HIV-1
Ribonucleotide reductase (RNR; Ki=10 μM for human RNR M2 subunit) [3]
- Deoxyribonucleotide (dNTP) synthesis (inhibition via RNR blockade; EC50 for human tumor cell lines: 50-200 μM) [3]
- Viral DNA replication (inhibition of HIV-1 reverse transcription via dNTP depletion; EC50=100 μM) [1]

Hydroxyurea can inhibit HIV-1 replication. The 90% inhibitory concentration (IC90) of hydroxyurea for laboratory strains of HIV-1 in activated PBMC has been demonstrated by in vitro experiments to be 0.4 mM. In activated PBMC, hydroxyurea was also found to inhibit HIV-1 replication and work in concert with didanosine, a nucleoside reverse transcriptase inhibitor; this inhibition may be brought about by a decrease in the amounts of deoxynucleoside triphosphate pools. Mutants resistant to didanosine have been shown to become sensitized to hydroxyurea[1][2]. In sickle cell anemia patients, hydroxyurea has been shown to be effective in reducing hemolysis by boosting the fetal hemoglobin production. Ribonucleotide reductase is the rate-limiting enzyme that converts ribonucleotides into deoxyribonucleotides, which are necessary for DNA synthesis. Hydroxyurea inhibits this enzyme to produce its cytostatic effect. Consequently, the S phase of cellular division is halted[1].

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Exhibited antiproliferative activity against human hematologic and solid tumor cell lines, including chronic myeloid leukemia (K562, IC50=80 μM), acute myeloid leukemia (HL-60, IC50=120 μM), and colorectal cancer (HCT116, IC50=150 μM) (72-hour exposure) [3]
- Selectively inhibited RNR activity; 100 μM Hydroxyurea (Hydroxycarbamide) reduced RNR activity by 75% in K562 cells, leading to 60% depletion of intracellular dATP/dGTP pools and inhibition of DNA synthesis ([3H]-thymidine incorporation reduced by 80%) [3]
- Induced S-phase cell cycle arrest and mild apoptosis in tumor cells; 200 μM treatment for 48 hours increased S-phase cell population by 3-fold and apoptotic rate by 30% (annexin V positivity) in HL-60 cells [3]
- Inhibited HIV-1 replication in peripheral blood mononuclear cells (PBMCs); 100 μM reduced viral p24 antigen levels by 50% without significant cytotoxicity to PBMCs (CC50 >500 μM) [1]
- Suppressed colony formation of K562 cells; 50 μM Hydroxyurea (Hydroxycarbamide) reduced colony formation efficiency by 70% compared to untreated controls [3]

Hydroxyurea treatment consistently lowers WBC and ANC without anemia improvement during a 17-week period. At 50 mg/kg, hydroxyurea creates a decreased white blood cell count, zero neutrophil count, and no improvement in anemia when compared to sickle cell mice receiving a vehicle treatment.

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Suppressed tumor growth in nude mice bearing K562 CML xenografts; oral administration of 200 mg/kg daily for 3 weeks resulted in 65% tumor growth inhibition (TGI) compared to vehicle control [3]
- Reduced HIV-1 viral load in a murine model of HIV-1 infection; oral dosing of 150 mg/kg twice daily for 2 weeks decreased plasma viral RNA levels by 1.5 log10 copies/mL [1]
- Ameliorated sickle cell anemia phenotype in transgenic mice; oral administration of 100 mg/kg daily for 4 weeks increased fetal hemoglobin (HbF) levels by 25% and reduced sickle cell polymerization [2]

Assayed human RNR activity using purified recombinant RNR M2 subunit; incubated 10-500 μM Hydroxyurea (Hydroxycarbamide) with enzyme, ribonucleoside diphosphates (substrates), and dithiothreitol (cofactor) at 37°C for 60 minutes; measured formation of deoxyribonucleoside diphosphates by HPLC to calculate Ki [3]
- Evaluated HIV-1 reverse transcriptase (RT) inhibition indirectly via dNTP depletion; treated PBMCs with 50-200 μM Hydroxyurea (Hydroxycarbamide) for 24 hours; isolated intracellular dNTPs and quantified by HPLC; correlated dNTP levels with HIV-1 p24 antigen production [1]

The same patients' erythroid cells from their peripheral blood are treated with hydroxyurea in vitro one year after they stopped taking it orally for two years, starting at a dose of 5 mg/kg/day for five days a week and increasing to a maximum of 10 mg/kg/day. Thirteen β-Thal/HbE patients receive this treatment.Cells are treated in primary culture for 96 hours with 30 μM hydroxyurea.

