Cyclophosphamide induces outer membrane blebbing, leading to DNA fragmentation, as shown by TUNEL staining of free 3'-OH DNA termination, and induction of caspase 3 and caspase 7 in 9L/P450 cells Complete Bcl-2 expression of the substrate PARP blocks activation of initiator caspases as well as effector caspase 3 in cells treated with the activator cyclophosphamide. Bcl-2 suppresses cytotoxicity, but does not inhibit cellular activation of cyclophosphamide. Cyclophosphamide can reversely transcribe AChE, with an IC50 of 511 μM [2]. Carbon tetrachloride does not impact the direct cytotoxicity of cyclophosphamide or 4-cyclophosphamide to cultivated cells [3].

In SW1 tumor-labeled C3H mice, cyclophosphamide (2 mg/mouse) administered intraperitoneally in 0.1 mL PBS raises the proportion of cells positive for CD3, CD4, or CD8 in the tumor and spleen [4].

Animal/Disease Models: Six to eight weeks of age Female C3H/HeN mice bearing SW1 tumors [4]
Doses: 2 mg/mouse
Route of Administration: intraperitoneal (ip) injection; ]. 2 mg/mouse in 0.1 mL PBS; 4-day
Experimental Results: Increased percentage of cells stained for CD3, CD4, or CD8 in spleen and tumors.

Absorption, Distribution and Excretion
After oral administration, peak concentrations occur at one hour.
Cyclophosphamide is eliminated primarily in the form of metabolites. 10-20% is excreted unchanged in the urine and 4% is excreted in the bile following IV administration.
30-50 L
Total body clearance = 63 ± 7.6 L/kg.
Cyclophosphamide is well absorbed orally.
PLACENTAL TRANSFER OF (14)CARBON-CYCLOPHOSPHAMIDE HAS BEEN DEMONSTRATED IN MICE; AND A POSITIVE CORRELATION BETWEEN THE ALKYLATION OF EMBRYONIC DNA AND PRODUCTION OF CONGENITAL ABNORMALITIES IN MICE HAS BEEN REPORTED. A SIMILAR CORRELATION HAS BEEN FOUND FOR NUCLEAR-DNA-DEPENDENT RNA POLYMERASES IN RAT EMBRYOS. IN MOST SPECIES, CYCLOSPHSPHAMIDE IS RAPIDLY ABSORBED, METABOLIZED AND EXCRETED. IN RATS, THE SPECIFIC ACTIVITY IN TISSUES IS HIGHEST WITHIN 20-30 MIN FOLLOWING IP INJECTION; UP TO 75% OF THE RADIOACTIVITY IS EXCRETED WITHIN 5-8 HR. /MONOHYDRATE/
AFTER ITS IV INJECTION, THE DRUG IS RAPIDLY ABSORBED FROM THE BLOOD. IN PATIENTS RECEIVING 6.7-80 MG/KG BODY WT PER DAY OF RING LABELLED CYCLOPHOSPHAMIDE, RADIOACTIVITY WAS DISTRIBUTED RAPIDLY TO ALL TISSUES: ITS HALF LIFE IN THE PLASMA WAS 6.5 HOURS. NO RADIOACTIVITY WAS FOUND IN THE EXPIRED AIR OR FECES. RECOVERY OF RADIOACTIVITY IN URINE HAS BEEN REPORTED TO BE BETWEEN 50-68%, MAINLY IN THE FORM OF CARBOXYPHOSPHAMIDE AND PHOSPHORAMIDE MUSTARD; 10-40% OF THE DRUG WAS EXCRETED UNCHANGED; AND 56% OF THE REACTIVE METABOLITES WERE BOUND TO PLASMA PROTEINS. /MONOHYDRATE/
In a cross sectional study, the urine of 20 hospital workers occupationally exposed to cyclophosphamide and 21 unexposed controls was monitored for excretion of cyclophosphamide. During the week in which samples were collected, most of the workers handled cyclophosphamide fewer than 5 times and the amount handled each time ranged from 100-1000 mg (mean + or - 350 mg). All workers claimed to have taken regular safety precautions; ie, at least wearing gloves during handling. The drug was identified in 5 cases (range: 0.7-2.5 ug cyclophosphamide excreted/24 hr urine). A clear relationship between cyclophosphamide handling and urinary detection was shown. 4 of 5 persons with detectable urinary cyclophosphamide had handled cyclophosphamide 10 times or more during the week.
For more Absorption, Distribution and Excretion (Complete) data for CYCLOPHOSPHAMIDE (7 total), please visit the HSDB record page.

