Ifosfamide (IF) is a widely used antitumor prodrug. It is in the oxazaphosphorine class of alkylating agents, and it is effective against solid tumors such as sarcomas and hematologic malignancies [Article:17464949]. IF's mechanism of crosslinking DNA plays a major role in preventing cancer cells from proliferating [Article:8555414]. Major clinical toxicities caused by IF include urotoxicity, nephrotoxicity, encephalopathy and cardiotoxicity, as well as neurotoxicity (which occurs in approximately 20% of patients) [Article:22247713]. IF has lower myelotoxicity relative to its structural analog cyclophosphamide but higher rates (45% compared to 10%) of the nephrotoxic metabolite chloroacetaldehyde (CAA) [Article:21569511]. Glomerular and tubular dysfunctions represent serious side effects, especially in children who are co-treated with other nephrotoxic drugs. Thus, children are often supplemented with the organosulfur compound Mesna as an adjuvant [Article:18781911].
IF activation is catalyzed by the hepatic cytochrome P450 (CYP) isoforms (shown in detail on the ifosfamide pharmacokinetics pathway), and IF is split into two oxidation pathways, with one leading to the formation of the active metabolite Isophosphoramide mustard (IPM). Both of these competing pathways result in toxic metabolites, the detoxification pathway producing chloroacetaldehyde (CAA) and the 4-hydroxylation pathway yielding acrolein as well as the active component IF mustard. 4-Hydroxyifosfamide rapidly interconverts with its tautomer, aldoifosfamide. It is likely that both of these metabolites passively diffuse out of hepatic cells, circulate, and then passively enter other cells [Article:1922424]. Aldoifosfamide partitions between ALDH1A1-mediated detoxification to the inactive metabolite carboxyifosfamide and a spontaneous (non-enzymatic) elimination reaction to yield the therapeutically active metabolite isophosphoramide mustard (IPM) and acrolein (associated with bladder toxicity) [Article:16393888]. IPM, the DNA crosslinking agent of clinical significance, is a circulating metabolite but the anionic IPM does not enter cells as readily as its metabolic precursors [Article:18028906]. Thus, the intracellular generation of IPM from aldoifosfamide is generally believed to be important for therapeutic efficacy [Article:18028906].
Like cyclophosphamide, IF is chiral at phosphorus but unlike the case for cyclophosphamide, enantioselectivity in IF metabolism may have clinical significance. ( R )-IF is subject to less dechloroethylation, a more rapid 4-hydroxylation, and increased toxicity relative to the (S)-IF [Article:17855037].
There are three processes by which IPM reduces the ability of cancer cells to proliferate. 1, direct interaction with DNA in the nucleus; 2, activation of pro-apoptotic pathways and 3, reduction of inflammatory and antiapoptotic pathways.
IPM is translocated into the nucleus by passive diffusion. In the nucleus, IPM reacts with DNA by covalently bonding its highly reactive alkyl group with nucleophilic groups on DNA forming intra and interstrand crosslinks. These DNA strand breaks result in an inability to synthesize DNA and leads to cell apoptosis by the caspase cascade.
IF employs the caspase cascade to induce tumor cell death. Early on following IF treatment there is an activation of caspases 3, 8, and 9. IF increases the gene expression levels of caspases 3 and 9 while it decreases the expression of BCL2, a caspase inhibitor. Not only does IF decrease the possibility of BCL2 blocking the release of cytochrome c from the mitochondria but IF can activate BAX and BAK which are pro- cytochrome c releasers [Article:23213347].
In studies involving MDCK cells, it has been identified that IF modulates a series of cell cycle and immune response regulators. IPM interacts with proliferation and apoptotic pathways by both modifying MAP kinase signaling by downregulating genes responsible for proliferation and apoptosis control, as well as downregulating heat shock protein activity. IPM also decreases expression of the genes TP53 and CIP1 which modulate the cell cycle regulator p53 activity. The 72Arg variant of TP53 has been shown to induce apoptosis at a higher frequency than the 72Pro form, thus leading to higher response rates and survival in chemo and radiation therapy patients [Articles:18028906, 16331344, 23165797].
In addition, IPM downregulates TXNRD1 in the NRF-2 pathway responsible for oxidative stress response. By IF inhibiting thioredoxin reductase activity the cell is unable to neither respond to oxidative stress nor transcribe proteins involved in the apoptotic and proliferation pathways. Lastly, IPM can interfere with both the innate and adaptive immune response by decreasing production of the transcription factor NF-kB [Articles:18028906, 22580237].
IER3, a radiation inducible early response gene that regulates apoptosis, is typically upregulated in cancer cells. In vitro, when the IER3 gene is silenced, cells gain sensitivity to IF, suggesting that IPM downregulates this gene as a mechanism of anti-tumor therapy. This also indicates that antiapoptotic mechanisms may be involved in a growing resistance to IF [Article:19920113].