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Seeded tumor cells (5×103 cells/well) in 96-well plates; allowed to adhere for 24 hours; treated with Hydroxyurea (Hydroxycarbamide) (10-1000 μM) for 72 hours; measured cell viability using MTT assay and calculated IC50 [3]
- Cultured K562 cells (1×105 cells/well) in 6-well plates; exposed to 50-200 μM Hydroxyurea (Hydroxycarbamide) for 24-48 hours; isolated intracellular dNTPs and quantified by HPLC; assessed DNA synthesis by [3H]-thymidine incorporation assay [3]
- Infected human PBMCs with HIV-1 (MOI=0.01) for 2 hours; treated with 50-200 μM Hydroxyurea (Hydroxycarbamide) for 7 days; quantified HIV-1 p24 antigen in supernatants by ELISA [1]

Mice: SCD mice are given hydroxyurea at doses of 25 mg/kg, 50 mg/kg, and 100 mg/kg, or vehicle, five days a week, to investigate whether hydroxyurea would improve anemia and/or prevent or lessen the development of organ damage in the absence of HbF induction.

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Nude mice (6-7 weeks old) were implanted subcutaneously with 3×106 K562 CML cells; when tumors reached 100 mm³, mice received oral Hydroxyurea (Hydroxycarbamide) suspended in 0.5% carboxymethylcellulose sodium at 200 mg/kg daily for 3 weeks; control mice received vehicle; tumor volume was measured every 3 days [3]
- HIV-1-infected mice (humanized PBMC model) were treated with oral Hydroxyurea (Hydroxycarbamide) at 150 mg/kg twice daily for 2 weeks; plasma viral RNA levels were quantified by RT-PCR [1]
- Transgenic sickle cell anemia mice were administered oral Hydroxyurea (Hydroxycarbamide) at 100 mg/kg daily for 4 weeks; blood samples were collected to measure HbF levels by HPLC and assess sickle cell polymerization [2]

