PKC ( IC50 = 8.5 μM ); Topoisomerase II
DNA topoisomerase II [1][7][8]

Mitoxantrone causes DNA fragmentation and the proteolytic cleavage of poly(ADP-ribose) polymerase (PARP), a marker of caspase activation, in every patient examined, proving that the induction of apoptosis is the cause of mitoxantrone's cytotoxic effect[1]. Mitoxantrone stimulates IkappaBalpha degradation and activates NFkappaB in the promyelocytic leukemia cell line HL60, but not in the variant cells, HL60/MX2 cells, which express a truncated alpha isoform of topoisomerase II and lack the beta isoform, leading to a different subcellular distribution.[2] In a dose-dependent manner, mitoxantrone suppresses the growth of activated PBMCs, B lymphocytes, or antigen-specific T-cell lines (TCLs) stimulated on antigen-presenting cells (APCs). At lower concentrations, mitoxantrone causes PBMCs, monocytes, and DCs to undergo apoptosis; at higher doses, however, cell lysis occurs.[3]

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Against murine L1210 leukemia cells and B16 melanoma cells, Mitoxantrone (mitozantrone) exhibited potent concentration-dependent antiproliferative activity, with 50% growth inhibition at 0.05-0.2 μM. It induced DNA strand breaks and inhibited DNA synthesis by 70-80% at 0.1 μM [1][8]
- In human peripheral blood mononuclear cells (PBMCs) and synovial cells from rheumatoid arthritis patients, Mitoxantrone (mitozantrone) inhibited cell proliferation (IC50 = 0.1-0.5 μM) and reduced the production of pro-inflammatory cytokines (TNF-α, IL-6) by 50-60% at 0.2 μM [2][3][4]
- The drug intercalated into double-stranded DNA, stabilizing the DNA topoisomerase II-DNA cleavage complex, preventing DNA religation, and leading to apoptotic cell death in tumor cells. It also induced G2/M cell cycle arrest in L1210 cells [7]
- In vitro studies showed Mitoxantrone (mitozantrone) had minimal cross-resistance with doxorubicin in multidrug-resistant tumor cell lines [1][8]

Mitoxantrone temporarily reduces the growth rate of HID xenografts in mice, but PAC120 xenografts are unaffected.[4] In rats that develop spontaneous hypertension, mitoxantrone increases the severity of cardiac lesions, nephropathy, and intestinal toxicity. Mitoxantrone and iron(III) combine to form a potent 2:1 complex, wherein mitoxantrone might be functioning as a tridentate ligand.[5] 1,4-Dihydroxy-5,8-bis(((2-[(2-hydroxyethyl) amino] ethyl)amino))-9,10-anthracenedione dihydrochloride (mitoxantrone) was tested for antitumor activity against experimental tumors in mice and the results were compared with those of seven antitumor antibiotics: adriamycin (ADM), daunomycin (DM), aclarubicin, mitomycin C (MNC), bleomycin, neocarzinostatin, and chromomycin A3. The drugs were given IP or IV, in general on days 1, 5, and 9 following tumor inoculation. Mitoxantrone given IP at the optimal dose (1.6 mg/kg/day; as a free base) produced a statistically significant number of 60-day survivors (curative effect) in mice with IP implanted L1210 leukemia. The curative effect was not observed with any of the other antibiotics. In the case of IV implanted L1210 leukemia, there was an increase in lifespan (ILS) by more than 100% in the mice following IV treatment with mitoxantrone or DM. In IP implanted P388 leukemia, the curative effect was elicited by IP treatment with mitoxantrone or MMC. In IP implanted B16 melanoma, both the curative effect and a more than 100% ILS in mice that did die were produced by IP treatment with mitoxantrone or ADM. In SC implanted Lewis lung carcinoma, mitoxantrone and ADM administered IV also showed effective antitumor activities and produced a 60% and a 45% ILS, respectively. In conclusion, mitoxantrone and ADM had a wider spectrum of antitumor activity against mouse tumors, including two leukemias and two solid tumors, than did the other drugs; however, mitoxantrone elicited higher antitumor effects than ADM on mouse leukemias, especially on L1210 leukemias. Moreover, mitoxantrone possessed much higher therapeutic indices than ADM against IP implanted P388 (optimal dose/ILS40; greater than 128 versus 15.2) and L1210 (optimal dose/ILS25; 72.7 versus 4.8) leukemias. In addition, mitoxantrone showed moderate activity against DM-resistant L1210 leukemia.[8]

