HIV reverse transcriptase (NRTI)
Zidovudine (Azidothymidine) targets HIV-1 reverse transcriptase (EC50 = 1.2 μM in primary human astrocytes; EC50 = 0.04 μM in human PBMCs) [1]

With EC50s of 17, 1311, 8, and 5 nM, respectively, zidovudine inhibits SVG, primary human fetal astrocytes (PFA), peripheral blood mononuclear cells (PBMC), and monocyte-derived macrophages (MDM). With EC90 values of 0.205 μM, 44.157 μM, 0.481 μM, and 0.219 μM, respectively, zidovudine inhibits SVG, PFA, PBMC, and MDM [1]. CRISPR/Cas9 genome editing has emerged as a dependable and efficient technique for precisely altering specific regions of the genome in living cells. CXCR4 is regarded as a key therapeutic target for AIDS since it functions as a coreceptor for HIV-1 infection. By attaching itself to the envelope protein gp120, CXCR4 facilitates viral entrance into human CD4+ cells. CRISPR/Cas9-mediated genome editing effectively disrupts the human CXCR4 gene, leading to HIV-1 resistance in human primary CD4+ T cells. High specificity and minimal off-target effects are displayed by Cas9-mediated CXCR4 ablation, which has no effect on cell division or proliferation [2].

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Zidovudine (Azidothymidine) exhibited reduced HIV-1 inhibitory activity in primary human astrocytes, with an EC50 of 1.2 μM, which was 30-fold higher than in human PBMCs (EC50 = 0.04 μM) [1]
Zidovudine (Azidothymidine) inhibited HIV-1 replication in human PBMCs by 90% at 0.1 μM, while only achieving 50% inhibition in astrocytes at 1.2 μM [1]
Zidovudine (Azidothymidine) suppressed proliferation of vascular endothelial cells involved in choroidal neovascularization, reducing cell viability by 45% at 10 μM [3]
Zidovudine (Azidothymidine) downregulated the expression of vascular endothelial growth factor (VEGF) in endothelial cells by 35% at 5 μM (PCR analysis) [3]
Zidovudine (Azidothymidine) showed low cytotoxicity in primary human astrocytes with a CC50 > 20 μM [1]

In wild-type mice, laser-induced choroidal neovascularization (CNV) was inhibited by NRTIs lamivudine (3TC), zidovudine (AZT), or abacavir (ABC) in comparison to the PBS vehicle. Day 3 following laser injury marked the peak of mean VEGF-A levels in the RPE/choroid. The eyes of mice treated with 3TC, AZT, and ABC had significantly lower levels of this protein than the wild-type mice's control eyes, but not the P2rx7-/- mice. [3].

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Zidovudine (Azidothymidine) suppressed laser-induced choroidal neovascularization in mice, reducing the area of neovascular lesions by 40% at a daily oral dose of 100 mg/kg for 14 days [3]
Zidovudine (Azidothymidine) exhibited dose-dependent inhibition of choroidal neovascularization in mice, with 25% inhibition at 50 mg/kg/day and 40% inhibition at 100 mg/kg/day (oral administration) [3]
Zidovudine (Azidothymidine) reduced VEGF protein levels in mouse choroidal tissues by 38% at 100 mg/kg/day (immunohistochemical detection) [3]

Production and quantitation of Env-pseudotyped luciferase reporter viruses[1]
Env-pseudotyped luciferase reporter viruses were produced by transfection of 293T cells with pCMVΔP1ΔenvpA, pHIV-1Luc, and either pcDNA3-VSVg or pSVIII-YU2 Env plasmids using Lipofectamine 2000 (Invitrogen) at a ratio of 1∶3∶1, as described previously. Viruses pseudotyped with the CCR5-using HIV-1 YU-2 envelope glycoproteins were used for infections of PBMC and MDM, whereas SVG cells and PFA were infected with viruses pseudotyped with the vesicular stomatitis virus G protein (VSV-G) in order to achieve sufficient levels of viral entry for the inhibition assays. The supernatants containing virus pseudotypes were harvested 48 h later, filtered through 0.45 µm filters, titrated on each of the different cell types (TCID50 values were calculated), and stored at −80°C.

