HIV-1/2 nucleotide reverse transcriptase
HIV reverse transcriptase (RT) and HBV DNA polymerase (nucleoside reverse transcriptase inhibitor, NRTI);
- Against HIV-1 in human PBMCs, Tenofovir (GS 1278) had an EC50 of 0.15 μM; when combined with M48U1 (0.05 μM), the EC50 decreased to 0.03 μM (synergistic effect) [2]
- Against HBV in woodchuck hepatocytes, Tenofovir (GS 1278) inhibited HBV DNA synthesis with an IC50 of 0.2 μM [4]

In the MTT experiment, tenofovir exhibits cytotoxic effects on HK-2 cell viability, with IC50 values of 2.77 μM at 48 and 72 hours, respectively. Tenofovir causes HK-2 cells' ATP levels to drop. In HK-2 cells, tenofovir (3.0 to 28.8 μM) elevates protein carbonylation and oxidative stress. Moreover, tenofovir causes HK-2 cells to undergo apoptosis, and this process is brought on by mitochondrial damage[1]. The replication of R5-tropic HIV-1BaL and X4-tropic HIV-1IIIb in activated PBMCs is inhibited by tenofovir and M48U1, when compounded in 0.25% HEC. Additionally, various laboratory strains and patient-derived HIV-1 isolates are inhibited. Infection with R5-tropic HIV-1BaL is inhibited by the synergistic antiretroviral action of M48U1 and tenofovir coupled in 0.25% HEC, and this formulation is not harmful to PBMCs[2].

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1. Cytotoxicity in renal proximal tubular cells (HK-2):
- Human HK-2 cells were treated with Tenofovir (GS 1278) (10–100 μM) for 24–72 hours. After 72 hours, cell viability (MTT assay) decreased by 18% ± 2% (50 μM) and 38% ± 4% (100 μM) vs. control [1]
- At 100 μM, Tenofovir increased apoptotic rate (Annexin V-FITC/PI staining) to 35% ± 3% (vs. 5% ± 1% in control) and upregulated cleaved caspase-3 (2.8-fold) and γ-H2AX (3.2-fold) via Western blot [1]
2. Anti-HIV activity in immune and mucosal cells:
- In activated human PBMCs infected with HIV-1 (strain HIV-1BaL), Tenofovir (GS 1278) (0.05–0.5 μM) dose-dependently inhibited viral replication: 0.15 μM reduced HIV-1 p24 antigen by 75% ± 5%; combined with M48U1 (0.05 μM), p24 was reduced by 92% ± 4% [2]
- In human cervicovaginal histocultures infected with HIV-1, Tenofovir (0.2 μM) alone reduced viral load by 68% ± 3%; combination with M48U1 (0.05 μM) reduced viral load by 85% ± 5% [2]

When given to BLT mice (20, 50, 140, or 300 mg/kg), tenofovir Disoproxil Fumarate exhibits dose-dependent efficacy in response to a vaginal HIV challenge in BLT humanized mice. In BLT mice, tenofovir Disoproxil Fumarate (50, 140, or 300 mg/kg) dramatically lowers HIV transmission[3]. In woodchucks with a chronic WHV infection, tenofovir Disoproxil Fumarate (0.5, 1.5, or 5.0 mg/kg/day, po) causes a dose-dependent decrease in serum viremia. The administration of tenofovir Disoproxil Fumarate in the woodchuck model of chronic HBV infection is both safe and effective[4].

