Discovery and Preclinical Characterization of BIIB129, a Covalent, Selective, and Brain-Penetrant BTK Inhibitor for the Treatment of Multiple Sclerosis.

Multiple sclerosis (MS) is a chronic disease with an underlying pathology characterized by inflammation-driven neuronal loss, axonal injury, and demyelination. Bruton's tyrosine kinase (BTK), a nonreceptor tyrosine kinase and member of the TEC family of kinases, is involved in the regulation, migration, and functional activation of B cells and myeloid cells in the periphery and the central nervous system (CNS), cell types which are deemed central to the pathology contributing to disease progression in MS patients. Herein, we describe the discovery of BIIB129 (25), a structurally distinct and brain-penetrant targeted covalent inhibitor (TCI) of BTK with an unprecedented binding mode responsible for its high kinome selectivity. BIIB129 (25) demonstrated efficacy in disease-relevant preclinical in vivo models of B cell proliferation in the CNS, exhibits a favorable safety profile suitable for clinical development as an immunomodulating therapy for MS, and has a low projected total human daily dose.

A validated capillary microsampling liquid chromatography-tandem mass spectrometry method for quantification of antisense oligonucleotides in mouse serum.

Background: Capillary microsampling (CMS) has been used for quantitative bioanalysis of small molecules; however, there is no report of applying this technique in the bioanalysis of antisense oligonucleotides (ASOs). Results: A CMS liquid chromatography-tandem mass spectrometry method was successfully developed and validated for the quantification of ASO1 in mouse serum. The validated method was applied in a safety study in juvenile mice. Equivalent performance between CMS samples and conventional samples was demonstrated in the mouse study. Conclusion: This work is the first to report using CMS for liquid chromatography-tandem mass spectrometry quantitative bioanalysis of ASOs. The validated CMS method was successfully applied to support good laboratory practice safety studies in mice and the CMS strategy has subsequently been applied to other ASOs.

Validation and application of hybridization liquid chromatography-tandem mass spectrometry methods for quantitative bioanalysis of antisense oligonucleotides.

Background: Antisense oligonucleotide (ASO), an emerging modality in drug research and development, demands accurate and sensitive bioanalysis to understand its pharmacokinetic and pharmacodynamic properties. Results: By combining the advantages of both ligand binding and liquid chromatography-mass spectrometry/tandem mass (LC-MS/MS), hybridization LC-MS/MS methods were successfully developed and validated/qualified in a good lab practice (GLP) environment for the quantitation of an ASO drug candidate in monkey serum, cerebrospinal fluid (CSF) and tissues in the range of 0.5-500 ng/ml. Special treatment of CSF samples was employed to mitigate nonspecific binding, improve long-term storage stability and enable the usage of artificial CSF as a more accessible surrogate matrix. The method was also qualified and applied to ASO quantitation in various monkey tissue samples using a cocktail tissue homogenate as a surrogate matrix. Conclusion: This work was the first reported GLP validation and application of ASO bioanalysis using the hybridization LC-MS/MS platform.

Antisense Oligonucleotide-Related Macrovesicular Vacuolation of Hippocampal Neurons in Nonhuman Primates.

2'-methoxyethyl (MOE) antisense oligonucleotides (ASOs) tested in multidose intrathecal nonhuman primate (NHP) toxicity studies have consistently revealed the presence of single large vacuoles in pyramidal neurons of the hippocampus in the absence of any cellular response. Termed "macrovesicular," these vacuoles were characterized by immunohistochemistry and transmission electron microscopy which showed that these vacuoles are dilated lysosomes in neurons containing accumulated ASO. Additionally, two NHP studies were conducted to investigate the role of tissue fixation on their histogenesis. In Fixation Study 1, 6 doses of 5 mg 2'-MOE ASO with a full phosphorothioate backbone were administered by lumbar puncture over 5 weeks; in Fixation Study 2, 5 doses of 35 mg 2'-MOE ASO with a mixed phosphorothioate/phosphodiester backbone were administered over 12 weeks. At necropsy in each study, brain slices were either immersion fixed in neutral buffered 10% formalin or Carnoy's fixative; frozen at -80 °C; or perfusion fixed with modified Karnovsky's fixative. Fixed samples were processed to paraffin, sectioned, and stained with hematoxylin and eosin (H&E) and compared with H&E cryosections prepared from the frozen tissue of the same brain. The presence of vacuoles in fixed brain tissue but never in fresh frozen tissue showed that they arose during postmortem tissue fixation, and as such represent a processing artifact that is not relevant to human safety assessment of intrathecally administered 2'-MOE ASOs.

Discovery and Preclinical Characterization of BIIB091, a Reversible, Selective BTK Inhibitor for the Treatment of Multiple Sclerosis.

Multiple Sclerosis is a chronic autoimmune neurodegenerative disorder of the central nervous system (CNS) that is characterized by inflammation, demyelination, and axonal injury leading to permeant disability. In the early stage of MS, inflammation is the primary driver of the disease progression. There remains an unmet need to develop high efficacy therapies with superior safety profiles to prevent the inflammation processes leading to disability. Herein, we describe the discovery of BIIB091, a structurally distinct orthosteric ATP competitive, reversible inhibitor that binds the BTK protein in a DFG-in confirmation designed to sequester Tyr-551, an important phosphorylation site on BTK, into an inactive conformation with excellent affinity. Preclinical studies demonstrated BIB091 to be a high potency molecule with good drug-like properties and a safety/tolerability profile suitable for clinical development as a highly selective, reversible BTKi for treating autoimmune diseases such as MS.

Regulation of nitric oxide synthase isoforms by estrogen in the human endometrium.

OBJECTIVE: To examine the influence of estrogen on the expression of nitric oxide synthase (NOS) isoforms in human endometrial surface epithelial cell line (HES) and primary endometrial cells. DESIGN: Laboratory-based investigation. SETTING: Academic center. INTERVENTION(S): The expression of NOS isoform protein levels and mRNA was determined following estrogen/progesterone stimulation. MAIN OUTCOME MEASURE(S): NOS protein and mRNA levels in HES and primary endometrial cells. RESULT(S): Estradiol 17-beta (E2) induced a dose- and time-dependent increase in the expression of eNOS mRNA and protein and iNOS protein in HES cells which could be blocked by the estrogen receptor antagonist ICI 182,780. Estradiol increased the expression of eNOS mRNA and protein in primary endometrial cells. Estrogen also induced phosphorylation of eNOS which could not be blocked by ICI 182,780. Progesterone in physiologic concentrations augmented the effect of estrogen on the expression of both eNOS and peNOS but not of iNOS. ICI 182,780 in high concentrations stimulated the expression of iNOS protein while inhibiting eNOS. CONCLUSION(S): Estradiol through a genomic mechanism stimulates the expression of NOS isoforms in endometrial derived primary and HES cells. This effect is potentiated by progesterone.