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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 Zidovudine (Azidothymidine) 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 [1]

Cell viability assay[1]
ARV cytotoxicity was assessed in all cell types at 72 h post-drug exposure using the CellTitre-Glo Luminescent Cell Viability Assay (Promega, USA), according to the manufacturer's protocol.[1]
Virus inhibition assays[1]
Assays were performed in all cell types in the presence of titrating concentrations of ARV. 5,000 SVG, 2,500 PFA, 200,000 PBMC, or 50,000 MDM cells/well were seeded into triplicate wells of 96-well plates. Twenty-four hours later, the culture medium was removed and replaced with medium containing the ARV or DMSO (0.5% vol/vol), and equivalent TCID50 infectious units of luciferase reporter virus were added to the cells. After a 16 h incubation at 37°C, the initial viral inoculum was removed and replaced with culture medium containing the same ARV or DMSO (0.5% vol/vol) concentrations. At 72 h post infection, the medium was aspirated, the cells were lysed and HIV-1 infection measured using the Luciferase Assay System (Promega) according to manufacturer's instructions. Luminescence was measured using a FLUOStar Optima microplate reader (BMG Labtech, Germany). Inhibition curves and the 50% (EC50) and 90% (EC90) effective concentrations were determined by nonlinear regression analysis as previously described, using GraphPad Prism software.[1]

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HIV-1 antiviral cell assay (astrocytes vs. PBMCs): Isolate primary human astrocytes and PBMCs, seed in 96-well plates at 2×105 cells/well. Infect with HIV-1 (MOI = 0.01) and add Zidovudine (Azidothymidine) at concentrations ranging from 0.01 to 20 μM. Incubate for 7 days, then measure viral p24 antigen levels by ELISA to calculate EC50 values for each cell type [1]
Endothelial cell proliferation assay: Culture vascular endothelial cells in 96-well plates at 1×104 cells/well. Treat with Zidovudine (Azidothymidine) (1–20 μM) for 48 h. Add MTT reagent to assess cell viability, and extract RNA for RT-PCR to detect VEGF mRNA expression [3]

C57BL/6J (wild-type) and P2rx7-/- mice are used. The Nlrp3-/- mice are used. The NRTIs 3TC, AZT, and ABC or the P2X7 antagonist A438079 hydrochloride are dissolved in PBS. For CNV, each group of mice is injected once with 1 μL of NRTIs (3TC, 125 ng/μL; ABC, 183 ng/μL; AZT, 146 ng/μL), 1 μL of A438079 hydrochloride (3, 30, or 300 ng/μL), or the same volume of vehicle (PBS) into the vitreous humor using a 33-gauge needle immediately after laser injury. Another group of mice is injected with 3TC (125 ng) in combination with an anti-mouse VEGF polyclonal antibody (10 ng). Goat whole IgG (10 ng) is used as a biological control for the anti-mouse VEGF antibody. .

Mice

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Laser-induced choroidal neovascularization mouse assay: Female C57BL/6 mice (8–10 weeks old) are anesthetized, and laser photocoagulation is performed to induce choroidal neovascularization. Starting 1 day post-laser, Zidovudine (Azidothymidine) is administered via oral gavage at 50 or 100 mg/kg/day for 14 days. The drug is formulated in 0.5% methylcellulose. At study end, mice are euthanized, eyes are enucleated, and choroidal flat mounts are prepared to measure neovascular lesion area; retinal tissues are collected for VEGF immunohistochemistry [3]

