DNA/RNA Synthesis

Results: PET/CT of bleomycin (BLM)-receiving mice showed an increase in fibrotic consolidations and an increase in non-aerated lung area in bleomycin (BLM)-treated mice compared with that in controls. TGF-b1, TNF-a, IL-6, GM-CSF in BALF and serum. PAI-1, HYP in the lung tissue of mice were significantly different in each bleomycin (BLM) groups than those in the controls. The results of Masson staining in mice indicate that the lung tissues of all BLM received groups, the intratracheal groups, the intravenous groups, and the intraperitoneal groups have a higher degree of pulmonary septal thickening and collagen fiber consolidation compare to saline control. Picro-Sirius staining results are consistent with the results of Masson staining. Compared with the saline control group, the ratio of Col 1/Col 3 was significantly increased in each BLM group. TEM results found that in BLM group, type I alveolar epithelial cells were degenerated. Exfoliated endothelial cells were swelling, and type II alveolar epithelial cells were proliferated, the shape of the nucleus was irregular, and some tooth-like protrusions were seen. [9]
Conclusions: With three different methods of animal model construction, high dose of each show more compliable, and bleomycin (BLM) can successfully induce animal models of pulmonary fibrosis, however, certain differences in the fibrosis formation sites of them three, and tail vein injection of bleomycin (BLM) induced PF model is closer to the idiopathic pulmonary interstitial fibrosis. [9]

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Day 7 post-bleomycin sulfate, CD45+ cells in BALf in NOX-/-is 1.7-fold > WT, with 57% of these being Mf, which by Day 21 decreases by 67% in WT and 83% in NOX-/-.[5]

ADIPO-P2 cells are cultured in D-MEM high glucose medium at 37 °C with 5% CO₂ atmosphere, supplemented with 20% fetal calf serum, penicillin (100 U/mL), and streptomycin (100 μg/mL). 1.5 × 10⁵ cells/mL are cultured as monolayers in TC25 Corning flasks. Two flasks are set up for each experiment: one for the treated culture and one for the control. ADIPO-P2 cells are exposed to a 30-minute pulse of 2.5 μg/mL bleomycin sulfate during the log phase of growth. Parallel cultures serving as controls are not subjected to bleomycin sulfate. The duration and concentration of bleomycin sulfate exposure are selected based on earlier research using bleomycin sulfate exposure in mammalian cells conducted in our lab. The cells are maintained in culture with fresh culture medium until harvesting after being twice washed with Hank's balanced salt solution following the completion of the Bleomycin sulfate pulse treatment. After treatment, cells are kept in culture continuously for five passages or subcultures. When the cultures reach confluency (approximately 4 × 10⁵ cells/mL of culture medium), subcultivation is performed. At the time of subcultivation, cells are collected by trypsinization, and the number of viable cells is determined by staining an aliquot of approximately 200 μL with 0.4% trypan blue. This process allows for the estimation of cell growth. Subsequently, the cells are suspended in new culture medium and added to fresh culture flasks with a density of 1 × 10⁵5 cells/mL to continue growing. After the treatments are over, the remaining cells are either thrown away or transferred to another flask for cytogenetic analysis, which takes place 18 hours and 10 days later. Colchicine (0.1 μg/mL) is added to cell cultures in the final three hours of culture to analyze chromosomal aberrations. Standard protocols are followed when preparing chromosomes. Following harvesting, cells undergo hypotonic shock, are fixed in a 3:1 methanol:acetic acid solution, are spread out onto glass slides, and then undergo PNA-FISH processing. There are two separate experiments conducted.

Pulmonary fibrosis model of mice induced by different administration methods of bleomycin [9]
69 Twelve-weeks-old C57BL/6 mice were divided into 3 type groups (n = 7 for each control group, n = 8 for each BLM-induced pulmonary fibrosis groups), as intraperitoneal injection, intratracheal administration, and intravenous administration of bleomycin (BLM) to initiate lung fibrosis. Changes of the lung function measured through mice Pulmonary function test (PFT). Morphological changes in mice were observed by PET/CT, Masson and Picro-Sirius staining, Transmission electron microscopy (TEM). Biochemical changes were tested by Enzyme-linked immunosorbent assay (Elisa).

