Causes and possibilities to circumvent cyclophosphamide toxicity.

Cyclophosphamide is an inert prodrug converted into 4-hydroxycyclophosphamide (OHCP) by hepatic hydroxylation. OHCP is in equilibrium with its tautomeric aldophosphamide (ALDO). From ALDO, the cytotoxic active metabolites are formed enzymatically by phosphodiesterases; these are the alkylating metabolite phosphoramide mustard (PAM) and the proapoptotic aldehyde 3-hydroxypropanal (HPA). PAM damages the DNA by alkylation; HPA amplifies the thereby induced apoptosis. The generally accepted view that acrolein, which is believed to be formed in the formation of PAM by ß-elimination from ALDO would be mainly responsible for the toxicity of cyclophosphamide, has to be revised because no acrolein is formed in the systemic circulation of patients after cyclophosphamide administration. It is shown that not acrolein, but OHCP itself is the true toxic metabolite of cyclophosphamide. Toxicity tests with OHCP and PAM were carried out, which demonstrated that OHCP unfolds its toxicity, not as a carrier of PAM but is toxic itself by reacting with nucleophilic groups of macromolecules, for example, thiol groups of membrane proteins. Further experiments demonstrate that the toxicity of oxazaphosphorine cytostatics may be drastically reduced if the formation of the pharmacologically active metabolite ALDO bypasses the formation of OHCP. Toxicity experiments in mice with S-ethanol-cyclophosphamide (SECP) that hydrolyzes to OHCP show that SECP is as toxic as OHCP, whereas the thiazolidine of ALDO, which hydrolyzes to ALDO bypassing OHCP is 7-9 times less toxic without loss of antitumor activity.

Assessment of hepatic metabolism-dependent nephrotoxicity on an organs-on-a-chip microdevice.

Drug-induced nephrotoxicity is one of the most frequent adverse events in pharmacotherapy. It has resulted in numerous clinical trial failures and high drug development costs. The predictive capabilities of existing in vitro models are limited by their inability to recapitulate the complex process of drug metabolism at the multi-organ level in vivo. We present a novel integrated liver-kidney chip that allows the evaluation of drug-induced nephrotoxicity following liver metabolism in vitro. The liver-kidney chip consists of two polydimethylsiloxane layers with compartmentalized micro-channels separated by a porous membrane. Hepatic and renal cells were co-cultured in separate micro-chambers on a single chip. Ifosfamide and verapamil were used as model drugs, and their metabolites produced by hepatic metabolism were identified using mass spectrometry, respectively. The metabolites triggered significantly distinct nephrotoxic effects as assessed by cell viability, lactate dehydrogenase leakage and permeability of renal cells. This in vitro liver-kidney model facilitates the characterization of drug metabolism in the liver as well as the assessment of subsequent nephrotoxicity in a single assay. Obviously, this multi-organ platform is simple and scalable, and maybe widely applicable to the evaluation of drug metabolism and safety during the early phases of drug development.

Role of metabolites of cyclophosphamide in cardiotoxicity.

BACKGROUND: The dose-limiting toxic effect of cyclophosphamide (CY) is cardiotoxicity. The pathogenesis of myocardial damage is poorly understood, and there is no established means of prevention. In previous studies, we suggested that for CY-induced cardiotoxicity, whereas acrolein is the key toxic metabolite, carboxyethylphosphoramide mustard (CEPM) is protective. We sought to verify that acrolein is the main cause of cardiotoxicity and to investigate whether aldehyde dehydrogenase (ALDH), which is associated with greater CEPM production, is involved in the protective effect for cardiotoxicity. We also evaluated the protective effect of N-acetylcysteine (NAC), an amino acid with antioxidant activity and a known acrolein scavenger. METHODS: H9c2 cells were exposed to CY metabolites HCY (4-hydroxy-cyclophosphamide), acrolein or CEPM. The degree of cytotoxicity was evaluated by MTT assay, lactate dehydrogenase (LDH) release, and the production of reactive oxygen species (ROS). We also investigated how the myocardial cellular protective effects of CY metabolites were modified by NAC. To quantify acrolein levels, we measured the culture supernatants using high performance liquid chromatography. We measured ALDH activity after exposure to HCY or acrolein and the same with pre-treatment with NAC. RESULTS: Exposure of H9c2 cells to CEPM did not cause cytotoxicity. Increased ROS levels and myocardial cytotoxicity, however, were induced by HCY and acrolein. In cell cultures, HCY was metabolized to acrolein. Less ALDH activity was observed after exposure to HCY or acrolein. Treatment with NAC reduced acrolein concentrations. CONCLUSIONS: Increased ROS generation and decreased ALDH activity confirmed that CY metabolites HCY and acrolein are strongly implicated in cardiotoxicity. By inhibiting ROS generation, increasing ALDH activity and decreasing the presence of acrolein, NAC has the potential to prevent CY-induced cardiotoxicity.