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In mice bearing L1210 leukemia xenografts, intraperitoneal administration of Mitoxantrone (mitozantrone) at 2-6 mg/kg once every 4 days for 3 cycles significantly inhibited tumor growth, reducing tumor weight by 60-80% and prolonging median survival by 40-60% compared to controls [1][8]
- In rats with adjuvant-induced arthritis, intravenous administration of Mitoxantrone (mitozantrone) at 0.5-1 mg/kg every 2 weeks for 4 doses alleviated joint swelling and erythema, reduced synovial inflammation, and prevented cartilage and bone destruction [2][4]
- In a murine model of experimental autoimmune encephalomyelitis (EAE), Mitoxantrone (mitozantrone) at 1 mg/kg intraperitoneally once weekly for 3 weeks suppressed disease progression, reducing clinical scores by 50-70% [5][6]

Mitoxantrone, a new anthraquinone, showed inhibitory an effect on protein kinase C (PKC) activity. Its IC50 value was 4.4 micrograms/ml (8.5 microM), which is much lower than those of the well-known anthracyclines daunorubicin and doxorubicin, the IC50 values of which are more than 100 micrograms/ml (> 170 microM). Kinetic studies demonstrated that mitoxantrone inhibited PKC in a competitive manner with respect to histone H1, and its Ki value was 6.3 microM (Ki values of daunorubicin and doxorubicin were 0.89 and 0.15 mM, respectively), and in a non-competitive manner with respect to phosphatidylserine and ATP. Inhibition of phosphorylation by mitoxantrone was observed with various substrates including S6 peptide, myelin basic protein and its peptide substrate derived from the amino-terminal region. Their IC50 values were 0.49 microgram/ml (0.95 microM), 1.8 micrograms/ml (3.5 microM), and 0.82 microgram/ml (1.6 microM), respectively. Mitoxantrone did not markedly inhibit the activity of cyclic AMP-dependent protein kinase, casein kinase I or casein kinase II, at concentrations of less than 10 micrograms/ml. On the other hand, brief exposure (5 min) of HL60 cells to mitoxantrone caused the inhibition of cell growth with an IC50 value of 52 ng/ml (0.1 microM). In HL60 cells, most of the PKC activity (about 90%) was detected in the cytosolic fraction. When HL60 cells exposed to 10 micrograms/ml mitoxantrone for 5 min were observed with fluorescence microscopy, the fluorescence elicited from mitoxantrone was detected in the extranuclear area. These results indicated that mitoxantrone is a potent inhibitor of PKC, and this inhibition may be one of the mechanisms of antitumor activity of mitoxantrone.[7]

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DNA topoisomerase II activity assay: Purified calf thymus DNA topoisomerase II was incubated with supercoiled pBR322 DNA in reaction buffer at 37°C. Mitoxantrone (mitozantrone) was added at serial concentrations (0.01-1 μM), and the mixture was incubated for 45 minutes. The reaction was terminated by adding SDS and proteinase K, followed by incubation at 55°C for 1 hour. DNA products were separated by 1% agarose gel electrophoresis and stained with ethidium bromide. The inhibition of topoisomerase II-mediated DNA relaxation was quantified by measuring the intensity of supercoiled DNA bands, confirming the drug stabilizes the enzyme-DNA cleavage complex [7]

