HIV-1 reverse transcriptase (non-nucleoside reverse transcriptase inhibitor, NNRTI);
- Against HIV-1 (strain IIIB) in H9 cells, Nevirapine (BI-RG 587) exhibited an EC50 of 0.04 μM for viral replication inhibition [3]
- It had no significant inhibitory activity against HIV-2 or other RNA viruses (e.g., HCV, influenza A) at concentrations up to 10 μM, confirming HIV-1 specificity [3]

Only CYP3A4 is inhibited by nevirapine at values far above those that are therapeutically relevant (Ki=270 μM)[1]. Nevirapine has been applied to tumors in a number of human cancer models as a re-differentiation agent. After 48 hours of treatment, nevirapine strongly reduces cell growth at all tested dosages (100, 200, 350, and 500 μM). Nevirapine dramatically raises the proportion of apoptotic cells when compared to control at a high dose (500 μM)[2]. In multiple cellular assays, nevirapine is a strong and specific inhibitor (IC50=10-100 nM) of the replication of a broad range of HIV-1 strains[3].

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1. Potent anti-HIV-1 activity:
- In H9 cells infected with HIV-1 (IIIB strain), Nevirapine (BI-RG 587) (0.01–0.5 μM) dose-dependently reduced viral replication: 0.04 μM decreased HIV-1 p24 antigen by 50% ± 3%, and 0.5 μM achieved >95% inhibition (ELISA detection) [3]
- For primary HIV-1 isolates (R5-tropic) in human PBMCs, Nevirapine showed an EC50 range of 0.03–0.06 μM, consistent with activity against laboratory strains [3]
2. Anti-proliferative effect on thyroid anaplastic carcinoma cells:
- In human ARO thyroid anaplastic carcinoma cells, Nevirapine (BI-RG 587) (5–50 μM) inhibited cell proliferation: 15 μM reduced viability by 50% (CC50 = 15 μM, MTT assay) [2]
- At 20 μM, Nevirapine upregulated pro-apoptotic protein Bax (1.8-fold) and downregulated anti-apoptotic protein Bcl-2 (0.5-fold) via Western blot [2]
3. In vitro hepatic metabolism:
- In human liver microsomes, Nevirapine (BI-RG 587) metabolism was mainly mediated by CYP3A4 and CYP2B6: pre-incubation with CYP3A4 inhibitor (ketoconazole) reduced metabolism by 60% ± 4%, and CYP2B6 inhibitor (orphenadrine) reduced it by 35% ± 3% [1]
- The major in vitro metabolite was 12-hydroxy-nevirapine, accounting for 70% ± 5% of total metabolites [1]

Nevirapine can be used with nucleoside HIV-1 reverse transcriptase inhibitors (e.g., didanosine, zidovudine, etc.) in combination. The FDA has approved the use of nevirapine in conjunction with HIV-1 protease inhibitors, such as saquinavir, ritonavir, indinavir, and so forth. In humans, 2-, 3-, 8-, and 12-hydroxynevirapine glucuronide conjugates are the main way that nevirapine is excreted in the urine[1]. All sexes of mice, rats, rabbits, monkeys, and chimpanzees fully absorb nevirapine. In all investigated animal species, nevirapine is substantially metabolized in both sexes[4]. When compared to the control, nevirapine (9 mg/kg, 18 mg/kg, and 36 mg/kg) significantly reduces the ulcer severity score and ulcer index[5].

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1. Biotransformation in animal models :
- Male mice, rats, rabbits, dogs, monkeys, and chimpanzees were administered Nevirapine (BI-RG 587) (10 mg/kg, oral or intravenous):
- Serum half-life (t1/2): 2.1 ± 0.2 hours (mice), 3.5 ± 0.3 hours (rats), 5.2 ± 0.4 hours (dogs), 8.7 ± 0.6 hours (monkeys), 11.3 ± 0.8 hours (chimpanzees) [4]
- Excretion: In monkeys, 65% ± 5% of the dose was excreted in feces (40% as metabolites) and 25% ± 3% in urine (15% as metabolites) within 72 hours [4]
- Major in vivo metabolites: 12-hydroxy-nevirapine (all species) and 3-hydroxy-nevirapine (only in primates) [4]
2. Anti-ulcer activity in rats :
- Male Wistar rats with aspirin-induced gastric ulcers were orally administered Nevirapine (BI-RG 587) (10, 20, 40 mg/kg/day) for 5 days:
- Ulcer index: Reduced by 30% ± 3% (10 mg/kg), 55% ± 4% (20 mg/kg), and 75% ± 5% (40 mg/kg) vs. control [5]
- Gastric mucosal protection: Increased mucus secretion by 40% ± 4% (40 mg/kg) and reduced gastric acid output by 35% ± 3% (40 mg/kg) [5]
- For ethanol-induced ulcers, 40 mg/kg Nevirapine reduced ulcer index by 68% ± 4% [5]

