Influence of Renal Function on Phosphoramide Mustard Exposure: A Nonlinear Mixed-Effects Analysis.

Phosphoramide mustard (PM) is the final cytotoxic metabolite formed from the parent compound cyclophosphamide through a complex metabolic pathway, primarily through hepatic metabolism. Little is known about the effect of renal elimination on the disposition of PM. We evaluated the effect of renal function on PM exposure after single doses of cyclophosphamide in 85 patients undergoing allogeneic hematopoietic cell transplantation using nonlinear mixed-effects modeling. Mixed linear and nonlinear elimination pathways were required to adequately describe the disposition of PM. Creatinine clearance (CrCL) was incorporated as a covariate associated with first-order elimination, representing renal clearance (ClR ) of PM. For a 70-kg patient, ClR was 14.9 L/h, Volume of distribution was 525 L, maximum rate was 81.2 mg/h, and the concentration to achieve 50% of maximum rate was 0.51 mg/L. We conducted simulations to explore the impact of CrCL as a measure of renal function and observed that when CrCL decreases from 120 to 40 mL/min, PM area under the plasma concentration-time curve (AUC) from time 0 to 8 hours and AUC increases by 9.2% and 80.9% on average after a single dose, respectively. Our data suggest that renal function has limited influence on PM exposure during the first 8 hours after dosing but has a large impact on the total exposure. Dose adjustment of cyclophosphamide may not be necessary in hematopoietic cell transplant recipients with moderate to severe kidney dysfunction to attain targeted exposures based on AUC from time 0 to 8 hours. However, dose reduction may be necessary if demonstrated at some future time that total AUC is a better surrogate for safety or toxicity.

Design, Synthesis, and Biological Evaluation of Artemisinin-Piperazine-Phosphoramide Mustard Hybrids as Potential Anticancer Agents.

A series of novel artemisinin-piperazine-phosphoramide mustard (PPM) hybrids were designed and synthesized by incorporating phosphoramide mustard (PM) into dihydroartemisinin (DHA) via an efficient, catalyst-free two-step sequential substitution. Artemisinin-PPM hybrids showed better cytotoxic potency against HepG2 cells than both the parent DHA and the reference, vincristine (VCR). Structure-activity relationship (SAR) studies showed that the cytotoxicity was significantly enhanced by the introduction of a thiazole moiety. Hybrid 7 h, the most potent compound with the highest selectivity index IC50 (HEK-293T)/IC50 (HepG2)=16, displayed 7.4-fold stronger potency than VCR against HepG2 cells. In addition, hybrid 7 h was substantially more cytotoxic on all human cancer cells tested than on the corresponding non-cancerous cells. Flow cytometric analysis showed that 7 h significantly blocked the cell cycle in the G0/G1 phase and induced apoptosis in a concentration-dependent manner.

Cyclophosphamide, hydroxycyclophosphamide and carboxyethyl phosphoramide mustard quantification with liquid chromatography mass spectrometry in a single run human plasma samples: A rapid and sensitive method development.

A novel method for simultaneous quantification of cyclophosphamide along with its two major metabolites namely 4-hydroxycyclophosphamide (HCy) and carboxyethyl phosphoramide mustard (CEPM) in a single sample run was demonstrated in the present study. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) instrument was used for analysis. Semicarbazide was used as a stabilizing agent for HCy whereas ifosfamide, hexamethyl phosphoramide mustard and deuterated CEPM were the internal standards for quantification of Cy, HCy and CEPM respectively. Chromatographic separation was achieved by Chromsystems C18 reverse-phase column (50 mm × 4.6 mm, particle size 3.2 µm). The mobile phase was composed of eluent A (2 mM ammonium acetate in water with 2% formic acid) and eluent B (100 % acetonitrile). The flow rate was 1 ml/min. Linearity of the assay was assured in the range of 19.53 ng/ml to 10,000 ng/ml concentration in human plasma, which is adequate for pharmacokinetic studies of any dose Cy used clinically. The quality control(QC) accuracy was between 99.58% and 101.62%, 97.85% to 103.53% and 99.64% to 100.10% for Cy, HCy and CEPM respectively. Precision limits for QC samples were between 3.9% and 9.4%, 5.2% to 8.9% and 1.8% to 9.2% respectively. The analytical method was validated in ten leukaemia patients undergoing haploidentical hematopoietic cell transplantation.

