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Introduction: BMS-986278 is being investigated as a treatment (tx) for idiopathic pulmonary fibrosis (IPF). In vitro studies demonstrated that OATP mediates human hepatic uptake of BMS-986278. Patients with IPF often receive concomitant medications, including OATPi. Aims and Objectives: To evaluate the effects of rifampin (rif) on the PK and tolerability of BMS-986278 in healthy adult participants (ppts). Methods: This was an open-label drug-interaction study. Fasted ppts received an oral dose of 30 mg BMS-986278 on d1 followed by a 7-d washout; on d8, ppts received an oral dose of 600 mg rif and 30 mg BMS-986278. Plasma PK was measured for BMS-986278 and rif; tolerability was monitored. Results: All 15 enrolled ppts completed the study (73% male, 73% white, mean [SD] age 46.1 [12.7] y and BMI 25.2 [2.7] kg/m2). Following coadministration with rif, BMS-986278 mean area under the curve from 0 to infinity (AUC0-inf) and maximum concentration (Cmax) were elevated by 3.13- and 2.75-fold, respectively, vs BMS-986278 alone. Median time to Cmax (Tmax) increased from 1.1 h (BMS-986278) to 2.0 h (BMS-986278+rif). Rif PK matched published data. BMS-986278+rif and BMS-986278 alone demonstrated a similar safety profile. Of 4 reported adverse events, 3 were mild (back pain, nausea, and presyncope) and 1 was moderate (constipation); none were deemed related to tx. Conclusions: BMS-986278+rif and BMS-986278 alone were generally well tolerated; increases in exposure up to 3x of BMS-986278 were observed with co-administration of rif. Concomitant OATPi medications anticipated to increase BMS-986278 exposure

As BMS-820836 causes dose-dependent heart rate increases, QTcI, QTcF, QTcB, and QT beat-to-beat methods were compared in this thorough QT study in healthy subjects. Two parallel groups of subjects (n = 60 per group) received 2 mg (maximum therapeutic) or 4 mg (supratherapeutic) of BMS-820836 once daily for 14 days with uptitration. Another 60 subjects received encapsulated moxifloxacin (400 mg) or matching placebo in a nested-crossover study design. Compared with QTcF and QTcB, baseline QTcI had the smallest and near-zero Pearson correlation coefficient with heart rate (0.0938), which supported the choice of QTcI as the primary electrocardiographic end point. BMS-820836 was not associated with prolongation of the QT interval at the doses tested; however, ΔΔQTbtb showed the smallest deviation from 0 milliseconds compared with ΔΔQTcI and ΔΔQTcF. The ΔΔQTbtb results appeared to be more consistent with the very low likelihood of any effect on the QT interval by BMS-820836 based on the wide margin (>400×) of the in vitro hERG IC50 and free plasma concentration. Further, this is the first published study that used the nested-crossover design and QTbtb analysis in a thorough QT study. Assay sensitivity was confirmed with encapsulated moxifloxacin.

The objectives of this study were to evaluate the safety, tolerability and pharmacokinetics (PK) of BMS-986001 as a single oral dose in healthy male subjects. Sixty-four healthy male subjects were randomized to receive a single dose of BMS-986001 or placebo in this single-blind, placebo-controlled, sequential ascending-dose study. There were eight treatment groups (10, 30, 100, 300, 600, and 900 mg fed; and 100 and 300 mg fasted) of eight subjects each (BMS-986001 n = 6/placebo n = 2). BMS-986001 was well tolerated, with no serious adverse events (AEs), deaths, or discontinuations due to AEs reported. AEs were experienced by 14.6% of subjects receiving BMS-986001; however, these did not appear to be dose related and were not considered to be related to study drug. BMS-986001 was rapidly absorbed and exhibited a linear dose-exposure relationship across the dose range studied. PK appeared similar whether administered with or without food. Administration of BMS-986001 as a single dose was generally safe and well tolerated. A linear dose-exposure relationship was seen across all doses studied, with no apparent food effect. Further clinical development is warranted.