Absorption, Distribution and Excretion
Following oral administration, zidovudine capsules and solutions are rapidly and almost completely absorbed in the gastrointestinal tract; however, due to first-pass metabolism, their systemic bioavailability is approximately 65% (range: 52% to 75%). Bioavailability is approximately 89% in newborns under 14 days of age, decreasing to approximately 61% in newborns over 14 days of age and 65% in children aged 3 months to 12 years. Concomitant administration with a high-fat meal may reduce the rate and extent of absorption. As in adult patients, the primary route of excretion is metabolism to GZDV. Following intravenous administration, approximately 29% of the dose is excreted unchanged in the urine, and approximately 45% is excreted as GZDV. Apparent volume of distribution, HIV-infected patients, intravenous administration = 1.6 ± 0.6 L/kg
0.65 ± 0.29 L/hr/kg [HIV infection, birth to 14 days]
1.14 ± 0.24 L/hr/kg [HIV infection, 14 days to 3 months]
1.85 ± 0.47 L/hr/kg [HIV infection, 3 months to 12 years]. Transporters ABCB1, ABCC4, ABCC5, and ABCG2 are involved in zidovudine clearance.
Plasma concentrations of zidovudine may be elevated and the half-life prolonged in patients with impaired renal function. A study in HIV-infected adults with impaired renal function (creatinine clearance range 6–31 ml/min) showed a mean β-half-life of zidovudine of 1.4 hours, similar to reported values in HIV-infected adults with normal renal function. However, the mean beta half-life of glucuronide in these adults with impaired renal function was 8 hours, significantly prolonged compared to reported values in HIV-infected adults with normal renal function. A study of adult patients with hemophilia and HIV infection and elevated serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels showed significant individual variability in pharmacokinetics after a single oral dose of 300 mg zidovudine. In HIV-infected patients, 63-95% of the oral dose of zidovudine is excreted in the urine; approximately 14-18% is excreted unchanged, and 72-74% is excreted as zidovudine 5'-O-glucuronide within 6 hours. In HIV-infected adults or children, approximately 18-29% of the dose of intravenously administered zidovudine is excreted unchanged in the urine, and 45-60% is excreted within 6 hours as zidovudine 5'-O-glucuronide. Zidovudine and 3'-azo-3'-deoxy-5'-O-β-D-glucuronide thymidine are primarily excreted in the urine via glomerular filtration and tubular secretion. In HIV-infected adults, the mean systemic clearance of zidovudine after oral or intravenous administration is 1.6 L/h/kg (range: 0.8-2.7 L/h/kg), and the mean renal clearance is 0.34 L/h/kg. In children aged 3 months to 12 years, the mean systemic clearance is 1.85 L/h/kg. In a limited study of neonates and infants under 3 months of age, the mean total drug clearance was 0.65 L/h/kg in infants 14 days and under, and 1.14 L/h/kg in infants older than 14 days. Zidovudine is rapidly metabolized in the liver via glucuronidation to zidovudine 5'-O-glucuronide (GAZT); the apparent elimination half-life of this metabolite is 1 hour (range: 0.6–1.7 hours). Zidovudine 5'-O-glucuronide appears to have no antiviral activity against HIV. For more complete data on the absorption, distribution, and excretion of zidovudine (21 items in total), please visit the HSDB records page. Metabolism/Metabolites Liver. Zidovudine is primarily metabolized via glucuronidation to its major inactive metabolite, 3'-azido-3'-deoxy-5'-O-β-D-pyranoglobulin-thymidine (GZDV). UGT2B7 is the main UGT isoenzyme responsible for glucuronidation. Compared to zidovudine, GZDV has approximately three times the area under the curve (AUC). Cytochrome P450 isoenzymes are responsible for reducing the azide group to 3'-amino-3'-deoxythymidine (AMT). Zidovudine is rapidly metabolized in the liver primarily via glucuronidation to 3-azido-3-deoxy-5-O-β-D-pyranoglobulin-thymidine (GZDV; formerly known as GAZT). Zidovudine is also metabolized to GZDV in renal microsomes. The apparent elimination half-life of GZDV is 1 hour (range: 0.6–1.7 hours), and it appears to have no antiviral activity against HIV. In addition, two other hepatic metabolites of zidovudine have been identified as 3-amino-3-deoxythymidine (AMT) and its glucuronide derivative (GAMT). Intracellularly, zidovudine is converted to zidovudine monophosphate by cellular thymidine kinase in both virus-infected and uninfected cells; this monophosphate derivative is phosphorylated to zidovudine diphosphate by cellular dTMP kinase (thymidine kinase), and then phosphorylated to zidovudine triphosphate by other cellular enzymes. The conversion of zidovudine to its triphosphate derivative within host cells is necessary for its antiviral activity. However, activation of its antibacterial activity does not depend on intracellular phosphorylation but on intracellular transformation within bacterial cells. The intestinal mucosal transport and metabolic mechanisms of zidovudine and other thymidine analogues were investigated. Zidovudine metabolites were not detected in any part of the gastrointestinal tract. Other thymidine analogues are rapidly metabolized in the upper digestive tract but not in the colon. Hepatic metabolism. It is primarily metabolized via glucuronidation to the inactive metabolite 3'-azido-3'-deoxy-5'-O-β-D-pyranoglobulin-thymidine (GZDV). UGT2B7 is the major UGT isoenzyme responsible for glucuronidation. Compared to zidovudine, GZDV has approximately three times the area under the curve (AUC). Cytochrome P450 isoenzymes are responsible for reducing the azide group to 3'-amino-3'-deoxythymidine (AMT). Elimination pathway: As in adult patients, the primary elimination pathway is via metabolism to GZDV. Following intravenous administration, approximately 29% of the dose is excreted unchanged in the urine, and approximately 45% is excreted as GZDV.
Half-life: Elimination half-life in HIV-infected patients, intravenous administration = 1.1 hours (range 0.5-2.9 hours)
Bio-half-life

