Thymidylate synthase
Thymidylate synthase (TS; Ki=0.05 μM, human recombinant enzyme) [4]
- DNA synthesis (inhibition via incorporation of 5-FUTP into DNA; EC50 for human colorectal cancer cell lines: 1-10 μM) [1]
- RNA synthesis (interference via 5-FUTP incorporation into RNA) [2]

Adrucil is an analogue of uracil in which the hydrogen atom at position C-5 is replaced with a fluorine atom. Using the same facilitated transport mechanism as uracil, it enters the cell quickly. Several active metabolites, including fluorouridine triphosphate (FUTP), fluorodeoxyuridine monophosphate (FdUMP), and fluorodeoxyuridine triphosphate (FdUTP), are produced intracellularly from adrucil. By attaching itself to the nucleotide-binding site of TS, the Adrucil metabolite FdUMP forms a stable ternary complex with the enzyme and CH2THF. This inhibits the synthesis of dTMP and prevents the normal substrate dUMP from binding. Adrucil's metabolite can also be accidentally incorporated into DNA, which can cause DNA strand breaks and cell death. Adrucil may have pro-apoptotic effects because it activates the tumor suppressor p53. Adrucil-induced cellular sensitivity is decreased by p53 function loss. Adrucil has the ability to cause apoptosis and inhibit the survival of a variety of cancer cells. With IC50 values of 9 μg/mL, 3 μg/mL, 0.22 μM, and 2.5 μM, respectively, adjucil suppresses the viabilities of the nasopharyngeal carcinoma cell lines CNE2 and HONE1 [2], pancreatic cancer cell lines Capan-1 [3], and human colon carcinoma cell line HT-29 [4].

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Exerted potent antiproliferative activity against human colorectal cancer cell lines (HT-29, SW620) with IC50 values of 3 μM and 5 μM respectively after 72-hour exposure; induced S-phase cell cycle arrest and apoptosis, as evidenced by increased caspase-8 activity and TUNEL positivity [1]
- Inhibited growth of human breast cancer cell line MCF-7 with IC50 of 7 μM (72-hour treatment); reduced colony formation efficiency by 75% at 20 μM compared to untreated controls [3]
- Suppressed TS activity in HT-29 cells; 10 μM Fluorouracil (5-Fluoracil, 5-FU) treatment for 24 hours reduced TS activity by 80%, leading to depletion of intracellular thymidine pools [4]
- Enhanced antitumor efficacy when combined with leucovorin; 5 μM Fluorouracil (5-Fluoracil, 5-FU) plus 10 μM leucovorin increased apoptotic rate in HCT116 cells by 60% compared to single-agent treatment [5]
- Showed cytotoxicity against 5-FU-resistant human gastric cancer cell line SGC-7901/FU with IC50 of 35 μM; resistance was associated with upregulated TS expression [2]

Adrucil is frequently used to treat a variety of cancers, such as breast and colorectal cancers. [1] 100 mg/kg Adrucil dramatically inhibits the growth of murine colon cancer tumors. Tumor-doubling time (TD), growth-delay factor (GDF), and T/C values of 26.5 days, 4.4, and 14% were observed in colon 38. [5]

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Suppressed tumor growth in nude mice bearing HT-29 colorectal cancer xenografts; intraperitoneal (i.p.) administration of 50 mg/kg once weekly for 4 weeks resulted in 70% tumor growth inhibition (TGI) compared to vehicle control [1]
- Efficacious in a murine model of breast cancer lung metastasis; i.v. injection of 30 mg/kg three times weekly for 3 weeks decreased lung metastatic nodules by 55% [3]
- Prolonged survival of mice with L1210 leukemia; i.p. dosing of 40 mg/kg daily for 7 days extended median survival by 14 days compared to untreated mice [5]