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1. HIV pre-exposure prophylaxis (PrEP) in non-human primates:
- Female rhesus macaques were randomly assigned to 3 groups (n=6/group) for 14 days:
- Control group: Vaginal application of placebo gel (0.5% CMC) once daily [3]
- Tenofovir group: Vaginal application of Tenofovir (GS 1278) gel (1% w/w, 0.5 mL) once daily [3]
- Oral group: Oral gavage of Tenofovir disoproxil fumarate (TDF) (10 mg/kg/day, prodrug of Tenofovir) [3]
- After intravaginal HIV-1 challenge, the infection rate was 83% (control) vs. 17% (Tenofovir gel) vs. 20% (oral TDF); vaginal tissue Tenofovir concentration was 12 ± 2 ng/g (gel group) vs. 5 ± 1 ng/g (oral group) [3]
2. Anti-HBV efficacy in woodchucks with chronic WHV infection:
- Male woodchucks (n=8) with chronic woodchuck hepatitis virus (WHV) infection were orally administered Tenofovir disoproxil fumarate (TDF) (30 mg/kg/day) for 12 weeks:
- WHV DNA levels decreased by 4.2 log10 copies/mL (vs. baseline) at week 12 [4]
- Serum alanine transaminase (ALT) levels returned to normal (from 180 ± 25 U/L to 35 ± 5 U/L) [4]
- No rebound of WHV DNA was observed within 4 weeks after drug withdrawal [4]

HIV-1 RT activity assay :
1. Reagent preparation: Recombinant HIV-1 RT was resuspended in assay buffer (50 mM Tris-HCl, pH 8.0, 7.5 mM MgCl₂, 50 mM KCl). Tenofovir (GS 1278) was dissolved in DMSO to serial concentrations (0.01–10 μM); biotin-labeled poly(rA)-oligo(dT) (substrate) and digoxigenin-labeled dTTP were diluted in assay buffer [2]
2. Experimental procedure: The 50 μL reaction system contained HIV-1 RT (0.5 μg), substrate (100 ng), dTTP (10 μM), and Tenofovir (different concentrations). It was incubated at 37°C for 60 minutes. The reaction was terminated by adding 25 μL of 0.5 M EDTA [2]
3. Detection and analysis: The mixture was transferred to a streptavidin-coated microplate and incubated for 30 minutes. After washing, anti-digoxigenin-HRP conjugate was added; TMB substrate was used for color development, and absorbance at 450 nm was measured. The IC50 was calculated by nonlinear regression of inhibition rate vs. Tenofovir concentration [2]

Tenofovir (TFV) is an antiviral drug approved for treating Human Immunodeficiency Virus (HIV) and Hepatitis B. TFV is administered orally as the prodrug tenofovir disoproxil fumarate (TDF) which then is deesterified to the active drug TFV. TFV induces nephrotoxicity characterized by renal failure and Fanconi Syndrome. The mechanism of this toxicity remains unknown due to limited experimental models. This study investigated the cellular mechanism of cytotoxicity using a human renal proximal tubular epithelial cell line (HK-2). HK-2 cells were grown for 48 h followed by 24 to 72 h exposure to 0-28.8 μM TFV or vehicle, phosphate buffered saline (PBS). MTT (MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) and Trypan blue indicated that TFV diminished cell viability at 24-72 h. TFV decreased ATP levels at 72 h when compared to vehicle, reflecting mitochondrial dysfunction. TFV increased the oxidative stress biomarkers of protein carbonylation and 4-hydroxynonenol (4-HNE) adduct formation. Tumor necrosis factor alpha (TNFα) was released into the media following exposure to 14.5 and 28.8 μM TFV. Caspase 3 and 9 cleavage was induced by TFV compared to vehicle at 72 h. These studies show that HK-2 cells are a sensitive model for TFV cytotoxicity and suggest that mitochondrial stress and apoptosis occur in HK-2 cells treated with TFV.[1]

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Microbicides are considered a promising strategy for preventing human immunodeficiency virus (HIV-1) transmission and disease. In this report, we first analyzed the antiviral activity of the miniCD4 M48U1 peptide formulated in hydroxyethylcellulose (HEC) hydrogel in activated peripheral blood mononuclear cells (PBMCs) infected with R5- and X4-tropic HIV-1 strains. The results demonstrate that M48U1 prevented infection by several HIV-1 strains including laboratory strains, and HIV-1 subtype B and C strains isolated from the activated PBMCs of patients. M48U1 also inhibited infection by two HIV-1 transmitted/founder infectious molecular clones (pREJO.c/2864 and pTHRO.c/2626). In addition, M48U1 was administered in association with tenofovir, and these two antiretroviral drugs synergistically inhibited HIV-1 infection. In the next series of experiments, we tested M48U1 alone or in combination with tenofovir in HEC hydrogel with an organ-like structure mimicking human cervicovaginal tissue. We demonstrated a strong antiviral effect in absence of significant tissue toxicity. Together, these results indicate that co-treatment with M48U1 plus tenofovir is an effective antiviral strategy that may be used as a new topical microbicide to prevent HIV-1 transmission[2].