Obesity alters phosphoramide mustard-induced ovarian DNA repair in mice.

Phosphoramide mustard (PM) destroys rapidly dividing cells and activates the DNA double strand break marker, γH2AX, and DNA repair in rat granulosa cells and neonatal ovaries. The effects of PM exposure on DNA damage and activation of DNA damage repair in lean and obese female mice were investigated. Wild type (lean) non agouti (a/a) and KK.Cg-Ay/J heterozygote (obese) mice received sesame oil or PM (95%; 25 mg/kg; intraperitoneal injection). Obesity increased (P < 0.05) hepatic and spleen but decreased (P < 0.05) uterine weight. PM exposure reduced (P < 0.05) spleen weight regardless of body composition, however, decreased (P < 0.05) ovarian and hepatic weight were observed in the obese PM-exposed females. PM decreased (P < 0.05) primordial and primary follicle number in lean females. Obesity and PM increased (P < 0.05) γH2AX protein. DNA damage repair genes Prkdc, Parp1, and Rad51 mRNA were unaltered by obesity, however, Atm and Xrcc6 mRNA were increased (P < 0.05) while Brca1 was reduced (P < 0.05). Obesity reduced (P < 0.05) PRKDC, XRCC6 and but increased (P < 0.05) ATM protein. ATM, BRCA1 and RAD51 protein levels were increased (P < 0.05) by PM exposure in both lean and obese mice, while PM-induced increased (P < 0.05) XRCC6 and PARP1 were observed only in lean mice. Thus, PM induces ovarian DNA damage in vivo; obesity alters DNA repair response gene mRNA and protein level; the ovary activates DNA repair proteins in response to PM; but obesity compromises the ovarian PM response.

Progressive obesity alters ovarian insulin, phosphatidylinositol-3 kinase, and chemical metabolism signaling pathways and potentiates ovotoxicity induced by phosphoramide mustard in mice.

Mechanisms underlying obesity-associated reproductive impairment are ill defined. Hyperinsulinemia is a metabolic perturbation often observed in obese subjects. Insulin activates phosphatidylinositol 3-kinase (PI3K) signaling, which regulates ovarian folliculogenesis, steroidogenesis, and xenobiotic metabolism. The impact of progressive obesity on ovarian genes encoding mRNA involved in insulin-mediated PI3K signaling and xenobiotic biotransformation [insulin receptor (Insr), insulin receptor substrate 1 (Irs1), 2 (Irs2), and 3 (Irs3); kit ligand (Kitlg), stem cell growth factor receptor (Kit), protein kinase B (AKT) alpha (Akt1), beta (Akt2), forkhead transcription factor (FOXO) subfamily 1 (Foxo1), and subfamily 3 (Foxo3a), microsomal epoxide hydrolase (Ephx1), cytochrome P450 family 2, subfamily E, polypeptide 1 (Cyp2e1), glutathione S-transferase (GST) class Pi (Gstp1) and class mu 1 (Gstm1)] was determined in normal wild-type nonagouti (a/a; lean) and lethal yellow mice (KK.CG-Ay/J; obese) at 6, 12, 18, or 24 weeks of age. At 6 weeks, ovaries from obese mice had increased (P < 0.05) Insr and Irs3 but decreased (P < 0.05) Kitlg, Foxo1, and Cyp2e1 mRNA levels. Interestingly, at 12 weeks, an increase (P < 0.05) in Kitlg and Kit mRNA, pIRS1Ser302, pAKTThr308, EPHX1, and GSTP1 protein level was observed due to obesity, while Cyp2e1 mRNA and protein were reduced. A phosphoramide mustard (PM) challenge increased (P < 0.05) ovarian EPHX1 protein abundance in lean but not obese females. In addition, lung tissue from PM-exposed animals had increased (P < 0.05) EPHX1 protein with no impact of obesity thereon. Taken together, progressive obesity affected ovarian signaling pathways potentially involved in obesity-associated reproductive disorders.

