ALBERT

Description: Mycophenolate mofetil (MMF) is frequently used as part of the immunosuppressive regimen after allogeneic nonmyeloablative HCT. We and others have previously shown that low mycophenolic acid (MPA) exposure, the active component of MMF, is associated with poorer rates of engraftment and increased risk of graft-vs-host disease (GVHD). As therapeutic plasma targets are difficult to achieve in adult HCT recipients with doses of MMF 2 gm/day, higher doses are required. To determine if higher MMF doses of 3 gm/day would achieve the therapeutic plasma targets we conducted a prospective pharmacokinetic study in adult recipients of nonmyeloablative HCT treated with cyclophosphamide 50 mg/kg, fludarabine 200 mg/m2 and total body irradiation 200 cGy in combination with cyclosporine and MMF as either 1.5 gm every 12 hours (n=15) or 1 gm every 8 hours (n=15). MMF was initiated on day -3 intravenously and switched to the oral form within the first week at the same dose. Pharmacokinetic sampling at steady state was performed once in each patient between days -1 and +5 while on intravenous therapy and then repeated once between days 5 and 14 posttransplant after oral administration. There were no differences in total or unbound MPA 24 hour cumulative area under the curve (AUC), concentration at steady state (Css) or troughs between the two dosing regimens. Total MPA 24 hour AUC (median, [range]) exposures were not different between intravenous 1 gm every 8 hours (53.59 [22.86–101.99] mcg*hr/mL) and intravenous 1.5 gm every 12 hours (60.90 [35.89–127.24] mcg*hr/mL) regimen (p=0.34). Unbound MPA 24 hour AUC exposures were also not different between the intravenous 1 gm every 8 hours (0.942 [0.389–1.722] mcg*hr/mL) and intravenous 1.5 gm every 12 hours (1.081 [0.610–2.194] mcg*hr/mL)(p=0.25). Following oral therapy, total and unbound MPA 24 hour AUC exposures after 1 gm every 8 hours and 1.5 gm every 12 hours were also not different (p≥0.43). Total MPA Css after intravenous 1 gm every 8 hours, oral 1 gm every 8 hours, intravenous 1.5 gm every 12 hours, oral 1.5 gm every 12 hours were 2.23 [0.95–4.25], 2.05 [1.32–3.24], 2.53 [1.46–5.24] and 2.35 [1.47–4.05] mcg/ml, respectively (p≥0.15). We previously found that an unbound 24 hour cumulative MPA AUC 0.600 mcg*hr/mL in 87–100% of subjects. All patients with the every 8 hour dosing had neutrophil recovery (ANC 〉500 cells/uL x 3 consecutive days) at a median [range] of day 10 [6–38] as compared to day 12 [0–31] in those with every 12 hour dosing. Acute GVHD grades II–IV developed in 3 (20%) and 6 (40%) patients in the MMF every 8 hour and every 12 hour dosing cohorts, respectively. When compared to our historical controls, MPA exposure is higher with MMF 3 gm/day compared to 2 gm/day regardless of dosing schedule and achieves adequate exposure in most adult patients. In conclusion, MMF 3 g/day should be considered the standard starting dose for adult patients undergoing nonmyeloablative HCT.

Description: Introduction 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. Methods 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. Results 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. AUC0-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, AUC0-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 AUC0-inf, and CrCl, SCr, BMI and BSA (all r2