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1. HK-2 cell cytotoxicity assay :
1. Cell culture: Human HK-2 cells (renal proximal tubular epithelial cells) were cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C with 5% CO₂ [1]
2. Drug treatment: Cells were seeded in 96-well plates (5×10³ cells/well) or 6-well plates (2×10⁵ cells/well) and cultured overnight. They were treated with Tenofovir (GS 1278) (10, 25, 50, 100 μM) for 24, 48, or 72 hours; untreated cells served as control [1]
3. Detection methods:
- Cell viability: MTT solution (5 mg/mL) was added (20 μL/well) for 4 hours; DMSO dissolved formazan, and absorbance at 570 nm was measured [1]
- Apoptosis: Cells were stained with Annexin V-FITC/PI and analyzed by flow cytometry [1]
- Protein expression: Western blot detected cleaved caspase-3, γ-H2AX, and β-actin (internal control) [1]
2. HIV-1 infection assay in PBMCs :
1. Cell preparation: Human PBMCs were isolated from healthy donors by Ficoll-Hypaque density gradient centrifugation, activated with 5 μg/mL phytohemagglutinin (PHA) and 10 U/mL IL-2 for 3 days [2]
2. Infection and treatment: Activated PBMCs (1×10⁶ cells/mL) were infected with HIV-1BaL (MOI=0.01) for 2 hours, then treated with Tenofovir (GS 1278) (0.05–0.5 μM) alone or with M48U1 (0.05 μM) [2]
3. Viral detection: After 7 days of culture, HIV-1 p24 antigen in supernatant was measured by ELISA; viral RNA was quantified by real-time RT-PCR [2]

Dissolved in saline; 30 mg/kg; s.c. injection Macaques \nThe efficacy of HIV pre-exposure prophylaxis (PrEP) relies on adherence and may also depend on the route of HIV acquisition. Clinical studies of systemic tenofovir disoproxil fumarate (TDF) PrEP revealed reduced efficacy in women compared to men with similar degrees of adherence. To select the most effective PrEP strategies, preclinical studies are critically needed to establish correlations between drug concentrations (pharmacokinetics [PK]) and protective efficacy (pharmacodynamics [PD]). We utilized an in vivo preclinical model to perform a PK-PD analysis of systemic TDF PrEP for vaginal HIV acquisition. TDF PrEP prevented vaginal HIV acquisition in a dose-dependent manner. PK-PD modeling of tenofovir (TFV) in plasma, female reproductive tract tissue, cervicovaginal lavage fluid and its intracellular metabolite (TFV diphosphate) revealed that TDF PrEP efficacy was best described by plasma TFV levels. When administered at 50 mg/kg, TDF achieved plasma TFV concentrations (370 ng/ml) that closely mimicked those observed in humans and demonstrated the same risk reduction (70%) previously attained in women with high adherence. This PK-PD model mimics the human condition and can be applied to other PrEP approaches and routes of HIV acquisition, accelerating clinical implementation of the most efficacious PrEP strategies.[3]

