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
Peak plasma concentrations occur 1 hour after oral administration. Cyclophosphamide is primarily excreted as metabolites. After intravenous administration, 10-20% is excreted unchanged in the urine and 4% in the bile. Total clearance = 63 ± 7.6 L/kg (30-50 L). Cyclophosphamide is well absorbed orally. Placental transfer of (14)-carbon-cyclophosphamide has been confirmed in mice; and there are reports of a positive correlation between DNA alkylation in mouse embryos and the occurrence of congenital abnormalities. Similar associations have been found with nuclear DNA-dependent RNA polymerase in rat embryos. In most species, cyclophosphamide is rapidly absorbed, metabolized, and excreted. In rats, the specific activity in tissues reaches its peak within 20-30 minutes after intraperitoneal injection; up to 75% of the radioactivity is eliminated within 5-8 hours. /Monohydrate/ The drug is rapidly absorbed from the bloodstream after intravenous injection. Following daily administration of 6.7–80 mg/kg body weight of cyclophosphamide, the radioactive material rapidly distributes to all tissues: its half-life in plasma is 6.5 hours. No radioactive material was detected in exhaled breath or feces. The recovery rate of radioactive material in urine has been reported to be 50–68%, primarily in the form of carboxyphosphamide and phosphoramide mustard; 10–40% of the drug is excreted unchanged; and 56% of the active metabolites are bound to plasma proteins. /Monohydrate/
In a cross-sectional study, researchers monitored the excretion of cyclophosphamide in the urine of 20 hospital workers with occupational exposure to cyclophosphamide and 21 unexposed control subjects. Within one week of sample collection, most workers were exposed to cyclophosphamide fewer than five times, with each exposure ranging from 100–1000 mg (mean ± 350 mg). All workers reported taking routine safety precautions, such as wearing gloves at least during procedures. The drug was detected in 5 samples (24-hour urinary excretion of cyclophosphamide ranged from 0.7 to 2.5 μg). The results showed a significant correlation between exposure to cyclophosphamide and detection of the drug in urine. Of the 5 patients with detectable cyclophosphamide in their urine, 4 had been exposed to cyclophosphamide 10 times or more within a week.
For more complete data on the absorption, distribution, and excretion of cyclophosphamide (7 items in total), please visit the HSDB record page.

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Metabolism/Metabolites
Cyclophosphamide is metabolized and activated in the liver. 75% of the drug is activated by cytochrome P450 isoenzymes CYP2A6, 2B6, 3A4, 3A5, 2C9, 2C18, and 2C19. The CYP2B6 isoenzyme exhibits the highest 4-hydroxylase activity. Cyclophosphamide activation ultimately forms active metabolites, including phosphoramide mustard and acrolein. Cyclophosphamide appears to induce its own metabolism, leading to an overall increase in clearance, increased production of the 4-hydroxy metabolite, and a shortened half-life (t1/2) after repeated dosing. Cyclophosphamide is activated by the hepatic cytochrome P450 system. It is first converted to 4-hydroxycyclophosphamide, which remains in a steady state with its acyclic tautomer, aldehyde phosphamide. In vitro studies have shown that cyclophosphamide is activated by CYP2B-type P450 isoenzymes, studied using human liver microsomes and cloned P450 isoenzymes. 4-Hydroxycyclophosphamide may be further oxidized in the liver or tumor tissue by aldehyde oxidases, or by other enzymes, to produce carboxyphosphamide and 4-ketocyclophosphamide, neither of which has significant biological activity. These secondary reactions appear to minimize liver damage, while large amounts of active metabolites, such as 4-hydroxycyclophosphamide and its tautomer, aldehyde phosphamide, are transported to the target site via the circulatory system. In tumor cells, aldehyde phosphamide spontaneously cleaves, generating equimolar amounts of phosphoramic acid mustard and acrolein. Phosphoramide mustard is considered a source of antitumor activity. Acrolein may be the cause of hemorrhagic cystitis during cyclophosphamide treatment. Parenteral administration of messo (a thiol compound that readily reacts with acrolein in the acidic environment of the urinary tract) can reduce or prevent the severity of cystitis. …After intravenous injection, very little unmetabolized cyclophosphamide is recovered in urine and feces. Plasma concentrations reach their maximum 1 hour after oral administration, with a plasma half-life of approximately 7 hours.
Other sheep were given oral cyclophosphamide. Two metabolites were observed in the collected urine and identified as O-(2-carboxyethyl)-N,N-bis(2-chloroethyl)phosphoryldiamine and 2-(bis(2-chloroethyl)amino)tetrahydro-2H-1,3,2-oxazolphosphine 2,4-dioxide (4-ketocyclophosphamide).
A potent alkylating and cytotoxic metabolite, N,N-bis(2-chloroethyl)phosphoryldiamine, has recently been isolated from the oxidation products of cyclophosphamide and mouse liver microchromosomes.
Cyclophosphamide is well absorbed orally, reaching peak plasma concentrations approximately one hour after oral administration. It can also be administered intravenously. The drug is metabolized in the liver to the cytotoxic metabolite 4-hydroxycyclophosphamide, which is in equilibrium with the acyclic tautomer aldehyde phosphamide. While most of these metabolites are further oxidized to inactive products, a portion of aldehyde phosphamide is converted to phosphoramic acid mustard (which can alkylate DNA) and acrolein.
For more complete metabolite/metabolite data on cyclophosphamide (a total of 8 metabolites), please visit the HSDB record page.
Metabolism and activation occur in the liver. 75% of cyclophosphamide is activated by cytochrome P450 isoenzymes CYP2A6, 2B6, 3A4, 3A5, 2C9, 2C18, and 2C19. Of these, the CYP2B6 isoenzyme exhibits the highest 4-hydroxylase activity. Cyclophosphamide, upon activation, ultimately produces active metabolites such as phosphoramide mustard and acrolein. Cyclophosphamide appears to induce its own metabolism, leading to an overall increase in clearance, increased production of 4-hydroxyl metabolites, and a shortened half-life (t1/2) after repeated dosing. Elimination pathway: Cyclophosphamide is primarily eliminated as metabolites. After intravenous administration, 10-20% is excreted unchanged in the urine, and 4% is excreted in the bile. Half-life: 3-12 hours. The maximum plasma concentration is reached 1 hour after oral administration, with a plasma half-life of approximately 7 hours.