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Metabolism / Metabolites
Metabolism and activation occurs at the liver. 75% of the drug is activated by cytochrome P450 isoforms, CYP2A6, 2B6, 3A4, 3A5, 2C9, 2C18, and 2C19. The CYP2B6 isoform is the enzyme with the highest 4-hydroxylase activity. Cyclophosphamide undergoes activation to eventually form active metabolites, phosphoramide mustard and acrolein. Cyclophosphamide appears to induce its own metabolism which results in an overall increase in clearance, increased formation of 4-hydroxyl metabolites, and shortened t1/2 values following repeated administration.
... /Cyclophosphamide/ is activated by the hepatic cytochrome P450 system. Cyclophosphamide is first converted to 4-hydroxycyclophosphamide, which is in a steady state with the acyclic tautomer aldophosphamide. In vitro studies with human liver microsones & cloned P450 isoenzymes have shown that cyclophosphamide is activated by the CYP2B group of P450 isoenzymes... . 4-hydroxycyclophosphamide may be oxidized further by aldehyde oxidase either in liver or in tumor tissue & perhaps by other enzymes, yielding the metabolites carboxyphosphamide & 4-ketocyclophsphamide, neither of which possesses significant biological activity. It appears that hepatic damage is minimized by theses secondary reactions, whereas significantl amoutns of the active metabolies, such as 4-hydroxycyclophosphamide & its tautomer, aldophosphamed, are transported to the target sites by the circulatory system. In tumor cells, the aldophosphamide cleaves spontaneously, generating stoichiometric amounts of phosphoramide mustard & acrolein. The former is believed to be responsible for antitumor effects. The latter cmpd may be responsible for the hemorrhagic cystitis seen during therapy with cyclophosphamide. Cystitis can be reduced in intensity or prevented by the pareneteral admin of mesna, a sulfhydryl cmpd that reacts readily with acrolein in the acid environment of the urinary tract. ... Urinary & fecal recovery of unchanged cyclophosphamide is minimal after iv admin. Maximal concns in plasma are achieved 1 hr after oral admin, & the half-life in plasma is about 7 hr.
SHEEP WERE ORALLY DOSED WITH CYCLOPHOSPHAMIDE. IN COLLECTED URINE, 2 METABOLITES WERE OBSERVED AND CHARACTERIZED AS O-(2-CARBOXYETHYL)-N,N-BIS (2-CHLOROETHYL)PHOSPHORODIAMIDATE & 2-(BIS (2-CHLOROETHYL)AMINO)TETRAHYDRO-2H-1,3,2-OXAZOPHOSPHORINE 2,4-DIOXIDE (4-KETOCYCLOPHOSPHAMIDE).
A REACTIVE METABOLITE, N,N-BIS-(2-CHLOROETHYL)PHOSPHORODIAMIDIC ACID, WHICH POSSESSES POTENT ALKYLATING & CYTOTOXIC PROPERTIES, HAS RECENTLY BEEN ISOLATED FROM THE OXYGENATION PRODUCTS OF CYCLOPHOSPHAMIDE AND MOUSE LIVER MICROCHROMOSOMES.
Cyclophosphamide is well absorbed orally, and peak plasma levels appear about one hour after oral use. It is also administered intravenously. This drug is metabolized in the liver to the cytotoxic metabolite, 4-hydroxycyclophosphamide, which is in equilibrium with the acyclic tautomer, aldophosphamide. Although the major fraction of these metabolites is oxidized further to inactive products, some aldophosphamide is converted to phophoramidemustard, which alkylates DNA, and to acrolein.
For more Metabolism/Metabolites (Complete) data for CYCLOPHOSPHAMIDE (8 total), please visit the HSDB record page.
Metabolism and activation occurs at the liver. 75% of the drug is activated by cytochrome P450 isoforms, CYP2A6, 2B6, 3A4, 3A5, 2C9, 2C18, and 2C19. The CYP2B6 isoform is the enzyme with the highest 4-hydroxylase activity. Cyclophosphamide undergoes activation to eventually form active metabolites, phosphoramide mustard and acrolein. Cyclophosphamide appears to induce its own metabolism which results in an overall increase in clearance, increased formation of 4-hydroxyl metabolites, and shortened t1/2 values following repeated administration.
Route of Elimination: Cyclophosphamide is eliminated primarily in the form of metabolites. 10-20% is excreted unchanged in the urine and 4% is excreted in the bile following IV administration.
Half Life: 3-12 hours