Phosphoramide mustard induces autophagy markers and mTOR inhibition prevents follicle loss due to phosphoramide mustard exposure.

Phosphoramide mustard (PM) is an ovotoxic metabolite of cyclophosphamide. Postnatal day 4 Fisher 344 rat ovaries were exposed to vehicle control (1% DMSO) or PM (60µM)±LY294002 or rapamycin for 2 or 4 d. Transmission election microscopy revealed abnormally large golgi apparatus and electron dense mitochondria in PM-exposed ovaries prior to and at the time of follicle depletion. PM exposure increased (P<0.05) mRNA abundance of Bbc3, Cdkn1a, Ctfr, Edn1, Gstp1, Nqo1, Tlr4, Tnfrsfla, Txnrd1 and decreased (P<0.05) Casp1 and Il1b after 4d. PM exposure increased (P<0.1) BECN1 and LAMP, decreased (P<0.1) ABCB1 and did not alter ABCC1 protein. LY294002 did not impact PM-induced ovotoxicity, but decreased ABCC1 and ABCB1 protein. Rapamycin prevented PM-induced follicle loss. These data suggest that the mammalian target of rapamycin, mTOR, may be a gatekeeper of PM-induced follicle loss.

The ovarian DNA damage repair response is induced prior to phosphoramide mustard-induced follicle depletion, and ataxia telangiectasia mutated inhibition prevents PM-induced follicle depletion.

Phosphoramide mustard (PM) is an ovotoxic metabolite of cyclophosphamide and destroys primordial and primary follicles potentially by DNA damage induction. The temporal pattern by which PM induces DNA damage and initiation of the ovarian response to DNA damage has not yet been well characterized. This study investigated DNA damage initiation, the DNA repair response, as well as induction of follicular demise using a neonatal rat ovarian culture system. Additionally, to delineate specific mechanisms involved in the ovarian response to PM exposure, utility was made of PKC delta (PKCδ) deficient mice as well as an ATM inhibitor (KU 55933; AI). Fisher 344 PND4 rat ovaries were cultured for 12, 24, 48 or 96h in medium containing DMSO ±60µM PM or KU 55933 (48h; 10nM). PM-induced activation of DNA damage repair genes was observed as early as 12h post-exposure. ATM, PARP1, E2F7, P73 and CASP3 abundance were increased but RAD51 and BCL2 protein decreased after 96h of PM exposure. PKCδ deficiency reduced numbers of all follicular stages, but did not have an additive impact on PM-induced ovotoxicity. ATM inhibition protected all follicle stages from PM-induced depletion. In conclusion, the ovarian DNA damage repair response is active post-PM exposure, supporting that DNA damage contributes to PM-induced ovotoxicity.

Phosphoramide mustard exposure induces DNA adduct formation and the DNA damage repair response in rat ovarian granulosa cells.