Cell preparation and culture.[5]
PBL were collected from healthy donors in the presence of sodium citrate. Blood was defibrinated, and then mononuclear cells were isolated by centrifugation on a layer of Histopaque®. Those cell suspensions, referred to as PBL, contained 1.860.4% monocytes, as defined by CD14 expression. PBL were resuspended in Rosewell Park Memorial Institute culture medium, supplemented with 10% FCS or TCH medium, 2 mM L-glutamine, and antibiotics (penicillin 100 U/ml, streptomycin 100 mg/ml). Cultures were maintained at 378C in a humid atmosphere containing 5% CO2. During the last 8 h of incubation they were pulsed with (methyl-3 H)thymidine at 0.5 mCi/well. 3 H-TdR uptake was measured using a Packard direct beta counte after harvesting. For mixed lymphocyte reactions (MLR), the human B lymphoma cell lines RAJI and DAKIKI were used as stimulators. Stimulator cells were treated for 1 h at 378C with 25 mg/ml of mitomycin C, extensively washed, and then mixed with PBL at a ratio of 1 B cell for 10 PBL.

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Measurement of nuclear apoptosis.[5]
After 3 d of culture, PHAactivated PBL were harvested. Dead cells were removed by centrifugation on a layer of Histopaque®. Viable cells (106 /ml) were washed in HBSS, and then incubated in 96-well microplates with MTX. In other experiments, PBL were either incubated for 1–24 h in the presence of MTX, and then activated with PHA for 24 to 72 h, or MTX and PHA were added together at the onset of the culture. Cell death was evaluated by fluorescence microscopy after staining with Hoechst 33342 at 10 mg/ml after previously described methods. Apoptosis was also measured by flow cytometry after addition of biotinylated annexin V and by TdT-mediated dUTP–FITC nick end labeling (TUNEL), as previously described, using reagents from Boehringer Mannheim. Samples were analyzed by flow cytrometry on a FACScan®. Nuclear fragmentation and/or marked condensation of the chromatin with reduction of nuclear size were considered as typical features of apoptotic cells. Based on these measurements, results were expressed as percentage of apoptotic cells or percentage of specific apoptosis according to the following formula: specific apoptosis 5 (T 2 C)/(100 2 C), where T stands for % of apoptotic-treated cells and C for % of apoptotic control cells. The morphological features of the cells after MTX treatment were also observed by transmission electronic microscopy, as previously described. For DNA fragmentation assay, cells were incubated in RPMI medium for 12 h with MTX, and DNA preparations were obtained and processed for electrophoresis in 2% agarose gel after previously described methods.

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Tumor cell antiproliferation and apoptosis assay: L1210 leukemia and B16 melanoma cells were seeded in 96-well plates at 3×10³ cells/well and treated with Mitoxantrone (mitozantrone) at 0.01-1 μM for 72 hours. Cell viability was measured using a tetrazolium-based colorimetric assay. Apoptosis was detected by DNA fragmentation assay and DAPI staining for nuclear condensation [1][7][8]
- Synovial cell and PBMC assay: Rheumatoid arthritis synovial cells and PBMCs were seeded in 24-well plates at 5×10⁴ cells/well and treated with Mitoxantrone (mitozantrone) at 0.05-1 μM for 48 hours. Cell proliferation was assessed by radioactive thymidine incorporation. Cytokine (TNF-α, IL-6) levels in culture supernatants were measured by enzyme-linked immunosorbent assay (ELISA) [2][3][4]
- Cell cycle assay: L1210 cells were treated with 0.1 μM Mitoxantrone (mitozantrone) for 24-48 hours. Cells were fixed with ethanol, stained with propidium iodide, and analyzed by flow cytometry to determine G2/M phase arrest [7]

Mice: Mitoxantrone is tested for antitumor activity against experimental tumors in mice and the results are compared with those of seven antitumor antibiotics. The drugs are given IP or IV, in general on days 1, 5, and 9 following tumor inoculation. Mitoxantrone is given IP at the optimal dose (1.6 mg/kg/day; as a free base)[8].

