HIV-1
Suppression of insulin signaling pathway components: Insulin Receptor-β (IR-β), Insulin Receptor Substrate-1 (IRS-1), Phosphatidylinositol 3-kinase (PI3K/p85α), Akt (Protein Kinase B), and Endothelial Nitric Oxide Synthase (eNOS).

Nelfinavir (AG1341) Mesylate (1-10 μM; 48 hours) suppresses the growth of several myeloma cells[4].
Nelfinavir Mesylate suppresses the activity of 26S chymotrypsin-like proteasomes, hinders myeloma cell line proliferation, and induces apoptosis in newly generated plasma cells[4].
Nelfinavir Mesylate (1-10 μM; 17 hours) cell lines undergo apoptosis when exposed to nelfinavir mesylate[4].
Nelfinavir Mesylate (5 μM; 0-24 hours) decreases the phosphorylation of AKT[4].
Nelfinavir Mesylate causes caspase-3 to cleave, phosphorylates AKT, STAT-3, and ERK1/2, and triggers the unfolded protein response system's pro-apoptotic pathway[4].
Nelfinavir has an IC50 of 35.93 μM, making it another SARS-CoV 3CLpro inhibitor[5].

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Chronic exposure (72 hours) of Human Aortic Endothelial Cells (HAECs) to Nelfinavir at physiological concentrations (0.25 - 2 μg/mL) significantly decreased both basal (2.5-fold) and insulin-induced nitric oxide (NO) production (4- to 5-fold), as measured by fluorimetric assays using DAF-FM DA and DAF-2 DA dyes.
Nelfinavir (1 and 2 μg/mL) suppressed insulin-induced phosphorylation of Akt at Ser473 and eNOS at Ser1177 in a dose-dependent manner, as determined by Western blot analysis.
Nelfinavir (1 and 2 μg/mL) suppressed insulin-induced tyrosine phosphorylation of upstream insulin signaling components: IR-β, IRS-1, and the PI3K/p85α subunit, as shown by immunoprecipitation followed by immunoblotting with anti-phosphotyrosine antibody.
The suppressive effects of Nelfinavir on insulin-induced Akt/eNOS phosphorylation and NO production were ameliorated by co-treatment with the thiazolidinedione insulin sensitizer Troglitazone (TRO, 250 nM).
Chronic Nelfinavir exposure (2 μg/mL) also suppressed insulin-induced eNOS gene (mRNA) expression, which was partially restored by TRO co-treatment. [1]

Nelfinavir Mesylate (75 mg/kg; i.p.; 5 days a week for 21 days) inhibits the growth of multiple myeloma cells in NOD/SCID mice.

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In H157 NSCLC xenograft models, intraperitoneal (i.p.) administration of nelfinavir (50 or 100 mg/kg, 5 days/week) significantly inhibited tumor growth by 61% and 63% respectively at day 11.
In A549 NSCLC xenograft models, i.p. administration of nelfinavir (50 mg/kg daily) inhibited tumor growth by 48% at day 19.
Oral administration (gavage) of nelfinavir (100 mg/kg, 5 days/week) also significantly inhibited H157 tumor growth.
Tumors from nelfinavir-treated mice showed increased apoptosis (TUNEL-positive cells), dilation of the ER, presence of autophagic vacuoles (electron microscopy), and increased levels of phospho-eIF2α, ATF3, and LC3-II by immunoblotting, confirming induction of ER stress, autophagy, and apoptosis in vivo.
Akt phosphorylation (S473) in tumors was not modulated by nelfinavir treatment in vivo.

26S Proteasome Activity Assay: Multiple myeloma cells were lysed. For each cell line, 30 µg of protein were collected in a Tris buffer and incubated with nelfinavir, bortezomib, or MG132 for 2 hours at 37°C. Subsequently, 1 mM of the fluorogenic substrate was added. The chymotrypsin-like activity was measured using the Z-Leu-Leu-Val-Tyr-AMC substrate, and the trypsin-like activity was measured using the Bz-Val-Gly-Arg-AMC substrate. Enzymatic activities were measured using a fluorescence plate reader. All experiments were performed in triplicate.