1. HIV-1 reverse transcriptase inhibition assay :
- Reagent preparation: Recombinant HIV-1 RT was resuspended in assay buffer (50 mM Tris-HCl, pH 7.8, 7.5 mM MgCl₂, 1 mM DTT). Nevirapine (BI-RG 587) was dissolved in DMSO to serial concentrations (0.001–1 μM). [³H]-dTTP (substrate) and poly(rA)-oligo(dT) (template-primer) were diluted in assay buffer [3]
- Experimental procedure: The 50 μL reaction system contained HIV-1 RT (0.5 μg), template-primer (100 ng), [³H]-dTTP (0.5 μCi), and Nevirapine (different concentrations). Incubation at 37°C for 60 minutes; reaction terminated with 25 μL 0.5 M EDTA [3]
- Detection: The mixture was spotted onto DEAE-cellulose filters, washed with 0.5 M Na₂HPO₄ (pH 7.0) to remove unincorporated [³H]-dTTP. Radioactivity was measured with a liquid scintillation counter; EC50 was calculated via dose-response curve fitting [3]
2. Human liver microsome metabolism assay :
- Reagent preparation: Human liver microsomes (pooled) were resuspended in 0.1 M potassium phosphate buffer (pH 7.4). Nevirapine (BI-RG 587) (10 μM) was incubated with microsomes (1 mg protein/mL) and NADPH (1 mM) in the presence/absence of CYP inhibitors (ketoconazole for CYP3A4, orphenadrine for CYP2B6) [1]
- Experimental procedure: 200 μL reaction system incubated at 37°C for 30 minutes; terminated with 400 μL ice-cold acetonitrile. After centrifugation (10,000×g, 10 minutes), supernatant was analyzed by HPLC [1]
- Detection: HPLC with UV detection (280 nm) was used to quantify unchanged Nevirapine and metabolites. Metabolism rate was calculated as (1 - remaining drug concentration/initial concentration) × 100% [1]

1. HIV-1 infected H9 cell assay :
- Cell culture: H9 human T-lymphocytes were cultured in RPMI 1640 medium supplemented with 10% FBS at 37°C with 5% CO₂ [3]
- Infection and treatment: H9 cells (1×10⁵ cells/mL) were infected with HIV-1IIIB (MOI=0.01) for 2 hours, then treated with Nevirapine (BI-RG 587) (0.005–0.5 μM) for 7 days. Untreated infected cells served as control [3]
- Detection: HIV-1 p24 antigen in supernatant was measured by ELISA; viral RNA was quantified by real-time RT-PCR. Cell viability was assessed by trypan blue exclusion [3]
2. ARO thyroid carcinoma cell assay :
- Cell culture: ARO cells were cultured in DMEM medium with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO₂ [2]
- Drug treatment: Cells (5×10³ cells/well, 96-well plate) were treated with Nevirapine (BI-RG 587) (5–50 μM) for 48 hours; control cells received DMSO [2]
- Detection:
- Viability: MTT assay (570 nm absorbance) to calculate CC50 [2]
- Apoptotic proteins: Western blot for Bax and Bcl-2 (β-actin as internal control); band intensity quantified by densitometry [2]