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Biological Half-Life
3-12 hours
Maximal concns in plasma are achieved 1 hr after oral admin, & the half-life in plasma is about 7 hr.

Hepatotoxicity
Mild and transient elevations in serum aminotransferase levels are found in up to 43% of patients with cancer who are treated with cyclophosphamide. The abnormalities are generally asymptomatic and transient and do not require dose modification. Enzyme elevations are more common with higher doses and with intravenous therapy. In some instances, marked elevations arise warranting dose modification or discontinuation of cyclophosphamide (Case 3).
Clinically apparent liver injury from standard doses of cyclophosphamide is uncommon, but several case reports of acute liver injury with jaundice have been published (Cases 1 and 2). The onset is within 2 to 8 weeks of starting cyclophosphamide and the pattern of serum enzyme elevations is hepatocellular. Immunoallergic and autoimmune features are uncommon. The injury in most cases is self-limited and resolves within 1 to 3 months of stopping; however, fatal instances have been reported. Recurrence on reexposure has been described.
High doses of cyclophosphamide given as chemotherapy of cancer or as myeloablative therapy in combination of total body irradiation or busulfan in preparation for hematopoietic cell transplantation can induce sinusoidal obstruction syndrome (veno-occlusive disease), which can be severe leading to acute liver failure and death. The onset of injury is usually within 10 to 20 days of the myeloablation and is characterized by a sudden onset of abdominal pain, weight gain, ascites, marked increase in serum aminotransferase levels (and lactic dehydrogenase), and subsequent jaundice and hepatic dysfunction. The severity of sinusoidal obstruction syndrome varies from a transient, self limited injury to acute liver failure. The diagnosis is usually based on clinical features of tenderness and enlargement of the liver, weight gain, ascites and jaundice. Liver biopsy is diagnostic but often contraindicated, because of severe thrombocytopenia after hematopoietic cell transplantation.
Likelihood score: B (highly likely cause of clinically apparent liver injury).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Cyclophosphamide appears in milk in potentially toxic amounts; additionally, highly toxic active metabolites could add to the risk to the infant. Neutropenia has been reported in 2 infants whose mothers breastfed them while receiving cyclophosphamide. Most sources consider breastfeeding to be contraindicated during maternal cytotoxic antineoplastic drug therapy, especially alkylating agents such as cyclophosphamide. Although some have suggested withholding breastfeeding for 1 to 3 days after a dose, it appears to take more than 21 days for the drug and its metabolites to be completely eliminated from breastmilk. Some authors’ data suggest that it might take 6 weeks for milk levels to drop to a safe level after a dose of cyclophosphamide 750 mg/sq. m.
Chemotherapy may adversely affect the normal microbiome and chemical makeup of breastmilk. Women who receive chemotherapy during pregnancy are more likely to have difficulty nursing their infant.
◉ Effects in Breastfed Infants
In one 23-day-old infant, neutropenia, thrombocytopenia and a low hemoglobin were possibly caused by cyclophosphamide after 3 days of maternal treatment with cyclophosphamide 6 mg/kg IV daily (total dose 300 mg).
In a 4-month-old, neutropenia was probably caused by cyclophosphamide in a mother 9 days after the last of 6 weekly doses of 800 mg cyclophosphamide intravenously, 2 mg vincristine intravenously and daily doses of 30 mg of prednisolone orally. Neutropenia persisted at least 12 days and was accompanied by a brief episode of diarrhea.
A woman was diagnosed with B-cell lymphoma at 27 weeks of pregnancy. Labor was induced at 34 4/7 weeks and treatment was begun with a standard regimen of rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone in unspecified doses on a 21-day cycle, starting on day 2 postpartum. She pumped and discarded her milk and fed her infant donor milk for the first 10 days of each cycle and then breastfed her infant for the remaining 10 days before the next treatment cycle. The 10-day period of breastfeeding abstinence was determined by using about 3 half-lives of vincristine. After completion of 4 cycles of chemotherapy, her infant was reportedly healthy and developing without any complications.
◉ Effects on Lactation and Breastmilk
A case of euprolactinemic galactorrhea was reported in a 55-year-old woman receiving cyclophosphamide for pemphigus vulgaris. One month after starting cyclophosphamide 50 mg daily she experienced fullness in both breasts and bilateral milky nipple discharge. No hormonal abnormalities were found. After cyclophosphamide was discontinued, her symptoms resolved completely and did not recur. Galactorrhea was probably caused by cyclophosphamide.
Telephone follow-up study was conducted on 74 women who received cancer chemotherapy at one center during the second or third trimester of pregnancy to determine if they were successful at breastfeeding postpartum. Only 34% of the women were able to exclusively breastfeed their infants, and 66% of the women reported experiencing breastfeeding difficulties. This was in comparison to a 91% breastfeeding success rate in 22 other mothers diagnosed during pregnancy, but not treated with chemotherapy. Other statistically significant correlations included: 1. mothers with breastfeeding difficulties had an average of 5.5 cycles of chemotherapy compared with 3.8 cycles among mothers who had no difficulties; and 2. mothers with breastfeeding difficulties received their first cycle of chemotherapy on average 3.4 weeks earlier in pregnancy. Of the 56 women who received a cyclophosphamide-containing regimen, 34 had breastfeeding difficulties.

