Background: High dose Methotrexate (HDMTX) has been a key element of standard therapies for lymphatic malignancy (ML), osteosarcoma (OS), etc. Despite optimal supportive care, delayed MTX excretion inevitably occur resulting in severe renal failure, mucositis, septicemia, etc. Glucarpidase (CPG2) is a recombinant enzyme hydrolyzing MTX into inactive metabolites. This is a first prospective clinical trial of CPG2 for patients (pts) with MTX nephropathy to assess clinical efficacy and clearance of MTX, Leucovorin (LV), DAMPA, 5MeTHF by LC/MS/MS method. Methods: Pts receiving HDMTX (MTX > 1g/m2) and confirmed high serum MTX concentration (cons.) after 15 hours from completing MTX administration were eligible. CPG2 50U/kg, defined by preceded phaseI, was administered with high dose LV (HDLV). If serum MTX after 46 hrs post-CPG2 was no less than 1µmol/L, additional CPG2 was planned. Primary objective is clinical important reduction (CIR: reduce and sustain serum MTX cons. under1µmol/L just after first CPG2 administration). Sample size of 16 patients provided 85% power at a one-sided alpha of 0.05 to reject the CIR rate of 42% under the expectation of 77%. Results: From 2012 to 2015, 13 pts (median age 14.5 yrs (7 pts: OS, 5 pts: ML, 1 pts: brain tumor)) were enrolled (15% had no HDMTX history). The median pre-CPG2 MTX cons. was 26.10 µmol/L (range: 1.81-486). The first dose of CPG2 was given from 1 to 4 days after starting MTX administration. Three pts was given CPG2 twice (2 of 3 was received over 24 hrs continuous MTX administration) and didn’t achieve CIR. CIR rate was 75% (95% CI, 42.8-94.5; p = 0.02). The median MTX reduction rate at 15 mins post-CPG2 was 99.1%. All adverse event except one Grade1 allergic reaction were not associated with CPG2 but with MTX. All pts with increased creatinine were normalized. Conclusions: CPG2 with HD-LV is confirmed safe and clinically effective and could rescue from irreversible renal damage for delayed MTX excretion. Although continuous MTX administration over 24 hrs may be high risk for MTX cons. rebound, additional CPG2 would possibly sustain low level serum MTX. Clinical trial information: JMA-IIA00097.Clinical trial information: #12288;.