Development and characterization of antibody reagents to assess anti-PEG IgG antibodies in clinical samples.

BACKGROUND: Polyethylene glycol (PEG) is a polymer that can be conjugated with therapeutic proteins. Monitoring anti-PEG antibodies in human subjects may be required as part of immunogenicity assessment. The lack of well-characterized anti-PEG reagents have limited our understanding of anti-PEG humoral response. RESULTS: Antibodies reactive to PEG were engineered with a human IgG1 Fc. Surface plasmon resonance and plate-based methods demonstrated that their binding was dependent on molecular weight (MW) of PEG. Specificity experiments using chemical analogs identified their specificity. CONCLUSION: Affinity, specificity and MW of PEG are critical characteristics that impact interactions of anti-PEG antibodies with PEG. These attributes especially MW of PEG and the assay formats may impact the ability to detect anti-PEG antibodies.

Identification and validation of phospho-SRC, a novel and potential pharmacodynamic biomarker for dasatinib (SPRYCEL), a multi-targeted kinase inhibitor.

PURPOSE: Dasatinib (BMS-354825) is a potent, oral multi-targeted kinase inhibitor. It is an effective therapy for patients with imatinib-resistant or -intolerant Ph+ leukemias,. It has demonstrated promising preclinical anti-tumor activity, and is under clinical evaluation in solid tumors. To support the clinical development of dasatinib, we identified a pharmacodynamic biomarker to assess in vivo SRC kinase inhibition, with subsequent evaluation in cancer patients. METHODS: The biomarker, phosphorylated SRC (phospho-SRC), was first identified in human prostate PC-3 tumor cells and peripheral blood mononuclear cells (PBMCs) in vitro. It was further assessed in nude mice bearing PC-3 xenografts. Phospho-SRC[pY418] in tumors and PBMC were measured by western blot analysis, and were quantified by ELISA assays. Dasatinib plasma concentrations were determined using LC/MS/MS. RESULTS: In PC-3 cells, dasatinib showed dose-dependent anti-proliferative effect, which correlated with the inhibition of phospho-SRC[pY418] and of SRC kinase activity. With a single oral dose of 50 or 15 mg/kg, tumoral phospho-SRC[pY418] was maximally inhibited at 3 h, partially reversed between 7 and 17 h, and completely recovered after 24 h post dose. At 5 mg/kg, tumoral phospho-SRC[pY418] inhibition was less pronounced and recovered more rapidly to baseline level within 24h. Dasatinib (1 mg/kg) resulted in little inhibition. In PBMCs, a similar time course and extent of phospho-SRC[pY418] inhibition was observed. Inhibition of phospho-SRC[pY418] in vivo appeared to correlate with the preclinical in vivo efficacy and PK profiles of dasatinib in mice. CONCLUSIONS: Phospho-SRC[pY418] may potentially be used as a biomarker to enable assessment of target inhibition in clinical studies exploring dasatinib antitumor activity.

Determination of a neuroprotective agent (S)-(+)-BMS-204352 in human, rat and dog plasma by enantioselective liquid chromatography-tandem mass spectrometry.

A sensitive liquid chromatography-electrospray ionization tandem mass spectrometry method (LC/ESI/MS/MS) for the enantioselective determination of (S)-(+)-BMS-204352, a potent and specific maxi-K channel opener, in human, rat and dog plasma was developed. (S)-(+)-BMS-204352, its enantiomer (R)-(--)-BMS-204353 and the internal standard (13C-deuterated racemate of (S)-(+)-BMS-204352) were extracted from plasma using toluene. Chromatographic separation for the enantiomers was achieved on a Chiralcel OD-H analytical column with a run time of 8 min. An aqueous mobile phase modifier was added post column to enhance the mass spectrometer sensitivity. ESI mass spectra were acquired in the negative mode with selected reaction monitoring. The limit of quantitation (LLOQ) is 0.10 ng/mL for human plasma assay. Samples from a clinical study and two animal studies were processed using these procedures. Based on the in vivo data, lack of inversion of (S)-(+)-BMS-204352 to (R)-(--)-BMS-204353 was demonstrated in human, rat and dog after administration of the drug. A sensitive non-enantioselective LC/ESI/MS/MS assay has also been developed for (S)-(+)-BMS-204352 which uses a similar extraction procedure with a C18 column with a limit of quantitation at 0.05 ng/mL. Human study samples were analyzed by both methods and the correlation coefficient between both sets of data is greater than 0.99.

Effect of dose and input rate on the brain penetration of BMS-204352 following intravenous administration to rats.

BMS-204352 is a novel maxi-K channel opener that is being developed for the treatment for stroke. The current study was designed to evaluate the plasma and brain pharmacokinetics of BMS-204352 in rats, in particular, assessing the effect of dose and input rate on brain penetration of BMS-204352. Rats (3 animals/group/time point) received a single intravenous dose of BMS-204352 as 5 mg/kg bolus, 5 mg/kg 30 min infusion, 5 mg/kg 60 min infusion, and 10 mg/kg bolus dose, into the jugular vein. Terminal blood (for plasma) and brain samples were collected for up to 9 h post-dose and samples were analyzed for the concentrations of intact BMS-204352 using a validated liquid chromatographic tandem mass spectrometric method (LC/MS/MS). As dose increased from 5 to 10 mg/kg, both BMS-204352 C(max) and AUC values increased in plasma and brain, somewhat greater in proportion to the increment in dose. Whereas the peak concentrations of BMS-204352 were affected by infusion time, overall AUCs were comparable across the bolus and infusion groups. Terminal disposition (T-half ranged from 1.6 to 2.7 h) of BMS-204352 was unaltered as a function of input rate. BMS-204352 crossed the blood-brain barrier with brain-to-plasma (B/P) ratios of approximately 7-11. Brain-to-plasma ratios appeared to be independent of dose and infusions produced somewhat higher brain penetration (B/P of ca. 11) as compared to bolus (B/P of ca. 7-8) dose. The decline of BMS-204352 in the brain paralleled that of plasma independent of the input rate and dose.