Cell Culture and Chronic Drug Treatment: Primary Human Aortic Endothelial Cells (HAECs) were maintained in endothelial growth medium. For experiments, cells at 70-80% confluency were treated with Nelfinavir (0.25, 0.5, 1, 2 μg/mL) and/or Troglitazone (TRO, 250 nM) for a total of 72 hours, with drugs replenished every 24 hours. Following chronic treatment, cells were stimulated with insulin (100 ng/mL) for 10 or 15 minutes prior to analysis.
Nitric Oxide (NO) Measurement - Fluorescence Microscopy: HAECs cultured on coverslips were loaded with the fluorescent dye DAF-2 DA (5 μM) for 10 minutes in the dark after drug treatments and insulin stimulation. Cells were then washed, fixed, and mounted. Fluorescence (indicative of NO) was visualized using a fluorescence microscope with excitation at 480 nm and emission at 505 nm.
Nitric Oxide (NO) Measurement - Fluorimetry: HAECs cultured in black 96-well plates were loaded with DAF-FM DA (5 μM) after treatments. Following incubation, cells were washed and incubated with L-arginine (100 μM) for 15 minutes. Fluorescence was measured using a fluorimeter (excitation 485 nm, emission 525 nm). Values were normalized to total protein content.
Western Blot Analysis: After treatments, HAECs were lysed. Protein samples (≈50 μg) were separated by SDS-PAGE, transferred to nitrocellulose membranes, and probed with specific primary antibodies against total and phosphorylated forms of Akt (Ser473), eNOS (Ser1177), IR-β, IRS-1, and PI3K/p85α, followed by HRP-conjugated secondary antibodies. Proteins were detected using chemiluminescence.
Immunoprecipitation: For analyzing tyrosine phosphorylation of IR-β and IRS-1, cell lysates (500 μg) were incubated overnight with respective antibodies. Immunocomplexes were captured using Protein A agarose beads, eluted, and then subjected to Western blotting using an anti-phosphotyrosine antibody.
Reverse Transcription Polymerase Chain Reaction (RT-PCR): Total RNA was extracted from treated HAECs using TRIzol reagent. cDNA was synthesized from 1 μg RNA. Semi-quantitative PCR was performed using specific primers for eNOS and GAPDH (housekeeping gene). PCR products were separated on agarose gels, stained, and band intensities were quantified. [1]

NOD/SCID mice (bearing U266-luc cells)[4]
75 mg/kg
I.p.; 5 days a week for 21 days

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H157 Xenograft - Intraperitoneal Administration: Six-week-old male athymic nude mice were injected subcutaneously with H157 cells. When tumors were palpable, mice were divided into groups receiving i.p. injections of vehicle (4% DMSO, 5% polyethylene glycol, 5% Tween 80 in saline) or nelfinavir (50 or 100 mg/kg dissolved in vehicle) once daily on specified days (days 1-4, 7, 8, 10-12). Mice were weighed and tumors measured every other day.
A549 Xenograft - Intraperitoneal Administration: Six-week-old male athymic nude mice were injected subcutaneously with A549 cells in Matrigel. When tumors reached ~200 mm³, mice received daily i.p. injections of vehicle or nelfinavir (50 mg/kg). Mice were weighed and tumors measured three times weekly.
H157 Xenograft - Oral (Gavage) Administration: Eight-week-old female athymic nude mice bearing H157 xenografts received daily gavage (5 days/week) of vehicle or nelfinavir (100 mg/kg dissolved in 35% ethanol). Tumor volume was calculated using the formula (a b²) / 2.
Pharmacokinetic Study in Mice: Six-week-old female athymic nude mice received a single i.p. injection of nelfinavir (50 or 100 mg/kg in vehicle). Blood was collected via cardiac puncture at various time points under anesthesia. Plasma was separated and stored at -80°C until analysis by HPLC.
Tissue Analysis: Formalin-fixed, paraffin-embedded tumor sections were used for TUNEL staining according to kit instructions. For immunoblotting, frozen tumors were homogenized in lysis buffer.

