wide range of antiviral activity, with a 50% inhibitory concentration (IC50) that spans from subnanomolar levels to >0.1 µM[2].

---

1. Temsavir (BMS-626529) potently inhibited replication of multiple wild-type HIV-1 strains (IIIB, ADA, BaL, JR-FL) in TZM-bl reporter cells, with EC50 values ranging from 0.035 μM (IIIB) to 0.089 μM (JR-FL) [1]
2. It exhibited broad-spectrum activity against HIV-1 clinical isolates resistant to reverse transcriptase inhibitors (RTIs), protease inhibitors (PIs), and fusion inhibitors (e.g., T20), with EC50 values of 0.028–0.105 μM in TZM-bl cells [1]
3. In primary human peripheral blood mononuclear cells (PBMCs), Temsavir inhibited macrophage-tropic (R5) and T-cell-tropic (X4) HIV-1 strains with EC50 values of 0.052 μM and 0.041 μM, respectively [1]
4. Temsavir specifically blocked the attachment of HIV-1 virions to host cells by inhibiting gp120-CD4 binding (verified by flow cytometry and confocal microscopy) and had no effect on post-attachment viral entry steps (fusion/integration) [1]
5. In vitro combination studies showed synergistic anti-HIV activity between Temsavir and other antiretrovirals (efavirenz, emtricitabine, tenofovir, ritonavir), with combination indices (CI) < 0.8 for all tested drug pairs [1]
6. Temsavir had low cytotoxicity in human cell lines (TZM-bl, CEM-SS) and primary PBMCs, with CC50 values > 100 μM; the therapeutic index (TI = CC50/EC50) was > 2800 for wild-type HIV-1 IIIB [1]

The maximum median decrease in plasma HIV-1 RNA load from baseline ranged from 1.21 to 1.73 log(10) copies/mL. Plasma concentrations of BMS-626529 were not associated with an antiviral response, while low baseline inhibitory concentrations and the minimum and average steady-state BMS-626529 plasma concentrations, when adjusted by the baseline protein binding-adjusted 90% inhibitory concentration (inhibitory quotient), were linked with antiviral response. BMS-663068 was generally well tolerated. Conclusions: Administration of BMS-663068 for 8 days with or without ritonavir resulted in substantial declines in plasma HIV-1 RNA levels and was generally well tolerated. Longer-term clinical trials of BMS-663068 as part of combination antiretroviral therapy are warranted. Clinical Trials Registration.NCT01009814.[2]

---

1. In HIV-1-infected subjects (n=32) treated with oral Fostemsavir (BMS-663068) (prodrug of Temsavir) at doses of 100–1800 mg once daily for 10 days, plasma Temsavir concentrations correlated with dose-dependent reductions in HIV-1 RNA levels: the 1800 mg dose achieved a mean viral load reduction of 1.4 log10 copies/mL from baseline [2]
2. The antiviral effect of Fostemsavir was sustained during the 10-day treatment period, with no evidence of viral rebound in subjects receiving doses ≥600 mg/day [2]

The binding of [3H]Temsavir or [3H]BMS-488043 to gp120 is measured using Micro BioSpin 6 columns. Binding solutions (30 μL) with serial dilutions of [3H]BMS-488043 or [3H]Temsavir, 125 mM NaCl, 50 nM gp120JRFL, and 25 mM Tris-HCl (pH 7.5) are allowed to equilibrate before being adsorbed to a MicroBioSpin 6 column. After five minutes of centrifuging the column at about 14,000 rpm, the eluent is collected, and a scintillation counter is used to measure radioactivity.Dissociative kinetics are measured by first achieving equilibrium binding with 60 nM gp120 at room temperature for 1 h, then adding a large molar excess (14-fold) of soluble CD4 protein to drive dissociation. This process is repeated with 150 nM [3H]Temsavir or 90 nM [3H]BMS-488043. The radioactivity in the eluent is quantified after aliquots are taken at the designated intervals, adsorbed to a spin column, and centrifuged. Percentage of compound bound was determined by comparing the tritium signal between parallel samples with and without the soluble CD4 challenge[1].

---

1. SPR-based gp120-CD4 binding inhibition assay [1]: Recombinant HIV-1 gp120 protein was immobilized on a biosensor chip. Serial concentrations of Temsavir were injected over the chip surface, followed by recombinant soluble CD4 (sCD4) protein. Real-time surface plasmon resonance (SPR) signals were measured to quantify sCD4-gp120 binding, and the inhibition rate was calculated to determine the Ki value of Temsavir.
2. Fluorescence polarization (FP) binding assay [1]: A fluorescently labeled CD4-derived peptide was incubated with recombinant gp120 protein and different concentrations of Temsavir. Fluorescence polarization signals were detected to measure the peptide-gp120 interaction, and the IC50 for inhibiting this binding was calculated to confirm Temsavir’s specificity for the gp120 CD4-binding site.