Phosphoramide mustard (PM), the ovotoxic metabolite of the anti-cancer agent cyclophosphamide (CPA), destroys rapidly dividing cells by forming NOR-G-OH, NOR-G and G-NOR-G adducts with DNA, potentially leading to DNA damage. A previous study demonstrated that PM induces ovarian DNA damage in rat ovaries. To investigate whether PM induces DNA adduct formation, DNA damage and induction of the DNA repair response, rat spontaneously immortalized granulosa cells (SIGCs) were treated with vehicle control (1% DMSO) or PM (3 or 6µM) for 24 or 48h. Cell viability was reduced (P<0.05) after 48h of exposure to 3 or 6µM PM. The NOR-G-OH DNA adduct was detected after 24h of 6µM PM exposure, while the more cytotoxic G-NOR-G DNA adduct was formed after 48h by exposure to both PM concentrations. Phosphorylated H2AX (γH2AX), a marker of DNA double stranded break occurrence, was also increased by PM exposure, coincident with DNA adduct formation. Additionally, induction of genes (Atm, Parp1, Prkdc, Xrcc6, and Brca1) and proteins (ATM, γH2AX, PARP-1, PRKDC, XRCC6, and BRCA1) involved in DNA repair were observed in both a time- and dose-dependent manner. These data support that PM induces DNA adduct formation in ovarian granulosa cells, induces DNA damage and elicits the ovarian DNA repair response.

Ovarian xenobiotic biotransformation enzymes are altered during phosphoramide mustard-induced ovotoxicity.

The anti-neoplastic prodrug, cyclophosphamide, requires biotransformation to phosphoramide mustard (PM), which partitions to volatile chloroethylaziridine (CEZ). PM and CEZ are ovotoxicants, however their ovarian biotransformation remains ill-defined. This study investigated PM and CEZ metabolism mechanisms through the utilization of cultured postnatal day 4 (PND4) Fisher 344 (F344) rat ovaries exposed to vehicle control (1% dimethyl sulfoxide (DMSO)) or PM (60µM) for 2 or 4 days. Quantification of mRNA levels via an RT(2) profiler PCR array and target-specific RT-PCR along with Western blotting found increased mRNA and protein levels of xenobiotic metabolism genes including microsomal epoxide hydrolase (Ephx1) and glutathione S-transferase isoform pi (Gstp). PND4 ovaries were treated with 1% DMSO, PM (60µM), cyclohexene oxide to inhibit EPHX1 (CHO; 2mM), or PM + CHO for 4 days. Lack of functional EPHX1 increased PM-induced ovotoxicity, suggesting a detoxification role for EPHX1. PND4 ovaries were also treated with 1% DMSO, PM (60µM), BSO (Glutathione (GSH) depletion; 100µM), GEE (GSH supplementation; 2.5mM), PM ± BSO, or PM ± GEE for 4 days. GSH supplementation prevented PM-induced follicle loss, whereas no impact of GSH depletion was observed. Lastly, the effect of ovarian GSH on CEZ liberation and ovotoxicity was evaluated. Both untreated and GEE-treated PND4 ovaries were plated adjacent to ovaries receiving PM + GEE or PM + BSO treatments. Less CEZ-induced ovotoxicity was observed with both GEE and BSO treatments indicating reduced CEZ liberation from PM. Collectively, this study supports ovarian biotransformation of PM, thereby influencing the ovotoxicity that ensues.

Involvement of a volatile metabolite during phosphoramide mustard-induced ovotoxicity.

The finite ovarian follicle reserve can be negatively impacted by exposure to chemicals including the anti-neoplastic agent, cyclophosphamide (CPA). CPA requires bioactivation to phosphoramide mustard (PM) to elicit its therapeutic effects however; in addition to being the tumor-targeting metabolite, PM is also ovotoxic. In addition, PM can break down to a cytotoxic, volatile metabolite, chloroethylaziridine (CEZ). The aim of this study was initially to characterize PM-induced ovotoxicity in growing follicles. Using PND4 Fisher 344 rats, ovaries were cultured for 4 days before being exposed once to PM (10 or 30 µM). Following eight additional days in culture, relative to control (1% DMSO), PM had no impact on primordial, small primary or large primary follicle number, but both PM concentrations induced secondary follicle depletion (P<0.05). Interestingly, a reduction in follicle number in the control-treated ovaries was observed. Thus, the involvement of a volatile, cytotoxic PM metabolite (VC) in PM-induced ovotoxicity was explored in cultured rat ovaries, with control ovaries physically separated from PM-treated ovaries during culture. Direct PM (60 µM) exposure destroyed all stage follicles after 4 days (P<0.05). VC from nearby wells depleted primordial follicles after 4 days (P<0.05), temporarily reduced secondary follicle number after 2 days, and did not impact other stage follicles at any other time point. VC was determined to spontaneously liberate from PM, which could contribute to degradation of PM during storage. Taken together, this study demonstrates that PM and VC are ovotoxicants, with different follicular targets, and that the VC may be a major player during PM-induced ovotoxicity observed in cancer survivors.