Absorption, Distribution and Excretion
Hydroxyurea is rapidly absorbed from the gastrointestinal tract after oral administration. Peak plasma concentrations are reached within 2 hours, and serum concentrations are almost zero after 24 hours. In cancer patients, the bioavailability of hydroxyurea is complete or near-complete. Rapid absorption is observed after oral administration of 20 mg/kg hydroxyurea, with peak plasma concentrations of approximately 30 mg/L reached in children with sickle cell syndrome and in adults after 0.75 hours and 1.2 hours, respectively. The total exposure within 24 hours after administration is 124 mg/L in children and adolescents, and 135 mg/L in adults. Oral bioavailability of hydroxyurea is also near-complete in other indications besides sickle cell syndrome. In a comparative bioavailability study in healthy adult volunteers (n=28), 500 mg hydroxyurea oral solution was bioequivalent to the reference 500 mg capsule in terms of peak concentration and area under the curve. Compared to the reference 500 mg capsules, the time to peak concentration (TTP) of the oral hydroxyurea solution was significantly shorter (0.5 h vs. 0.75 h, p = 0.0467), indicating faster absorption. [L47137] In a study of children with sickle cell disease, the area under the curve, peak concentration, and half-life of the liquid and capsule formulations were similar. The most significant difference in pharmacokinetic characteristics was a trend toward a shorter TTP after administration of the liquid formulation compared to the capsules, but this difference did not reach statistical significance (0.74 h vs. 0.97 h, p = 0.14). Hydroxyurea is largely eliminated via non-renal (primarily hepatic) pathways. It has been reported that approximately 37% of the unchanged drug is recovered in urine in adults with normal renal function. In children, approximately 50% of the unchanged hydroxyurea is excreted in urine. Hydroxyurea distributes rapidly in the body, entering the cerebrospinal fluid, appearing in the peritoneal fluid and ascites, and accumulating in leukocytes and erythrocytes. The estimated volume of distribution of hydroxyurea is roughly equivalent to the total body water volume. The estimated volume of distribution after oral administration of hydroxyurea is approximately equal to the total body water volume: reported values are 0.48–0.90 L/kg for adults and an estimated 0.7 L/kg for children. The total clearance of hydroxyurea in adult patients with sickle cell disease is 0.17 L/h/kg. The corresponding value for children is similar, at 0.22 L/h/kg. Hydroxyurea is readily absorbed from the gastrointestinal tract. Peak serum concentrations are reached within 1–4 hours after oral administration. Plasma concentrations decline rapidly, and repeated administration has no cumulative effect. Therefore, a single high-dose oral administration of a conventional dose achieves higher plasma concentrations compared to divided doses. Increasing the drug dose results in a disproportionate increase in peak plasma concentration and area under the concentration-time curve (AUC). The effect of food on hydroxyurea absorption has not been determined. Hydroxyurea distributes rapidly in the body and concentrates in leukocytes and erythrocytes. The estimated volume of distribution of this drug is close to the total body fluid volume. Hydroxyurea can cross the blood-brain barrier; peak concentrations in cerebrospinal fluid are reached within 3 hours after oral administration. The drug can distribute into ascites, resulting in ascites concentrations 2-7.5 times lower than plasma concentrations. Studies using 14C-labeled hydroxyurea have shown that approximately half of the oral dose is degraded in the liver and excreted in the urine as respirable carbon dioxide and urea. The remaining drug is excreted unchanged in the urine. Approximately 30-60% of oral hydroxyurea is excreted unchanged by the kidneys, but usually about 35% is excreted. For more complete data on the absorption, distribution, and excretion of hydroxyureas (7 types), please visit the HSDB record page. Metabolites: Up to 60% of the oral dose is metabolized to acetylhydroxyxamic acid by saturated hepatic metabolism. A smaller degradation pathway occurs through urease produced by intestinal bacteria, which degrades the drug to acetylhydroxyxamic acid. Studies have shown that up to 50% of orally administered hydroxyurea is metabolized in the liver; however, the exact metabolic pathway has not been determined. A smaller metabolic pathway may involve the degradation of the drug by urease (an enzyme produced by intestinal bacteria). Acetylhydroxyxamic acid was detected in the serum of three leukemia patients treated with hydroxyurea, which may be a product of urease breakdown of hydroxyurea. Hepatic metabolism. Excretion pathway: Renal excretion is one of its excretion pathways. Half-life: 3-4 hours. In adult cancer patients, the half-life of hydroxyurea is approximately 2-3 hours. In a single-dose study in children with sickle cell disease, a mean half-life of 1.7 hours was reported. Hydroxyurea has a short half-life: the initial half-life after intravenous administration is 0.63 hours, the initial half-life after oral administration is 1.78 hours, the terminal half-life after oral administration is 3.32 hours, and the terminal half-life after intravenous administration is 3.39 hours (for humans). In rats, daily intraperitoneal injection of 137 mg/kg body weight of hydroxyurea during days 9-12 of gestation resulted in a half-life of 15 minutes in the mother and 85 minutes in the embryo. In rhesus monkeys, intravenous injection of 100 mg/kg body weight/day during days 23-32 of gestation resulted in a half-life of 120 minutes in the mother and 265 minutes in the fetus after the last injection.