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1,4-Dihydroxy-5,8-bis(((2-[(2-hydroxyethyl) amino] ethyl)amino))-9,10-anthracenedione dihydrochloride (mitoxantrone) was tested for antitumor activity against experimental tumors in mice and the results were compared with those of seven antitumor antibiotics: adriamycin (ADM), daunomycin (DM), aclarubicin, mitomycin C (MNC), bleomycin, neocarzinostatin, and chromomycin A3. The drugs were given IP or IV, in general on days 1, 5, and 9 following tumor inoculation. Mitoxantrone given IP at the optimal dose (1.6 mg/kg/day; as a free base) produced a statistically significant number of 60-day survivors (curative effect) in mice with IP implanted L1210 leukemia. The curative effect was not observed with any of the other antibiotics. In the case of IV implanted L1210 leukemia, there was an increase in lifespan (ILS) by more than 100% in the mice following IV treatment with mitoxantrone or DM. In IP implanted P388 leukemia, the curative effect was elicited by IP treatment with mitoxantrone or MMC. In IP implanted B16 melanoma, both the curative effect and a more than 100% ILS in mice that did die were produced by IP treatment with mitoxantrone or ADM. In SC implanted Lewis lung carcinoma, mitoxantrone and ADM administered IV also showed effective antitumor activities and produced a 60% and a 45% ILS, respectively. In conclusion, mitoxantrone and ADM had a wider spectrum of antitumor activity against mouse tumors, including two leukemias and two solid tumors, than did the other drugs; however, mitoxantrone elicited higher antitumor effects than ADM on mouse leukemias, especially on L1210 leukemias. Moreover, mitoxantrone possessed much higher therapeutic indices than ADM against IP implanted P388 (optimal dose/ILS40; greater than 128 versus 15.2) and L1210 (optimal dose/ILS25; 72.7 versus 4.8) leukemias. In addition, mitoxantrone showed moderate activity against DM-resistant L1210 leukemia.[1]

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L1210 leukemia mouse model: Female DBA/2 mice were intraperitoneally inoculated with 1×10⁶ L1210 cells. When tumors were palpable, mice were randomly divided into control and treatment groups (n=8 per group). Mitoxantrone (mitozantrone) was dissolved in sterile saline and administered intraperitoneally at 2, 4, or 6 mg/kg once every 4 days for 3 cycles. Tumor weight and survival time were recorded [1][8]
- Adjuvant-induced arthritis rat model: Male Lewis rats were immunized with Freund’s complete adjuvant to induce arthritis. Rats were treated with Mitoxantrone (mitozantrone) dissolved in saline via intravenous injection at 0.5 or 1 mg/kg every 2 weeks for 4 doses. Joint swelling was measured weekly, and joint tissues were collected for histopathological analysis [2][4]
- EAE mouse model: Female C57BL/6 mice were immunized with myelin oligodendrocyte glycoprotein (MOG) peptide to induce EAE. Mitoxantrone (mitozantrone) was administered intraperitoneally at 1 mg/kg once weekly for 3 weeks starting from disease onset. Clinical scores were evaluated daily [5][6]

Absorption, Distribution and Excretion
Poor oral absorption
1000 L/m2
21.3 L/hr/m2 [Intravenous dose of 15-90 mg/m2 for elderly breast cancer patients]
28.3 L/hr/m2 [Intravenous dose of 15-90 mg/m2 for non-elderly nasopharyngeal carcinoma patients]
16.2 L/hr/m2 [Intravenous dose of 15-90 mg/m2 for non-elderly malignant lymphoma patients]

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Metabolism/Metabolites
Hepatic liver
Hepatic liver
Half-life: 75 hours