Pharmacokinetics of TH-302: a hypoxically activated prodrug of bromo-isophosphoramide mustard in mice, rats, dogs and monkeys.

PURPOSE: To characterize the pharmacokinetics of the prodrug, TH-302, and its active metabolite, bromo-IPM (Br-IPM), in nonclinical species. METHODS: TH-302 was administered in single oral, intraperitoneal and intravenous bolus doses to mice, rats, dogs and monkeys as well as in acute and chronic safety studies in rats and dogs as a 30-min intravenous infusion given once a week for 3 weeks. Assessments were made using liquid chromatography-tandem mass spectrometry. RESULTS: TH-302 was extensively distributed with high systemic clearance exceeding hepatic plasma flow in all species studied, resulting in half-lives ranging between 8 min (mice) and over 4 h (rats). In rats, TH-302 exhibited linear kinetics following intravenous administration and good oral bioavailability. In acute and chronic safety studies, there was no accumulation of TH-302 following once weekly dosing for 3 weeks in the rat and dog. Br-IPM plasma concentrations were a small fraction of the TH-302 plasma concentrations with significantly smaller percentages present in dogs than in rats. Allometric scaling predicted that the systemic clearance and steady-state volume of distribution in humans would be 38.8 l/h/m(2) and 34.3 l/m(2), respectively, resulting in a terminal elimination half-life of about 36 min. These values were similar to those observed in patients with solid tumors (27.1 l/h/m(2), 23.5 l/m(2) and 47 min). CONCLUSIONS: TH-302 exhibited good safety, efficacy and pharmacokinetic properties in nonclinical species, translating into favorable properties in humans.

Metabolism, pharmacokinetics and excretion of a novel hypoxia activated cytotoxic prodrug, TH-302, in rats.

The metabolism, pharmacokinetics and excretion of a hypoxically activating prodrug developed for the treatment of cancer, TH-302, were studied in rats following intravenous administration of 50 mg/kg [(14)C]-TH-302. The pharmacokinetics of TH-302 was characterized by a short half-life of 12.3 min, a high clearance of 2.29 L/h/kg and a volume of distribution of 0.627 L/kg. In intact and bile duct-cannulated rats, TH-302 was extensively metabolized with total recovery in excreta of 68.1% and 85.8%, respectively, with equal amounts excreted through urine and bile. Quantitative whole body autoradiography showed rapid distribution of [(14)C]-TH-302 associated radioactivity with the highest concentrations in the kidney and small intestinal content, suggesting significant biliary excretion and/or gut secretion. TH-302 was metabolized via (i) hydrolysis to form 2-bromoethyl amine RM3 (7.5%); (ii) monoglutathione conjugation and subsequently to the mercapturic acid RM13 (7.5%); and (iii) diglutathione conjugation followed by hydrolysis to form the dicysteine conjugate RM5 (6.5%). A large percentage (19.7%) of the dose in the excreta was associated with unidentified polar metabolites RM1 and RM2. TH-302 was the predominant circulating component in plasma and the two major metabolites in plasma were the cysteine conjugate RM8 and mercapturic acid RM13.

Detection of DNA damage in oocytes of small ovarian follicles following phosphoramide mustard exposures of cultured rodent ovaries in vitro.