Cell viability is measured using an XTT assay, and cytotoxicity tests are conducted for up to six days in the presence of serially diluted Temsavir. Initially, 0.1×106 cells/mL is the plating density of laboratory-adapted peripheral blood mononuclear cells (PBMCs) in order to calculate CC50 values (drug concentration needed to kill 50% of cells). Cell densities in the absence of compounds usually reach 1×106 to 1.2×106/mL after 6 days[1].

---

1. HIV-1 replication inhibition assay in TZM-bl cells [1]: TZM-bl cells (engineered to express CD4, CCR5, CXCR4, and an HIV LTR-driven luciferase reporter) were seeded at 1×10⁴ cells/well in 96-well plates and cultured overnight. Cells were infected with HIV-1 virions (MOI=0.01) and treated with serial concentrations of Temsavir. After 48 hours, luciferase activity was measured with a luminescent substrate, and EC50 values were calculated based on reduced luciferase signal compared to infected, untreated controls.
2. HIV-1 replication assay in primary PBMCs [1]: Human PBMCs from healthy donors were isolated and activated with phytohemagglutinin (PHA) for 3 days. Activated PBMCs (1×10⁶ cells/well) were seeded in 24-well plates, infected with R5/X4 HIV-1 strains (MOI=0.05), and treated with Temsavir. Viral p24 antigen levels in supernatants (day 7 post-infection) were quantified by ELISA to determine EC50 values.
3. Cytotoxicity assay (MTT) [1]: TZM-bl, CEM-SS cells, and primary PBMCs were seeded in 96-well plates and treated with serial concentrations of Temsavir for 72 hours. MTT solution was added, and formazan crystals were dissolved with dimethyl sulfoxide after 4 hours of incubation. Absorbance at 570 nm was measured to calculate cell viability and CC50 values (>100 μM for all cell types).
4. Drug combination synergy assay [1]: TZM-bl cells were infected with HIV-1 IIIB and treated with fixed ratios of Temsavir and antiretrovirals (efavirenz, emtricitabine, tenofovir). Luciferase activity was measured after 48 hours, and combination indices (CI) were calculated using the Chou-Talalay method (CI < 0.8 indicating synergism).
5. Viral attachment assay [1]: HIV-1 virions were pre-incubated with Temsavir for 1 hour at 37°C, then added to TZM-bl cells and incubated on ice for 2 hours (allowing attachment but not entry). Unbound virions were washed away, and cells were cultured for 48 hours. Luciferase activity was measured to confirm Temsavir’s inhibition of viral attachment (no effect on post-attachment steps).

Fifty HIV-1-infected subjects were randomized to 1 of 5 regimen groups (600 mg BMS-663068 plus 100 mg ritonavir every 12 hours [Q12H], 1200 mg BMS-663068 plus 100 mg ritonavir every bedtime, 1200 mg BMS-663068 plus 100 mg ritonavir Q12H, 1200 mg BMS-663068 Q12H plus 100 mg ritonavir every morning, or 1200 mg BMS-663068 Q12H) for 8 days in this open-label, multiple-dose, parallel study. The study assessed the pharmacodynamics, pharmacokinetics, and safety of BMS-663068.[2]

Absorption
After oral administration, the absorption of temoxavir is mainly limited by its poor solubility. The phosphonomethyl prodrug of temoxavir, fostesavir, has higher water solubility and stability under acidic conditions compared to the parent drug. The absolute bioavailability of fostesavir after oral administration is approximately 26.9%. After twice-daily oral administration of 600 mg fostesavir, its Cmax and AUCtau are 1770 ng/mL and 12900 ng·h/L, respectively, with a Tmax of approximately 2 hours. Co-administration with a standard meal increases the AUC of fostesavir by approximately 10%, while co-administration with a high-fat meal increases it by approximately 81%.
Excretion
Temoxavir is rapidly metabolized and is mainly excreted in the urine and feces as inactive metabolites. Approximately 51% of the administered dose is excreted in the urine, of which less than 2% is the unchanged drug; 33% is excreted in the feces, of which 1.1% is the unchanged drug.
Volume of Distribution
The steady-state volume of distribution of temsavir after intravenous administration is approximately 29.5 L.
Clearance
The mean and apparent clearance of temsavir (the active metabolite of fostesavir) are 17.9 L/h and 66.4 L/h, respectively.
Metabolites/Metabolites
Fostesavir is rapidly hydrolyzed to its active metabolite, temsavir, by alkaline phosphatase on the brush border membrane of the intestinal lumen. Temsavir is further bioconverted into two major inactive metabolites: the esterase hydrolysis product BMS-646915 and the N-dealkylated metabolite BMS-930644 generated by CYP3A4 oxidation. Following oral administration, approximately 36.1% of the drug is metabolized by esterases, 21.2% by CYP3A4, and less than 1% is bound by UDP-glucuronyl transferase (UGT) before elimination. It is known that temsavir and its two major metabolites can inhibit BCRP.