Absorption, Distribution and Excretion
The distribution of nelfinavir in tissues and fluids is not fully understood. The volume of distribution after oral administration of nelfinavir in animals was 27 L/kg, indicating widespread tissue distribution. Rat studies showed that 4 hours after oral administration of radiolabeled nelfinavir, drug concentrations in the liver, lymph nodes, pancreas, kidneys, lungs, submandibular glands, heart, and spleen were all higher than the corresponding plasma concentrations. Nelfinavir was also detected in rat brain tissue. Nelfinavir binds to plasma proteins at a rate exceeding 98%, primarily to albumin and α1-acid glycoprotein. The concentration of nelfinavir in cerebrospinal fluid is less than 1% of its plasma concentration, at least partly due to its extensive binding to plasma proteins, but possibly also related to P-glycoproteins on the blood-brain barrier. Nelfinavir and its metabolites are primarily excreted in feces, with less than 2% excreted in urine. Moderate or severe hepatitis may prolong the half-life of nelfinavir and increase its plasma concentration, while decreasing the plasma concentration of its major metabolite M8 (hydroxytert-butylamide). Nelfinavir absorption is highly sensitive to food; a moderately fatty meal can increase AUC by 2 to 3 times, while a high-fat meal can achieve even higher concentrations. For more complete data on the absorption, distribution, and excretion of nelfinavir mesylate (a total of 8 metabolites), please visit the HSDB record page. Metabolism/Metabolites Nelfinavir is primarily metabolized oxidatively in the liver via CYP3A4, but is also metabolized via CYP2C19 and CYP2D6. Its major metabolite, hydroxytert-butylamide (M8), exhibits in vitro antiretroviral activity comparable to the parent drug, but its plasma concentration is only 40% of that of nelfinavir. M8 metabolites are primarily generated by CYP2C19.
Biological Half-Life
The plasma elimination half-life of nelfinavir in individuals aged 13 years and older is 3.5–5 hours.
Following a single intraperitoneal injection of nelfinavir in mice, peak plasma concentrations (Cmax) were reached at 30 minutes: 23.21 μg/mL (~34.96 μmol/L) in the 50 mg/kg group and 54.33 μg/mL (~81.83 μmol/L) in the 100 mg/kg group.
The drug was rapidly eliminated; 4 hours post-injection, plasma concentrations in the 50 mg/kg and 100 mg/kg groups decreased to 0.1 μg/mL (~0.15 μmol/L) and 3 μg/mL (~4.5 μmol/L), respectively. Compared to the 50 mg/kg group (1398.26 min μg/mL), the area under the plasma concentration-time curve (AUC) of the 100 mg/kg group was approximately 2.5 times higher (3445.53 min μg/mL). This study indicates that the Cmax of nelfinavir in HIV patients is approximately 7-9 μmol/L, exceeding the in vitro average GI50 value of 5.2 μmol/L. The oral half-life in humans is 3-5 hours.

Effects During Pregnancy and Lactation
◉ Overview of Use During Lactation
Nelfinavir is present in low concentrations in breast milk and is usually undetectable in the serum of breastfed infants. However, there is evidence that nelfinavir may cause adverse reactions in breastfed infants. Nelfinavir is not recommended for use during lactation. Achieving and maintaining viral suppression through antiretroviral therapy can reduce the risk of breast milk transmission to below 1%, but not zero. For HIV-infected individuals receiving antiretroviral therapy with a persistently low viral load, breastfeeding should be encouraged if they choose to do so. If viral load is not suppressed, pasteurized donor breast milk or formula is recommended.
◉ Impact on Breastfed Infants
One study compared the incidence of rash, hepatotoxicity, and hyperbilirubinemia in 464 breastfed infants. The mothers of these infants were taking nelfinavir (n = 206) or nevirapine (n = 258) during pregnancy and postpartum, while also taking zidovudine and lamivudine to treat HIV infection. Researchers examined the infants at weeks 1, 2, and 6 postpartum. Among the infants exposed to nevirapine, 7 (2.7%) developed a moderate rash; among the infants exposed to nelfinavir, 1 (0.5%) developed a moderate rash. The median time of rash onset was 2 weeks postpartum. Four infants exposed to nelfinavir (1.9%) developed hepatotoxicity (3 moderate, 1 severe), while no cases of hepatotoxicity were found among the infants exposed to nevirapine. Twenty-one infants (4.5%) developed high-risk hyperbilirubinemia, all within 48 hours of birth, but there was no difference in exposure levels between the two drugs.
◉ Effects on Lactation and Breast Milk
Gynecomastia has been reported in men receiving highly active antiretroviral therapy. Gynecomastia initially occurs unilaterally, but about half of cases progress to bilateral gynecomastia. No changes in serum prolactin levels have been observed, and it usually resolves spontaneously within a year even with continued treatment. Some case reports and in vitro studies suggest that protease inhibitors may cause hyperprolactinemia and galactorrhea in some male patients, but this view remains controversial. The implications of these findings for lactating mothers are unclear. Prolactin levels in established lactating mothers may not affect their ability to breastfeed.

