Abstract Pralatrexate is a rationally designed antifolate with high affinity for reduced folate carrier-1 (RFC-1) and folyl poly-glutamyl synthetase (FPGS), resulting in efficient uptake and retention in cancer cells. Pralatrexate is being developed for various indications, including peripheral T-cell lymphoma (PTCL), cutaneous T-cell lymphoma, non-small cell lung cancer (NSCLC), and bladder cancer. Pralatrexate is a 1:1 racemic mixture of R- and S- diastereomers. Here we summarize the pharmacokinetics (PK) of pralatrexate from 3 Phase 1–2 studies. Pralatrexate was administered weekly or bi-weekly by IV push over 3–5 minutes (n=44) or as a 60-minute infusion (n=10) at doses ranging from 30–325 mg/m2. Serial plasma and urine samples, collected for up to 72 hours after dosing, were analyzed using a validated chiral LC-MS/MS method (lower limit of quantitation: 0.5 ng/ml). PK parameters were estimated by noncompartmental PK analysis. Clinical covariates were captured at screening to explore relationships with pralatrexate PK. The final PK results were based on 54 patients; 10 with relapsed/refractory PTCL (PDX-008), 38 with advanced NSCLC (PDX-007), and 6 with advanced solid tumors and concurrent docetaxel administration (PDX-99-083). The PK population was predominantlyWhite (81%) and balanced by gender (52% female). Age ranged from 24 to 77 (mean 60) years, body weight from 43–127 (77) kg, and estimated creatinine clearance from 53 to 130 (89) mL/min. Patients had adequate hepatic and hematologic function. Both pralatrexate diastereomers showed a similar multiphasic decline in plasma concentrations with a slow terminal phase (t½term = 12∓18h, PDX-008), likely reflecting the return from deep intracellular compartments. There was no evidence of PK nonlinearities in the dose range of 30–325 mg/m2. On a weekly or biweekly administration schedule, there was no significant drug accumulation in plasma over the first 2–6 doses. Other than expected changes in Cmax and Tmax, there were no apparent differences in PK between IV administration over 3–5 minutes and 60-minute infusion. Mean (COV) values (n=41−54) for total body clearance, renal clearance and volume of distribution at steady-state were 495 mL/min (57%), 125 mL/min (71%) and 106 L (121%) for the S-diastereomer and 218 mL/min (44%), 74 mL/min (60%) and 36 L (78%) for the R-diastereomer, respectively. These results suggest net renal tubular secretion, low hepatic extraction, and limited tissue distribution for both diastereomers. Consistent with approximately 30% contribution of renal excretion to overall drug elimination, creatinine clearance and age accounted for only 10% of the observed variability in total body clearance. In conclusion, this analysis suggests that pralatrexate PK is linear and dose-proportional across the dose range tested, with no accumulation with repeated dosing. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B211.