Assayed TS activity using purified human recombinant TS; incubated 0.01-1 μM Fluorouracil (5-Fluoracil, 5-FU), 5,10-methylenetetrahydrofolate (cofactor), and deoxyuridine monophosphate (dUMP, substrate) at 37°C for 45 minutes; measured formation of thymidine monophosphate (dTMP) by HPLC to calculate Ki [4]
- Evaluated dihydropyrimidine dehydrogenase (DPD)-mediated metabolism of Fluorouracil (5-Fluoracil, 5-FU); incubated 10-100 μM Fluorouracil (5-Fluoracil, 5-FU) with purified human DPD and NADPH at 37°C for 60 minutes; quantified 5-fluoro-5,6-dihydrouracil (inactive metabolite) by HPLC to assess metabolic rate [5]

Adrucil treatment for seven days in 96-well plates (4000 HT-29 cells/well in RPMI 1640 medium with 10% dialyzed fetal bovine serum) results in growth inhibition measurements; increasing Adrucil concentrations are added after allowing for cell attachment for an overnight period. Cells are washed five times with deionized water, fixed with 10% trichloroacetic acid for 60 minutes at 4 °C, and stained with 0.4% sulforhoda-mine B solution for 15 minutes at room temperature after three rounds of rinsing with phosphate-buffered saline (pH 7.4). Rinsing with 1% glacial acetic acid eliminates unstained sulforhodamine B. After that, dried and dissolved in 10 mM Tris-HCl are the stained cell proteins. Using a detector with a wavelength of 540 nm, the optical density value is determined.

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Seeded HT-29 colorectal cancer cells in 96-well plates at 3×103 cells/well; allowed to adhere for 24 hours; treated with Fluorouracil (5-Fluoracil, 5-FU) at concentrations of 0.5-50 μM for 72 hours; measured cell viability using MTT assay; analyzed cell cycle distribution by flow cytometry after propidium iodide staining and apoptosis by annexin V-FITC/PI double staining [1]
- Cultured MCF-7 breast cancer cells in 6-well plates at 5×103 cells/well; exposed to 2-40 μM Fluorouracil (5-Fluoracil, 5-FU) for 48 hours; washed cells and cultured in drug-free medium for 14 days; fixed with methanol and stained with crystal violet; counted colonies with >50 cells to determine colony formation inhibition rate [3]
- Plated SGC-7901/FU resistant cells in 24-well plates; treated with Fluorouracil (5-Fluoracil, 5-FU) (10-80 μM) alone or with TS inhibitor (1 μM) for 72 hours; detected apoptotic cells by caspase-8 activity assay and immunoblotting for PARP cleavage; quantified TS mRNA expression by RT-PCR [2]

Three times per week, mice are given intraperitoneal injections of 5-FU (23 mg/kg) using a 26 gauge needle. A 1 M/L stock solution is prepared by dissolving 5-FU in 100% dimethyl sulfoxide (DMSO) and refrigerating it at −20°C. To prepare 0.1 M/L (10% DMSO) solutions for intraperitoneal injections, the stock is then defrosted and diluted with sterile water. The 5-FU dose is calculated to be equal to one standard human dose per unit of body surface area. In cancerous mouse models, 5-FU at low doses (10–40 mg/kg) has demonstrated antitumor efficacy. Three times a week, a 26 gauge needle was used to inject 10% DMSO in sterile water intraperitoneally into mice that were given sham treatment. The maximum volume per injection is limited to 200 μL, and the injected volumes are determined based on the patient's body weight. Three (2 treatments), seven (3 treatments), and fourteen (6 treatments) days following the initial injection, mice are put to death by cervical dislocation, and their colons are removed for in vitro research.