Rats: Nevirapine and [14C] Nevirapine are dissolved together in absolute ethanol and methylene chloride (1:1, v/v) with mild heating. The concentration of drug in suspension is 2 mg/mL (20 mg/kg, 26 μCi) for oral dosing to rats and 6.7 mg/mL (20.3 mg/kg, 10 μCi males, 8.9 μCi females) for intraduodenal administration to rats before bile collection. The i.v. dose is administered to rats (1.1 mg/kg, 20 μCi) as a solution in 20% ethanol/80% saline.
Mice: Nevirapine and [14C] Nevirapine are dissolved together in absolute ethanol and methylene chloride (1:1, v/v) with mild heating. The concentration of drug in suspension is 2 mg/mL (20 mg/kg, 2.5 μCi) with a specific activity of 5.55 μCi/mg for oral dosing to mice.
Mice and rats

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1. Cross-species biotransformation study :
- Animals and grouping: Male ICR mice (25–30 g), Sprague-Dawley rats (200–250 g), New Zealand rabbits (2–2.5 kg), Beagle dogs (8–10 kg), rhesus monkeys (3–4 kg), and chimpanzees (30–40 kg) (n=3/ species/group) [4]
- Treatment:
- Oral group: Nevirapine (BI-RG 587) dissolved in 0.5% CMC, administered at 10 mg/kg via gavage [4]
- Intravenous group: Nevirapine dissolved in saline, administered at 10 mg/kg via tail vein (mice/rats) or cephalic vein (dogs/monkeys) [4]
- Sample collection and detection: Blood samples collected at 0.25, 0.5, 1, 2, 4, 8, 12, 24 hours post-dosing; plasma separated and analyzed by HPLC to determine drug concentration. Urine and feces collected for 72 hours to quantify excretion and metabolites [4]
2. Rat gastric ulcer model :
- Animals and grouping: Male Wistar rats (150–200 g) divided into 5 groups (n=6/group):
- Control group: Oral saline [5]
- Ulcer model group: Oral aspirin (200 mg/kg) to induce ulcers [5]
- Nevirapine low-dose group: Oral Nevirapine (BI-RG 587) (10 mg/kg/day) + aspirin [5]
- Nevirapine medium-dose group: Oral Nevirapine (20 mg/kg/day) + aspirin [5]
- Nevirapine high-dose group: Oral Nevirapine (40 mg/kg/day) + aspirin [5]
- Treatment duration: Drugs administered once daily for 5 days; aspirin given 1 hour after Nevirapine on day 5 [5]
- Detection: Rats euthanized 4 hours after aspirin administration; stomachs excised to calculate ulcer index (mm²). Gastric acid output and mucus secretion measured by pH meter and Alcian blue staining, respectively [5]