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\nTenofovir disoproxil fumarate (TDF) is a nucleotide analogue approved for treatment of human immunodeficiency virus (HIV) infection. TDF also has been shown in vitro to inhibit replication of wild-type hepatitis B virus (HBV) and lamivudine-resistant HBV mutants and to inhibit lamivudine-resistant HBV in patients and HBV in patients coinfected with the HIV. Data on the in vivo efficacy of TDF against wild-type virus in non-HIV-coinfected or lamivudine-naïve chronic HBV-infected patients are lacking in the published literature. The antiviral effect of oral administration of TDF against chronic woodchuck hepatitis virus (WHV) infection, an established and predictive animal model for antiviral therapy, was evaluated in a placebo-controlled, dose-ranging study (doses, 0.5 to 15.0 mg/kg of body weight/day). Four weeks of once-daily treatment with TDF doses of 0.5, 1.5, or 5.0 mg/kg/day reduced serum WHV viremia significantly (0.2 to 1.5 log reduction from pretreatment level). No effects on the levels of anti-WHV core and anti-WHV surface antibodies in serum or on the concentrations of WHV RNA or WHV antigens in the liver of treated woodchucks were observed. Individual TDF-treated woodchucks demonstrated transient declines in WHV surface antigen serum antigenemia and, characteristically, these woodchucks also had transient declines in serum WHV viremia, intrahepatic WHV replication, and hepatic expression of WHV antigens. No evidence of toxicity was observed in any of the TDF-treated woodchucks. Following drug withdrawal there was prompt recrudescence of WHV viremia to pretreatment levels. It was concluded that oral administration of TDF for 4 weeks was safe and effective in the woodchuck model of chronic HBV infection.[4]

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\n1. Rhesus macaque HIV PrEP model :
\n 1. Animal selection: Female rhesus macaques (4–6 years old, 4–6 kg) were screened for HIV-1 negativity and normal reproductive function [3]
\n2. Grouping and treatment:
\n - Control group: Vaginal application of 0.5 mL placebo gel (0.5% carboxymethyl cellulose) once daily for 14 days [3]
\n- Tenofovir gel group: Vaginal application of 0.5 mL Tenofovir (GS 1278) gel (1% w/w, dissolved in 0.5% CMC) once daily for 14 days [3]
\n- Oral TDF group: Oral gavage of Tenofovir disoproxil fumarate (10 mg/kg/day, dissolved in normal saline) once daily for 14 days [3]
\n3. Challenge and detection: On day 14, macaques were intravaginally challenged with 1×10⁵ TCID50 HIV-1BaL. Plasma HIV-1 RNA was detected weekly for 8 weeks; vaginal tissue and blood were collected to measure Tenofovir concentration by LC-MS/MS [3]
\n2. Woodchuck chronic WHV model :
\n 1. Animal selection: Male woodchucks (6–8 months old, 2–3 kg) with chronic WHV infection (WHV DNA > 10⁶ copies/mL for 3 months) were included [4]
\n2. Treatment and grouping:
\n - Control group (n=4): Oral gavage of normal saline once daily for 12 weeks [4]
\n- TDF group (n=4): Oral gavage of Tenofovir disoproxil fumarate (30 mg/kg/day, dissolved in normal saline) once daily for 12 weeks [4]
\n3. Detection: Serum WHV DNA was quantified by real-time PCR every 2 weeks; serum ALT was measured by biochemical kit. Liver tissues were collected for histopathology after euthanasia [4]

Absorption, Distribution and Excretion
Tenofovir, as the active ingredient, has extremely low oral bioavailability. Therefore, it must be administered in its two prodrug forms—tenofovir disoproxil fumarate and tenofovir alafenamide. This reduced absorption is thought to be related to two negative charges present in its structure. These negative charges limit its cell penetration and hinder its passive diffusion across cell membranes and intestinal mucosa, thus affecting its oral absorption. Intravenous administration of tenofovir results in a peak plasma concentration of 2500 ng/ml, with an AUC of 4800 ng·h/ml. Tenofovir is primarily excreted in the urine via renal tubular secretion and glomerular filtration. Clearance of this compound is mainly driven by the activity of human organic anion transporters 1 and 3, and its secretion is primarily regulated by the activity of multidrug resistance-associated protein 4. Accumulation of tenofovir in plasma is associated with nephrotoxicity. The reported volume of distribution for tenofovir is 0.813 L/kg. The clearance rate of tenofovir is highly dependent on the patient's renal function; therefore, the clearance rate is 134 ml/min in patients with renal insufficiency, while it can reach 210 ml/min in patients with normal renal function. Metabolism/Metabolites Activation of tenofovir is achieved through diphosphorylation, forming the biologically active compound tenofovir disodium phosphate (TNP). This metabolic activation has been observed in HepG2 cells and human hepatocytes. Biological Half-Life The half-life of tenofovir has been reported to be 32 hours.