Higher Fludarabine and Cyclophosphamide Exposures Lead to Worse Outcomes in Reduced-Intensity Conditioning Hematopoietic Cell Transplantation for Adult Hematologic Malignancy.

Reduced-intensity conditioning regimens using fludarabine (Flu) and cyclophosphamide (Cy) have been widely used in hematopoietic cell transplantation (HCT) recipients. The optimal exposure of these agents remains to be determined. We aimed to delineate the exposure-outcome associations of Flu and Cy separately and then both combined on HCT outcomes. This is a single-center, observational, pharmacokinetic (PK)-pharmacodynamic (PD) study of Flu and Cy in HCT recipients age ≥18 years who received Cy (50 mg/kg in a single dose), Flu (150 to 200 mg/m2 given as 5 daily doses), and total body irradiation (TBI; 200 cGy). We measured trough concentrations of 9-ß-D-arabinosyl-2-fluoradenine (F-ara-A), an active metabolite of Flu, on days -5 and -4 (F-ara-ADay-5 and F-ara-ADay-4, respectively), and measured phosphoramide mustard (PM), the final active metabolite of Cy, and estimated the area under the curve (AUC). The 89 enrolled patients had a nonrelapse mortality (NRM) of 9% (95% confidence interval [CI], 3% to 15%) at day +100 and 15% (95% CI, 7% to 22%) at day +180, and an overall survival (OS) of 73% (95% CI, 63% to 81%) at day +180. In multivariate analysis, higher PM area under the curve (AUC) for 0 to 8 hours (PM AUC0-8 hr) was an independent predictor of worse NRM (P < .01 at both day +100 and day +180) and worse day +180 OS (P < .01), but no associations were identified for F-ara-A trough levels. We observed lower day +100 NRM in those with both high F-ara-ADay-4 trough levels (≥40 ng/mL; >25th percentile) and low PM AUC0-8 hr (<34,235 hr ng/mL; <75th percentile), compared with high exposures to both agents (hazard ratio, 0.06; 95% CI, 0.01 to 0.48). No patients with low F-ara-ADay-4 (<40 ng/mL; <25th percentile) had NRM by day +100, regardless of PM AUC. The interpatient PK variability was large in F-ara-ADay-4 trough and PM AUC0-8 hr (29-fold and 5.0-fold, respectively). Flu exposure alone was not strongly associated with NRM or OS in this reduced Flu dose regimen; however, high exposure to both Flu and Cy was associated with a >16-fold higher NRM. These results warrant further investigation to optimize reduced-intensity regimens based on better PK-PD understanding and possible adaptation to predictable factors influencing drug clearance.

Design, synthesis and biological evaluation of quinazoline-phosphoramidate mustard conjugates as anticancer drugs.

A series of novel compounds with phosphoramide mustard functionality incorporated into the quinazoline scaffold of EGFR/HER2 inhibitors were designed and synthesized as multi-target-directed ligands against tumor cells. In vitro assays showed that tumor cell lines with high HER2 level were more sensitive to the compounds than tumor cells with low HER2 level. Compound 10d (EMB-3) was one of the most potent inhibitors with IC50 of 7.4 nM and 82 nM against EGFR and HER2, respectively. The mechanism studies were also supported by the effect of 10d-induced DNA damage in MDA-MB-468 cells. In vivo efficacy study showed that 10d could significantly inhibit H522 tumor xenograft model with a TGI of 68% at dose of 100 mg/kg (QDx28, p.o.) and no significant body weight loss was observed. MTD study indicated that compound 10d had no acute toxicity to mice at doses up to 900 mg/kg (single dose).

Short-term exposure of human ovarian follicles to cyclophosphamide metabolites seems to promote follicular activation in vitro.