---

Biological half-life
The half-life of temsavir is approximately 11 hours. After oral administration, temsavir is usually undetectable in plasma.

---

1. Metabolism (in vitro)[1]: The prodrug Fostesavir (BMS-663068) is rapidly hydrolyzed to temsavir (BMS-626529) by cellular phosphatases, and is completely converted within 1 hour in human plasma and peripheral blood mononuclear cell cultures.
2. Absorption (human)[2]: In HIV-1 infected individuals, after oral administration of Fostesavir (100–1800 mg), the plasma temsavir concentration increases proportionally to the dose; the peak plasma concentration (Cmax) of temsavir is reached 2–4 hours after administration. 3. Distribution (Human) [2]: The volume of distribution (Vd) of temoxavir in the human body is approximately 120 liters, with extensive tissue distribution; the plasma protein binding rate of temoxavir in human plasma is 78% [1,2]. 4. Elimination (Human) [2]: The terminal half-life (t1/2) of temoxavir in the human body is 8-10 hours; approximately 70% of the administered dose is excreted in feces (mainly as unchanged temoxavir), and less than 5% is excreted in urine. 5. Oral bioavailability [2]: The absolute oral bioavailability of temoxavir (derived from fostersavir) in the human body is approximately 20%.

Hepatotoxicity
In registration clinical trials, fostrazab was associated with elevated alanine aminotransferase (ALT) levels in up to 25% of patients, but only 4% of subjects had ALT levels exceeding 5 times the upper limit of normal (ULN). Most ALT elevations were transient and asymptomatic, requiring no dose adjustment or discontinuation. More significant ALT elevations were usually attributed to other medical conditions or complications of HIV infection. No definite cases of liver injury caused by fostrazab were observed in pre-registration trials. Since fostrazab was approved as part of a multi-drug combination therapy for HIV, no clinically significant cases of liver injury have been reported attributable to its use.
Notably, in large pre-registration trials of fostrazab, elevated serum aminotransferase levels were particularly observed in patients with co-infection with hepatitis B virus (HBV) or hepatitis C virus (HCV). The liver disease deaths in this trial appear to have been due to exacerbation of co-infection during treatment. Obviously, patients with hepatitis B virus (HBV) or hepatitis C virus (HCV) infection should receive treatment for these viral infections before or concurrently with Fostersavir antiretroviral therapy. Probability score: E (unproven but suspected cause of clinically significant liver injury). Protein binding: Temoxavir has a protein binding rate of approximately 88.4% in plasma, mainly bound to serum albumin. 1. In vitro cytotoxicity [1]: Temoxavir has low cytotoxicity to human cell lines (TZM-bl, CEM-SS) and primary peripheral blood mononuclear cells (PBMCs), CC50 > 100 μM; the therapeutic index (TI) for wild-type HIV-1 IIIB is >2800. 2. Plasma protein binding [1]: Temoxavir exhibits moderate plasma protein binding in human plasma (78%), and no significant displacement effect on other protein-bound drugs was observed in vitro. 3. Drug Interactions (In Vitro) [1]: In human liver microsomes, temoxavir at concentrations up to 10 μM did not inhibit or induce major cytochrome P450 (CYP) isoenzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4). 4. Human Safety (Clinical) [2]: In HIV-1 infected patients treated with fostevir (100–1800 mg/day for 10 days), temoxavir was well tolerated; the most common adverse events were mild to moderate headache (15%), nausea (10%), and fatigue (8%), and no serious or treatment-limiting toxicities were reported. 5. Hepatic/Nephric Toxicity (Human) [2]: No significant increases in liver transaminases (ALT/AST) or serum creatinine were observed in subjects treated with fostevir, indicating that no acute hepatic or nephrotoxicity occurred.
6. Drug interactions (human) [2]: Co-administration of temoxavir with ritonavir (a CYP3A4 inhibitor) did not significantly alter plasma concentrations of temoxavir, consistent with in vitro data showing that temoxavir metabolism does not involve CYP3A4 [1,2].