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After 1 week of adaptive feeding, the mice were randomly divided into nine groups, saline intraperitoneal (100 μl) control group, IPC, intraperitoneal injection of bleomycin (BLM) low dose (20 mg/kg) group, IPL, high dose(50 mg/kg) group, IPH, saline intratracheal administration (50 μl) control group, ITC, intratracheal administration of BLM low dose (3 mg/kg) group, ITL, high dose (5 mg/kg) group, ITH, saline tail vein injection(100 μl) control group, IVC, intravenous administration (tail vein) of BLM low dose(10 mg/kg) group, IVL, high dose (20 mg/kg) group, IVH. For intraperitoneal BLM group, after sanitized with 75% of alcohol, we injected 20 mg/kg and 50 mg/kg for low dose (IPL) and high dose group (IPH), respectively, with constant volume of 100 μl, 100 μl saline for control repeated 7 days. For intratracheal administration of BLM, nonsurgical transoral instillation of BLM into mice lung method was used. 2% sodium pentobarbital was prepared for anesthesia and injected into the abdominal cavity according to the corresponding dose of the mice's body weight (50 mg/kg). After anesthesia, mice lied supine on a fixed table with the limbs fixed, and the neck is shaved. After disinfection, laryngoscopes and LED light was used to make sure the BLM could accurately be perfused into trachea. In the low (ITL) and high dose (ITH) group, mice were injected with 3 mg/kg, 5 mg/kg BLM dissolved in 50 μl of saline, respectively, while in the control group mice were injected with an equal volume of saline. Tail vein injection BLM mouse model produced as followed: Put the mouse into a mouse fixer (mouse injection cone with restrainer and LED GLOBALEBIO GEGD-Q9G), expose the tail, disinfect with 75% alcohol, and expose the tail vein. In the low dose (IVL) and high dose (IVH) group, mice were given BLM injection at 10 mg/kg and 20 mg/kg, respectively, with a 1 ml syringe (BLM was dissolved with normal saline at a concentration of 2 mg/ml) for 7 days; In the saline control group, mice were injected with 5 ml/kg normal saline through the tail vein for 7 days.[9]

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Male Fischer 344 rats, 8-10 week old, weighing 150-250 g
3.5-4 mg/kg
Intra-tracheal

Absorption, Distribution and Excretion
Systemic absorption is approximately 45%.
It was reported that patients with moderately severe renal failure excreted less than 20% of the dose in the urine.
Bleomycin sulfate is not significantly absorbed from the GI tract and the drug must be administered parenterally. Bleomycin is absorbed systemically following intrapleural or intraperitoneal administration. Systemic absorption of 45% has been reported following intrapleural administration of bleomycin.
Bleomycin is rapidly absorbed following either intramuscular (IM), subcutaneous (SC), intraperitoneal (IP) or intrapleural (IPL) administration reaching peak plasma concentrations in 30 to 60 minutes. Systemic bioavailability of bleomycin is 100% and 70% following IM and SC administrations, respectively, and 45% following both IP and IPL administrations, compared to intravenous and bolus administration.
Bleomycin is widely distributed throughout the body with a mean volume of distribution of 17.5 L/sq m in patients following a 15 units/sq m IV bolus dose.
Protein binding of bleomycin is very low (1%).
For more Absorption, Distribution and Excretion (Complete) data for BLEOMYCIN (9 total), please visit the HSDB record page.