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Protein Binding
20% of cyclophosphamide is protein bound with no dose dependent changes. Some metabolites are protein bound to an extent greater than 60%.

[1]. Cyclophosphamide induces caspase 9-dependent apoptosis in 9L tumor cells. Mol Pharmacol. 2001 Dec;60(6):1268-1279.

[2]. Inhibition of human acetylcholinesterase by cyclophosphamide. Toxicology. 1995 Jan 19;96(1):1-6.

[3]. Carbon tetrachloride-induced increase in the antitumor activity of cyclophosphamide in mice: a pharmacokineticstudy. Cancer Chemother Pharmacol. 1984;12(3):167-72.

[4]. Administration of cyclophosphamide changes the immune profile of tumor-bearing mice. J Immunother. 2010 Jan;33(1):53-9.

[5]. Kinetics of Cyclophosphamide Metabolism in Humans, Dogs, Cats, and Mice and Relationship to Cytotoxic Activity and Pharmacokinetics. Drug Metab Dispos. 2019, 47, 3.

[6]. Pharmacological administration of recombinant human AMH rescues ovarian reserve and preserves fertility in a mouse model of chemotherapy, without interfering with anti-tumoural effects. J Assist Reprod Genet. 2019, 36, 9.

[7]. Probiotic Lactobacillus strains protect against myelosuppression and immunosuppression in cyclophosphamide-treated mice. Int Immunopharmacol. 2014, 22, 1.