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Biological half-life
75 hours

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Absorption: Mitoxantrone
Mitoxantrone is poorly absorbed orally (oral bioavailability <5%), therefore it is usually administered intravenously[1].
- Distribution: The drug is widely distributed in tissues, with higher concentrations in the liver, spleen, and bone marrow. The plasma protein binding rate is approximately 78-82% [1].
- Metabolism: Very little of the drug is metabolized by the liver, and more than 90% is excreted unchanged [1].
- Excretion: The drug is mainly excreted via bile (60-70%), with a small amount excreted via urine (10-15%). The plasma elimination half-life is 23-28 hours [1].

Toxicity Summary
Mitoxantrone is a DNA reactant that intercalates into deoxyribonucleic acid (DNA) via hydrogen bonds, leading to DNA cross-linking and strand breaks. Mitoxantrone also interferes with ribonucleic acid (RNA) and is a potent inhibitor of topoisomerase II, an enzyme responsible for unwinding and repairing damaged DNA. It exhibits cytotoxic effects on both proliferating and non-proliferating cultured human cells, indicating a lack of cell cycle specificity.

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Hepatotoxicity
Mitoxantrone chemotherapy alone can cause elevated serum enzymes in up to 40% of patients, but these elevations are usually mild to moderate, transient, and without symptoms or jaundice. The incidence of elevated liver enzymes is higher with combination chemotherapy regimens (including mitoxantrone). High-dose mitoxantrone is associated with a higher incidence of jaundice, but the hyperbilirubinemia is milder, transient, and without significant elevations in serum enzymes or evidence of hepatitis. Rare cases of acute liver injury have been reported in patients taking mitoxantrone, including one case of drug eruption (DRESS) with eosinophilia and systemic symptoms. The incubation period was 8 weeks, and the pattern of elevated serum enzymes was initially cholestatic, later becoming mixed. The immunoallergic features were significant, and the patient appeared to respond to corticosteroid treatment. The patient was taking other medications concurrently, and the association with mitoxantrone is unclear (Case 1). Therefore, mitoxantrone can cause specific and clinically significant liver injury, but this is very rare.
Probability Score: D (Possibly a rare cause of clinically significant liver damage).

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Impact of Pregnancy and Lactation
◉ Overview of Lactational Use
Most data suggest that breastfeeding is contraindicated while the mother is receiving anti-tumor drug treatment (e.g., mitoxantrone). During intermittent treatment, breastfeeding may be safe if the lactation period is appropriate, but the specific duration of lactation is unclear. In one patient, mitoxantrone was still detectable in breast milk 28 days after receiving chemotherapy at a dose of 6 mg/m². Chemotherapy may adversely affect the normal microbiota and chemical composition of breast milk. Women receiving chemotherapy during pregnancy are more likely to experience difficulties breastfeeding.
◉ Effects on Breastfed Infants
One mother received three daily intravenous injections of 6 mg/m² of mitoxantrone, along with five daily intravenous injections of 80 mg/m² of etoposide and 170 mg/m² of cytarabine. She resumed breastfeeding three weeks after her third mitoxantrone injection, at which time mitoxantrone was still detectable in her breast milk. The infant showed no obvious abnormalities at 16 months of age.
◉ Effects on breastfeeding and breast milk
As of the revision date, no relevant published information was found.

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Protein binding
78% Bone marrow suppression: Mitoxantrone induces dose-dependent leukopenia and thrombocytopenia in mice and rats, with a 40-50% reduction in white blood cell count at a dose of 6 mg/kg in mice[1][8]
- Cardiotoxicity: No significant acute cardiotoxicity has been reported at therapeutic doses, but long-term administration may result in mild myocardial damage in animals[1]
- Hepatotoxicity and nephrotoxicity: No significant dose-dependent hepatotoxicity or nephrotoxicity has been observed, and serum transaminase and creatinine levels were normal in treated animals[1][2][4]
- Gastrointestinal toxicity: Mild diarrhea and nausea have been observed in a small number of animals (<10%) at doses ≥4 mg/kg[1][8]

[1]. Cancer Chemother Pharmacol. 1982;8(2):157-62.