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Interactions
Utilizing protein homeostasis is a novel approach in cancer treatment. Nelfinavir (NFV), an HIV protease inhibitor, induces endoplasmic reticulum (ER) stress in cancer cells. Under ER stress, misfolded proteins are transported from the ER back to the cytosol and subsequently degraded via the ubiquitin-proteasome system. Bortezomib (BZ) is a proteasome inhibitor that interferes with the degradation of misfolded proteins. This study demonstrates that NFV and BZ enhance proteotoxicity in non-small cell lung cancer (NSCLC) and multiple myeloma (MM) cells. The combination of these two drugs synergistically inhibits cell proliferation and induces cell death. Expression of activating transcription factor (ATF)3 and CCAAT enhancer-binding protein homolog (CHOP), markers of endoplasmic reticulum stress, rapidly increased, while siRNA-mediated knockdown inhibited cell death. Knockdown of double-stranded RNA-activated protein kinase-like endoplasmic reticulum kinase (ER kinase, an endoplasmic reticulum stress signaling molecule) significantly reduced apoptosis. Pretreatment with the protein synthesis inhibitor cycloheximide reduced levels of ubiquitinated protein, ATF3, CHOP, and total cell death, indicating that inhibiting protein synthesis can improve cell survival by alleviating proteotoxic stress. The combination of NFV/BZ inhibited the growth of NSCLC xenograft tumors, which is associated with the induction of endoplasmic reticulum stress and apoptosis markers. These data collectively indicate that nelfinavir (NFV) and benzimidazole (BZ) enhance protein toxicity in non-small cell lung cancer (NSCLC) and multiple myeloma (MM) cells, suggesting that this combination may disrupt the fragile balance of protein homeostasis in cancer cells, thereby providing therapeutic benefit. Nelfinavir competes with the cytochrome P450 enzyme CYP3A, potentially inhibiting the metabolism of these drugs (amiodarone, astemizole, cisapride, ergot derivatives, midazolam, quinidine, terfenadine, triazolam), and may cause serious and/or life-threatening arrhythmias or prolonged sedation; concomitant use is not recommended. Concomitant use with lamivudine increases the AUC of lamivudine by 10%. Concomitant use of oral contraceptives (e.g., ethinylestradiol or norethindrone) with nelfinavir may decrease the plasma concentrations of these drugs; other or additional contraceptive methods should be used. For more complete data on interactions of nelfinavir mesylate (10 in total), please visit the HSDB record page. This study indicates that nelfinavir (a first-generation HIV protease inhibitor) is associated with serious metabolic side effects, including lipid metabolism disorders, dyslipidemia, insulin resistance, and an increased risk of cardiovascular disease (such as atherosclerosis and coronary artery disease) in HIV patients receiving highly active antiretroviral therapy (HAART). In vitro studies used physiologically relevant concentrations of nelfinavir (0.25–2 μg/mL), concentrations achievable in human plasma during treatment. [1]

[1]. Nelfinavir suppresses signaling and nitric oxide production by human aortic endothelial cells: protective effects of thiazolidinediones. Ochsner J. 2013 Spring;13(1):76-90.

[2]. Nelfinavir, A lead HIV protease inhibitor, is a broad-spectrum, anticancer agent that inducesendoplasmic reticulum stress, autophagy, and apoptosis in vitro and in vivo. Clin Cancer Res. 2007 Sep 1;13(17):5183-94.

