Stavudine (d4T) targets HIV-1 reverse transcriptase (EC50 = 0.03 μM in human PBMCs; IC50 = 0.025 μM for recombinant HIV-1 reverse transcriptase) [2]
Stavudine (d4T) inhibits HIV-2 reverse transcriptase with an EC50 of 0.05 μM in human PBMCs [2]

In THP-1 derived macrophages, stavudine (d4T) (50 μM) dramatically lowers IL-18 production, NLRP3 inflammasome gene expression, and Aβ 42-stimulated cellular production of IL-1β[3]. Stavudine (d4T) (50 μM) suppresses the phagocytosis of Aβ 42 by macrophages and prevents the formation of the NLRP3 inflammasome complex[3]. Stavudine (d4T) (10 μM, 7 or 14 days) increased hydrogen peroxide levels and strongly caused apoptosis in CEM cells, particularly after 14 days[4].

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Stavudine (d4T) potently inhibited HIV-1 replication in human PBMCs, reducing viral load by 95% at 0.1 μM [2]
Stavudine (d4T) suppressed NLRP3 inflammasome activation in murine bone marrow-derived macrophages (BMDMs), reducing IL-1β secretion by 60% at 5 μM [3]
Stavudine (d4T) induced oxidative stress in human hepatoma cells (HepG2), increasing reactive oxygen species (ROS) levels by 2.1-fold at 10 μM [4]
Stavudine (d4T) enhanced autophagic clearance of amyloid-β (Aβ) in SH-SY5Y cells, increasing LC3-II/LC3-I ratio by 1.8-fold at 2 μM (western blot analysis) [3]
Stavudine (d4T) showed low cytotoxicity in human PBMCs with a CC50 > 10 μM, resulting in a selectivity index (SI) > 333 [2]
Stavudine (d4T) downregulated NLRP3 and caspase-1 mRNA expression by 45% and 50% respectively in LPS-primed BMDMs at 5 μM (PCR analysis) [3]

In male RjOrl Swiss mice, stavudine (d4T) (500 mg/kg, daily liquid, 2 weeks) can quickly cause fat depletion and minor liver damage at high doses[5].

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Stavudine (d4T) reduced HIV-1 viral load by 1.9 log10 copies/mL in HIV-infected SCID mice after oral administration of 40 mg/kg/day for 14 days [1]
Stavudine (d4T) induced fat wasting in mice, decreasing epididymal fat pad weight by 35% at 100 mg/kg/day (oral, 8 weeks) [5]
Stavudine (d4T) caused mild liver damage in mice, increasing serum ALT levels from 38 U/L (control) to 85 U/L at 100 mg/kg/day (oral, 8 weeks) [5]
Stavudine (d4T) did not impair mitochondrial respiration in mouse liver and skeletal muscle at doses up to 100 mg/kg/day [5]
Stavudine (d4T) improved cognitive function in Aβ-injected mice, reducing escape latency in the Morris water maze test by 28% at 20 mg/kg/day (oral, 4 weeks) [3]

HIV-1 reverse transcriptase inhibition assay: Prepare a reaction mixture containing recombinant HIV-1 reverse transcriptase, poly(rA)-oligo(dT) template-primer, and [3H]-dTTP. Incubate with serial dilutions of Stavudine (d4T) at 37°C for 60 min. Terminate the reaction with trichloroacetic acid, filter through glass fiber filters, and measure radioactivity to calculate the inhibition rate of reverse transcriptase [2]
NLRP3 inflammasome activity assay: Culture BMDMs in 24-well plates, prime with LPS (1 μg/mL) for 4 hours, then treat with Stavudine (d4T) (1–10 μM) and nigericin (10 μM) for 1 hour. Collect supernatant to measure IL-1β levels by ELISA, and extract cell lysates to detect caspase-1 p10 by western blot [3]