Elimination half-life in HIV-infected patients, intravenous administration = 1.1 hours (range 0.5-2.9 hours)
The mean plasma half-life after oral or intravenous administration of zidovudine in adults is approximately 0.53 hours. Following intravenous administration of zidovudine in adults or children, plasma drug concentrations show a biphasic decline. The initial half-life in adults is less than 10 minutes, and the terminal half-life is 1 hour. In symptomatic HIV-infected children aged 1-13 years, the mean α-half-life after a single intravenous dose of 80, 120, or 160 mg/m² of zidovudine is 0.16-0.25 hours, and the mean β-half-life is 1-1.7 hours. The plasma half-life of zidovudine in newborns is generally longer than in older children and adults, but it decreases with increasing neonatal maturity. In a small study of newborns and infants under 3 months of age, the mean plasma half-life of zidovudine in infants 14 days and under was 3.1 hours, while the mean plasma half-life in infants older than 14 days was 1.9 hours. In a study of preterm newborns (26–32 weeks of gestation; birth weight 0.7–1.9 kg), the mean serum half-life of zidovudine was 7.3 hours at mean 6.3 days after birth and 4.4 hours at mean 17.7 days after birth. The half-life of zidovudine is generally 1–2 hours.

Toxicity Summary
Zidovudine is a structural analogue of thymidine, a prodrug that must be phosphorylated to convert into its active metabolite 5'-dGTP, namely zidovudine triphosphate (ZDV-TP). It inhibits the activity of HIV-1 reverse transcriptase (RT) by terminating the DNA chain after incorporation into the DNA chain via a nucleotide analogue. It competes with the natural substrate dGTP for incorporation into viral DNA. It also has weak inhibitory effects on cellular DNA polymerases '±' and '_. Toxicity Data
Male rats (oral): LD50 = 3.1 g/kg Female rats (oral): LD50 = 3.7 g/kg Male mice: LD50 = 3.6 g/kg Female mice (oral): LD50 = 3.1 g/kg The oral LD50 for mice is 3084 mg/kg.
Interactions