[3]. Nelfinavir mesylate (AG1343): a potent, orally bioavailable inhibitor of HIV-1 protease. J Med Chem. 1997 Nov 21;40(24):3979-85.

[4]. The human immunodeficiency virus-1 protease inhibitor nelfinavir impairs proteasome activity and inhibits the proliferation of multiple myeloma cells in vitro and in vivo. Haematologica. 2012;97(7):1101‐1109.

[5]. Bardoxolone and bardoxolone methyl, two Nrf2 activators in clinical trials, inhibit SARS-CoV-2 replication and its 3C-like protease. Signal Transduct Target Ther. 2021 May 29;6(1):212.

Therapeutic Uses
HIV protease inhibitor. HIV protease inhibitors are associated with HIV protease inhibitor-associated lipid metabolism disorder syndrome. Researchers hypothesized that liposarcomas are also susceptible to the apoptotic effects of the HIV protease inhibitor nelfinavir. We conducted a phase I clinical trial of nelfinavir for the treatment of liposarcoma. Prior chemotherapy history was not limited. The starting dose was 1250 mg twice daily (level 1). The dose was increased in groups of three up to a maximum evaluable dose of 4250 mg (level 5). One course of treatment lasted 28 days. Steady-state pharmacokinetics (PK) of nelfinavir and its major active metabolite M8 were determined at levels 4 (3000 mg) and 5. A total of 20 subjects (13 men) were enrolled. The median age of the subjects was 64 years (range 37–81 years). One subject in level 1 developed reversible grade 3 pancreatitis after 1 week of treatment and was subsequently replaced. No other dose-limiting toxicities were observed. The median number of treatment cycles was 3 (range 0.6–13.5). The best observed efficacy was: 1 partial remission, 1 mild remission, 4 stable disease, and 13 disease progression. The mean peak plasma concentration and AUC of nelfinavir in grade 4 subjects (7.3 mg/L and 60.9 mg/L·h, respectively) were higher than those in grade 5 subjects (6.3 mg/L and 37.7 mg/L·h, respectively). The mean ratio of M8 to nelfinavir AUC was approximately 1:3 in both grades. Pharmacokinetic studies indicated self-induction of nelfinavir clearance within the studied dose range, but the mechanism is unclear. Peak plasma concentrations were within the range of concentrations that have demonstrated anticancer activity in vitro. The presence of the M8 metabolite, approximately one-third that of nelfinavir, may also contribute to the observed anticancer activity. Nelfinavir is indicated for HIV-infected patients requiring antiretroviral therapy. The US product label includes:
A Phase I/II dose-range open-label monotherapy study was conducted to evaluate the safety, pharmacokinetics, and antiviral activity of nelfinavir mesylate (Viracept, a human immunodeficiency virus (HIV)-1 protease inhibitor). The study enrolled 65 HIV-1 infected individuals. After 28 days, 54 responding subjects enrolled in an open-label extension study, allowing the addition of a nucleoside reverse transcriptase inhibitor and dose increases to maintain durable efficacy. The drug was well tolerated and demonstrated potent antiviral activity, with the 750 mg and 1000 mg three times daily regimens showing significantly better efficacy than other regimens. Thirty subjects continued treatment at 12 months, with a sustained decrease in HIV RNA levels of 1.6 log10 and an average increase in CD4 cells of 180-200 cells/mm³. Post-rebound viral genotypic studies have shown that the initial active site mutation leading to nelfinavir resistance is mediated by a unique amino acid substitution in HIV-1 protease D30N, which does not result in extrinsic cross-resistance to currently available protease inhibitors. For more complete data on the therapeutic uses of nelfinavir mesylates (6 in total), please visit the HSDB record page.
Drug Warnings In adults, the most common adverse reaction to nelfinavir treatment is mild to moderate diarrhea. In Phase II/III clinical studies, 13% of adults receiving the recommended dose of nelfinavir reported a rash. In clinical studies, the incidence of allergic reactions, dermatitis, folliculitis, fungal dermatitis, maculopapular rash, pruritus, sweating, and urticaria in adult patients receiving nelfinavir was less than 2%. During post-marketing surveillance, hypersensitivity reactions possibly associated with nelfinavir have been reported, including bronchospasm, moderate to severe rash, fever, and edema. In Phase II/III clinical studies, fatigue occurred in 1% of adult patients receiving standard-dose nelfinavir in combination with two nucleoside reverse transcriptase inhibitors. In clinical studies, less than 2% of adults receiving nelfinavir reported anxiety, depression, dizziness, mood swings, headache (including migraine), ADHD, insomnia, malaise, paresthesia, seizures, sleep disturbances, somnolence, and suicidal ideation. In clinical studies, up to 3% of adults receiving nelfinavir experienced significantly elevated serum AST (SGOT) or ALT (SGPT) levels (5.1–10 times higher than baseline, or more than 10 times higher than baseline). In clinical studies, less than 2% of adults treated with nelfinavir reported hepatitis, elevated serum alkaline phosphatase levels, elevated gamma-glutamyl transferase (GGT, GGTP) levels, or abnormal liver function test results. More complete data on drug warnings for nelfinavir mesylate (19 in total) can be found on the HSDB record page. Nelfinavir is an HIV-1 protease inhibitor used in highly active antiretroviral therapy (HAART). This study investigated one of the major side effects of nelfinavir: induction of endothelial dysfunction. The mechanism involves nelfinavir inhibiting the insulin-PI3K/Akt-eNOS signaling pathway in endothelial cells, leading to reduced nitric oxide production, which is essential for vascular homeostasis and vasodilation. This study suggests that the combined use of insulin-sensitizing thiazolidinediones (e.g., troglitazone) may help prevent nelfinavir-induced endothelial dysfunction, suggesting a potential adjunctive therapy strategy. [1]