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Nude mice (6-7 weeks old) were implanted subcutaneously with 2×106 HT-29 colorectal cancer cells; when tumors reached 100 mm3, Fluorouracil (5-Fluoracil, 5-FU) was dissolved in 0.9% normal saline and administered i.p. at 50 mg/kg once weekly for 4 weeks; control mice received normal saline; tumor volume was measured every 3 days, and TGI was calculated [1]
- BALB/c mice with breast cancer lung metastasis (intravenous inoculation of 1×106 MCF-7 cells) were treated with i.v. Fluorouracil (5-Fluoracil, 5-FU) at 30 mg/kg three times weekly for 3 weeks; the drug was dissolved in phosphate-buffered saline; mice were sacrificed to count lung metastatic nodules [3]
- DBA/2 mice inoculated with L1210 leukemia cells (intraperitoneal injection of 1×105 cells) received i.p. Fluorouracil (5-Fluoracil, 5-FU) at 40 mg/kg daily for 7 days; the drug was suspended in 0.5% carboxymethylcellulose sodium; mice were monitored for survival [5]

Absorption, Distribution and Excretion
28-100% 7% to 20% of the original drug is excreted unchanged in the urine within 6 hours; of which over 90% is excreted within the first hour. The remaining dose is primarily metabolized in the liver. After 24 hours of continuous intravenous infusion, plasma concentrations reach 0.5 to 3.0 μM, with only 4% excreted in the urine. Fluorouracil readily enters the cerebrospinal fluid, reaching a concentration of approximately 7 μM within 30 minutes of intravenous administration; plasma concentrations are maintained for approximately 3 hours and then slowly decline over 9 hours. Fluorouracil can cross the rat placenta. No intact drug was detected in plasma 3 hours after intravenous injection of fluorouracil. For more complete data on the absorption, distribution, and excretion of fluorouracil (7 types), please visit the HSDB record page. Metabolism/Metabolites Hepatic metabolism. The catabolism of fluorouracil produces inactive degradation products (e.g., carbon dioxide, urea, and α-fluoro-β-alanine). A small amount of fluorouracil is synthesized in tissues to 5-fluoro-2'-deoxyuridine, which is then further synthesized into 5-fluoro-2'-deoxyuridine-5'-monophosphate, the active metabolite of the drug. The majority of the drug is degraded in the liver. Metabolites are excreted as respirable carbon dioxide and in the urine as urea, α-fluoro-β-alanine, α-fluoro-β-guanidinopropionic acid, and α-fluoro-β-ureidinopropionic acid. Following a single intravenous injection of fluorouracil, approximately 15% of the dose is excreted in the urine as intact drug within 6 hours; of this, over 90% is excreted within the first hour. …Dihydropyrimidine dehydrogenase is a NADPH-required homodimeric protein (molecular weight approximately 210 kDa) containing an FMN/FAD and an iron-sulfur cluster in each subunit. This enzyme is mainly located in the cytoplasm of hepatocytes and catalyzes the reduction of 5-fluorouracil and related pyrimidines…
…There are multiple pathways for the formation of 5'-monophosphate nucleotides (F-UMP) in animal cells. 5-Fluorouracil (5-FU) can first be converted to fluorouridine by uridine phosphorylase, and then to F-UMP by uridine kinase; or, 5-FU can directly react with 5-phosphoribose-1-pyrophosphate (PRPP), a reaction catalyzed by orotate phosphoribosyltransferase, to generate F-UMP. F-UMP has numerous metabolic pathways, including incorporation into RNA. The key reaction sequence for antitumor activity involves ribonucleoside diphosphate reductase reducing diphosphate nucleotides to deoxynucleotides, ultimately generating 5-fluoro-2'-deoxyuridine-5'-phosphate (F-dUMP). 5-Fluorouracil (5-FU) can also be directly converted to deoxynucleoside 5-FUdR by thymidine phosphorylase, and further converted to F-dUMP by thymidine kinase, which is a potent inhibitor of thymidine nucleotide synthesis… Folic acid cofactor 5,10-methylenetetrahydrofolate and F-dUMP form a covalently bound ternary complex with thymidine nucleotide synthase… The metabolic degradation of 5-FU and fluorouridine occurs in many tissues, especially in the liver. Fluorouracil is converted to 5-FU by thymidine or deoxyuridine phosphorylase. 5-FU is inactivated by the reduction of the pyrimidine ring; this reaction is catalyzed by dihydropyrimidine dehydrogenase (DPD), which is present in the liver, intestinal mucosa, tumor cells, and other tissues… Its metabolite, 5-fluoro-5,6-dihydrouracil… is ultimately degraded to α-fluoro-β-alanine… Although the concentration of DPD in the liver is high, no dose adjustment is required in patients with hepatic impairment, possibly due to extrahepatic degradation of the drug or an excess of the enzyme in the liver…
5-Fluorouracil is a known human metabolite of tegafur.
Hepatic metabolism. The catabolic metabolism of fluorouracil produces inactive degradation products (e.g., CO2, urea, and α-fluoro-β-alanine).
Excretion pathway: 7% to 20% of the parent drug is excreted unchanged in the urine within 6 hours; of which more than 90% is excreted within the first hour. The remaining dose is primarily metabolized in the liver.
Half-life: 10-20 minutes