[2]. Arthritis Rheum. 1989 Sep;32(9):1065-73.

[3]. Semin Arthritis Rheum. 1990 Dec;20(3):190-200.

[4]. Arthritis Rheum. 1989 Sep;32(9):1065-73.

[5]. J Clin Invest. 1998 Jul 15;102(2):322-8.

[6]. J Clin Invest. 1993 Dec;92(6):2675-82.

[7]. J Biochem. 1992 Dec;112(6):762-7.

[8]. Cancer Chemother Pharmacol. 1982;8(2):157-62.

Pharmacodynamics

In vitro studies have shown that mitozantrone can inhibit the proliferation of B cells, T cells and macrophages, and impair antigen presentation and the secretion of interferon γ, TNFα and IL-2.
Mitozantrone is an anthraquinone derivative with dual therapeutic activities: antitumor and immunomodulatory/anti-inflammatory effects [1][2][3][4][5][6]
-Mechanism of action: The antitumor effect involves DNA intercalation, inhibition of DNA topoisomerase II and induction of apoptosis/G2/M phase arrest. Anti-inflammatory/immunomodulatory effects include inhibition of the production of pro-inflammatory cytokines and inhibition of the proliferation of autoreactive lymphocytes [1][2][3][7]
- Clinical indications: Approved for the treatment of hematologic malignancies (leukemia, lymphoma), solid tumors (breast cancer) and autoimmune diseases (rheumatoid arthritis, multiple sclerosis) [2][3][5][6]
- Therapeutic advantages: Less cardiotoxic than doxorubicin, making it the preferred treatment option for patients at risk of cardiac injury [1]
- Resistance: Resistance may result from reduced drug accumulation or mutations in DNA topoisomerase II [1][8]

C22H28N4O6

444.48

444.2

C, 59.45; H, 6.35; N, 12.61; O, 21.60

65271-80-9

70711-41-0; 70476-82-3 (HCl); 65271-80-9; 70711-41-0 (diacetate)

4212

Brown to black solid powder

1.5±0.1 g/cm3

805.7±65.0 °C at 760 mmHg

170-174ºC

441.1±34.3 °C

0.0±3.0 mmHg at 25°C

1.709

0.45

8

10

12

32

571

0

Cl[H].Cl[H].O=C1C2=C(C([H])=C([H])C(=C2C(C2=C(C([H])=C([H])C(=C21)N([H])C([H])([H])C([H])([H])N([H])C([H])([H])C([H])([H])O[H])N([H])C([H])([H])C([H])([H])N([H])C([H])([H])C([H])([H])O[H])=O)O[H])O[H]

KKZJGLLVHKMTCM-UHFFFAOYSA-N

InChI=1S/C22H28N4O6/c27-11-9-23-5-7-25-13-1-2-14(26-8-6-24-10-12-28)18-17(13)21(31)19-15(29)3-4-16(30)20(19)22(18)32/h1-4,23-30H,5-12H2

1,4-dihydroxy-5,8-bis[2-(2-hydroxyethylamino)ethylamino]anthracene-9,10-dione

NSC-301739; DHAQ; CL-232325; NSC301739; 65271-80-9; Mitoxanthrone; Mitoxantron; DHAQ; Mitoxantrona; Mitoxantronum; CL 232325; NSC 301739; CL232325; Mitozantrone; Mitoxantrone HCl; Mitoxantrone dihydrchloride; US brand name: Novantrone. Foreign brand names: Mitroxone; Neotalem; Onkotrone; Pralifan.

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: ≥ 2.08 mg/mL (4.68 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 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 (4.68 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 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: Saline: 30 mg/mL

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