Hepatotoxicity
Up to 43% of cancer patients receiving cyclophosphamide treatment experience mild, transient elevations in serum transaminase levels. These abnormalities are usually asymptomatic and transient, requiring no dose adjustment. Enzyme elevations are more common at high doses and with intravenous administration. In some cases, significant elevations occur, requiring dose adjustment or discontinuation of cyclophosphamide (Case 3). Clinically significant liver injury caused by standard doses of cyclophosphamide is uncommon, but several cases of acute liver injury with jaundice have been reported (Case 1 and 2). Liver injury typically occurs within 2 to 8 weeks of initiation of cyclophosphamide, with a hepatocellular pattern of serum enzyme elevation. Immune hypersensitivity and autoimmune features are uncommon. Most cases of liver injury are self-limiting, recovering within 1 to 3 months after discontinuation; however, fatal cases have been reported. Relapses following re-exposure have been reported. High-dose cyclophosphamide used in cancer chemotherapy or as part of bone marrow ablation therapy combined with total body irradiation, or busulfan (used for preparation before hematopoietic stem cell transplantation), can induce hepatic sinusoidal obstruction syndrome (hepatic venous occlusion), which can lead to acute liver failure and even death in severe cases. The injury usually occurs within 10 to 20 days after bone marrow ablation, characterized by sudden abdominal pain, weight gain, ascites, and significantly elevated serum transaminase (and lactate dehydrogenase) levels, followed by jaundice and liver dysfunction. The severity of hepatic sinusoidal obstruction syndrome varies, ranging from transient, self-limiting injury to acute liver failure. Diagnosis is usually based on clinical manifestations such as liver tenderness and enlargement, weight gain, ascites, and jaundice. Liver biopsy has diagnostic value, but it is generally not recommended due to the potential for severe thrombocytopenia after hematopoietic stem cell transplantation. Probability score: B (Very likely the cause of clinically apparent liver injury).