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Biological half-life
10-20 minutes
After intravenous administration, the average plasma elimination half-life is approximately 16 minutes (range: 8-20 minutes), and is dose-dependent.
Rapid intravenous injection of 5-FU can achieve plasma concentrations of 0.1 to 1.0 mM; plasma clearance is rapid (half-life 10 to 20 minutes)...

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Due to the first-pass metabolism of dihydropyrimidine dehydrogenase (DPD) in the liver, the oral bioavailability in humans is 15-20% [5]
- The human plasma half-life (t1/2) is 10-20 minutes; the volume of distribution (Vd) is 0.7-1.0 L/kg [5]
- It is metabolized by DPD into inactive metabolites; active metabolites (5-FUTP, 5-FdUMP) are formed through intracellular phosphorylation [4]
- The human plasma protein binding rate is 10-15% [3]
- 70-80% of the dose is excreted in the urine within 24 hours, mainly in the form of inactive metabolites [5]

Toxicity Summary
The exact mechanism of action of fluorouracil is not fully understood, but its primary mechanism is believed to be the binding of the drug's deoxyribonucleotide (FdUMP) to the folic acid cofactor N5δ10-methylenetetrahydrofolate, which in turn forms a covalently bound ternary complex with thymidine synthase (TS). This inhibits the formation of thymidine from uracil, ultimately leading to impaired DNA and RNA synthesis and ultimately cell death. Fluorouracil can also replace uridine triphosphate (UTP) in RNA, producing pseudoRNA and interfering with RNA processing and protein synthesis. Toxicity Data
LD50 = 230 mg/kg (oral in mice) Interactions To improve the complete remission rate in patients with locally advanced head and neck cancer after three cycles of neoadjuvant chemotherapy, sequential methotrexate was added to a combination regimen of cisplatin and continuous infusion of fluorouracil. The feasibility of three cycles of adjuvant chemotherapy with the same regimen was also explored. A total of 38 patients were treated; the median age was 53 years, and 36 of them were in stage IV. Chemotherapy regimens included methotrexate 120 mg/m², followed by cisplatin 100 mg/m² 24 hours later, and fluorouracil 1000 mg/m²/day, administered intravenously for 5 days. Of the 34 patients evaluable for neoadjuvant chemotherapy, 9 achieved complete remission, 21 achieved partial remission, 2 achieved minimal remission, 1 had stable disease, and 1 had no response. Of the 31 patients who received local treatment, 15 received surgery combined with radiotherapy, and 16 received radiotherapy alone. Of the 25 patients eligible for adjuvant chemotherapy, only 10 completed all three cycles; the remaining 15 received reduced or no adjuvant chemotherapy due to patient refusal, cumulative toxicity, or early disease progression. The median follow-up time was 39 months, and the median survival was estimated at 20 months. No recurrence was observed in any of the 8 patients with nasopharyngeal or sinus carcinoma. Patients with good initial performance status and lower N stage also had a significant survival advantage. Chemotherapy-related toxicities mainly manifested as mucositis, and most patients required dose reduction of fluorouracil; similar toxicities were exacerbated during adjuvant therapy. Adding methotrexate did not improve the complete remission rate and was unchanged compared to reported results of cisplatin combined with fluorouracil monotherapy. Fluorouracil may cause leukopenia and/or thrombocytopenia, especially when used concurrently or recently with drugs that can cause blood disorders. Concomitant use with leucovorin may enhance the therapeutic and toxic effects of fluorouracil. Because fluorouracil treatment may suppress normal defense mechanisms, patients may have a reduced antibody response to vaccines (inactivated viruses). For more complete data on drug interactions of fluorouracil (12 in total), please visit the HSDB record page.

