Bruton’s tyrosine kinase (BTK), a non-receptor tyrosine kinase, is a member of the Tec family of kinases and is essential for B cell receptor (BCR) mediated signaling. BTK also plays a critical role in the downstream signaling pathways for the Fcγ receptor in monocytes, the Fcε receptor in granulocytes, and the RANK receptor in osteoclasts. As a result, pharmacological inhibition of BTK is anticipated to provide an effective strategy for the clinical treatment of autoimmune diseases such as rheumatoid arthritis and lupus. This article will outline the evolution of our strategy to identify a covalent, irreversible inhibitor of BTK that has the intrinsic potency, selectivity, and pharmacokinetic properties necessary to provide a rapid rate of inactivation systemically following a very low dose. With excellent in vivo efficacy and a very desirable tolerability profile, 5a (branebrutinib, BMS-986195) has advanced into clinical studies.
A novel methodology of in-sample calibration curves (ISCC) using multiple isotopologue reaction monitoring (MIRM) of multiple naturally occurring isotopologue transitions of a stable isotopically labeled (SIL) analyte for instant liquid chromatography-tandem mass spectrometry (LC-MS/MS) bioanalysis of biomarkers, biotherapeutics, and small-molecule compounds is proposed and demonstrated for the first time. The theoretical isotopic abundances of the SIL analyte in its MIRM channels can be accurately calculated based on the isotopic distributions of its daughter ion and neutral loss. The isotopic abundances in these MIRM channels can also be accurately measured with a triple quadrupole mass spectrometer. By spiking a known amount of a SIL analyte into each study sample, an ISCC can be established based on the relationship between the calculated theoretical isotopic abundances (analyte concentration equivalents) in the selected MIRM channels of the SIL analyte and the measured MS/MS peak areas in the corresponding MIRM channels in each individual study sample. The analyte concentration of each study sample can then be calculated individually with the ISCC instantly without using an external calibration curve. The MIRM− ISCC−LC-MS/MS methodology was evaluated and demonstrated in this work with the examples of quantitation of a protein biomarker in human and monkey serum processed with immunocapture and trypsin digestion; three surrogate peptides in trypsin-digested human colon tissue homogenates; and a small-molecule drug in human and rat plasma extracted with liquid− liquid extraction. The potential applications of the MIRM−ISCC−LC-MS/MS methodology in quantitative proteomics, clinical laboratories, and other areas are also discussed in this paper. Without the need for using external calibration curves, this novel MIRM−ISCC−LC-MS/MS methodology can provide accurate and reliable bioanalysis in many potential applications, especially for cases where authentic matrices for external calibration curves are not available.
We analyzed the der(11) and der(4) genomic breakpoint junctions of a t(4;11) in the leukemia of a patient previously administered etoposide and dactinomycin by molecular and biochemical approaches to gain insights about the translocation mechanism and the relevant drug exposure. The genomic breakpoint junctions were amplified by PCR. Cleavage of DNA substrates containing the normal homologues of the MLL and AF-4 translocation breakpoints was examined in vitro upon incubation with human DNA topoisomerase II␣ and etoposide, etoposide catechol, etoposide quinone, or dactinomycin. The der(11) and der(4) genomic breakpoint junctions both involved MLL intron 6 and AF-4 intron 3. Recombination was precise at the sequence level except for the overall gain of a single templated nucleotide. The translocation breakpoints in MLL and AF-4 were DNA topoisomerase II cleavage sites. Etoposide and its metabolites, but not dactinomycin, enhanced cleavage at these sites. Assuming that DNA topoisomerase II was the mediator of the breakage, processing of the staggered nicks induced by DNA topoisomerase II, including exonucleolytic deletion and templatedirected polymerization, would have been required before ligation of the ends to generate the observed genomic breakpoint junctions. These data are inconsistent with a translocation mechanism involving interchromosomal recombination by simple exchange of DNA topoisomerase II subunits and DNA-strand transfer; however, consistent with reciprocal DNA topoisomerase II cleavage events in MLL and AF-4 in which both breaks became stable, the DNA ends were processed and underwent ligation. Etoposide and͞or its metabolites, but not dactinomycin, likely were the relevant exposures in this patient. L eukemia has become an increasingly common complication of effective chemotherapy (reviewed in ref. 1). Two classes of chemotherapy are associated with leukemia-alkylating agents and DNA topoisomerase II inhibitors (reviewed in refs. 1-3). The hallmarks of leukemias associated with DNA topoisomerase II inhibitors are balanced chromosomal translocations, many of which involve the MLL gene at chromosome band 11q23 (reviewed in refs. 2, 4, and 5). It has been suggested that the MLL translocation mechanism may involve DNA topoisomerase IImediated chromosomal breakage and formation of the translocations when the breakage is repaired (2, 4, 5).DNA topoisomerase II catalyzes the relaxation of supercoiled DNA by transiently cleaving and religating both strands of the double helix (6). The DNA topoisomerase II homodimer introduces four-base staggered nicks in DNA as each subunit covalently binds and cleaves one strand (6). In the presence of ATP, the DNA open gate allows passage of a second DNA helix through the cleaved strands (6). Next, the cleaved DNA strands are religated, and after ATP hydrolysis, the enzyme homodimer attains its original conformational state to catalyze another cycle (6). DNA topoisomerase II has been implicated in MLL translocations, because several anticancer drugs interfere with its c...
