Cyclophosphamide, an inactive prodrug, is a kind of nitrogen mustard alkylating agent. Cyclophosphamide requires enzymatic and chemical activation. As a result, nitrogen mustard is produced. It causes DNA cross-linking that accounts for its cytotoxic properties. ^[1] IC₅₀ of cytotoxicity in mouse embryo BALB/c 3T3 cells is 37.6 μM, ^[2] IC₅₀ of cytotoxicity against human HL60 cells is 8.79 μM measured by MTT assay. ^[3]?

Cyclophosphamide attaches the alkyl group to the guanine base of DNA causing its crosslinking, strand breakage and inducing mutations.?

In vitro, cyclophosphamide has a dose-dependent, bimodal effect on the immune system.?Low-dose cyclophosphamide not only decreases cell number but leads to decreased functionality of regulatory T cells (T_REGs). Cyclophosphamide treatment enhances apoptosis and decreases homeostatic proliferation of these cells. Expression of GITR and FoxP3, which are involved in the suppressive activity of T_REGs, is down-regulated after cyclophosphamide administration.^[4] In primary human hepatocyte cultures, cyclophosphamide increases CYP3A4, CYP2C8, and CYP2C9 protein levels, causing its 4-hydroxylation rate enhance.^[5]?In somatic cells, cyclophosphamide produces gene mutations, chromosome aberrations, micronuclei and sister chromatid exchanges in a variety of cultured cells in the presence of metabolic activation as well as sister chromatid exchanges without metabolic activation. ^[6]?

In vivo, it has produced chromosome damage and micronuclei in rats, mice and Chinese hamsters, and gene mutations in the mouse spot test and in the transgenic lacZ construct of Muta^?Mouse. ^[6]?

References:

[1] Emadi A,?Jones RJ,?Brodsky RA. Cyclophosphamide?and?cancer:?golden?anniversary. Nat Rev Clin Oncol.?2009 Nov; 6 (11):638-47.?

[2] Moon KY,?Kwon CH. N3-methyl-mafosfamide as a chemically stable, alternative prodrug of mafosfamide. Bioorg Med Chem Lett.?1998 Jul 7; 8 (13):1673-8.

[3] Patel MM,?Mali MD,?Patel SK. Bernthsen synthesis, antimicrobial activities and cytotoxicity of acridine derivatives. Bioorg Med Chem Lett.?2010 Nov 1; 20 (21):6324-6.

[4] Lutsiak ME,?Semnani RT,?De Pascalis R,et al. Inhibition?of?CD4(+)25+?T?regulatory?cell?function?implicated?in?enhanced?immune?response?by?low-dose?cyclophosphamide. Blood.?2005 Apr 1; 105 (7):2862-8. Epub 2004 Dec 9.

[5] Chang TK,?Yu L,?Maurel P,?Waxman DJ. Enhanced?cyclophosphamide?and?ifosfamide?activation?in?primary?human?hepatocyte?cultures: response?to?cytochrome?P-450?inducers?and?autoinduction?by oxazaphosphorines. Cancer Res.?1997 May 15; 57 (10):1946-54.

[6] Anderson D,?Bishop JB,?Garner RC, et al. Cyclophosphamide: review of its mutagenicity for an assessment of potential germ cell risks. Mutat Res.?1995 Aug; 330 (1-2):115-81.