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Impact of Pregnancy and Lactation
◉ Overview of Drug Use During Lactation
Cyclophosphamide can appear in breast milk at concentrations that can reach toxic levels; furthermore, its highly toxic active metabolites may increase the risk to the infant. There have been two reports of infants developing neutropenia, both of whose mothers continued breastfeeding while receiving cyclophosphamide treatment. Most data suggest that breastfeeding should be avoided while the mother is receiving cytotoxic antitumor drugs, especially alkylating agents such as cyclophosphamide. Although some studies recommend suspending breastfeeding for 1 to 3 days after administration, the drug and its metabolites appear to take more than 21 days to be completely cleared from breast milk. Data from some authors indicate that after an injection of 750 mg/m² of cyclophosphamide, the cyclophosphamide content in breast milk may take up to 6 weeks to reach safe levels.
Chemotherapy can adversely affect the normal microbiota and chemical composition of breast milk. Women receiving chemotherapy during pregnancy are more likely to experience breastfeeding difficulties.
◉ Effects on Breastfed Infants
A 23-day-old infant developed neutropenia, thrombocytopenia, and hypohemoglobinemia, possibly due to the mother receiving intravenous cyclophosphamide (6 mg/kg, total dose 300 mg) daily after 3 days of cyclophosphamide treatment.
A 4-month-old infant developed neutropenia, possibly due to the mother receiving cyclophosphamide (800 mg) intravenously once weekly, vincristine (2 mg), and prednisolone (30 mg) orally daily for 6 weeks after 9 days of cyclophosphamide treatment. Neutropenia persisted for at least 12 days and was accompanied by transient diarrhea.
A woman was diagnosed with B-cell lymphoma at 27 weeks of gestation. She was induced at 34 weeks and 4 days of gestation and began standard treatment with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (dosage not specified), for 21 days, starting on day 2 postpartum. For the first 10 days of each treatment cycle, she expressed and discarded breast milk, feeding the baby with donated breast milk, and then breastfed for 10 days before the start of the next cycle. The 10-day breastfeeding pause was determined based on the half-life of vincristine (approximately 3 half-lives). After completing four cycles of chemotherapy, the baby was reported to be developing well without any complications.
◉ Effects on Lactation and Breast Milk
A case of normoprolactinemic galactorrhea was reported in a 55-year-old woman receiving cyclophosphamide treatment for pemphigus vulgaris. After one month of taking cyclophosphamide 50 mg/day, she developed bilateral breast engorgement and bilateral nipple discharge. No hormonal abnormalities were found. After discontinuing cyclophosphamide, her symptoms completely disappeared without recurrence. The galactorrhea was likely caused by cyclophosphamide.
A telephone follow-up study investigated 74 women who received cancer chemotherapy at the same center during mid- or late-pregnancy to determine their postpartum breastfeeding success rates. Only 34% of women were able to exclusively breastfeed their infants, and 66% reported breastfeeding difficulties. In contrast, the breastfeeding success rate was 91% among 22 mothers who were diagnosed during pregnancy but did not receive chemotherapy. Other statistically significant correlations included: 1. Mothers experiencing breastfeeding difficulties received an average of 5.5 cycles of chemotherapy, while mothers without breastfeeding difficulties received an average of 3.8 cycles; 2. Mothers experiencing breastfeeding difficulties received their first chemotherapy cycle an average of 3.4 weeks earlier. Of the 56 women receiving cyclophosphamide-containing regimens, 34 experienced breastfeeding difficulties. Protein Binding: Cyclophosphamide has a protein binding rate of 20%, which did not change in a dose-dependent manner. Some metabolites have protein binding rates exceeding 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.

According to California labor law, cyclophosphamide (hydrate) may be carcinogenic. It may also have developmental toxicity, according to an independent committee of scientific and health experts. It may also have female and male reproductive toxicity depending on state or federal labeling requirements. Cyclophosphamide is a white, crystalline, fine powder, odorless, and slightly bitter. Its melting point is 41-45°C. A 2% solution has a pH of 4 to 6. It is used medically as an antitumor drug. Cyclophosphamide is a diphosphamide with the structure 1,3,2-oxazolylphosphine-2-amine-2-oxide, in which the amino nitrogen atom is replaced by two 2-chloroethyl groups. It has multiple functions, including carcinogenicity, alkylation, immunosuppression, antitumor, antirheumatic, environmental pollution, xenobiotic, and drug allergen. It is a diphosphamide, nitrogen mustard, and organochlorine compound. It is a precursor to alkyl nitrogen mustard antitumor and immunosuppressant drugs and must be activated in the liver to form active aldehyde phosphamide. It has been used to treat lymphoma and leukemia. Its side effects—hair loss—have been used to remove wool from sheep. Cyclophosphamide may also cause infertility, birth defects, gene mutations, and cancer. Anhydrous cyclophosphamide is an alkylating agent. The mechanism of action of anhydrous cyclophosphamide is alkylating activity. Cyclophosphamide is an alkylating agent used to treat a variety of cancers, including leukemia, lymphoma, and breast cancer. Cyclophosphamide treatment is associated with mild, transient elevations in serum enzymes and is linked to rare cases of acute liver injury. Furthermore, high doses of cyclophosphamide, when used as part of myeloablative therapy, can cause acute hepatic sinusoidal obstruction syndrome. Cyclophosphamide is a synthetic alkylating agent with a chemical structure related to nitrogen mustard compounds, possessing antitumor and immunosuppressive activities. In the liver, cyclophosphamide is converted to the active metabolites aldehydephosphamide and phosphoramide mustard, which can bind to DNA, thereby inhibiting DNA replication and initiating cell death. Anhydrous cyclophosphamide is the anhydrous form of cyclophosphamide, a synthetic nitrogen mustard alkylating agent with antitumor and immunosuppressive activities. In the liver, cyclophosphamide is converted into active metabolites, including phosphoramic acid mustard, which bind to and cross-link DNA and RNA, thereby inhibiting DNA replication and protein synthesis. At low doses, this drug is also a potent immunosuppressant, primarily acting by depleting regulatory T cells. It is a precursor to alkyl nitrogen mustard antitumor and immunosuppressant drugs and must be activated in the liver to form active aldehyde phosphoramide. It has been used to treat lymphoma and leukemia. A side effect is hair loss, so it has also been used for dehairing sheep. Cyclophosphamide may also cause infertility, birth defects, gene mutations, and cancer.