Healthy oocytes are critical for producing healthy children, but little is known about whether or not oocytes have the capacity to identify and recover from injury. Using a model ovotoxic alkylating drug, cyclophosphamide (CPA), and its active metabolite, phosphoramide mustard (PM), we previously showed that PM (≥3µM) caused significant follicle loss in postnatal day 4 (PND4) mouse ovaries in vitro. We now investigate whether PM induces DNA damage in oocytes, examining histone H2AX phosphorylation (γH2AX), a marker of DNA double-strand breaks (DSBs). Exposure of cultured PND4 mouse ovaries to 3 and 0.1µM PM induced significant losses of primordial and small primary follicles, respectively. PM-induced γH2AX was observed predominantly in oocytes, in which foci of γH2AX staining increased in a concentration-dependent manner and peaked 18-24h after exposure to 3-10µMPM. Numbers of oocytes with ≥5 γH2AX foci were significantly increased both 1 and 8days after exposure to ≥1µMPM compared to controls. Inhibiting the kinases that phosphorylate H2AX significantly increased follicle loss relative to PM alone. In adult mice, CPA also induced follicle loss in vivo. PM also significantly decreased primordial follicle numbers (≥30µM) and increased γH2AX foci (≥3µM) in cultured PND4 Sprague-Dawley rat ovaries. Results suggest oocytes can detect PM-induced damage at or below concentrations which cause significant follicle loss, and there are quantitative species-specific differences in sensitivity. Surviving oocytes with DNA damage may represent an increased risk for fertility problems or unhealthy offspring.

Exploiting the drug-activating properties of a novel trypanosomal nitroreductase.

Nitroheterocyclic prodrugs have been used to treat trypanosomal diseases for more than 40 years. Recently, the key step involved in the activation of these compounds has been elucidated and shown to be catalyzed by a type I nitroreductase (NTR). This class of enzyme is normally associated with bacteria and is absent from most eukaryotes, with trypanosomes being a major exception. Here we exploit this difference by evaluating the trypanocidal activity of a library of nitrobenzylphosphoramide mustards against bloodstream-form Trypanosoma brucei parasites. Biochemical screening against the purified enzyme revealed that a subset of halogenated nitroaromatic compounds were effective substrates for T. brucei NTR (TbNTR), having apparent K(cat)/K(m) values approximately 100 times greater than nifurtimox. When tested against T. brucei, cytotoxicity mirrored enzyme activity, with 50% inhibitory concentrations of the most potent substrates being less than 10 nM. T. brucei NTR plays a key role in parasite killing: heterozygous lines displayed resistance to the compounds, while parasites overexpressing the enzyme showed hypersensitivity. We also evaluated the cytotoxicities of substrates with the highest trypanocidal activities by using mammalian THP-1 cells. The relative toxicities of these newly identified compounds were much lower than that of nifurtimox. We conclude that halogenated nitrobenzylphosphoramide mustards represent a novel class of antitrypanosomal agents, and their efficacy validates the strategy of specifically targeting NTR activity to develop new therapeutics.

Design of new oxazaphosphorine anticancer drugs.

The oxazaphosphorines including cyclophosphamide (CPA, Cytoxan, or Neosar), ifosfamide (IFO, Ifex) and trofosfamide (Ixoten) represent an important group of therapeutic agents due to their substantial antitumor and immunomodulating activity. However, several intrinsic limitations have been uncounted during the clinical use of these oxazaphosphorines, including substantial pharmacokinetic variability, resistance and severe host toxicity. To circumvent these problems, new oxazaphosphorines derivatives have been designed and evaluated with an attempt to improve the selectivity and response with reduced host toxicity. These include mafosfamide (NSC 345842), glufosfamide (D19575, beta-D-glucosylisophosphoramide mustard), S-(-)-bromofosfamide (CBM-11), NSC 612567 (aldophosphamide perhydrothiazine) and NSC 613060 (aldophosphamide thiazolidine). Mafosfamide is an oxazaphosphorine analog that is a chemically stable 4-thioethane sulfonic acid salt of 4-hydroxy-CPA. Glufosfamide is IFO derivative in which the isophosphoramide mustard, the alkylating metabolite of IFO, is glycosidically linked to a beta-D-glucose molecule. Phase II studies of glufosfamide in the treatment of pancreatic cancer, non-small cell lung cancer (NCSLC), and recurrent glioblastoma multiform (GBM) have recently completed and Phase III trials are ongoing, while Phase I studies of intrathecal mafosfamide have recently completed for the treatment of meningeal malignancy secondary to leukemia, lymphoma, or solid tumors. S-(-)-bromofosfamide is a bromine-substituted IFO analog being evaluated in a few Phase I clinical trials. The synthesis and development of novel oxazaphosphorine analogs with favourable pharmacokinetic and pharmacodynamic properties still constitutes a great challenge for medicinal chemists and cancer pharmacologists.