Cyclophosphamide (Hydrated) can cause cancer according to California Labor Code. It can cause developmental toxicity according to an independent committee of scientific and health experts. It can cause female reproductive toxicity and male reproductive toxicity according to state or federal government labeling requirements.
Cyclophosphamide is a fine white crystalline powder. Odorless with a slightly bitter taste. Melting point 41-45 °C. A 2% solution has pH of 4 to 6. Used medicinally as an antineoplastic agent.
Cyclophosphamide is a phosphorodiamide that is 1,3,2-oxazaphosphinan-2-amine 2-oxide substituted by two 2-chloroethyl groups at the amino nitrogen atom. It has a role as a carcinogenic agent, an alkylating agent, an immunosuppressive agent, an antineoplastic agent, an antirheumatic drug, an environmental contaminant, a xenobiotic and a drug allergen. It is a phosphorodiamide, a nitrogen mustard and an organochlorine compound.
Precursor of an alkylating nitrogen mustard antineoplastic and immunosuppressive agent that must be activated in the liver to form the active aldophosphamide. It has been used in the treatment of lymphoma and leukemia. Its side effect, alopecia, has been used for defleecing sheep. Cyclophosphamide may also cause sterility, birth defects, mutations, and cancer.
Cyclophosphamide anhydrous is an Alkylating Drug. The mechanism of action of cyclophosphamide anhydrous is as an Alkylating Activity.
Cyclophosphamide is an alkylating agent used in the treatment of several forms of cancer including leukemias, lymphomas and breast cancer. Cyclophosphamide therapy is associated with minor transient serum enzyme elevations and has been linked to rare cases of acute liver injury. In addition, when given in high doses as a part of a myeloablative therapy, cyclophosphamide can cause acute sinusoidal obstruction syndrome.
Cyclophosphamide is a synthetic alkylating agent chemically related to the nitrogen mustards with antineoplastic and immunosuppressive activities. In the liver, cyclophosphamide is converted to the active metabolites aldophosphamide and phosphoramide mustard, which bind to DNA, thereby inhibiting DNA replication and initiating cell death.
Cyclophosphamide Anhydrous is the anhydrous form of cyclophosphamide, a synthetic nitrogen mustard alkylating agent, with antineoplastic and immunosuppressive activities. In the liver, cyclophosphamide is converted to active metabolites including phosphoramide mustard, which binds to and crosslinks DNA and RNA, thereby inhibiting DNA replication and protein synthesis. This agent, at low doses, is also a potent immunosuppressant primarily by depleting T-regulatory cells.
Precursor of an alkylating nitrogen mustard antineoplastic and immunosuppressive agent that must be activated in the liver to form the active aldophosphamide. It has been used in the treatment of lymphoma and leukemia. Its side effect, alopecia, has been used for defleecing sheep. Cyclophosphamide may also cause sterility, birth defects, mutations, and cancer.
Precursor of an alkylating nitrogen mustard antineoplastic and immunosuppressive agent that must be activated in the LIVER to form the active aldophosphamide. It has been used in the treatment of LYMPHOMA and LEUKEMIA. Its side effect, ALOPECIA, has been used for defleecing sheep. Cyclophosphamide may also cause sterility, birth defects, mutations, and cancer.

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Drug Indication
Cyclophosphamide is indicated for the treatment of malignant lymphomas, multiple myeloma, leukemias, mycosis fungoides (advanced disease), neuroblastoma (disseminated disease), adenocarcinoma of the ovary, retinoblastoma, and carcinoma of the breast. It is also indicated for the treatment of biopsy-proven minimal change nephrotic syndrome in pediatric patients.
Treatment of all malignant neoplasms
Treatment of malignant diseases