Not only can IPM's ability to crosslink DNA inhibit proliferation of tumor cells, but it also alters the human immune response. IPM causes a depletion of intracellular glutathione (GSH), a major antioxidant. Multiple IF metabolites can react with GSH resulting in the formation of various conjugates at different sites along the pathway [Article:8555414]. This decreased level of GSH is evident in T cells and natural killer cells that reduces functionality of the immune response. IF influences differential dendritic cell mediated effector activities specifically natural killer cells. However, this suppression of dendritic cell mediated natural killer cell proliferation is indirect and through T cells. The low levels of GSH reduce the ability for dendritic cells to stimulate T cell IL-2 production [Article:18647323]. In addition, GSH conjugates with toxic metabolites, so a decrease in the levels of GSH results in increased toxicity [Article:23220588].
Acrolein, a toxic byproduct of the IPM activation pathway is an unsaturated and highly reactive aldehyde. Upon activation acrolein can enter uroepithelial cells and activate intracellular reactive oxidative genes, leading to peroxynitrite production which can damage proteins, DNA, and lipids. Accumulation of acrolein in the bladder results in hemorrhagic cystitis [Article:23220588].
It is estimated that 25-60% of IF is metabolized into CAA. CAA suppresses activation of complex I in the mitochondrial respiratory chain resulting in decreased levels of GSH and ATP, and can induce cell death. CAA can cross the blood brain barrier and this may cause encephalopathy. Local accumulation of CAA in the kidney is also a cause of nephrotoxicity. At toxic concentrations, CAA can deplete levels of GSH which is typically an antagonist of the toxic metabolite [Articles:20541539, 20972373, 2247713, 23220588].
Aldehyde dehydrogenase enzymes can detoxify acrolein and CAA via oxidation to less toxic carboxylic acids, acrylic acid, and chloroacetic acid. Therefore, inhibition of aldehyde dehydrogenase contributes to increased toxicity. Overexpression of ALDH1A1 and ALDH3A1 are thought to contribute to IF resistance. It is believed that the overexpression directs the metabolism of 4-Hydroxyifosfamide to the inactive metabolite instead of the active IPM. It has been demonstrated in cyclophosphamide (CP), an analog to IF, that ALDH3A1 contributes to increased detoxification and tumor resistance. A variant allele ALDH3A1*2 has been noted but the effects of this polymorphism on CP or IF metabolism is unknown. In addition, it has been documented that increased ALDH1A1 and ALDH3A1 expression mediates resistance to chemotherapy treatment in breast cancer cells [Articles:19224214, 18496131].
Another possible contributor to IF resistance is the MGMT enzyme, responsible for repairing pre-carcinogenic and pre-toxic DNA damage. MGMT has the ability to repair larger adducts that have formed due to methylating or chlorethylating agents, such as acrolein. Thus, MGMT may both recognize and repair acrolein induced DNA adducts. Conversely, studies have indicated that when there is high concentrations of MGMT in tumor cells, those cells are less sensitive to the growth inhibitory mechanisms of IF [Articles:17485251, 1745538].
IF interferes with both tumor cell regulators as well as the human immune response to combat tumor proliferation. Although much is known about the cellular PD of IF, the pharmacogenetics have not been well characterized. Future studies should integrate the known pharmacokinetics pharmacogenetics to look for connections between the increased toxicity, the immune response and cell cycle modulation. In addition, it would be beneficial to further investigate the mechanisms of resistance to IF.
Lowenberg Daniella, Thorn Caroline F, Desta Zeruesenay, Flockhart David A, Altman Russ B, Klein Teri E. "PharmGKB summary: ifosfamide pathways, pharmacokinetics and pharmacodynamics" Pharmacogenetics and genomics (2014).
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Entities in the Pathway
Relationships in the Pathway
|Arrow From||Arrow To||Controllers||PMID|
|4-hydroxyifosfamide||4-ketoifosfamide||ADH1A, ADH1B, ADH1C, ADH4, ADH5, ADH6, ADH7||12136253, 8699325|
|acrolein||acrylic acid||ALDH1A1, ALDH3A1||11306050|
|aldoifosfamide||alcoifosfamide||AKR1A1, AKR1B1, AKR1B10|
|aldoifosfamide||acrolein, isophosphoramide mustard||2295063, 2754703, 8699325|
|isophosphoramide mustard||2-chloroethylamine||6821629, 8699325|
|4-hydroxyifosfamide||Ifosfamide Pathway, Pharmacokinetics||19224214|
|aldoifosfamide||Ifosfamide Pathway, Pharmacokinetics||19224214|
|chloro-acetaldehyde||Ifosfamide Pathway, Pharmacokinetics||19224214|
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