Preparation of multisample external calibration curves and dilution of study samples are critical steps in bioanalytical sample processing for quantitative liquid chromatography–tandem mass spectrometry (LC-MS/MS) based bioanalysis of small-molecule compounds, biotherapeutics, and biomarkers, but they can be time-consuming and prone to error. It is highly desired to simplify or eliminate these two steps in order to improve the assay throughput and robustness. While multisample external calibration curve preparation using authentic matrices can be eliminated with a previously reported in-sample calibration curve (ISCC) approach using multiple isotopologue reaction monitoring (MIRM) of a stable isotopically labeled (SIL) analyte, dilution of study samples is still inevitable due to limited LC-MS/MS assay ranges. In this work, a one-sample multipoint external calibration curve and isotope sample dilution, both using MIRM of an analyte, for quantitative LC-MS/MS based bioanalysis are proposed and demonstrated. By spiking a known amount of an analyte into one blank authentic matrix sample, a one-sample multipoint external calibration curve in an authentic matrix can be established on the basis of the relationship between the calculated theoretical isotopic abundances (analyte concentration equivalents) and the MS/MS responses in the corresponding MIRM channels. This one-sample multipoint external calibration curve can be used in the same way as the traditional multisample external calibration curve for quantitative LC-MS/MS-based bioanalysis. As isotopic abundance in each MIRM channel can be calculated and measured accurately, isotope sample dilution can be achieved by simply monitoring one or a few of the MIRM channels of the analyte in addition to the most abundant MIRM channel for study samples. While the most abundant MIRM channel (isotopic abundance of 100%) is used for the quantitation of samples having concentrations within the assay calibration curve range, less abundant MIRM channels (isotopic abundance of IA%) can be used for the quantitation of samples having concentrations beyond the assay upper limit of quantitation (ULOQ), resulting in isotope dilution factors (IDF) of 100%/IA%. The approaches of one-sample multipoint external calibration curve and isotope sample dilution were evaluated and demonstrated in this work with an example of the quantitation of daclatasvir in human plasma extracted with liquid–liquid extraction. Using these approaches together with the MIRM-ISCC methodology, accurate and reliable LC-MS/MS bioanalysis can be achieved without the need of preparation of multisample external calibration curve and dilution of study samples.
The pharmacokinetics, pharmacodynamics, safety, and tolerability of BMS-932481, a γ-secretase modulator (GSM), were tested in healthy young and elderly volunteers after single and multiple doses. BMS-932481 was orally absorbed, showed dose proportionality after a single dose administration, and had approximately 3-fold accumulation after multiple dosing. High-fat/caloric meals doubled the Cmax and area under the curve and prolonged Tmax by 1.5 hours. Consistent with the preclinical pharmacology of GSMs, BMS-932481 decreased cerebrospinal fluid (CSF) Aβ39, Aβ40, and Aβ42 while increasing Aβ37 and Aβ38, thereby providing evidence of γ-secretase enzyme modulation rather than inhibition. In plasma, reductions in Aβ40 and Aβ42 were observed with no change in total Aβ; in CSF, modest decreases in total Aβ were observed at higher dose levels. Increases in liver enzymes were observed at exposures associated with greater than 70% CSF Aβ42 lowering after multiple dosing. Although further development was halted due to an insufficient safety margin to test the hypothesis for efficacy of Aβ lowering in Alzheimer’s disease, this study demonstrates that γ-secretase modulation is achievable in healthy human volunteers and supports further efforts to discover well tolerated GSMs for testing in Alzheimer’s disease and other indications.
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