Description: CY and FLU are chemotherapeutic agents often used in RIC regimens. RIC regimens may yield lower transplant related complications and toxicities as compared to myeloablative conditioning. However TRM at 12 months still approximates 30%. We previously reported that high F-ara-A (active metabolite of FLU) exposure was associated with more TRM at a FLU dose of 40 mg/m2, but the relationship is unknown at lower doses. Similarly, phosphoramide mustard (PM, the active metabolite of CY) may influence clinical outcomes. Thus we studied the relationship between clinical outcomes and systemic exposure of F-ara-A at lower doses and PM. Forty adults undergoing allogeneic RIC HCT were prospectively studied from March 2013 to May 2014. All patients received FLU, CY and TBI as conditioning and cyclosporine or sirolimus, plus mycophenolate for posttransplant immunosuppression. Median age was 62 years (21-72). 22 were male and 18 female. The stem cell sources were sibling or unrelated donor PBSC (n=22, 55%), cord blood (n=13, 32.5%) or bone marrow (n=5, 12.5%). CY 50mg/kg was administered IV over 2 hrs on day -6. Pharmacokinetic samples were obtained at predose and 2, 4, 6, 21, 24 and 45 hrs after the end of infusion. PM was derivatized with diethyldithiocarbamate and measured by ultraviolet detection with HPLC. PM area under the curve (AUC0-last), AUC0-6 and AUC0-24 were calculated using non-compartmental methods (Phoenix WinNonlin Professional 6.3). Fludarabine dosing was 30 mg/m2/d (n=35), 32 mg/m2/d (n=1), 35 mg/m2/d (n=2) and 40 mg/m2/day (n=2) x 5 doses depending on protocol and was administered IV over 1 hr on days -6 to -2. In addition, F-ara-A troughs were obtained at 24 hours after the start of the first infusion on day -6 and 24 hours after start of 2nd infusion. F-ara-A quantification was performed using HPLC-UV. TRM was defined as death due to any cause other than relapse or disease progression. GVHD was staged and graded according to the standard GVHD criteria based on clinical and pathological criteria Recursive partitioning regression analysis was used to determine optimal cut points for PM and F-ara-A pharmacokinetic measures towards TRM at day 100, 6 months and 12 months and the incidence of acute graft vs host disease (GVHD). The cumulative incidence of engraftment, TRM and acute GVHD (II-IV and III-IV) was calculated using death prior to event as a competing risk. The proportional hazards model of Fine and Gray was used to assess the association of F-ara-A exposures towards TRM, acute GVHD and engraftment. The incidence of TRM at day 100 was 13%, 20% at 6 months and 47% at 12 months. The incidence of grades 2-4 and 3-4 acute GVHD was 39% and 25%. In univariate analysis, day 100 TRM risk was significantly higher in patients with a PM AUC0-24 ≥85 ug-hr/ml (relative risk (RR) 36%, [95% CI 9-64%]) vs. AUC0-24

Description: Chemotherapy related complications and risk of graft vs host disease limit HCT to younger patients with good clinical status and organ function. Non-myeloablative (NMA) transplant is now being widely used to extend access to HCT for patients not fit for conventional allografting. However, the incidence of engraftment and toxicity of this procedure can vary. Most NMA preparative therapies include fludarabine, a purine analog antimetabolite infrequently associated with neurotoxicity. In vivo, fludarabine undergoes rapid dephosphorylation to the active compound, F-ara-A which is 40 % renally eliminated and accumulates in renal dysfunction. Given that patients receiving a NMA HCT are often older or have poor renal function we hypothesized that variability in toxicities and clinical outcomes may be related to differences in exposure to F-ara-A. We evaluated the pharmacokinetics of F-ara-A in 29 subjects receiving a fludarabine based NMA HCT and the relationship between pharmacokinetics, engraftment and neurotoxicity. All received fludarabine 40 mg/m2/day IV x 5 days (infused over 1 hour) on days −6 to −2 in combination with cyclophosphamide 50 mg/kg/day IV on day −6 and TBI 200cGy single fraction day −1. Sampling was performed with the 1st and 5th dose of fludarabine. F-ara-A plasma concentrations were measured by HPLC. Patients underwent weekly neurotoxicity evaluation. The median age and weight of subjects was 54.8 years (range, 36–69) years and 79 kg (range, 54.6–134), respectively. Diagnoses were leukemia (n=13), lymphoma (n=7), myelodysplastic syndrome (n=5) and other (n=4). Subjects were transplanted with peripheral blood stem cells from related donors (n=10) and unrelated donor umbilical cord blood (n=19). Median CrCl and total bilirubin on admission was 100 ml/min (range, 49–190) and 0.5 mg/dL (range, 0.2–1.2), respectively. Median F-ara-A area under the curve (AUC0-∞) was 5261 ng hr/mL (range, 2935–7762), half life 9.6 hr (range, 3.1–26.6), clearance 14.5 L/hr (range, 9.4–25.6) and Cmax 851 ng/mL (range, 409–1146). F-ara-A plasma concentrations were slightly higher with dose 5. Correlation (r2) between AUC and age, weight, CrCl, SCr and total bilirubin were all ≤0.1. F-ara-A concentrations were detectable, 13.8 ng/mL (range, 5.7–38.2), on day 0 in all patients. Twenty five (cumulative incidence 89%, 95% CI: 77–100%) patients engrafted at a median of day 7. While neutrophil engraftment occurred in 100% with an AUC 〉5261 ng hr/mL (n=14), the incidence was 80% (95% CI 60–100%) for those with an AUC

