Phosphoramide Mustard As a Biomarker Of Cyclophosphamide Exposure In Adults Receiving Reduced Intensity Conditioning Hematopoietic Cell Transplantation

The use of RIC reduced intensity conditioning (RIC) hematopoietic cell transplant (HCT) has extended the availability of transplantation to older and less medically fit patients. Despite the increased tolerability of RIC HCT, treatment related mortality (TRM) still remains a significant problem. Biomarkers of chemotherapy exposure which are predictive of toxicity or efficacy would be clinically useful in making treatment decisions. We studied phosphoramide mustard (PM), one of the final active metabolites in the complex metabolic pathway of cyclophosphamide (CY), as a biomarker of toxicity and/or efficacy. PM is highly active and undergoes further non-enzymatic degradation to produce nornitrogen mustard which alkylates the guanine on DNA, thereby preventing DNA replication and ultimately leading to cellular death. This is the first report of PM in RIC HCT recipients.

We studied 10 patients undergoing allogeneic RIC HCT at the University of Minnesota from March 2013 to June 2013 for PM pharmacokinetics. Median age was 60 years (range 34 - 72). Four patients were male and six female. Patients were transplanted for MDS (n=3), AML (n=2), CLL, CMML, B-cell ALL, Hodgkins lymphoma and mantle cell lymphoma. Allogeneic donor sources were double umbilical cord blood (n=4), sibling peripheral blood (n=4) and matched unrelated donor peripheral blood (n=2). RIC preparative regimen consisted of CY 50 mg/kg x1 dose infused over 2 hours on day-6. CY doses were based on actual body weight except for 2 obese patients whose doses were based on an adjusted body weight and received 15 and 25% dose reductions. Preparative regimen also included fludarabine 30-40 mg/m2/day x5 on days -6 to -2 plus single fraction TBI 200 cGy. PM plasma pharmacokinetics were conducted with the single CY dose at times 0 (prior to infusion), 4, 6, 8 and 26 hours after start of CY infusion. PM was derivatised with DDTC and measured by HPLC with ultraviolet detection. Pharmacokinetic measures were calculated using noncompartmental analysis.

Median (range) total body weight was 76.7 kg (47.9-108.7) and CY dose was 3680mg (2395 - 5210mg). PM was readily measurable in all plasma samples. AUC_0-inf was 108.6 hr*mcg/mL (54.0 – 133.9), Cmax was 6.3 mcg/mL (3.7-7.5), concentration at 24 hours was 0.87 mcg/mL (0.06-2.0) and half-life 7.5 hr (2.9 - 12.9). Since not all patients received the same dose, AUC_0-inf was normalized to total CY dose and was 0.028 hr*mcg/mL/mg (0.019 - 0.037). There was a weak correlation between dose normalized AUC_0-inf, and CrCl, SCr, BMI and BSA (all r²<0.17) but a good inverse correlation with total protein at time of transplant (r²=0.63). There was a good correlation between PM concentration at 26 hours and AUC_{0-inf (}r² = 0.73).

Seven patients are alive. Two patients died with TRM at days 37 and 55 posttransplant. Causes of death were graft failure, septic shock secondary to multiple infectious organisms (HHV6, VRE, mucor), and progressive neurological decline, HHV6 encephalopathy and recurrent PRES, respectively. A third patient died of disease progression at posttransplant day 39. The two patients with TRM had 2 of the 3 highest AUCs; 133.9 and 129.8 hr*mcg/mL, respectively.

We have demonstrated that PM is readily measurable for at least 26 hours in the plasma after CY treatment. Preliminary analysis suggests that PM exposure may be associated with pretransplant total protein. Renal function appears to play a small role in PM exposure although the range of renal function in this cohort was limited. Additional data are needed to determine the relationship between PM exposure and clinical outcomes.

Special acknowledgment for financial support from the American Society of Hematology and the Trainee Research Award program (AW).

No relevant conflicts of interest to declare.