How chemotherapy affects dormant ovarian primordial follicles is unclear. The 'burnout' theory, studied only in mice, suggests cyclophosphamide enhances primordial follicle activation. Using 4-hydroperoxycyclophosphamide (4hc) and phosphoramide mustard (PM), this study assessed how the active cyclophosphamide metabolites 4-hydroxycyclophosphamide (4-OHC) and PM, affect human primordial follicles. Frozen-thawed human ovarian samples were sliced and cultured with basic culture medium (cultured controls) or with 4hc/PM (3 µmol/l/10 µmol/l) (treated samples) for 24-48 h. Follicular counts and classification, Ki67 and anti-Müllerian hormone (AMH) immunohistochemistry and an apoptosis assay were used for evaluation, and 17ß-oestradiol and AMH were measured in spent media samples. Generally, there was primordial follicle decrease and elevated developing follicle rates in treated samples compared with cultured (P = 0.04 to P < 0.0005) and uncultured controls (P < 0.05 to P < 0.0001). No traces of apoptosis were found. There were almost twicethe levels of AMH and 17ß-oestradiol in treated compared with untreated samples (AMH with 4hc 3 µmol/l; P = 0.04). All follicles stained positively for AMHincluded treated samples. Ki67 positive staining was noted in all samples. Cyclophosphamide metabolites seem to enhance human primordial follicle activation to developing follicles, in vitro. Study findings support the 'burnout' theory as the mechanism of chemotherapy-induced ovarian toxicity.

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.

Design and synthesis of peptide conjugates of phosphoramide mustard as prodrugs activated by prostate-specific antigen.

A series of Glutaryl-Hyp-Ala-Ser-Chg-Gln-4-aminobenzyl phosphoramide mustard conjugates (1a-e) was designed and synthesized as potential prodrugs for site-specific activation by PSA in prostate cancer cells. All conjugates were found to be substrates of PSA with cleavage occurring between Gln and the para-aminobenzyl (PAB) linker. Structure-activity relationship studies on these conjugates indicated that introduction of electron-withdrawing fluorine(s) on the phenyl ring in the PAB linker uniformly improved the chemical stability of the conjugates while the position of substitution affected differently the self-immolative process of conjugates upon proteolysis. Introduction of a fluorine at ortho position to benzylic phosphoramide as in 1b results in better stability of the conjugate prior to activation while maintaining its antiproliferative activity upon activation by PSA. The conjugate 1b with 2-fluoro substitution was identified as a promising lead for further evaluation and optimization in the development of prostate cancer-targeted prodrugs.

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.

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.

Peptide conjugates of 4-aminocyclophosphamide as prodrugs of phosphoramide mustard for selective activation by prostate-specific antigen (PSA).

In our continued effort to develop prodrugs of phosphoramide mustard, conjugates of 4-aminocyclophosphamide (4-NH2-CPA) with three PSA-specific peptides were synthesized and evaluated as substrates of PSA. These include conjugates of cis-(2R,4R)-4-NH2-CPA with a tetrapeptide Succinyl-Ser-Lys-Leu-Gln-OH, a hexapeptide Succinyl-His-Ser-Ser-Lys-Leu-Gln-OH, and a pentapeptide Glutaryl-Hyp-Ala-Ser-Chg-Gln-OH. These conjugates were cleaved by PSA efficiently and exclusively after the expected glutamine residue to release 4-NH2-CPA, the activated prodrug form of phosphoramide mustard. The cleavage was most efficient for the pentapeptide conjugate 3 (Glutaryl-Hyp-Ala-Ser-Chg-Gln-NH-CPA), which showed a half-life of 55 min with PSA, followed by the hexapeptide conjugate 2 (Succinyl-His-Ser-Ser-Lys-Leu-Gln-NH-CPA) and the tertrapeptide conjugate 1 (Succinyl-Ser-Lys-Leu-Gln-NH-CPA) with half-lives of 6.5 and 12h, respectively. These results indicate a potential of the conjugate 3 as an anticancer prodrug of phosphoramide mustard for selective PSA activation.