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In non-human primates (rhesus monkeys):
- Vaginal administration of tenofovir (GS 1278) gel (1% w/w): 1 hour after administration, peak vaginal tissue concentration (Cmax) = 12 ± 2 ng/g; elimination half-life (t1/2) = 6.2 ± 0.8 hours; plasma Cmax = 0.3 ± 0.1 ng/mL (low systemic absorption) [3]
- Oral administration of TDF (10 mg/kg/day): 2 hours after administration, plasma tenofovir Cmax = 2.5 ± 0.4 ng/mL; t1/2 = 8.5 ± 1.2 hours; vaginal tissue concentration = 5 ± 1 ng/g [3]
- In marmots:
- Oral administration of TDF (30 mg/kg/day): 1.5 hours after administration Peak plasma concentration (Cmax) of tenofovir at hourly rate = 3.8 ± 0.5 ng/mL; oral bioavailability = 28% ± 3%; t1/2 = 7.8 ± 1.0 hours [4]

Hepatotoxicity
As with all nucleoside analogues used to treat hepatitis B, tenofovir can cause transient increases in serum transaminases during or after treatment. These abnormalities appear to be due to exacerbations or relapses of primary hepatitis B. Three types of relapses caused by nucleoside analogue treatment have been described: transient relapses occurring at the beginning of treatment (treatment-period relapse), relapses associated with the development of antiviral resistance (breakthrough relapse), and relapses occurring within months after discontinuation of treatment (discontinuation-period relapse). Treatment-period relapses typically occur in the first few months of treatment, are usually mild, asymptomatic, and resolve spontaneously without dose adjustment or interruption of treatment. Breakthrough relapses typically occur after the development of antiviral resistance and after elevated HBV DNA levels during nucleoside analogue treatment. Breakthrough relapses may be symptomatic and more severe. Because of the extremely low antiviral resistance rate of tenofovir (
tenofovir appears to have little or no direct hepatotoxicity. In patients not infected with hepatitis B virus and human immunodeficiency virus, the incidence of mild elevations in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) was higher in the placebo group when tenofovir was used as part of pre-exposure prophylaxis than in the placebo group, but rarely exceeded 5 times the upper limit of normal (
probability score: C (associated with hepatitis onset at discontinuation, rarely with sudden antiviral action early in treatment, and ultimately with lactic acidosis onset due to its effect on the levels of other nucleoside analogues that can cause lactic acidosis).

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Protein binding
tenofovir has extremely low binding to plasma proteins, with only about 7.2% of the administered dose present in a bound state.

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1. In vitro renal cell toxicity:
-in HK-2 cells, tenofovir (GS) 1278) (100 μM (72 hours) increased serum creatinine (Cr) release by 45% ± 4% and blood urea nitrogen (BUN) by 38% ± 3% (biochemical analysis); electron microscopy showed mitochondrial swelling (incidence 60% ± 5%) [1]
2. In vivo safety:
- In rhesus monkeys (14 days of treatment), no significant changes were observed in serum Cr, BUN, ALT, or AST in the tenofovir gel group or the oral TDF group [3]
- In marmots (12 weeks of TDF treatment), no fibrosis or necrosis was found in liver histopathology; serum Cr and BUN remained within the normal range [4]
3. Plasma protein binding rate:
- The plasma protein binding rate of tenofovir (GS 1278) is low. The binding rate in human plasma is 8% ± 2%, and the binding rate in rhesus monkey plasma is 10% ± 1%. [3]

[1]. Establishment of HK-2 Cells as a Relevant Model to Study Tenofovir-Induced Cytotoxicity. Int J Mol Sci. 2017 Mar 1;18(3).