C33H49N3O7S2

663.8881

663.301

C, 59.70; H, 7.44; N, 6.33; O, 16.87; S, 9.66

159989-65-8

Nelfinavir;159989-64-7

64142

White to yellow solid powder

786.8ºC at 760 mmHg

131-135ºC

429.7ºC

6.052

5

9

10

45

922

5

S(C1C([H])=C([H])C([H])=C([H])C=1[H])C([H])([H])[C@@]([H])([C@@]([H])(C([H])([H])N1[C@]([H])(C(N([H])C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H])=O)C([H])([H])[C@]2([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[C@]2([H])C1([H])[H])O[H])N([H])C(C1C([H])=C([H])C([H])=C(C=1C([H])([H])[H])O[H])=O.S(C([H])([H])[H])(=O)(=O)O[H]

NQHXCOAXSHGTIA-SKXNDZRYSA-N

InChI=1S/C32H45N3O4S.CH4O3S/c1-21-25(15-10-16-28(21)36)30(38)33-26(20-40-24-13-6-5-7-14-24)29(37)19-35-18-23-12-9-8-11-22(23)17-27(35)31(39)34-32(2,3)4;1-5(2,3)4/h5-7,10,13-16,22-23,26-27,29,36-37H,8-9,11-12,17-20H2,1-4H3,(H,33,38)(H,34,39);1H3,(H,2,3,4)/t22-,23+,26-,27-,29+;/m0./s1

(3S,4aS,8aS)-N-tert-butyl-2-[(2R,3R)-2-hydroxy-3-[(3-hydroxy-2-methylbenzoyl)amino]-4-phenylsulfanylbutyl]-3,4,4a,5,6,7,8,8a-octahydro-1H-isoquinoline-3-carboxamide;methanesulfonic acid

Nelfinavir mesylate hydrate; AG-1343; AG1343; AG 1343; Nelfinavir; Viracept

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~100 mg/mL (~150.6 mM)
Ethanol: ~100 mg/mL (~150.6 mM)

Solubility in Formulation 1: ≥ 5 mg/mL (7.53 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 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: ≥ 5 mg/mL (7.53 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 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: ≥ 5 mg/mL (7.53 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: ≥ 2.5 mg/mL (3.77 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 5: ≥ 2.5 mg/mL (3.77 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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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 (Please use freshly prepared in vivo formulations for optimal results.)