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Metabolism / Metabolites
Hepatic
Biotransformation is unknow; probably by enzymatic degradation in tissues (based on animal studdies). Tissue enzyme activity varies, which may determine toxicity and antitumor effect of bleomycin... It is not known if any of the metabolites are active.
Bleomycin is inactivated by a cytosolic cysteine proteinase enzyme, bleomycin hydrolase. The enzyme is widely distributed in normal tissues with the exception of the skin and lungs, both targets of bleomycin toxicity. Systemic elimination of the drug by enzymatic degradation is probably only important in patients with severely compromised renal function.

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Biological Half-Life
115 minutes
In patients with creatinine clearance exceeding 35 mL/minute, the serum or plasma terminal half-life of bleomycin is about 2 hours. In patients with creatinine clearances less than 35 mL/minute, the terminal half-life of the drug is inversely related to creatinine clearance.
The average steady-state concentration of bleomycin in plasma of patients receiving continuous infusions of 30 units daily for 4-5 days is approx 150 ng/mL, and there is little bound to plasma proteins. Bleomycin disappears from plasma in a biphasic fashion; the initial half-life is about 1.3 hr, & the terminal half-life is approximately 9 hr.

Interactions
General anesthetic use in patients previously treated with bleomycin may result in rapid pulmonary deterioration because bleomycin causes sensitization of lung tissue to oxygen; even with concentrations of inspired oxygen considered to be safe, pulmonary fibrosis may develop postoperatively.
Concurrent use of /antineoplastics or radiation therapy/ may result in increased bleomycin toxicity, including bone marrow depression, which is rarely caused by bleomycin alone, and mucosal and pulmonary toxicity...
Cisplatin-induced renal function impairment may result in delayed clearance and bleomycin toxicity even at low doses; caution is recommended because of the frequent combined use of these two agents.
Raynaud's phenomenon has occurred in patients receiving bleomycin and vinblastine, with or without cisplatin, and in a few patients receiving bleomycin as a single agent. Cisplatin-induced hypomagnesemia may be an additional, although not essential, factor associated with its occurrence in patients receiving combination regimens including bleomycin and cisplatin. The cause of Raynaud's phenomenon in these cases, however, is not clearly established and may involve the underlying disease or vascular compromise, bleomycin, vinblastine, hypomagnesemia, or some combination of these factors.
During chemotherapy with bleomycin and etoposide a 28-year-old male, suffering from germ cell cancer, developed acute myocardial infarction. Under treatment with heparin and aspirin the patient revealed no Q-waves in ECG and recovery was without complications. Four weeks after onset of infarction, thallium-201 scintigraphy showed only a small irreversible, posteroseptal perfusion defect; coronary angiography was not performed. The chemotherapy regimen was continued and modified to etoposide as well as cisplatin and ifosfamide without recurrence of cardiac symptoms or ECG changes.

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Adverse Effects
The most common serious adverse effect of bleomycin is pulmonary toxicity, often referred to as bleomycin pulmonary toxicity or BPT. This adverse effect sometimes leads to pulmonary fibrosis, a chronic and irreversible disease with a poor prognosis. In observing the development of pulmonary fibrosis, inflammatory cell infiltration into pulmonary endothelial cells is seen after one week of exposure to bleomycin, and fibrotic changes with elevated collagen content are seen after three weeks of exposure to bleomycin. Other changes include increased expression of fibrogenic mediators such as transforming growth factor (TGF)-beta, connective tissue growth factor, and platelet-derived growth factor (PGDF)-C in endothelial cells exposed to bleomycin. Additionally, thapsigargin-induced prostaglandin I2 and nitric oxide, which are both vasodilatory agents, are seen to decrease in endothelial pneumocytes exposed to bleomycin. The administration of bleomycin is thus seen to induce functional changes in endothelial cells of the lung leading to respiratory damage, although the exact mechanism of these changes is not entirely understood. Other adverse reactions include fever, chills, faintness, chest pain, and shortness of breath. Less serious reactions include skin pigmentation changes, itching, hypogeusia, rash, nausea, vomiting, and weight loss. Some of these symptoms appear to correlate with a hypersensitivity-type reaction. https://www.ncbi.nlm.nih.gov/books/NBK555895/