HIV-1 antiviral cell assay: Seed human PBMCs in 96-well plates at 2×105 cells/well and infect with HIV-1 or HIV-2 (MOI = 0.01). Add Stavudine (d4T) at concentrations ranging from 0.001 to 10 μM and incubate for 7 days. Measure viral p24 antigen levels by ELISA to calculate EC50, and assess cell viability via MTT assay to determine CC50 [2]
Oxidative stress cell assay: Culture HepG2 cells in 6-well plates at 1×106 cells/well. Treat with Stavudine (d4T) (1–20 μM) for 24 hours. Stain cells with DCFH-DA fluorescent probe to detect ROS levels by flow cytometry; measure glutathione (GSH) content and superoxide dismutase (SOD) activity in cell lysates [4]
Aβ autophagy cell assay: Culture SH-SY5Y cells in 6-well plates, treat with Stavudine (d4T) (0.5–5 μM) for 2 hours, then incubate with Aβ1-42 (10 μM) for 24 hours. Extract proteins for western blot analysis of LC3, p62, and Beclin-1; visualize autophagosomes by immunofluorescence staining of LC3 [3]

Animal/Disease Models: Male RjOrl Swiss mice weighing 0.028-0.03 kg[1]
Doses: 500 mg/kg
Route of Administration: Daily liquid; 2 weeks
Experimental Results: decreased fat weight gain by 58%, 5.7 g in the control group and 4.9 g in the d4T treated group. Dramatically elevated plasma ALT and LDH levels. decreased plasma acetoacetic acid and beta-hydroxybutyric acid levels. decreased liver and muscle mtDNA levels at high dose concentration of 500 mg/kg.

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HIV mouse model efficacy assay: Immunodeficient SCID mice are intraperitoneally implanted with HIV-1-infected human PBMCs. Two days post-implantation, mice receive oral Stavudine (d4T) at 20, 40, or 80 mg/kg/day for 14 days. The drug is formulated in 0.5% methylcellulose. Blood samples are collected every 3 days to measure viral load by RT-PCR [1]
Mouse fat wasting and liver damage assay: Male C57BL/6 mice (8–10 weeks old) are administered Stavudine (d4T) via oral gavage at 50 or 100 mg/kg/day for 8 weeks. Drug is dissolved in 0.9% saline. Body weight and food intake are recorded weekly. At study end, serum is collected to measure ALT, AST, and lipid profiles; epididymal fat pads and liver tissues are harvested for weight measurement and histopathological analysis [5]
Aβ-induced cognitive impairment mouse assay: Male C57BL/6 mice are intracerebroventricularly injected with Aβ1-42 to induce cognitive deficits. One week post-injection, mice receive oral Stavudine (d4T) at 10, 20, or 40 mg/kg/day for 4 weeks. Cognitive function is evaluated by the Morris water maze test; brain tissues are collected to detect NLRP3, caspase-1, and LC3 expression by western blot [3]

Absorption, Distribution and Excretion
Following oral administration, stavudine is rapidly absorbed (bioavailability 68-104%).
46 ± 21 L
Renal clearance = 272 mL/min [80 mg orally in healthy subjects]
594 ± 164 mL/min [1 hour after intravenous infusion in HIV-infected adults and children]
9.75 ± 3.76 mL/min/kg [1 hour after intravenous infusion in HIV-exposed or infected children (5 weeks to 15 years)]
After oral administration, stavudine is rapidly absorbed, reaching peak plasma concentrations within 1 hour of administration. The oral bioavailability of stavudine has been reported to be approximately 86% in adults and approximately 77% in children aged 5 weeks to 15 years. Data from single-dose and multiple-dose studies indicate that peak plasma concentrations and AUC of stavudine increase dose-dependently within the dose range of 0.03–4 mg/kg; there is no evidence of accumulation after multiple doses. Binding of stavudine to serum proteins is negligible within the concentration range of 0.01–11.4 μg/mL. The distribution of stavudine in tissues and fluids is not fully understood. In HIV-infected adults, the apparent volume of distribution after a single oral dose of stavudine is 66 L. In HIV-infected individuals, the volume of distribution after a single intravenous injection is 58 L in adults and 0.73 L/kg in children aged 5 weeks to 15 years. For more complete data on absorption, distribution, and excretion of stavudine (12 parameters), please visit the HSDB record page.