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The oral bioavailability in humans is 80-100%; after oral administration of 80 mg/kg, the peak plasma concentration (Cmax) is 30 μg/mL [2]
- The plasma half-life (t1/2) in humans is 3-4 hours; the volume of distribution (Vd) is 0.5-1.0 L/kg [2]
- Very little liver metabolism; 80-90% of the dose is excreted unchanged in the urine within 24 hours [2]
- Human plasma protein binding rate is <10% [2]

Toxicity Summary
Identification and Uses: Hydroxyurea is a white crystalline powder. It is a drug indicated for the following uses: treatment of refractory chronic myeloid leukemia; treatment in combination with chemoradiotherapy for locally advanced squamous cell carcinoma of the head and neck (excluding the lip); and palliative treatment of sickle cell anemia, typically in patients who have experienced at least three moderate to severe recurrent painful crises within the past 12 months (designated as an orphan drug by the FDA for this purpose). Human Exposure and Toxicity: Over a two-year period, 26 patients who had taken hydroxyurea for more than six months and presented to a dermatologist underwent a systematic examination to evaluate skin side effects. With one exception, all patients experienced skin side effects, including dry skin, moderate alopecia, increased skin pigmentation, melanonychia, skin atrophy, leg ulcers, plantar hyperkeratosis, pseudodermatomyositis, lichen planus-like rash on the dorsum of the hands, actinic keratosis, squamous cell carcinoma, and oral ulcers. Post-marketing surveillance has shown that HIV-infected patients receiving hydroxyurea and other antiretroviral drugs have experienced hepatotoxicity, liver failure, and even death. Fatal liver failure most commonly occurs in patients receiving combination therapy with hydroxyurea, didanosin, and stavudine (note: hydroxyurea is not indicated for HIV treatment). An 85-year-old male patient with chronic myelomonocytic leukemia developed severe interstitial pneumonia after taking hydroxyurea. Toe and gangrene are rare but very serious complications of long-term hydroxyurea treatment. A retrospective study of semen analysis in four adult men during hydroxyurea treatment and in three of them after discontinuation showed that the drug typically reduces sperm count and motility and causes abnormal sperm morphology. Hydroxyurea can induce DNA hypermethylation in normal human embryonic lung fibroblasts and their corresponding cells transformed with simian virus 40. Although cancer has been observed in some patients receiving long-term hydroxyurea treatment, the carcinogenicity of this drug in humans has not been established. Animal studies: Symptoms of hydroxyurea exposure in dogs include vomiting, ataxia, methemoglobinemia, tachycardia, lethargy, and hypothermia. Fifty male and female mice in each group were intraperitoneally injected with hydroxyurea starting two days after birth, followed by weekly injections for one year. The incidence of lung tumors was 30/50 (60%) in the control group and 16/35 (46%) in the treatment group. Hydroxyurea is a rapidly acting developmental toxicant that inhibits DNA synthesis and is teratogenic in all studied mammals. Data from a mouse study showed that hydroxyurea impairs follicle development and the ability of resulting embryos to develop. A group of 27 pregnant golden hamsters received an intravenous injection of 50 mg/kg body weight of hydroxyurea on day 8 of gestation. High fetal mortality and malformation rates, particularly central nervous system malformations, were observed. Hydroxyurea caused cell transformation in large cultures of mouse embryonic cells but did not induce transformation in embryonic cell cultures or BALB/c 3T3 cells from two other mouse strains. Hydroxyurea treatment induced DNA hypermethylation in hamster fibrosarcoma cells. Hydroxyurea showed no frameshift or base substitution mutagenic activity in Salmonella Typhimurium strains TA1537, TA1535, TA98, and TA100, and the addition of exogenous metabolic activation systems did not affect these results. Hydroxyurea induced SOS repair in E. coli K12 cells. In various Saccharomyces cerevisiae strains, hydroxyurea induced mitotic crossing over, mitotic gene conversion, intrachromosomal recombination, and aneuploidy, but did not induce small mutations. It also increased the frequency of UV-induced mitotic gene conversion and induced recombination in dividing cells of the RS112 yeast strain (but not in G1 or G2 arrested cells). In meiotic yeast cells, hydroxyurea increased the frequency of meiotic recombination. Hydroxyurea induced micronuclei in the bone marrow of tumor-free male NMRI mice, but did not induce micronucleated cells in female C57BL/6 C3H/He hybrid mice, although it caused sperm abnormalities in male mice of this strain. Hydroxyurea is converted into the free radical nitrooxygen (NO) in vivo and diffuses into cells, quenching the tyrosine radical at the active site of the M2 protein subunit of ribonucleotide reductase, thereby inactivating the enzyme. The entire replicase complex, including ribonucleotide reductase, is inactivated, DNA synthesis is selectively inhibited, leading to S-phase cell death and synchronizing the cell cycle of surviving cells. Hydroxyurea also inhibits the repair of DNA damage caused by chemicals or radiation, suggesting a possible synergistic effect between hydroxyurea and radiation or alkylating agents. Hydroxyurea can also increase fetal hemoglobin levels, thereby reducing the incidence of sickle cell anemia vascular occlusion crisis. The increased fetal hemoglobin levels are due to hydroxyurea-derived NO activation of soluble guanylate cyclase (sGC).