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Mechanism of Action
Alkylating agents work by three different mechanisms: 1) attachment of alkyl groups to DNA bases, resulting in the DNA being fragmented by repair enzymes in their attempts to replace the alkylated bases, preventing DNA synthesis and RNA transcription from the affected DNA, 2) DNA damage via the formation of cross-links (bonds between atoms in the DNA) which prevents DNA from being separated for synthesis or transcription, and 3) the induction of mispairing of the nucleotides leading to mutations.
The chemotherapeutic alkylating agents have in common the property of becoming strong electrophiles through the formation of carbonium ion intermediates or of transition complexes with the target molecules. These reactions result in the formation of covalent linkages by alkylation of various nucleophilic moieties such as phosphate, amino, sulfhydryl, hydroxyl, carboxyl, & imidazole groups. The chemotherapeutic & cytotoxic effects are directly related to the alkylation of DNA. The 7 nitrogen atom of guanine is particularly susceptible to the formation of a covalent bond with bifunctional alkylating agents & may well represent the key target that determines their biological effects. It must be appreciated, however, that other atoms in the purine & pyrimidine bases of DNA- particularly, the 1 & 3 nitrogens of adenine, the 3 nitrogen of cytosine, & the 6 oxygen of guanine- also may be alkylated, as will be the phosphate atoms of the DNA chains & amino & sulfhydryl groups of proteins. /Alkylating agents/
Cyclophosphamide can be used to cause immunologically mediated regression of the immunogenic, cyclophosphamide-resistant L5178Y lymphoma in syngeneic and semisyngeneic mice (B6D2F1 (C57BL/6 x DBA/2) females). In order to cause tumor regression it was necessary to give cyclophosphamide (125-200 mg/kg of body wt, iv shortly before or shortly after tumor implantation. Regardless of whether cyclophosphamide was given before or after tumor implantation, tumor regression was associated with the presence in the spleen of an incr number of Lyt-2+ T-cells capable of passively transferring immunity to tumor bearing recipients. This augmented level of immunity was sustained throughout the period of tumor regression. In contrast, a lower level of concomitant immunity generated by control tumor bearers decayed after day 12 of tumor growth. Because the therapeutic effect of cyclophosphamide could be inhibited by passive transfer of L3T4+ T-cells from normal donor mice it is apparent that the therapeutic effect of cyclophosphamide is based on its ability to preferentially destroy L3T4+ suppressor T-cells. These putative precursor suppressor T-cells were regenerated 4 days after being destroyed by cyclophosphamide.
These studies enable the pattern of emesis and nausea for 3 days following high-dose cyclophosphamide to be described and give some insight into the mechanisms of emesis which may be operating. Nausea and vomiting induced by cyclophosphamide-based chemotherapy has long latency of onset (8-13 hr) and continues for at least 3 days. These findings are of particular importance as many of these patients receive chemotherapy as outpatients and emphasize the need for appropriate anti-emetic prophylaxis for patients at home. Ondansetron was extremely effective over this time in the control of emesis and nausea. These results suggest that high-dose cyclophosphamide-induced emesis over days 1-3 is largely mediated via 5-hydroxytryptamine (5-HT) and 5-HT3 receptors.
The most likely mechanism by which cyclophosphamide augments immune responses relates to preferential elimination of suppressor and relative sparing of effector and helper cells. Thus, precursors and mature murine suppressor cells are very sensitive to cyclophosphamide whereas the mature effector cells are relatively insensitive ... . Cyclophosphamide induced immunological regression of murine leukemia is reversed by the infusion of normal spleen cells as a source of precursors of suppressor cells ... . Memory and helper T cells are relatively resistant to the cytotoxic effect of cyclophosphamide ... . NK activity against YAC lymphoma targets by non T and non B cells is depressed by cyclophosphamide ... .

C7H15CL2N2O2P

261.08

260.024

50-18-0

Cyclophosphamide hydrate;6055-19-2;Cyclophosphamide-d4;173547-45-0;Cyclophosphamide-d8;1178903-96-2

2907

White to off-white solid powder

1.3±0.1 g/cm3

336.1±52.0 °C at 760 mmHg

41-45ºC

157.1±30.7 °C

0.0±0.7 mmHg at 25°C

1.506

0.23

1

4

5

14

212

0

CMSMOCZEIVJLDB-UHFFFAOYSA-N

InChI=1S/C7H15Cl2N2O2P/c8-2-5-11(6-3-9)14(12)10-4-1-7-13-14/h1-7H2,(H,10,12)

N,N-bis(2-chloroethyl)-2-oxo-1,3,2λ5-oxazaphosphinan-2-amine

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). This product is not stable in solution, please use freshly prepared working solution for optimal results.

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

DMSO : ≥ 38 mg/mL (~145.54 mM)
H2O : ~33.33 mg/mL (~127.66 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (9.58 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 (9.58 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 (9.58 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: 25 mg/mL (95.75 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.)