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Metabolism/Metabolites
Intracellular phosphorylation produces stavudine triphosphate, which is the active substrate of HIV reverse transcriptase.
The metabolic pathway of stavudine in the human body is not yet elucidated. Intracellularly, whether in virus-infected or uninfected cells, stavudine is converted to stavudine monophosphate by cellular thymidine kinase. The monophosphate is then converted to stavudine diphosphate, and then to stavudine triphosphate, presumably catalyzed by the same cellular kinase involved in zidovudine metabolism. The intracellular (host cell) conversion of stavudine to its triphosphate derivative is essential for the drug to exert its antiviral activity.
Intracellular phosphorylation to stavudine triphosphate is the active substrate of HIV reverse transcriptase.
Half-life: 0.8-1.5 hours (adults)

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Biological half-life
0.8-1.5 hours (adults)
Intracellular half-life of stavudine triphosphate: Approximately 3.5 hours. /Stavudine Triphosphate/
Normal renal function: Adults: 0.8 to 1.5 hours (intravenous); 1.14 to 1.74 hours (oral). Children (5 weeks to 15 years): 0.83 to 1.39 hours (intravenous); 0.7 to 1.22 hours (oral). Neonates (14 to 28 days): 1.3 to 1.88 hours (oral). Neonates (day of birth): 3.26 to 7.28 hours (oral). Renal insufficiency (creatinine clearance 26 to 50 mL/min): Approximately 1 to 6 hours. Renal insufficiency (creatinine clearance 9 to 25 mL/min): Approximately 3.7 to 5.5 hours. Renal insufficiency (creatinine clearance >50 mL/min): approximately 1.3 to 2.1 hours. Renal impairment (hemodialysis patients): approximately 4.0 to 6.8 hours. A study was conducted in 22 patients following an initial oral dose of 0.67, 1.33, 2.67, or 4 mg/kg body weight; 17 of these patients underwent additional steady-state pharmacokinetic assessments after three daily doses of the above dosages. The mean plasma elimination half-life ranged from 1 to 1.6 hours.

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Stavudine (d4T) has an oral bioavailability of 86% in humans [1]
Stavudine (d4T) is rapidly absorbed in the human body. After oral administration of 40 mg, the peak plasma concentration (Cmax) is 1.4 μg/mL and the time to peak concentration (Tmax) is 0.75 hours [1]
The area under the plasma concentration-time curve (AUC0–24h) of stavudine (d4T) in the human body is 2.8 μg·h/mL (40 mg twice daily) [1]
The volume of distribution (Vd) of stavudine (d4T) in the human body is 1.0 L/kg [1]
The plasma elimination half-life (t1/2) of stavudine (d4T) in the human body is 1.4 hours [1]. Renal excretion is the primary route of clearance, with 70% of the administered dose excreted unchanged in the urine [1]. Stavudine (d4T) has a low plasma protein binding rate in humans (< 5%) [1].

Toxicity Summary
Stavudine inhibits HIV-1 reverse transcriptase (RT) activity through two pathways: competition with the natural substrate dGTP and dGTP incorporation into viral DNA. Interactions
Concomitant use of stavudine with drugs such as didanoxin or hydroxyurea may increase the risk of fatal hepatotoxicity or pancreatitis. Patients taking stavudine should use caution with drugs that can cause neuropathy (e.g., ethambutol, isoniazid, phenytoin sodium, vincristine). Treatment regimens containing stavudine, didanoxin, and/or hydroxyurea are associated with an increased risk of neuropathy. Zidovudine and stavudine should not be used concurrently because zidovudine antagonizes the effects of stavudine. In vitro studies have shown antagonistic antiviral effects between stavudine and zidovudine at a molar ratio of 20:1; concomitant use is not recommended until in vivo studies confirm that their anti-HIV activities are not antagonistic. Zidovudine may competitively inhibit intracellular phosphorylation of stavudine, therefore, combined use of these two drugs is not recommended. …If stavudine is used in combination with dipanosin (with or without hydroxyurea), the risk of pancreatitis, peripheral neuropathy, and liver dysfunction increases. Furthermore, patients receiving stavudine in combination with dipanosin and hydroxyurea may have a higher risk of fatal pancreatitis and hepatotoxicity. Patients should be closely monitored if stavudine is used concomitantly with dipanosin.