RATIONALE Nucleotide analogs are highly polar and ionic, which impose great challenges on bioanalysis. Ion-pairing liquid chromatography/tandem mass spectrometry (LC/MS/MS) is the predominant reported approach for such compounds. Assay ruggedness of ion-pairing LC/MS/MS methods was often a challenge due to the potential contamination of the ion source of the mass spectrometer and LC column performance deterioration caused by ion-pairing reagents. METHODS An ion-pairing reagent was only added to the reconstitution solution to minimize its exposure to the MS ion source. To achieve optimum sensitivity, high pH mobile phases and negative ion ESI were needed for the LC/MS/MS method. However, high pH mobile phases led to the accumulation of ion-pairing reagent on the analytical column, which was washed off with an acidic solution to restore the column performance. In addition, isopropanol was used as a mobile phase modifier to improve peak shape and sensitivity. RESULTS The limit of detection was established at 1.0 ng/mL in the cell lysate. The calibration curve showed good linearity over the range of 1.0 to 100 ng/mL. The overall accuracy was no less than 87.7% based on four levels of quality control samples. Inter-run precision and intra-run precision across four analytical runs for low, geometric, medium and high QCs were less than 12.9. CONCLUSIONS By identifying and addressing the root cause of the assay ruggedness problem, we have developed a rugged ion-pairing LC/MS/MS method for a triphosphate (TP) metabolite of BMS-986001 in peripheral blood mononuclear cells. The new method overcame challenges such as a rapid deterioration of the peak shape, increased carryover and extremely poor column life. The peak shape was well maintained throughout multiple analytical runs. This method has been successfully applied to a toxicology study in cynomolgus monkey. Copyright © 2012 John Wiley & Sons, Ltd.

Dasatinib (Sprycel) is a potent antitumor agent prescribed for patients with chronic myeloid leukemia (CML). To enable reliable quantification of dasatinib and its pharmacologically active metabolites in human plasma during clinical testing, a sensitive and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated. Samples were prepared using solid phase extraction on Oasis HLB 96-well plates. Chromatographic separation was achieved isocratically on a Luna phenyl-hexyl analytical column. Analytes and the stable labeled internal standards were detected by positive ion electrospray tandem mass spectrometry. The assay was validated over a concentration range of 1.00-1000 ng/mL for dasatinib and its two active metabolites. Intra- and inter-assay precision values for replicate QC control samples were within 5.3% for all analytes during the assay validation. Mean QC control accuracy values were within ± 9.0% of nominal values for all analytes. Assay recoveries were high (>79%) and internal standard normalized matrix effects were minimal. The three analytes were stable in human plasma for at least 22 h at room temperature, for at least 123 days at -20°C, and following at least six freeze-thaw cycles. The validated method was successfully applied to the quantification of dasatinib and two active metabolites in a human pharmacokinetic study.

High performance liquid chromatoraphy/ionspray mass spectra of N-ethylmaleimide (NEM) and acrylate derivatives of the sulfhydryl compound BMS186716 exhibited a variety of fragmentation patterns, in positive- or negative-ion mode, providing a wide choice of conditions for developing a bioanalytical method based on selected-ion monitoring. Because the NEM derivative gave two chromatographic peaks (due to two diastereomers) whereas the acrylate derivative gave a single peak, the acrylate derivatization was chosen for further investigations. While BMS186716 in blood or plasma was extremely unstable, the compound could be stabilized by adding methyl acrylate (MA) to the biological matrix, owing to instantaneous reaction to form the stable BMS186716-MA derivative. This stabilization scheme is being investigated for developing a sensitive bioanalytical liquid chromatographic/mass spectrometric method for BMS186716.

Omapatrilat, the most clinically advanced member of a new class of cardiovascular agents, vasopeptidase inhibitors, is under development at Bristol-Myers Squibb Pharmaceutical Research Institute for the treatment of hypertension and heart failure. An electrospray LC/MS/MS method has been developed and validated for the simultaneous determination of omapatrilat and its four metabolites in human plasma. Since omapatrilat and two of the metabolites are sulfhydryl-containing compounds, methyl acrylate was used to stabilize these compounds in human blood and plasma samples. Methyl acrylate reacted instantly with the sulfhydryl group to form a derivative that was stable in blood and plasma. Extraction of the analytes from plasma samples was achieved by semiautomated liquid-liquid extraction, where a robotic liquid handler performed the liquid-transferring steps. The mass spectrometer was operated in the negative ion selected-reaction-monitoring mode. The calibration curve ranges were 0.5-250 ng/mL for omapatrilat and one metabolite and 2.0-250 ng/mL for the other three metabolites.

