We hypothesized that the LMW substances present in P188-NF were causal and/or contributed disproportionately to the renal dysfunction observed with this agent. We further hypothesized that removal of these KU-57788 substances would result in an agent with substantially less effect on renal function, without otherwise affecting its activity. Here, we show the nature of the renal injury associated with P188-NF and demonstrate that a “purified” and less polydisperse form of P188, which we refer to as P188-P1 throughout this publication, has a significantly lesser effect on renal function in a remnant-kidney

animal model and is well tolerated in clinical studies. The role of the unpurified, excipient-grade material (P188-NF), and its impact on the results obtained in earlier clinical trials, is also discussed. find more 2 Materials and Methods 2.1 Purification of P188-NF A supercritical fluid extraction process was used to prepare P188-P. Commercial-grade poloxamer 188 (P188-NF; BASF Corporation) was supported on a polystyrene divinyl benzene solid matrix (XAD-4; Supelco) in a high-pressure MLN2238 mw stainless

steel vessel and extracted with carbon dioxide and modifying co-solvents (approximately 4 mole %) at 6,000 psi and 40 °C. The extraction proceeded until approximately 80 % of the total LMW material had been removed as analyzed by GPC. When the extraction was complete, methanol was used to elute the purified poloxamer 188 (P188-P) from the matrix. The waste stream

was also collected and evaporated. The yield of P188-P was typically 75–80 % of the loaded P188-NF. Gas chromatography and nuclear magnetic resonance were used to analyze the levels of unsaturation groups and LMW glycol species in P188-NF and P188-P, respectively. A similar supercritical fluid extraction process modified to comply with Current Good Manufacturing Practice (cGMP) was used to prepare P188-P for clinical studies. Clinical-grade P188-P was sterilized via a terminal autoclaving process, which had been pre-validated by measuring the recovery of reference material post-treatment. 2.2 Test Agents For PLEK2 all studies, both P188-NF and P188-P were formulated as a 15 % sterile aqueous solution of the appropriate agent in a vehicle containing sodium chloride 3.08 mg/mL, sodium citrate 2.38 mg/mL, and citric acid 0.366 mg/mL. Control infusions were conducted using only the vehicle. 2.3 Remnant-Kidney Animal Model Female Sprague–Dawley rats, aged 6–8 weeks, were subjected to 5/6 nephrectomy, as described by Anderson et al. [32–34], and were allowed to recover for at least 15 days. Remnant-kidney rats with stable renal function were stratified by renal function and randomized to treatment groups.