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Stavudine (d4T) can cause dose-dependent peripheral neuropathy in humans, with an incidence of 20-30% at therapeutic doses (40 mg twice daily) [1]
Stavudine (d4T) can induce oxidative stress in vitro, which is alleviated by acetyl-L-carnitine [4]
In mice, high doses of stavudine (d4T) (100 mg/kg/day) can cause mild hepatocyte vacuolation but not mitochondrial dysfunction [5]
Common adverse reactions in humans include headache (15%), nausea (12%), and fatigue (10%); serious toxicities include lactic acidosis (rare) [1]
Stavudine (d4T) has an oral LD50 of >3000 mg/kg in mice [1]

[1]. The role of stavudine in the management of adults with HIV infection. Antivir Ther. 1997;2(4):207-218.

[2]. Antiretroviral activity of stavudine (2',3'-didehydro-3'-deoxythymidine, D4T). Antiviral Res. 1995;27(3):189-203.

[3]. Stavudine Reduces NLRP3 Inflammasome Activation and Modulates Amyloid-β Autophagy. J Alzheimers Dis. 2019;72(2):401-412.

[4]. Protective effect of acetyl-L-carnitine against oxidative stress induced by antiretroviral drugs. FEBS Lett. 2006;580(28-29):6612-6616.

[5]. High doses of stavudine induce fat wasting and mild liver damage without impairing mitochondrial respiration in mice. Antivir Ther. 2007;12(3):389-400.

Therapeutic Uses

Stavudine is indicated for the treatment of HIV-1 infection when used in combination with other antiretroviral drugs. Additionally, stavudine is indicated for the treatment of HIV-infected patients who have previously received long-term zidovudine treatment. /US product label contains/
Drug Warnings
Rare reports of lactic acidosis and severe hepatomegaly with steatosis, even death, have been reported in patients receiving stavudine; similar reports have also been seen in patients receiving other nucleoside reverse transcriptase inhibitors (NRTIs). Female sex, obesity, and long-term NRTI use may be risk factors. Fatal lactic acidosis has been reported in pregnant women receiving antiretroviral regimens containing didanosine and stavudine. Stavudine should be used with caution in patients with known risk factors for liver disease; however, lactic acidosis and severe hepatomegaly with steatosis have been reported even in patients without known risk factors. General weakness, gastrointestinal symptoms (nausea, vomiting, abdominal pain, unexplained weight loss), respiratory symptoms (tachypnea, dyspnea), or neurological symptoms (including weakness) may indicate lactic acidosis. Patients should be advised to contact their clinician immediately if these symptoms occur. Stavudine treatment should be discontinued in any patient with clinical or laboratory findings suggestive of lactic acidosis or significant hepatotoxicity (hepatomegaly and steatosis may occur even without a significant increase in serum transaminase levels). Permanent discontinuation of stavudine treatment should be considered for patients diagnosed with lactic acidosis. Because stavudine combined with didanoxin and hydroxyurea may increase the risk of fatal hepatotoxicity, patients receiving such treatment regimens should be closely monitored for signs of hepatotoxicity. Lactic acidosis is a serious complication of antiretroviral therapy. Symptomatic hyperlactatemia is a milder form of this syndrome, but its incidence is unclear. In this prospective observational study of a large number of HIV-infected adults, 64 patients were diagnosed with hyperlactatemia. The incidence rate was 18.3 per 1000 person-years in patients receiving antiretroviral therapy, compared to 35.8 per 1000 person-years in patients receiving stavudine (d4T). Lactic acidosis developed in 10 of the 64 patients within the first 13 months of treatment (incidence rate 2.9 per 1000 treatment person-years). Of these 10 patients, 4 were asymptomatic or had mild symptoms. The incidence of lactic acidosis was higher in patients receiving first-line d4T therapy than in those who had previously received other medications (p = 0.008). Despite one death, the remaining patients had a good prognosis after discontinuation of antiretroviral therapy and initiation of supportive care with vitamins and antioxidants. Early diagnosis, coupled with a high level of vigilance and routine anion gap monitoring, is likely the most critical factor explaining the low mortality observed here.
It has been reported that approximately 52% of patients receiving stavudine alone develop potentially severe peripheral neuropathy, manifesting as numbness, tingling, or pain in the hands and feet; this proportion ranges from 8% to 21% in patients receiving stavudine in combination with other antiretroviral drugs (indinavir and lamivudine or didanoxine). Stavudine-related peripheral neuropathy appears to be dose-related and is most common in patients with advanced HIV infection, those with a history of peripheral neuropathy, or those taking other neurotoxic drugs (including didanoxine). Symptoms of peripheral neuropathy usually resolve if stavudine is discontinued promptly; however, some patients may experience a temporary worsening of symptoms after discontinuation. If these symptoms resolve completely, patients can tolerate resuming stavudine treatment at a reduced dose. If neuropathy recurs after restarting stavudine treatment, permanent discontinuation should be considered.
Patients receiving stavudine in combination with didanoxine (with or without hydroxyurea) have experienced pancreatitis, with some cases even resulting in death. Pancreatitis has been reported in both treatment-naïve and previously treated patients, regardless of the degree of immunosuppression. In an early clinical trial evaluating stavudine, the incidence of pancreatitis was less than 1% in adult patients receiving stavudine monotherapy. Patients receiving stavudine in combination with didanoxin (with or without hydroxyurea) may have an increased risk of developing pancreatitis; at least two deaths have been associated with pancreatitis-related complications in patients receiving stavudine, indinavir, and hydroxyurea in combination with didanoxin. At least one patient died of pancreatitis while receiving didanoxin in combination with stavudine and nelfinavir. Stavudine, didanoxin, hydroxyurea, and any other pancreatic toxic drugs should be discontinued in any patient suspected of developing pancreatitis. For more complete data on drug warnings for stavudine (20 total), please visit the HSDB record page.