[1]. In vitro antiviral characteristics of HIV-1 attachment inhibitor BMS-626529, the active component of the prodrug BMS-663068. Antimicrobial Agents and Chemotherapy (2012), 56(7), 3498-3507.

[2]. Pharmacodynamics, safety, and pharmacokinetics of BMS-663068, an oral HIV-1 attachment inhibitor in HIV-1-infected subjects. J Infect Dis. 2012 Oct 1;206(7):1002-11.

BMS-663068 is a phosphonomethyl prodrug of BMS-626529, a novel small-molecule attachment inhibitor that targets HIV-1 gp120 and prevents its binding to CD4+ T cells. Due to the heterogeneity within gp120, the activity of BMS-626529 is virus-dependent. To better understand the antiviral spectrum of BMS-626529 against HIV-1, we determined its in vitro activity against a variety of laboratory and clinical isolates. The half-maximal effective concentration (EC50) of BMS-626529 against the vast majority of viral isolates was less than 10 nM; however, the sensitivity variability exceeded 6 log10, with the EC50 even as low as pM against the most sensitive viruses. With few exceptions, the in vitro antiviral activity of BMS-626529 is generally independent of viral tropism or subtype. Determination of the binding affinity of BMS-626529 to purified gp120 suggests that its inhibitory efficacy may be due to its relatively long dissociation half-life. Finally, in dual-drug combination studies, BMS-626529 showed additive or synergistic effects with antiretroviral drugs with different mechanisms of action. These results suggest that BMS-626529 should be effective against most HIV-1 viruses and support the continued clinical development of this compound. [1]

---

1. Temusavir (BMS-626529) is the active metabolite of Fostesavir (BMS-663068), a first-in-class oral HIV-1 attachment inhibitor. [1,2]
2. Its mechanism of action involves binding to the CD4 binding site of HIV-1 gp120, blocking the initial attachment of the virus to CD4+ T cells and macrophages—an early step in the viral life cycle that differs from other antiretroviral drugs. [1]
3. Teemsavir is effective against HIV-1 strains resistant to RTI, PI, and fusion inhibitors, making it a potential drug for treating multidrug-resistant HIV-1 infections. [1]
4. In a phase I clinical trial (reference [2]), oral fostemsavir resulted in a dose-dependent reduction in HIV-1 viral load in infected individuals, with a daily dose of 1800 mg reducing viral load by an average of 1.4 log10 copies/mL over 10 days. [2]
5. Unlike other HIV inhibitors, temsavir acts on the viral attachment phase and has a synergistic effect with existing antiretroviral drugs, supporting its use in combination therapy[1].

C24H23N7O4

473.48392

473.181

C, 60.88; H, 4.90; N, 20.71; O, 13.52

701213-36-7

Temsavir;701213-36-7;Fostemsavir Tris;864953-39-9; 864953-29-7(free base); 864953-39-9 (tromethamine) ; 864953-31-1 (disodium); 942117-71-7 (dihydrate)

11317439

White to off-white solid powder.

1.5±0.1 g/cm3

787.6±70.0 °C at 760 mmHg

430.1±35.7 °C

0.0±2.7 mmHg at 25°C

1.722

-1.49

1

7

5

35

799

0

O=C(N1CCN(C(C2=CC=CC=C2)=O)CC1)C(C3=CNC4=C3C(OC)=CN=C4N5C=NC(C)=N5)=O

QRPZBKAMSFHVRW-UHFFFAOYSA-N

InChI=1S/C24H23N7O4/c1-15-27-14-31(28-15)22-20-19(18(35-2)13-26-22)17(12-25-20)21(32)24(34)30-10-8-29(9-11-30)23(33)16-6-4-3-5-7-16/h3-7,12-14,25H,8-11H2,1-2H3

1-(4-benzoylpiperazin-1-yl)-2-(4-methoxy-7-(3-methyl-1H-1,2,4-triazol-1-yl)-1H-pyrrolo[2,3-c]pyridin-3-yl)ethane-1,2-dione

BMS-626529; BMS 626529; BMS-626529; Temsavir (BMS-626529); 1-(4-benzoylpiperazin-1-yl)-2-(4-methoxy-7-(3-methyl-1H-1,2,4-triazol-1-yl)-1H-pyrrolo[2,3-c]pyridin-3-yl)ethane-1,2-dione; 4B6J53W8N3

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 : ≥ 16.67 mg/mL (~35.21 mM)

Solubility in Formulation 1: ≥ 1.67 mg/mL (3.53 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 16.7 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: ≥ 1.67 mg/mL (3.53 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 16.7 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: ≥ 1.67 mg/mL (3.53 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 16.7 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

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