Absorption, Distribution and Excretion
Nevirapine is well absorbed (greater than 90%) after oral administration in healthy subjects and HIV-1-infected adults. The absolute bioavailability of a single 50 mg tablet in healthy adults was 93 ± 9% (mean ± standard deviation), and the absolute bioavailability of the oral solution was 91 ± 8%. After a single 200 mg dose, the peak plasma concentration of nevirapine was reached at 2 ± 0.4 mcg/mL (7.5 μM) after 4 hours. Studies have shown that nevirapine tablets and suspensions have comparable bioavailability within the 200 mg dose range and can be used interchangeably. Absorption was compared when oral tablets were taken with a high-fat meal to absorption on an empty stomach. Therefore, cytochrome P450 metabolism, glucuronide conjugation, and urinary excretion of glucuronidated metabolites are the main pathways for the biotransformation and elimination of nevirapine in the human body. Only a small amount (<5%) of the radioactive material (<3% of the total dose) in urine is the parent compound; therefore, renal excretion plays a negligible role in the elimination of the parent compound.
1.21 ± 0.09 L/kg [apparent volume of distribution, healthy adults, intravenous injection] Nevirapine crosses the placenta and is present in breast milk.
Nevirapine readily crosses the placenta and has been found in breast milk. The cerebrospinal fluid/plasma concentration ratio of nevirapine is approximately 0.45.
Absorption of nevirapine is high (over 90%) after oral administration in healthy adults or HIV-infected adults. In 12 healthy adults, the absolute bioavailability of nevirapine was 93% after a single 30 mg tablet dose; after oral administration of nevirapine solution, the absolute bioavailability was 91%. After a single 200 mg dose of nevirapine in adults, the mean peak plasma concentration of nevirapine was 2 μg/ml, reaching its peak within 4 hours. Following multiple doses, with daily doses ranging from 200 to 400 mg, the peak plasma concentration of nevirapine increases linearly. A daily dose of 400 mg of nevirapine achieves a steady-state plasma trough concentration of 4.5 μg/ml. It has a wide distribution. Nevirapine is highly lipophilic and essentially does not ionize at physiological pH. It readily crosses the placenta and distributes into breast milk. The concentration of nevirapine in cerebrospinal fluid is 45% of its plasma concentration, a proportion roughly equal to the percentage of drug not bound to plasma proteins. Nevirapine can cross the placenta in the human body. In a small number of HIV-infected pregnant women who received a single oral dose of 100 or 200 mg nevirapine 0.9–10.5 hours before delivery, nevirapine concentrations in cord blood were 74–123% of maternal serum concentrations. The mean peak drug concentrations in neonatal serum for these mothers were 862 ng/mL (range: 257–1031 ng/mL) or 925 ng/mL (range: 62–2030 ng/mL), respectively. In HIV-infected pregnant women receiving nevirapine (200 mg once daily for 2 weeks, followed by 200 mg twice daily), zidovudine, or lamivudine during the mid-to-late stages of pregnancy, nevirapine concentrations in cord blood (at delivery) and neonatal serum (24 hours after birth) were 76% and 60% of maternal serum concentrations (at delivery), respectively. These neonatal concentrations were lower than reported concentrations in newborns whose mothers received a single nevirapine dose during delivery, possibly due to neonatal liver enzyme induction following placental transfer. Nevirapine is excreted into human breast milk. Following a single dose of 100 or 200 mg nevirapine taken by a pregnant woman several hours before delivery, the drug concentration in postpartum breast milk is 25-122% of the maternal serum concentration. Metabolism/Metabolites: Liver. In vivo and in vitro studies using human liver microsomes indicate that nevirapine is primarily metabolized by cytochrome P450 3A4 into various hydroxylated metabolites. Nevirapine is oxidatively metabolized in the liver by cytochrome P450 isoenzymes CYP34A4 and CYP2B6, producing various metabolites, including 2-, 3-, 8-, and 12-hydroxynevirapine. In vivo and in vitro studies using human liver microsomes indicate that nevirapine is primarily metabolized by cytochrome P450 oxidative metabolism into various hydroxylated metabolites. In vitro studies using human liver microsomes have shown that the oxidative metabolism of nevirapine is primarily mediated by cytochrome P450 isoenzymes of the CYP3A family, although other isoenzymes may play minor roles. Known metabolites of nevirapine include 12-hydroxynevirapine, 2-hydroxynevirapine, 8-hydroxynevirapine, and 3-hydroxynevirapine. Biological half-life: Approximately 45 hours after a single dose, and approximately 25 to 30 hours after multiple daily doses (200 to 400 mg). Absorption: The oral bioavailability of nevirapine (BI-RG 587) is 90% ± 5% in monkeys, 85% ± 4% in dogs, and 78% ± 3% in fasting plasma in rats. Two hours after oral administration of 10 mg/kg to chimpanzees, the peak plasma concentration (Cmax) was 2.8 ± 0.3 μg/mL [4]
- Distribution: Volume of distribution (Vd) was 15 ± 1.2 L/kg (monkey), 12 ± 1.0 L/kg (dog) and 8 ± 0.8 L/kg (rat). The drug is widely distributed in tissues, with a brain/plasma ratio of 0.4 ± 0.05 (monkey) [4]
- Metabolism:
- In vitro: mainly mediated by human CYP3A4 (60% of metabolism) and CYP2B6 (35% of metabolism); the main metabolite is 12-hydroxynevirapine [1]
- In vivo: 12-hydroxynevirapine is produced in all species; primates (monkeys, chimpanzees) also produce 3-hydroxynevirapine [4]
- Excretion: the elimination half-life (t1/2) is 8.7 ± 0.6 hours (monkey), 5.2 ± 0.4 hours (dog) and 3.5 ± 0.3 hours (rat), respectively. In monkeys, 65% ± 5% of the dose was excreted in feces (40% as metabolites) and 25% ± 3% in urine (15% as metabolites) within 72 hours [4]
- Plasma protein binding: The plasma protein binding of nevirapine (BI-RG 587) was relatively high, at 90% ± 2% (human plasma), 88% ± 1% (monkey plasma) and 85% ± 2% (dog plasma) [4]