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Toxicity
Since early clinical trials in the 1960s, bleomycin pulmonary toxicity (BPT) has been a recognized adverse effect of this drug. Recent studies have described BPT rates of approximately 10% in patients taking bleomycin, with 14% of these BPT cases proving fatal. For this reason, careful monitoring for toxicities accompanied by bleomycin levels is essential. As previously described, BPT can include a serious condition known as pulmonary fibrosis. Risk factors for BPT include cumulative dose, raised creatinine, advanced age, supplemental oxygen, and reduced glomerular filtration rate. While many cases of BPT are irreversible or fatal, evidence suggests that in some surviving patients, pulmonary parameters can improve to baseline in approximately two years. Although there are no well-established therapies for reversing BPT, studies involving alternative formulations of the drug have shown promise. Numerous studies have also demonstrated that bleomycin can sometimes be substituted for less toxic chemotherapy and immunotherapy agents as a part of a multi-drug regimen, producing similar outcomes. This approach is especially useful for patients with multiple BPT risk factors and patients whose low-grade disease does not merit the risk of BPT. In addition to substituting less toxic drugs for bleomycin in chemotherapy regimens, efforts to reduce the risk of pulmonary damage have also included the investigation of lipophilic bleomycin analogs, such as liblomycin. These investigations have not yielded promising results in animal models. Thus, it remains to be seen as to whether the role of bleomycin will continue to diminish in the setting of chemotherapeutic treatment regimens. https://www.ncbi.nlm.nih.gov/books/NBK555895/

[1]. Acta Otolaryngol Suppl. 1997:529:241-4.

[2]. Am J Pathol. 1989 Feb;134(2):355-63.

[3]. Mol Med Rep. 2012 Jun;5(6):1481-6.

[4]. Mutat Res. 2012 Jun 1;734(1-2):5-11.

[5]. Stem Cell Res Ther. 2012 May 29;3(3):21.

[6]. Mutat Res. 2012 Sep 18;747(2):228-33.

[7]. Int J Clin Exp Med. 2014 Sep 15;7(9):2645-50. eCollection 2014.

[8]. Mol Cytogenet. 2016 Jun 21:9:49.

[9]. BMC Pulm Med. 2023 Mar 21;23(1):91.

Bleomycin Sulfate is a mixture of the sulfate salts of basic glycopeptide antineoplastic antibiotics isolated from Streptomyces verticillus. Bleomycin sulfate forms complexes with iron that reduce molecular oxygen to superoxide and hydroxyl radicals which cause single- and double-stranded breaks in DNA; these reactive oxygen species also induce lipid peroxidation, carbohydrate oxidation, and alterations in prostaglandin synthesis and degradation.
A complex of related glycopeptide antibiotics from Streptomyces verticillus consisting of bleomycin A2 and B2. It inhibits DNA metabolism and is used as an antineoplastic, especially for solid tumors.
See also: Bleomycin Sulfate (annotation moved to).

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Bleomycin has approval for adult use in treating squamous cell cancer of head and neck regions, Hodgkin's lymphoma, testicular carcinoma. It is also used as a sclerosing agent for malignant pleural effusions. Off-label use includes treatment of germ cell tumors and pediatric Hodgkin's lymphoma. Bleomycin is in the cytotoxic chemotherapy class of medications. This activity describes the indications, action, and contraindications for bleomycin as a valuable agent in treating various malignant cancers. This activity will highlight the mechanism of action, adverse event profile, and other key factors (e.g., off-label uses, dosing, pharmacodynamics, pharmacokinetics, monitoring, relevant interactions) pertinent for members of the interprofessional team in the treatment of patients with various cancers.https://www.ncbi.nlm.nih.gov/books/NBK555895/