During method development in support of non-clinical studies in animal models, BMS-186716 was found to be extremely unstable in blood and plasma. Stabilization of the compound was achieved by reacting the compound with methyl acrylate (MA) in blood, from which the plasma was then prepared. While the resulting BMS-186716-MA adduct was found to be stable in dog plasma, and hence it was used as the basis for the method developed for analysis of dog plasma samples, the BMS-186716-MA adduct was found to be unstable in rat plasma as it was readily hydrolyzed to BMS-186716-acrylic acid (AA) by native esterases found in rat plasma. Although the finding of the instability of BMS-186716-MA in rat plasma was not the result of prospective planning, we were able to successfully develop a quantitative bioanalytical method using BMS-186716-AA as the analyte instead of the originally planned BMS-186716-MA analyte. The standard and quality-control (QC) samples were prepared by spiking blank plasma with BMS-186716-MA, and then allowing them to stand at room temperature for 1 h to convert BMS-186716-MA to BMS-186716-AA. After adding the internal standard BMS-188035-AA, each sample was acidified with HCl and then extracted with methyl tert.-butyl ether. The reconstituted extract was injected into a HPLC-electrospray ionization mass spectrometric system for detection by positive ion electrospray ionization. A lower limit of quantitation (LLQ) of 5 ng/ml was achieved, using 0.1 ml plasma and a standard curve range of 5-5000 ng/ml.

As it is extremely unstable in blood, the thiol compound BMS186716 was stabilized by the addition of methyl acrylate (MA) to blood samples. The blood samples were then kept in ice for 10–15 min for completion of the Michael addition reaction to occur between the thiol group of BMS186716 and MA, after which the plasma was separated by centrifugation under refrigeration. For sample analysis, the standard and quality control samples were prepared by spiking blank plasma with the BMS186716–MA adduct. After addition of the internal standard, BMS188035–MA, each sample was acidified with HCl and then extracted with methyl tert .-butyl ether. Each reconstituted extract was injected into a high-performance liquid chromatography–positive ion electrospray ionization mass spectrometric system. The electrospray condition was chosen to enhance the [M+NH 4 ] + signal at the expense of the [M+H] + signal. Monitoring the [M+NH 4 ] + signal, a lower limit of quantitation of 2.5 ng/ml was achieved, with 0.5 ml plasma. We have thus shown that a sulfhydryl compound (BMS186716) in blood can successfully be stabilized by reacting it with MA and that the adduct produced is adequately stable in blood and plasma to allow the development of a rugged quantitative bioanalytical method.

BMS-186318 is a member of the recently discovered “aminodiol” class of HIV protease inhibitors. A simple but sensitive method was developed for the determination of BMS-186318 in dog plasma and then applied to monkey and rat plasma. The compound was extracted from dog plasma with methyl tert -butyl ether at basic pH. The dried extract was reconstituted in mobile phase and injected into a 150 × 2.1 mm i.d. Zorbax Rx-C 18 HPLC column. A portion of the effluent was directed into the LC-ionspray MS system, where the [M + H] + ion of the secondary amine compound was monitored. The HPLC conditions were chosen in order to achieve a short run time and large sample throughput, with both analyte and internal standard eluting within 1.5 min. The liquid-liquid extraction procedure provided very clean extracts so that sufficient signal-to-noise ratio was obtained with single-stage mass spectrometry instead of the more costly tandem mass spectrometry. The required lower limit of quantitation of 2.5 ng ml −1 was easily achieved. The method has also been validated for BMS-186318 in monkey plasma without modification. The method has been modified for rat plasma. Owing to irreproducibility observed when applying the liquid-liquid extraction method to rat plasma, a solid-phase extraction method was developed. The addition of phenylmethylsulfonyl fluoride was necessary to stabilize BMS-186318 in rat blood and plasma.