Concomitant use with probenecid may lead to a significant increase and prolonged zidovudine serum concentrations.
In at least one study, concomitant use with acetaminophen was reported to increase the risk of granulocytopenia in patients receiving zidovudine; the enhancement of hematologic toxicity appears to be related to the duration of acetaminophen use.
At least one HIV-infected patient experienced neurotoxicity (extreme somnolence and stupor) after co-treatment with acyclovir and zidovudine, and relapsed upon re-administration. This patient developed neurotoxicity 30–60 days after initiation of intravenous acyclovir treatment, with symptoms improving after oral acyclovir administration and disappearing upon discontinuation of acyclovir. In other HIV-infected patients, acyclovir and zidovudine have also been used concomitantly without evidence of increased toxicity. Although its clinical significance is unclear, there is evidence that acyclovir may enhance the antiretroviral activity of zidovudine in vitro; acyclovir monotherapy has very low antiretroviral activity. Both ganciclovir and zidovudine, as monotherapy, directly and dose-dependently inhibit myeloid and erythroid progenitor cells. Combination therapy increases the risk of hematologic toxicity and may lead to additive or synergistic myelotoxicity. In multiple studies of HIV-positive patients with cytomegalovirus infection, all patients receiving ganciclovir (5 mg/kg, IV, 1–4 times daily) in combination with zidovudine (200 mg, oral, every 4 hours) experienced severe, intolerable myelosuppression, primarily manifested as severe granulocytopenia; many also developed anemia. More than 80% of patients receiving both ganciclovir (5 mg/kg, IV, 1–2 times daily) and zidovudine (100 mg, oral, every 4 hours) experienced severe hematologic toxicity, requiring dose reduction of zidovudine. For more complete data on zidovudine interactions (18 in total), please visit the HSDB records page.

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Non-human toxicity values
Rat intravenous LD50 > 750 mg/kg
Mouse intravenous LD50 > 3000 mg/kg

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Zidovudine (azidothymidine) showed no significant cytotoxicity to primary human astrocytes at concentrations up to 20 μM[1]
In mice, oral administration of zidovudine (azidothymidine) at a dose of 100 mg/kg/day for 14 consecutive days did not cause significant changes in serum ALT, AST, or creatinine levels[3]
The plasma protein binding rate of zidovudine (azidothymidine) in humans is 34%–38%[1]

[1]. The NRTIs lamivudine, stavudine and zidovudine have reduced HIV-1 inhibitory activity in astrocytes. PLoS One. 2013 Apr 16;8(4):e62196.

[2]. Genome editing of CXCR4 by CRISPR/cas9 confers cells resistant to HIV-1 infection. Sci Rep. 2015 Oct 20;5:15577.

[3]. Nucleoside Reverse Transcriptase Inhibitors Suppress Laser-Induced Choroidal Neovascularization in Mice. Invest Ophthalmol Vis Sci. 2015 Nov;56(12):7122-9.

Therapeutic Uses
Anti-HIV drug; antimetabolite; antitumor antimetabolite; reverse transcriptase inhibitor Zidovudine is used in combination with other antiretroviral drugs to treat HIV infection. .../US product label includes/ Zidovudine is indicated for the prevention of mother-to-child transmission of HIV-1. Treatment regimens include: oral zidovudine starting at 14 to 34 weeks of gestation, continuous intravenous infusion of zidovudine during delivery, and zidovudine syrup for the first 6 weeks after birth. However, even with this treatment regimen, mother-to-child transmission can still occur in some cases. /US product label includes/ Zidovudine has been used to prevent HIV infection in healthcare workers at risk of occupational exposure to HIV. The risk of transmission from a single needlestick injury is approximately 0.3%. The efficacy, optimal dosage, and duration of prophylactic treatment are currently unknown; however, HIV infection has occurred in individuals who received zidovudine prophylaxis following needlestick injuries or other parenteral exposures. /Not included in the US product label/
For more complete data on the therapeutic uses of zidovudine (7 of these), please visit the HSDB record page.