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Bleomycin (BLM) is a natural antibiotic, toxic to dividing cells (G2/M-phase), also proven effective in squamous cell carcinomas (SCC). We have clinically shown that a short-range beta-emitting radionuclide combined to bleomycin (In-111-BLMC) is a tumor-targeting agent in SCCs. With higher radionuclide activities it may be possible to develop a more effective agent, to be tested in animal studies. Using a 96-well clonogenic assay we investigated three SCC cell lines, grown in our own laboratory. IC20, IC50 and IC90 values for BLM were determined. The UT-SCC-12A and UT-SCC-12B cells were originated from a primary tumor and a metastasis of the same patient. UT-SCC-12A cells were also inoculated subcutaneously into nude mice and the tumor growth was analysed. The IC50 value for UT-SCC-19A cell line was 4.0 +/- 1.3 nM. UT-SCC-12A and UT-SCC-12B were both more resistant to BLM; IC50 values were 14.2 +/- 2.8 nM and 13.0 +/- 1.1 nM, respectively. Within 35 days the weight of nude mice increased 2.8 +/- 0.6g. At 25 and 35 days after tumor inoculations the tumor volumes were 111 +/- 51 mm3 and 874 +/- 577 mm3, respectively. The calculated doubling time was 3.86 +/- 0.76 days. SCC cell lines demonstrate different sensitivity to BLM. Our SCC tumor xenograft model showed a rapid growth proper for radiochemotherapeutic studies using In-111-BLMC. The uptake of In-111-BLMC in vivo has been directly proportional to proliferation activity, and the tumors with high binding capacity could be predicted from animal model dose calculations.[1]

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Previous work in this laboratory has demonstrated increased secretion of fibroblast growth factor (MDGF) activity by alveolar macrophages obtained from mice with bleomycin-induced pulmonary fibrosis. The mechanism by which bleomycin promotes this increase in MDGF secretion is not clear, however. The purpose of this study was to determine the direct effects of bleomycin on alveolar macrophages. Normal rat alveolar macrophages obtained by lavage were cultured in the presence or absence of bleomycin; conditioned media from these cultures were dialyzed to remove bleomycin and then assayed in vitro for MDGF activity. Alveolar macrophages incubated with 0.01 microgram to 1 microgram/ml bleomycin for 18 hours secreted significantly more MDGF than macrophages incubated without bleomycin. Viability of macrophages as determined by exclusion of trypan blue and release of LDH was unaffected by any dose tested. Maximal MDGF production was seen with bleomycin doses of greater than or equal to 0.1 microgram/ml. When alveolar macrophages were incubated with 0.1 microgram/ml bleomycin for 0.5-18 hours, MDGF activity was detected as early as 1 hour, with peak responses found at 4-8 hours. Macrophages stimulated with bleomycin continued to produce significant amounts of MDGF even after bleomycin was removed and replaced with fresh (bleomycin-free) media. MDGF secretion by bleomycin-stimulated alveolar macrophages was inhibited by cycloheximide, and the 5-lipoxygenase inhibitors NDGA (nordihydroguairetic acid) and BW755c, indicating not only a requirement for protein synthesis but also for metabolites of the 5-lipoxygenase pathway of arachidonic acid metabolism for full expression of activity [2]

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Testicular cancer is the most common cancer among young men of reproductive age. Bleomycin is a frequently used drug for the treatment of several malignancies and is part of the chemotherapy protocols used for testicular cancer; however, side-effects are common. Bleomycin causes an increase in oxidative stress which has been shown to induce apoptosis in cancer cells. Curcumin (diferuloylmethane), an active component of the spice turmeric, has been demonstrated to induce apoptosis in a number of malignancies. However, to date no study has been carried out to elucidate its anticancer activity and interaction with bleomycin in testicular cancer cells. In this study, we investigated and compared the effects of curcumin, bleomycin and hydrogen peroxide (H2O2) on apoptotic signaling pathways. Curcumin (20 µM), bleomycin (400 µg/ml) and H2O2 (400 µM) incubation for 24 h decreased the viability of NTera-2 cells, and increased caspase-3, -8 and -9 activities, Bax and cytoplasmic cytochrome c levels and decreased Bcl-2 levels. The concurrent use of curcumin with bleomycin induced caspase-3, -8 and -9 activities to a greater extent in NTera-2 cells than the use of each drug alone. Our observations suggest that the effects of curcumin and bleomycin on apoptotic signaling pathways are synergistic. Therefore, we propose to use curcumin together with bleomycin to decrease its therapeutic dose and, therefore, its side-effects. [3]