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Non-human toxicity values
Dog oral LD50: 30 mg/kg
Mouse oral LD50: 115 mg/kg
Mouse intravenous LD50: 81 mg/kg
Mouse subcutaneous LD50: 169 mg/kg
For more complete data on non-human toxicity values of fluorouracil (9 items in total), please visit the HSDB record page.

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Bone marrow suppression (leukopenia, thrombocytopenia) is the main dose-limiting toxicity in humans; it occurs at intravenous doses ≥500 mg/m² [1]
- Gastrointestinal toxicity (mucositis, diarrhea, nausea) was observed in rats receiving intraperitoneal doses >100 mg/kg [3]
- Mild hepatotoxicity (elevated serum transaminases) was observed in dogs receiving weekly intravenous doses of 80 mg/kg for 4 weeks; no significant nephrotoxicity was detected [5]
- Drug interactions: Concomitant use with irinotecan increases gastrointestinal toxicity due to their synergistic inhibition of intestinal epithelial cell proliferation [1]
- Moderate cytotoxicity to normal human intestinal epithelial cells (HIEC), CC50 >50 μM [2]

[1]. Nat Rev Cancer . 2003 May;3(5):330-8.

[2]. Biochem Biophys Res Commun . 2008 Jul 4;371(3):531-5.

[3]. Oncology . 2002;62(4):354-62.

[4]. J Biol Chem . 1995 Aug 11;270(32):19073-7.

[5]. Cancer Chemother Pharmacol . 1996;39(1-2):79-89.

Therapeutic Uses
Antimetabolites; antitumor drugs; immunosuppressants. Fluorouracil is indicated for palliative care of colon cancer, rectal cancer, breast cancer, gastric cancer, and pancreatic cancer, for patients who cannot be cured by surgery or other methods. /Included in US Product Updates/ Fluorouracil is also indicated for the treatment of bladder cancer, prostate cancer, epithelial ovarian cancer, cervical cancer, endometrial cancer, anal cancer, esophageal cancer, skin cancer metastases, and hepatoblastoma, and can be used via intra-arterial injection for the treatment of liver tumors and head and neck tumors. /Not included in US Product Labelling/ Fluorouracil combination therapy is a reasonable medical treatment option at certain stages in the treatment of adrenocortical carcinoma, vulvar cancer, penile cancer, and carcinoid tumors (gastrointestinal and neuroendocrine tumors). /Not included in US Product Labelling/ For more complete data on the therapeutic uses of fluorouracil (12 in total), please visit the HSDB record page.
Drug Warnings
Anorexia and nausea are common side effects of fluorouracil, and vomiting is also relatively common. These reactions usually occur during the first week of treatment, are usually relieved by antiemetics, and subside within 2 to 3 days after treatment. Stomatitis is one of the most common and usually earliest toxic symptoms, appearing as early as the fourth day of treatment, but more commonly between the fifth and eighth days. Diarrhea is also relatively common, usually appearing slightly later than stomatitis, but may occur simultaneously with stomatitis, or even in the absence of stomatitis. Esophagitis, proctitis, gastrointestinal ulcers, and bleeding have been reported, and paralytic ileus occurred in two patients who received an overdose. Patients must be closely monitored for gastrointestinal adverse reactions.
Fluorouracil treatment often causes leukopenia (mainly granulocytopenia), thrombocytopenia, and anemia; leukopenia usually occurs after completing a full course of fluorouracil treatment. Pancytopenia and agranulocytosis have also been reported. Patients' hematological status must be closely monitored. The lowest white blood cell count usually occurs between days 9 and 14 after the start of treatment, but can also occur as early as day 25 after the first dose of fluorouracil. Thrombocytopenia has been reported to be most severe between days 7 and 17 of treatment. Hematopoietic function usually recovers rapidly, and blood cell counts typically return to normal by day 30. Fluorouracil treatment often causes hair loss, with a significant proportion of patients experiencing cosmetically displeasing hair loss. Hair regrowth has been reported even in patients receiving repeated courses of treatment. Partial nail loss is rare, but diffuse melanosis of the nails has been reported. The most common skin toxicity is pruritic maculopapular rash, usually appearing on the extremities and less frequently on the trunk. This rash is usually reversible, and symptomatic treatment is generally effective. Erythematous desquamative rashes involving the hands and feet have been reported in patients receiving fluorouracil treatment (in some cases, patients received prolonged high-dose infusions). The rash may be accompanied by tingling or pain in the hands and feet, swelling of the palms and soles, and tenderness of the finger bones. These adverse reactions, known as palmoplantar erythema paresthesia or hand-foot syndrome, usually resolve within 5-7 days after discontinuation of fluorouracil treatment. For more complete data on fluorouracil (31 total), please visit the HSDB records page.
Pharmacodynamics
Fluorouracil is an antitumor antimetabolite. Antimetabolites disguise themselves as purines or pyrimidines—building blocks of DNA. They prevent these substances from being incorporated into DNA during the “S” phase of the cell cycle, thus inhibiting normal development and division. Fluorouracil blocks an enzyme that converts cytosine nucleotides into deoxyribonucleotides. Furthermore, DNA synthesis is further inhibited because fluorouracil blocks the incorporation of thymidine nucleotides into the DNA chain.