Description: INTRODUCTION: Patients with severe acute graft-versus-host disease (aGVHD) are at high risk of death due to damaged organs and tissues. We have previously shown that severe aGVHD is characterized by an imbalance of circulating tissue repair factors, with elevated amphiregulin (AREG) and very low (

Description: Intestinal damage from prior chemotherapy or infections can contribute to the risk of gastrointestinal acute graft-versus-host disease (GI GVHD) and non-relapse mortality (NRM) after allogeneic hematopoietic cell transplantation (HCT). However, identifying patients who appear healthy but are at increased risk of GI GVHD and NRM due to pre-transplant intestinal tissue damage is challenging. The primary objective of this study was to determine the independent association of serum pre-transplant biomarkers associated with intestinal damage and GI GVHD on the incidence of GI GVHD and 1-year NRM. Specifically, we analyzed amphiregulin (AREG), epidermal growth factor (EGF), ratio of AREG/EGF, regenerating islet-derived 3a (REG3a), suppressor of tumorigenicity 2 (ST2), claudin-3, citrulline, and T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) in pre-transplant serum samples from 528 unique pediatric and adult (median age 41, range

Description: Reduced intensity conditioning regimend have successfully extended the use of HCT to older individuals and in those with co-morbidities. Cyclophosphamide (CY) is a commonly used alkylating agent in RIC regimens. It is an inactive pro-drug that is hepatically metabolized to the active form, PM. It exerts its cytoreductive action by further non-enzymatic conversions leading to formation of DNA adducts and cellular death. There is wide inter-patient variability in toxicity of CY, which may be due to variability in systemic exposure. The aim of our study was to characterize pharmacokinetics of PM and to identify clinical factors associated with pharmacokinetic variability. Forty-one adults undergoing allogeneic RIC HCT with CY, fludarabine and TBI were prospectively studied for PM pharmacokinetics from March 2013 to May 2014. CY 50mg/kg x one dose was administered intravenously over 2 hr at constant rate on days -6 and pharmacokinetic sampling was conducted at 2, 4, 6, 21, 24 and 45 hrs after the end of infusion. PM was derivatized with diethyldithiocarbamate and measured by a validated HPLC assay with ultraviolet detection. The lower limit of quantification was 50ng/ml. A population pharmacokinetic analysis was conducted using non-linear mixed effects model to obtain typical value of apparent clearance (Cl/fm), apparent volume of distribution (V/fm) of the metabolite and conversion rate constant (kf) of conversion to the metabolite. Clinical covariates such as weight, ideal body weight, age, gender, CrCl, total bilirubin, albumin, previous transplant, SCr, ALT, AST, and alkaline phosphatase were tested to explain the observed variability in kf, Cl/fm and V/fm. A step-wise covariate model building strategy of forward inclusion and backward elimination was used to identify the effect of clinical covariates on PM pharmacokinetics. A first order one-compartment absorption model described PM kinetics. The typical kf from parent CY to PM was 0.189 hr-1, with inter-individual variability of 42%. The typical Cl/fm and V/fm in the central comapartment were 39.1 L/hr and 264 L, respectively. The inter-individual variability in Cl/fm was 22.4% and 33.2% for V/fm. Gender was a significant covariate affecting kf, where females had 59% higher conversion rate than males, thus showing higher metabolite concentrations. Cl/fm and V/fm were allometrically scaled using total body weight. CrCl was also an important covariate affecting Cl/fm where 32% of PM was renally cleared. No other tested covariates were important within the range of our data. Model evaluations performed using visual predictive checks and non-parametric bootstrap determined that the pharmacokinetic model adequately described the observed data. Gender and CrCl significantly affected PM pharmacokinetics. Females had a significantly higher formation of PM than males. PM appears to be approximately 32% renally cleared and is affected by CrCl. These data may ultimately guide dose reduction decisions in patient with organ dysfunction. Disclosures No relevant conflicts of interest to declare.