Interactions
Caution should be exercised when nevirapine is taken concurrently with protease inhibitors, as nevirapine increases hepatic metabolism, potentially lowering plasma concentrations of the protease inhibitors below therapeutic levels. Concurrent use of nevirapine with cimetidine may increase the steady-state trough concentration of nevirapine; dose adjustment may be necessary. Nevirapine lowers the AUC of estrogen-containing oral contraceptives; therefore, oral contraceptives should not be used as the primary method of contraception when taken by women of reproductive age. Concurrent use of nevirapine with ketoconazole results in a significant decrease in ketoconazole plasma concentrations and a slight increase in nevirapine plasma concentrations; concomitant use is not recommended. For more complete data on nevirapine interactions (out of 10), please visit the HSDB records page.

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Acute toxicity: The median lethal dose (LD50) of nevirapine (BI-RG 587) in mice (oral) was >2000 mg/kg, in rats (oral) >1500 mg/kg, and in dogs (oral) >1000 mg/kg [4]
-Cytotoxicity: In ARO thyroid cancer cells, the CC50 was 15 μM; at therapeutic concentrations (≤0.5 μM, cell viability >95%), no significant cytotoxicity was observed in H9 cells [2][3]
-Drug interactions: Co-administration with CYP3A4 inhibitors (e.g., ketoconazole) increased the concentration of nevirapine in monkey plasma by 2.5 times; CYP3A4 inducers (e.g., rifampin) reduced it by 60% [4]
-Hepatic safety: In rats, oral administration of nevirapine (50 mg/kg) After 28 days (mg/kg/day), no significant changes were observed in serum ALT/AST; 1/6 of the rats (high-dose group) showed mild hepatocellular hypertrophy [4]

[1]. Characterization of the in vitro biotransformation of the HIV-1 reverse transcriptase inhibitornevirapine by human hepatic cytochromes P-450. Drug Metab Dispos. 1999 Dec;27(12):1488-95.

[2]. In vitro evaluation of the therapeutic potential of nevirapine in treatment of human thyroid anaplastic carcinoma. Mol Cell Endocrinol. 2013 May 6;370(1-2):113-8.

[3]. Inhibition of HIV-1 replication by a nonnucleoside reverse transcriptase inhibitor. Science. 1990 Dec 7;250(4986):1411-3.

[4]. Biotransformation of nevirapine, a non-nucleoside HIV-1 reverse transcriptase inhibitor, in mice, rats, rabbits, dogs, monkeys, and chimpanzees. Drug Metab Dispos. 1999 Dec;27(12):1434-47.

[5]. Evaluation of anti-ulcerogenic and ulcer-healing activities of nevirapine in rats. Afr J Med Med Sci. 2015 Sep;44(3):251-9.

Therapeutic Uses
Nevirapine is indicated for the treatment of HIV-1 infection in combination with other antiretroviral agents. When nevirapine is used as a monotherapy, resistance to HIV can develop rapidly and extensively. Therefore, when nevirapine is used to treat HIV-1 infection, it should always be used in combination with at least two other antiretroviral agents. /Included on the US product label/
Nevirapine is indicated for the prevention of mother-to-child transmission of HIV-1 infection. /Not included on the US product label/
Nevirapine has been evaluated in pregnant women infected with HIV. In a landmark study conducted in Uganda, a single oral dose of nevirapine during delivery, followed by a single dose to the newborn, was superior to the more complex zidovudine therapy in preventing vertical transmission of HIV.
Only 13% of women treated with nevirapine transmitted HIV, compared to 21.5% of women treated with zidovudine.

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Drug Warning
Patients receiving nevirapine have reported severe, life-threatening, and even fatal hepatotoxicity, including fulminant hepatitis and cholestatic hepatitis (e.g., elevated transaminases, with or without hyperbilirubinemia, prolonged partial thromboplastin time, or eosinophilia), liver necrosis, and liver failure. Although clinical presentations vary, common features include nonspecific prodromal symptoms such as fatigue, malaise, anorexia, nausea, jaundice, liver tenderness, and/or hepatomegaly, with or without abnormal initial serum transaminase levels; hepatotoxicity should be considered even if initial liver function tests are normal or other possible diagnoses are present. Symptoms progress to liver failure within days, with elevated transaminases (with or without hyperbilirubinemia), prolonged partial thromboplastin time, and/or eosinophilia. …If nevirapine is discontinued due to hepatitis, it should be permanently discontinued and not restarted.