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Drug Indications
Cyclophosphamide is indicated for the treatment of malignant lymphoma, multiple myeloma, leukemia, mycosis fungoides (advanced stage), neuroblastoma (disseminated disease), ovarian adenocarcinoma, retinoblastoma, and breast cancer. It is also indicated for the treatment of biopsy-confirmed minimal change disease nephropathy in children.
Treatment of all malignancies
Treatment of malignant diseases

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Mechanism of Action
Alkylating agents act through three different mechanisms: 1) Alkyl groups are attached to DNA bases, causing DNA to break as repair enzymes attempt to replace the alkylated bases, thereby preventing DNA synthesis and RNA transcription from damaged DNA; 2) By forming cross-links (bonds between atoms in DNA), they damage DNA, preventing DNA separation for synthesis or transcription; 3) They induce nucleotide mismatches leading to mutations.
A common characteristic of chemotherapeutic alkylating agents is that they become strongly electrophilic by forming carbocation intermediates or transition state complexes with target molecules. These reactions form covalent bonds by alkylating various nucleophilic groups, such as phosphate groups, amino groups, thiol groups, hydroxyl groups, carboxyl groups, and imidazole groups. Chemotherapy and cytotoxic effects are directly related to DNA alkylation. The nitrogen atom at position 7 of guanine is particularly prone to covalent bonding with bifunctional alkylating agents and is likely a key target determining its biological effects. However, it must be recognized that other atoms in the purine and pyrimidine bases of DNA—particularly the nitrogen atoms at positions 1 and 3 of adenine, the nitrogen atom at position 3 of cytosine, and the oxygen atom at position 6 of guanine—can also be alkylated, as can the phosphate groups of the DNA strand and the amino and thiol groups of proteins. /Alkylating Agents/
Cyclophosphamide can be used to induce immunomediated regression of immunogenic, cyclophosphamide-resistant L5178Y lymphoma in syngeneic and semi-syngeneic mice (female B6D2F1 (C57BL/6 x DBA/2)). To induce tumor regression, cyclophosphamide (125-200 mg/kg body weight) was administered intravenously before or shortly after tumor implantation. Regardless of whether cyclophosphamide was administered before or after tumor implantation, tumor regression was associated with an increase in the number of Lyt-2+ T cells in the spleen, which are capable of passively transferring immunity to the tumor-bearing receptor. This enhanced immunity persisted throughout the tumor regression process. In contrast, control tumor-bearing mice generated lower concomitant immunity, which began to decline 12 days after tumor growth. Since the therapeutic effect of cyclophosphamide can be suppressed by passively transferring L3T4+ T cells from normal donor mice, it is clear that the therapeutic effect of cyclophosphamide is based on its ability to preferentially destroy L3T4+ suppressor T cells. These probable precursor suppressor T cells regenerated 4 days after being destroyed by cyclophosphamide. These studies will describe the pattern of vomiting and nausea within 3 days after high-dose cyclophosphamide treatment and explore the possible mechanisms of vomiting. Nausea and vomiting induced by cyclophosphamide chemotherapy have a long latency period (8-13 hours) and last for at least 3 days. These findings are particularly important because many such patients receive outpatient chemotherapy and underscore the necessity of appropriate antiemetic prophylaxis for patients at home. During this period, ondansetron is highly effective in controlling vomiting and nausea. These results suggest that high-dose cyclophosphamide-induced vomiting on days 1-3 is primarily mediated by serotonin (5-HT) and 5-HT3 receptors. The most likely mechanism by which cyclophosphamide enhances the immune response is related to the preferential clearance of suppressor cells and the relative preservation of effector and helper cells. Therefore, precursors and mature suppressor cells in mice are highly sensitive to cyclophosphamide. Cyclophosphamide inhibits the proliferation of mature effector cells, which are relatively insensitive… Cyclophosphamide-induced immune remission in mouse leukemia can be reversed by infusion of normal spleen cells (as a precursor source of suppressor cells)… Memory T cells and helper T cells are relatively tolerant to the cytotoxic effects of cyclophosphamide… Cyclophosphamide can inhibit the NK activity of non-T cells and non-B cells against YAC lymphoma target cells...

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.)