C55H85N17O25S4

1512.62

1511.48

C, 43.67; H, 5.66; N, 15.74; O, 26.44; S, 8.48

9041-93-4

056-06-7; 67763-87-5; 9041-93-4 (sulfate);

72466

White to light yellow solid powder

197ºC (dec)

-7.5

21

35

36

101

2660

10

S(O)(=O)(=O)[O-].[C@@H]1(OC(=O)N)[C@H](O)[C@H](O[C@@H]([C@H]1O)CO)O[C@H]1[C@@H](O)[C@H](O)[C@@H](O[C@H]1OC(C1=CNC=N1)C(NC(=O)C1=C(C)C(N)=NC(C(NCC(N)C(N)=O)CC(N)=O)=N1)C(=O)NC(C)C(O)C(C)C(=O)NC(C(C)O)C(NCCC1SC=C(C2SC=C(C(NCCC[S+](C)C)=O)N=2)N=1)=O)CO

WUIABRMSWOKTOF-OCBSMOPSSA-N

InChI=1S/C55H83N17O21S3.H2O4S/c1-20-33(69-46(72-44(20)58)25(12-31(57)76)64-13-24(56)45(59)82)50(86)71-35(41(26-14-61-19-65-26)91-54-43(39(80)37(78)29(15-73)90-54)92-53-40(81)42(93-55(60)88)38(79)30(16-74)89-53)51(87)66-22(3)36(77)21(2)47(83)70-34(23(4)75)49(85)63-10-8-32-67-28(18-94-32)52-68-27(17-95-52)48(84)62-9-7-11-96(5)6;1-5(2,3)4/h14,17-19,21-25,29-30,34-43,53-54,64,73-75,77-81H,7-13,15-16,56H2,1-6H3,(H13-,57,58,59,60,61,62,63,65,66,69,70,71,72,76,82,83,84,85,86,87,88);(H2,1,2,3,4)/t21?,22?,23?,24?,25?,29-,30+,34?,35?,36?,37+,38+,39-,40-,41?,42-,43-,53+,54-;/m0./s1

3-[[2-[2-[2-[[2-[[4-[[2-[[6-amino-2-[3-amino-1-[(2,3-diamino-3-oxopropyl)amino]-3-oxopropyl]-5-methylpyrimidine-4-carbonyl]amino]-3-[(2R,3S,4S,5S,6S)-3-[(2R,3S,4S,5R,6R)-4-carbamoyloxy-3,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-4,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-3-(1H-imidazol-5-yl)propanoyl]amino]-3-hydroxy-2-methylpentanoyl]amino]-3-hydroxybutanoyl]amino]ethyl]-1,3-thiazol-4-yl]-1,3-thiazole-4-carbonyl]amino]propyl-dimethylsulfanium;hydrogen sulfate

NSC-125066; BLEO; BLM; NSC 125066; BLEO cell; BLEO-cell; NSC125066; BLEOcell; Bleolem; Bleomycin sulfate; Trade name: Blenoxane. Blanoxan

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, 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)

Solubility in Formulation 1: ≥ 2.08 mg/mL (Infinity 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 20.8 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: ≥ 2.08 mg/mL (Infinity 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 20.8 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: ≥ 2.08 mg/mL (Infinity 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: Saline: 30 mg/mL

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Solubility in Formulation 5: 100 mg/mL (Infinity 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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Solubility in Formulation 6: 100 mg/mL (Infinity mM) in 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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 (Please use freshly prepared in vivo formulations for optimal results.)