Characterizing the ovotoxicity of cyclophosphamide metabolites on cultured mouse ovaries.

Cyclophosphamide (CPA) is reported to target dormant primordial ovarian follicles in rodents and humans. However, mechanistic studies are complicated due to the complex ovarian structure. We present here the characterization of the sensitivity of ovaries to CPA metabolites and the timing of morphological alterations induced by phosphoramide mustard (PM) in an in vitro system. Intact mouse ovaries (postnatal-day-4) were cultured in vitro and exposed to multiple breakdown products of CPA on day 0 (d0). Tissues were cultured up to d8, and then follicle counts and immunohistochemistry were performed. 4-Hydroperoxy-CPA (4-HC), a precursor of an activated form of CPA, and PM depleted primordial and primary follicles (> or =1 microM and > or =3 microM, respectively, p < 0.05); acrolein had effects on follicle numbers only under continuous exposure (> =30 microM); carboxycyclophosphamide and 4-ketocyclophosphamide reduced primordial and small primary follicles only at high concentrations (100 microM). PM-induced follicle loss became significant (p < 0.05) by d1 or d2 following exposures to 10 microM or 3 microM PM, respectively, as determined by the numbers of pyknotic or TUNEL-positive follicles. Cellular targets were oocytes in the smallest follicles, but granulosa cells in large primary follicles. TUNEL staining was observed in both cell types, but caspase-3, a marker of apoptosis, was absent from primordial follicles. In addition, a pan-caspase inhibitor could not prevent follicle losses when administered prior to PM. Thus, brief exposures to 4-HC or PM are sufficient to induce permanent follicle loss in ovaries, and PM is likely the ultimate ovotoxicant. Furthermore, the cell death pathway is likely caspase-independent.

Comparative preclinical toxicology and pharmacology of isophosphoramide mustard, the active metabolite of ifosfamide.

BACKGROUND: Isophosphoramide mustard (IPM) is the cytotoxic alkylating metabolite of Ifosfamide (IFOS). IPM is being readied for a phase I clinical trial. In the present preclinical study, IPM was evaluated for usage in multidose intravenous (IV) infusion protocols. METHODS: Mice and dogs received IV IPM daily for 3 days. Single-day dosing-oral and IV-to mice, rats, and monkeys is also reviewed for comparison. Complete toxicology studies were completed in the mice and dogs. For mice, dogs and monkeys, IV pharmacokinetic studies were conducted and compared. RESULTS: For mice, the LD(10) for the 3-day IV schedule for IPM was calculated to be 119 mg/kg (with 95% confidence limits of 87-134 mg/kg) (combined sexes), and for adult male dogs the maximum tolerated dose (MTD) was 5 mg/kg. Pharmacokinetic studies in mice, dogs and monkeys were compared and projected to human dosing. For dogs that received 10 mg/kg of IPM, T(1/2beta) was 0.99 h, and clearance was constant (1.01 l/h/kg). IPM was detected from 0 h to 1.5 h after the 5 mg/kg dose and from 0 h to 2 h after the 10 mg/kg dose; none was detected after 2 h. The IV MTD in dogs was 5 mg/kg per day for 3 days. Renal tubular necrosis and bone marrow failure were the causes of death. Transient liver, renal and bone marrow toxicity and gastrointestinal dysfunction were seen at low doses (<5 mg/kg) in dogs. In mice (receiving 100 mg/kg IV) plasma concentrations disappeared in less than 1 h (T(1/2alpha) 2 min), with a clearance of 8.44 l/h/kg. For monkeys, the mean T(1/2) was 4.2 h. Median clearance was 1.65 l/h/kg and no IPM was detected 4 h after dosing. No potential IPM metabolites could be detected in any of the studies. In vitro, plasma protein bound 90% of IPM within 5 min of incubation. CONCLUSIONS: Predictions for human pharmacokinetic parameters and dosing are made from allometric analysis using the above three species. Data predicted an acceptable starting dose of 30 mg/m(2) with a clearance of 39.5 l/h, and a T(1/2) of 1 h 45 min for a 70-kg patient.