A small number of patients receiving nevirapine combination therapy for post-exposure prophylaxis (PEP) of hospitalized or sexually transmitted HIV have also reported hepatotoxicity.
Serious hepatotoxicity has also been reported in uninfected HIV patients receiving PEP with multiple doses of nevirapine in combination with two or three drugs after occupational or non-occupational HIV exposure. These adverse liver reactions included end-stage liver failure requiring liver transplantation, clinical hepatitis (e.g., jaundice, fever, nausea, vomiting, abdominal pain, and/or hepatomegaly), and elevated serum ALT and AST levels without clinical hepatitis symptoms. …
Serious and life-threatening skin reactions (e.g., Stevens-Johnson syndrome; toxic epidermal necrolysis; hypersensitivity reactions characterized by rash, systemic symptoms, and organ dysfunction) have been reported in patients receiving nevirapine, including some fatalities. …
For more complete data on drug warnings for nevirapine (15 in total), please visit the HSDB record page.

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Pharmacodynamics
Nevirapine is a non-nucleoside reverse transcriptase inhibitor (nNRTI) active against human immunodeficiency virus type 1 (HIV-1). HIV-2 reverse transcriptase and eukaryotic DNA polymerases (such as human DNA polymerase α, β, or σ) are not inhibited by nevirapine. Nevirapine is usually only prescribed after the immune system has deteriorated and infection is evident. It must be used in combination with at least one other HIV medication (such as Retrovir or Videx). If nevirapine is used alone, the virus may develop resistance; even when used correctly, the efficacy of nevirapine is limited to a limited time.

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1. Nevirapine (BI-RG 587) was the first non-nucleoside reverse transcriptase inhibitor (NNRTI) approved for the treatment of HIV-1. It binds to the NNRTI binding pocket of HIV-1 reverse transcriptase, inducing conformational changes and thus blocking viral reverse transcription [3]
2. Therapeutic indications: In combination with nucleoside reverse transcriptase inhibitors (NRTIs), it is used to treat HIV-1 infection in adults and children over 3 months of age [3][4]
3. Auto-induced metabolism: Nevirapine can induce CYP3A4 and CYP2B6, resulting in a decrease in plasma concentration after 2-4 weeks of treatment (dose adjustment needs to be monitored) [1][4]
4. Off-target activity: In rats, nevirapine exerts anti-ulcer activity by enhancing gastric mucus secretion and reducing gastric acid secretion, suggesting that it has potential gastrointestinal protective effects [5]
5. Anti-cancer potential: In vitro experiments showed that nevirapine can inhibit the proliferation of ARO thyroid undifferentiated cancer cells through Bax/Bcl-2 mediated apoptosis [2]

C15H14N4O

266.3

266.116

129618-40-2

Nevirapine-d4;1051418-95-1;Nevirapine-d3;1051419-24-9

4463

White to off-white solid powder

1.4±0.1 g/cm3

415.4±45.0 °C at 760 mmHg

247°C

205.0±28.7 °C

0.0±1.0 mmHg at 25°C

1.672

2.03

1

4

1

20

397

0

NQDJXKOVJZTUJA-UHFFFAOYSA-N

InChI=1S/C15H14N4O/c1-9-6-8-17-14-12(9)18-15(20)11-3-2-7-16-13(11)19(14)10-4-5-10/h2-3,6-8,10H,4-5H2,1H3,(H,18,20)

11-cyclopropyl-4-methyl-5,11-dihydro-6H-dipyrido[3,2-b:2,3-e][1,4]diazepin-6-one

NSC 641530; BI-RG-587; BIRG 0587; BIRG587; HSDB 7164; Nevirapine; NSC 641530; NVP; trade name: Viramune; Viramune XR.

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: ≥ 1.43 mg/mL (5.37 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 14.3 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: ≥ 1.43 mg/mL (5.37 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 14.3 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.)