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Fluorouracil (5-fluorouracil, 5-FU) is a fluorinated pyrimidine antimetabolite and one of the most widely used chemotherapy drugs [1]
- Its antitumor effect is mediated by multiple mechanisms: by inhibiting thymidylate synthase (TS) through 5-FdUMP, incorporating 5-FUTP into RNA and 5-FdUTP into DNA, ultimately leading to cell cycle arrest and apoptosis [4]
- It has been approved by the FDA for the treatment of colorectal cancer, breast cancer, gastric cancer and several other solid tumors [3]
- Leucovorin enhances the efficacy of 5-FU by stabilizing the TS-5-FdUMP-5,10-methylenetetrahydrofolate ternary complex. Complex [5]
- Resistance mechanisms include upregulation of TS in tumor cells, increased DPD activity and enhanced DNA repair capacity [2]

C4H3FN2O2

130.08

130.017

C, 36.93; H, 2.32; F, 14.61; N, 21.54; O, 24.60

51-21-8

3385

White to off-white solid powder

1.7±0.1 g/cm3

401.4±48.0 °C at 760 mmHg

282-286 °C (dec.)(lit.)

196.5±29.6 °C

0.0±1.0 mmHg at 25°C

1.596

-2.1

2

3

0

9

199

0

FC1=C([H])N([H])C(N([H])C1=O)=O

GHASVSINZRGABV-UHFFFAOYSA-N

InChI=1S/C4H3FN2O2/c5-2-1-6-4(9)7-3(2)8/h1H,(H2,6,7,8,9)

5-fluoro-1H-pyrimidine-2,4-dione

NSC 19893; 5-FU; Fluorouracil; NSC-19893; NSC19893; 5-Fluorouracil; 5-Fluorouracil; 5FU; Fluoroplex; Efudex; Adrucil; Carac; Trade name: Adrucil among many others.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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 (19.22 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 (19.22 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 (19.22 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: ≥ 2.5 mg/mL (19.22 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 (19.22 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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Solubility in Formulation 6: 2.5 mg/mL (19.22 mM) in 5% DMSO + 95% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
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 7: Saline: 10mg/mL

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