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Toxicity Data
Mice (oral): LD50 = 7330 mg/kg; Rats (oral): LD50 = 5760 mg/kg
Interactions
This analysis aimed to investigate whether the combination of didanoxin and stavudine (with or without hydroxyurea) had a higher incidence of neuropathy than a single-drug therapy. Data were obtained from patients followed up long-term at the Johns Hopkins AIDS Service. The incidence of neuropathy was calculated for five treatment regimens: didanoxin (+/- hydroxyurea), didanoxin + stavudine (+/- hydroxyurea), and stavudine. The relative risk of neuropathy for each regimen was compared using a Cox proportional hazards regression model, adjusted for factors such as CD4 cell count, other medications, and treatment duration. A total of 1116 patients received at least one treatment regimen. Of these, 117 developed neuropathy.
This analysis aimed to investigate whether the combination of didanoxin and stavudine (with or without hydroxyurea) had a higher incidence of neuropathy than single-drug therapy.
The crude incidence of neuropathy ranged from 6.8 cases per 100 person-years in the didanoxin group to 28.6 cases per 100 person-years in the didanoxin + stavudine + hydroxyurea group. After adjusting for CD4 cell count and other variables, the relative risk of neuropathy with stavudine alone was 1.39 [95% confidence interval (CI): 0.84–2.32], with didanoxin + hydroxyurea 2.35 (95% CI: 0.69–8.07), with didanoxin + stavudine 3.50 (95% CI: 1.81–6.77), and with didanoxin + stavudine + hydroxyurea 7.80 (95% CI: 3.92–15.5). Based on these data, the risk of neuropathy with didanoxin + stavudine + hydroxyurea has an additive or even synergistic effect compared to didanoxin or stavudine alone. Didanosine combined with stavudine also increases the risk of neuropathy, but the risk is lower than when hydroxyurea is used concurrently.
Concurrent use of hydroxyurea with other myelosuppressants or radiotherapy may increase the incidence of myelosuppression or other adverse reactions, and dose adjustment may be necessary.
Because hydroxyurea treatment may cause an increase in serum uric acid levels, the dose of uricosuric drugs may need to be adjusted.
Adding iron chelators enhances the antitumor activity of hydroxyurea, while adding Fe2+ to the culture medium inhibits its antitumor activity.
For more complete data on interactions of hydroxyurea (7 in total), please visit the HSDB record page.

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Bone marrow suppression (leukopenia, thrombocytopenia, anemia) is the main dose-limiting toxicity in humans; it can occur at oral doses ≥30 mg/kg/day[2]
- Mild gastrointestinal toxicity (nausea, vomiting, diarrhea) was observed in rats at oral doses >400 mg/kg[3]
- Skin toxicity (rash, hyperpigmentation) has been reported in humans at long-term therapeutic doses[2]
- No significant hepatotoxicity or nephrotoxicity was detected in humans at recommended doses[2]
- Drug interactions: Co-administration with other bone marrow suppressants or HIV-1 nucleoside reverse transcriptase inhibitors (NRTIs) increases the risk of hematologic toxicity[1]

[1]. Clin Infect Dis . 2000 Jun:30 Suppl 2:S193-7.

[2]. Clin Infect Dis . 2000 Jun:30 Suppl 2:S143-50.

[3]. Exp Hematol . 2005 Dec;33(12):1486-92.

[4]. Gene Ther . 2002 Aug;9(15):1023-30.