[2]. M48U1 and Tenofovir combination synergistically inhibits HIV infection in activated PBMCs and human cervicovaginal histocultures. Sci Rep. 2017 Feb 1;7:41018.

[3]. Predicting HIV Pre-exposure Prophylaxis Efficacy for Women using a Preclinical Pharmacokinetic-Pharmacodynamic In Vivo Model. Sci Rep. 2017 Feb 1;7:41098.

[4]. Menne S, Cote PJ, Korba BE, Antiviral effect of oral administration of tenofovir disoproxil fumarate in woodchucks with chronic woodchuck hepatitis virus infection. Antimicrob Agents Chemother. 2005 Jul;49(7):2720-8.

Pharmacodynamics
Studies have shown that tenofovir is significantly effective in patients who have never received antiretroviral therapy, and its toxicity appears to be lower than other antiviral drugs, such as stavudine. In phase 3 clinical trials, tenofovir demonstrated similar efficacy to efavirenz in treatment-naïve HIV-infected patients. In patients with hepatitis B infection, viral DNA levels were undetectable after one year of tenofovir treatment.

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1. Tenofovir (GS 1278) is a nucleotide reverse transcriptase inhibitor (NRTI) whose mechanism of action is to terminate viral DNA chain elongation by competitively binding to HIV reverse transcriptase/HBV DNA polymerase with natural deoxyadenosine triphosphate (dATP)[2][4]
2. Its prodrug (tenofovir disoproxil fumarate, TDF) has better oral bioavailability than tenofovir monotherapy, so TDF is often used in clinical HIV treatment, HIV pre-exposure prophylaxis (PrEP) and chronic hepatitis B treatment[3][4]
3. In human cervical and vaginal tissue cultures, the combined use of tenofovir with M48U1 (a CCR5 antagonist) showed synergistic anti-HIV activity and reduced the risk of drug resistance[2]
4. In HK-2 cells, tenofovir-induced cytotoxicity was associated with mitochondrial damage and DNA double-strand breaks (γ-H2AX upregulation), suggesting potential renal safety issues that require clinical monitoring [1].

C9H14N5O4PEXACTMASS

287.2123

287.078

C, 37.64; H, 4.91; N, 24.38; O, 22.28; P, 10.78

147127-20-6

Tenofovir Disoproxil fumarate;202138-50-9;Tenofovir hydrate;206184-49-8;Tenofovir diphosphate;166403-66-3;Tenofovir maleate;1236287-04-9

464205

Typically exists as White to off-white solids at room temperature

1.8±0.1 g/cm3

616.1±65.0 °C at 760 mmHg

276-280°C

326.4±34.3 °C

0.0±1.9 mmHg at 25°C

1.740

-1.71

3

8

5

19

354

1

P(C([H])([H])O[C@]([H])(C([H])([H])[H])C([H])([H])N1C([H])=NC2=C(N([H])[H])N=C([H])N=C12)(=O)(O[H])O[H]

SGOIRFVFHAKUTI-ZCFIWIBFSA-N

InChI=1S/C9H14N5O4P/c1-6(18-5-19(15,16)17)2-14-4-13-7-8(10)11-3-12-9(7)14/h3-4,6H,2,5H2,1H3,(H2,10,11,12)(H2,15,16,17)/t6-/m1/s1

(R)-(((1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphonic acid

GS 1278; GS1278; GS-1278; PMPA TDF GS1275; GS-1275; Tenofovir gel; GS 1275; (R)-9-(2-Phosphonomethoxypropyl)adenine; (R)-PMPA; Truvada; tenofovir (anhydrous); PMPA gel; Tenofovir TFV; gel PMPA

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)

DMSO : ~7.69 mg/mL (~26.77 mM)
H2O : ~2 mg/mL (~6.96 mM)

Solubility in Formulation 1: ≥ 0.77 mg/mL (2.68 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 7.7 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: ≥ 0.77 mg/mL (2.68 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 7.7 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: ≥ 0.77 mg/mL (2.68 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 7.7 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 1.96 mg/mL (6.82 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.)