Ifosfamide (NSC-109724) alkylates and forms DNA crosslinks, thereby preventing DNA strand separation and DNA replication. Ifosfamide is a synthetic analog of the nitrogen mustard cyclophosphamide with antineoplastic activity. This agent is a prodrug that must be activated through hydroxylation by hepatic microsomal enzymes.

Ifosfamide induced bladder edema, which peaked 12 hours after Ifosfamide injection. Microscopic analysis showed vascular congestion, edema, hemorrhage, fibrin deposition, neutrophil infiltration and epithelial denudation. Inducible nitric oxide synthase immunoreactivity was strongly reactive in the cytoplasm of bladder epithelial cells, and diffuse necrosis was seen. Intraperitoneal administration of 100 mg/kg, 200 mg/kg and 400 mg/kg Ifosfamide to mice induced a dose-dependent increase in bladder wet weight and Evans blue extravasation. Pretreatment with mesna reduced the increase in bladder edema, whereas treatment with l - ng -nitroarginine methyl ester, antisera TNF-alpha or IL-1beta, thalidomide, or pentoxifylline inhibited bladder edema and microscopic changes. Antiserum treatment also inhibited the expression of inducible nitric oxide synthase within the uroepithelium. Nitric oxide produced by inducible nitric oxide synthase was involved in uroepithelial cell injury and in the inflammatory response leading to hemorrhagic cystitis after ifosfamide administration in mice.

In the liver, Ifosfamide it is a prodrug converted to active alkylated compounds by cytochrome P450 mixed function oxidase. Ifosfamide has shown promising antitumor effects in pediatric solid tumors, ovarian cancer, small cell lung cancer, non-Hodgkin's and Hodgkin's lymphomas.Ifosfamide (50 mM) increases the levels of CYP2C8/9, CYP3A4 proteins in hepatocytes, which in turn elevates the rate of 4-hydroxylation of the hepatocytes themselves. In hepatocytes with higher CYP3A4 expression than CYP3A5, Ifosfamide induced only CYP3A4 expression.Ifosfamide was highly cytotoxic to MCF-7 cells stably transfected with CYP2B1 (which could be significantly reduced by the CYP2B1 inhibitor, metipraminexone), but did not affect the expression of β-galactosidase and the pro-tumor cells of MCF- 7 cells. In the prevention of tumor recurrence, the combination of Ifosfamide and zoledronic acid was more effective than the drug alone in increasing bone formation and improving tissue repair.

cAMP kinase assay: Diced epididymal fat pads from fed Wistar rats (175-225 gm) are obtained after decapitation and incubated at 37 °C for two hours in Krebs-bicarbonate buffer containing 1.27 mM CaCl2. When added, Tolbutamide is present only during the incubation. After incubation fat pads are rinsed and sonicated in cold Krebs-bicarbonate buffer. The aqueous supematants from centrifugation at 50,000 × g for 30 minutes at 4 °C contained 0.75 to 1.25 mg protein per mL and are assayed for cyclic AMP-stimulated protein kinase activity. The assay is performed in 0.2 mL with these additions, 10 μmoles sodium glycerofiosphate pH 7.0, 2 μmoles sodium fluoride, 0.4 μmoles theophylline, 0.1 μmoles ethylene glyool bis (β-aminoethyl ether)-N, N'-tetraaoetic acid, 3 μmoles magnesium chloride, 0.3 mg mixed histone, 2 nmoles (γ- 32P) ATP, 1 nmoles cyclic AMP when indicated, and 0.05 ml of supernatant.