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Drug Warnings
Systemic adverse reactions reported with intravenous zidovudine are similar to those reported with oral zidovudine. However, clinical experience with intravenous zidovudine is more limited than with oral zidovudine, and this drug is usually administered intravenously only for short periods. Long-term intravenous zidovudine treatment (i.e., longer than 2–4 weeks) has not been evaluated in adults and may exacerbate hematologic adverse reactions.
The most common adverse reactions to zidovudine are hematologic adverse reactions (e.g., anemia, neutropenia), nausea, and headache. Because HIV-infected patients receiving zidovudine often have serious underlying diseases, multiple baseline symptoms, and clinical abnormalities, and many adverse reactions that occur in patients treated with zidovudine also occur in patients receiving placebo, many reported adverse reactions may not be directly attributable to zidovudine. In adult patients, the more severe the disease at the start of treatment, the higher the incidence and severity of zidovudine-related adverse reactions. In a study of asymptomatic patients, who received oral zidovudine 100 mg five times daily for a mean duration of more than one year (range: 4 months to 2 years), nausea was reported more frequently in the zidovudine group compared to the placebo group. Adverse reactions reported in women, intravenous drug users, and minorities were similar to those reported in white men. Four patients with acquired immunodeficiency syndrome and a history of Pneumocystis carinii pneumonia developed severe pancytopenia (hemoglobin <85 g/L; granulocytes ≤0.5 × 10⁹/L; platelets ≤30 × 10⁹/L) 12 to 17 weeks after initiating azidothymidine (AZT) treatment. Three patients had significant bone marrow reduction, and the fourth had moderate bone marrow reduction. Three patients experienced partial bone marrow recovery within 4 to 5 weeks, but one patient did not show bone marrow recovery 6 months after discontinuing zidovudine treatment. Hematologic toxicity was causally related to zidovudine treatment and directly related to drug dosage and duration, most commonly occurring in patients with advanced symptomatic HIV infection or those with low pre-treatment hemoglobin concentrations, neutrophil counts, and helper/inducer (CD4+, T4+) T cell counts. Patients with low serum folate or vitamin B12 concentrations may have an increased risk of bone marrow toxicity during zidovudine treatment. Limited data suggest that the bone marrow of patients with fulminant acquired immunodeficiency syndrome (AIDS) may be more sensitive to zidovudine-induced toxicity than in patients with milder disease (e.g., AIDS-related syndromes (ARC)).
For more complete data on zidovudine (43 total), please visit the HSDB records page.

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Pharmacodynamics
Zidovudine is a nucleoside reverse transcriptase inhibitor (NRTI) active against human immunodeficiency virus type 1 (HIV-1). Zidovudine 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 lack of a 3'-OH group in the incorporated nucleoside analogue prevents the formation of the 5' to 3' phosphodiester bonds necessary for DNA chain elongation, thereby terminating viral DNA growth.

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Zidovudine (azidothymidine) is the first nucleoside reverse transcriptase inhibitor (NRTI) approved for the treatment of HIV-1 infection [1]
Zidovudine (azidothymidine) exerts its anti-HIV activity by being converted into zidovudine triphosphate in the cell. Zidovudine triphosphate competes with thymidine triphosphate (dTTP) for incorporation into viral DNA, thereby terminating the synthesis of HIV-1 DNA [1]
Zidovudine (azidothymidine) has shown potential efficacy in inhibiting choroidal angiogenesis, a pathological process of age-related macular degeneration [3]
Reduced anti-HIV activityThe effect of zidovudine (azidothymidine) in astrocytes is attributed to the reduced degree to which the drug is phosphorylated into its active triphosphate form in the cell [1]

C10H13N5O4

267.24

267.096

C, 44.94; H, 4.90; N, 26.21; O, 23.95

30516-87-1

117675-21-5 (glucuronide); 106060-89-3 (diphosphate); 92586-35-1 (triphosphate)

35370

White to off-white solid

113-115 °C(lit.)

47 ° (C=1, H2O)

-0.53

2

6

3

19

484

3

O=C(C(C)=CN1[C@@H](C2)O[C@@H]([C@H]2N=[N+]=[N-])CO)NC1=O

HBOMLICNUCNMMY-XLPZGREQSA-N

InChI=1S/C10H13N5O4/c1-5-3-15(10(18)12-9(5)17)8-2-6(13-14-11)7(4-16)19-8/h3,6-8,16H,2,4H2,1H3,(H,12,17,18)/t6-,7+,8+/m0/s1

1-[(2R,4S,5S)-4-azido-5-(hydroxymethyl)oxolan-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 (9.35 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 (9.35 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 (9.35 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: 18 mg/mL (67.4 mM)

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Solubility in Formulation 5: 20 mg/mL (74.84 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C).

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