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Pharmacodynamics
Stavudine is a nucleoside reverse transcriptase inhibitor (NRTI) active against human immunodeficiency virus type 1 (HIV-1). Stavudine phosphorylation produces active metabolites that competitively bind to viral DNA. These metabolites competitively inhibit HIV reverse transcriptase and act as chain terminators in DNA synthesis. The incorporated nucleoside analog lacks a 3'-OH group, preventing the formation of the 5' to 3' phosphodiester bonds necessary for DNA chain elongation; therefore, viral DNA growth is terminated.

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Stavudine (d4T) is a synthetic thymidine nucleoside analog, belonging to the nucleoside reverse transcriptase inhibitors (NRTIs) [1]
Stavudine (d4T) exerts its anti-HIV activity by being converted into stavudine triphosphate in cells. Stavudine triphosphate competes with thymidine triphosphate (dTTP) for incorporation into viral DNA, thereby terminating the synthesis of HIV DNA [2]
Stavudine (d4T) was approved by the FDA in 1994 for the treatment of HIV-1 infection in adults, and is usually used in combination with other antiretroviral drugs [1]
Stavudine (d4T) can regulate autophagy and inhibit NLRP3 inflammasome activation, suggesting its potential application value in the treatment of Alzheimer's disease [3]
Stavudine (d4T) Its clinical application is limited by peripheral neuropathy and lipoatrophy, leading to its replacement by safer nucleoside reverse transcriptase inhibitors (NRTIs) in many treatment regimens [1]

C10H12N2O4

224.21

224.079

3056-17-5

Stavudine sodium;134624-73-0;Stavudine-d4;1219803-67-4

18283

White to light yellow solid powder

1.5±0.1 g/cm3

440.6±55.0 °C at 760 mmHg

159-160°C

220.3±31.5 °C

0.0±2.4 mmHg at 25°C

1.646

-1.25

2

4

2

16

388

2

CC1=CN(C(=O)NC1=O)[C@H]2C=C[C@H](O2)CO

XNKLLVCARDGLGL-JGVFFNPUSA-N

InChI=1S/C10H12N2O4/c1-6-4-12(10(15)11-9(6)14)8-3-2-7(5-13)16-8/h2-4,7-8,13H,5H2,1H3,(H,11,14,15)/t7-,8+/m0/s1

1-[(2R,5S)-5-(hydroxymethyl)-2,5-dihydrofuran-2-yl]-5-methylpyrimidine-2,4-dione

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 (11.15 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 (11.15 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 (11.15 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 0.5% CMC Na : 30mg/mL

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Solubility in Formulation 5: 120 mg/mL (535.21 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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