Therapeutic Uses
Anti-tumor drugs; anti-sickle cell anemia drugs; enzyme inhibitors; nucleic acid synthesis inhibitors. ClinicalTrials.gov is a registry and results database that lists human clinical studies funded by public and private institutions worldwide. The website is maintained by the National Library of Medicine (NLM) and the National Institutes of Health (NIH). Each record on ClinicalTrials.gov includes a summary of the study protocol, including: disease or condition; intervention (e.g., the medical product, behavior, or procedure under investigation); the title, description, and design of the study; participation requirements (eligibility criteria); the location of the study; contact information for the study location; and links to relevant information from other health websites, such as the NLM's MedlinePlus (for providing patient health information) and PubMed (for providing citations and abstracts of academic articles in the medical field). Hydroxyurea is listed in the database. Hydroxyurea capsules (USP) are indicated for the treatment of: refractory chronic myeloid leukemia; locally advanced squamous cell carcinoma of the head and neck (excluding the lip), requiring combination therapy with chemoradiotherapy. /Listed on US Product Labels/
Hydroxyurea has been used to treat psoriasis and has been reported to be effective in hypereosinophilic syndrome unresponsive to corticosteroids. /Not Listed on US Product Labels/
For more complete therapeutic use data on hydroxyurea (12 types in total), please visit the HSDB record page.

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Drug Warnings
Hydroxyurea is a highly toxic drug with a low therapeutic index; a therapeutic response is unlikely if no toxic reaction occurs. Treatment with hydroxyurea can result in serious, sometimes life-threatening or fatal adverse reactions. This drug must be used under the close supervision of an experienced clinician with experience using cytotoxic drugs or treating sickle cell anemia with this drug. Patients who have recently received other cytotoxic drugs or radiation therapy should use hydroxyurea with caution, as these patients may experience bone marrow suppression. Additionally, post-radiation erythema exacerbation may occur. Hepatotoxicity has been reported in HIV-infected patients receiving hydroxyurea in combination with antiretroviral drugs, with some cases even leading to fatal liver failure. Fatal hepatotoxicity most commonly occurs in patients receiving combination therapy with hydroxyurea, didanosine, and stavudine. Elevated serum liver enzyme levels have been reported in patients taking hydroxyurea. Cutaneous vasculitis toxicity, including vasculitic ulcers and gangrene, has been reported in patients receiving hydroxyurea for myeloproliferative disorders, particularly those who have received or are currently receiving interferon therapy. For more complete data on hydroxyurea (37 total), please visit the HSDB record page.

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Pharmacodynamics
The correlation between hydroxyurea concentration, decreased incidence of crisis, and increased HbF is unclear.

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Hydroxyurea (hydroxyurea) is an antimetabolite targeting ribonucleotide reductase (RNR)[3]
- Its mechanism of action involves RNR inhibition, leading to dNTP depletion, DNA synthesis arrest, and cell cycle arrest; it can also induce erythroid cells to produce fetal hemoglobin (HbF)[2]
- It has been approved by the U.S. Food and Drug Administration (FDA) for the treatment of chronic myeloid leukemia (CML), sickle cell anemia, and essential thrombocytosis; it has also been used off-label for the treatment of HIV-1 infection (in combination with antiretroviral drugs)[1,2]
- The oral formulation is convenient to administer; due to bone marrow suppression, regular hematological monitoring is required[2]
- Its efficacy in treating sickle cell anemia is attributed to the induction of HbF, thereby reducing the aggregation of sickle hemoglobin (HbS) and sickling of erythrocytes[2]

CH4N2O2

76.05

76.027

C, 15.79; H, 5.30; N, 36.83; O, 42.07

127-07-1

3657

White to off-white solid powder

1.5±0.1 g/cm3

222.1±23.0 °C at 760 mmHg

135-140 °C

88.1±22.6 °C

0.0±1.0 mmHg at 25°C

1.501

-1.8

3

2

0

5

42.9

0

O([H])N([H])C(N([H])[H])=O

VSNHCAURESNICA-UHFFFAOYSA-N

InChI=1S/CH4N2O2/c2-1(4)3-5/h5H,(H3,2,3,4)

hydroxyurea

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 (32.87 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 (32.87 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 (32.87 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: 100 mg/mL (1314.92 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.)