Description: Fludarabine is a purine analog antimetabolite with antitumor and potent immunosuppressive activity. It is a prodrug that undergoes rapid dephosphorylation to the systemically circulating active compound, F-ara-A. Despite its common use in nonmyeloablative preparative regimens, the pharmacokinetics of F-ara-A are poorly characterized in HCT recipients and exposure-response relationships remain undefined. Our objective of this study was to study the association between F-ara-A systemic exposures and engraftment, acute graft vs host disease (GVHD) and treatment related mortality (TRM). Eighty seven adult patients with a median age of 55 (range 20–69) years undergoing sibling peripheral blood or bone marrow (n=22) or unrelated cord blood (n=65) donor HCT were enrolled in this pharmacokinetic-pharmacodynamic study. Underlying diseases were ALL (n=6), AML (n=26), CML (n=1), myelodysplasia (n=14), NHL (n=17), Hodgkins (n=8) and other (n=15). Patients received a nonmyeloablative regimen of fludarabine 40 mg/m2/day intravenously (IV) as a single daily dose × 5 days on days -6 to -2 given over one hour (9 were dose reduced and received 30–35 mg/m2/day), cyclophosphamide 50 mg/kg/day IV day -6 and TBI 200cGy single fraction on day -1. Equine antithymocyte globulin 15 mg/kg every 12 hours from days -3 to -1 was administered to 40 subjects. Cyclosporine and mycophenolate mofetil were given beginning on day –3. Pharmacokinetic sampling was performed with the first dose of fludarabine at times 0, 1.6, 2, 3, 4, 6, 8, 12, 24 after the start of the infusion and quantified with HPLC. Median (range) F-ara-A area under the curve (AUC)0-∞ was 5,000 (2,000–11,5000) ng hr/mL, clearance (CL) 15.3 (6.2–36.6) L/hour, concentration 24 hours after the first dose 55 (17–166) ng/mL and concentration on day zero 16.0 (0.1–144.1) ng/mL. Median serum creatinine and creatinine clearance on the first day of fludarabine was 0.9 (0.4–1.5) mg/dL and 82.1 (49.5–153.2) ml/min, respectively. Median time to neutrophil recovery was day 11 (1–38) posttransplant. Graft failure occurred in 14 (16%) individuals. Acute GVHD II-IV and III-IV developed in 47 (54%) and 15 (17%), respectively. TRM occurred in 18 (21%) individuals by 6 months posttransplant. Primary causes of death were organ failure, infection, hemorrhage and ARDS. Higher F-ara-A exposure was associated with greater TRM. The cumulative incidence (95% CI) of TRM at 6 months for individuals with an F-ara-A AUC 〉6500 ng hr/mL was 50% (23–77) vs 15% (7–23) for AUC ≤ 6500 ng hr/mL (p=0.0005), CL ≤ 12.5 L/hr 45% (24–67%) vs for CL 〉12.5 L/hr 12% (4–20%)(p=0.0002), concentration at 24 hours 〉80 ng/mL 64% (33–94%) vs ≤ 80 ng/mL 15% (7–24%) (p30 ng/mL 53% (28–78) vs ≤ 30 14% (5–22%) (p=0.0004). Sixteen percent of subjects had an F-ara-A AUC 〉6,500 ng hr/ml, 25% had an F-ara-A clearance 80 ng/mL and 20% with a day zero concentration 〉30 ng/mL. The F-ara-A exposures remained significant towards TRM in multivariate analysis. There was no association between F-ara-A exposures and engraftment or acute GVHD. These data suggest that elevated F-ara-A concentrations are associated with a greater risk of TRM. Prospective testing of fludarabine dose modifications based on pharmacokinetics is needed and may protect patients from the toxicities of fludarabine overexposure.