1,3- vs 1,5-intramolecular alkylation reactions in isophosphoramide and phosphoramide mustards.

It is well-established that at pH 7.4, intramolecular 1,3-N-alkylation reactions in isophosphoramide mustard (IPM) and phosphoramide mustard (PM) produce electrophilic alkylating agents with aziridinyl moieties. To investigate the role of 1,5-intramolecular cyclizations in the chemistry of IPM and PM, the five-membered ring phospholidine products of these reactions were independently synthesized and characterized by (31)P NMR. In 0.33 M BisTris, pH 7.4, 37 degrees C, the intramolecular O-alkylation product of IPM [2-(2-chloroethylamino)-2-tetrahydro-2H-1,3,2-oxazaphospholidine-2-oxide (11)] had a chemical shift of delta 33.0 and a half-life of 3.3 h. The O-alkylation product of PM [2-amino-3-(2-chloroethyl)tetrahydro-2H-1,3,2-oxazaphospholidine-2-oxide (12)] displayed a chemical shift of delta 30.6 and a half-life of 26.9 h. For both IPM and PM, 1,5-N-alkylation provides the same product [1-(2-chloroethyl)-2-hydroxy-tetrahydro-2H-1,3,2-diazaphospholidine-2-oxide (13)]. Because of its instability, 13 was generated in situ and was not isolated; however, the chemical shift (delta 33.0) and reactivity (half-life 0.3 h at 25 degrees C) of the species attributed to 13 were consistent with the assigned structure. Resonances with (31)P NMR chemical shifts indicative of 11 or 12 did not appear in reaction solutions of IPM or PM. The compound assigned as 13 gave hydrolysis products that were not found in reaction solutions of IPM or PM. The collective data supported the conclusion that intramolecular 1,5-alkylations do not contribute to the chemistry of IPM or PM in aqueous solutions at pH 7.4, 37 degrees C. Conversely, 11 and 12 were found to be the major if not exclusive products formed in DMSO solutions of the respective cyclohexylammonium salts of IPM and PM. Both 11 and 12 were relatively noncytotoxic against a series of cell lines, but there were differences in mutagenicities. Chinese hamster ovary cells were exposed to 11 or 12 for one half-life of each compound; 11 was nonmutagenic up to 500 microM, while 12 (500 microM) was mutagenic with 246 mutant colonies/10(6) surviving cells.

Phosphate prodrugs of isophosphoramide mustard.

Dibenzylphosphorobenzyl and phosphorobenzyl analogues of isophosphoramide mustard, an active metabolite of ifosfamide were synthesized. Phosphorobenzyl analogue posseses stronger cytotoxic activity than isophosphoroamide mustard against the cells of several cancer cell lines suggesting the possibility of the use of this compound in Gene-Directed Enzyme-Prodrug Therapy (GDEPT).

Antitumor activity and toxicity of novel nitroheterocyclic phosphoramidates.

A series of novel nitroheterocyclic phosphoramidates has been evaluated for antitumor activity in murine and xenograft tumor models and for toxicity in mice. Significant increases in lifespan and long-term survivors were noted in L1210 leukemia and B16 melanoma models, and both complete and partial tumor regressions were observed in the MX-1 breast cancer xenograft model. All compounds exhibited some degree of toxicity to granulocyte/macrophage progenitors in the bone marrow of mice. Two drugs were selected for further toxicologic, histopathologic, and pharmacokinetic evaluations. Toxicity of potential clinical significance was observed only in the bone marrow at the highest drug dose; otherwise no significant abnormalities in blood chemistries or organ histopathology were noted. The bone marrow lesions consisted of reduced numbers of progenitor cells in the myeloid and erythroid series; platelets were not affected. The compounds were eliminated rapidly by first-order kinetics, with half-lives in the 4-12 min range. The best of these compounds exhibits excellent antitumor activity and minimal toxicity at therapeutically effective doses in mice.