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.

Radiolabel Uncovers Nonintuitive Metabolites of BIIB104: Novel Release of [14C]Cyanide from 2-Cyanothiophene and Subsequent Formation of [14C]Thiocyanate.

BIIB104 (formerly PF-04958242), N-((3S,4S)-4-(4-(5-cyanothiophen-2-yl)phenoxy)tetrahydrofuran-3-yl)propane-2-sulfonamide, is an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor potentiator investigated for the treatment of cognitive impairment associated with schizophrenia. Preliminary in vitro metabolism studies with non-radiolabeled BIIB104 in rat, dog, and human liver microsomes (RLM, DLM, and HLM) showed O-dealkylation in all three species, tetrahydrofuran hydroxylation dominating in DLM and HLM, and thiophene hydroxylation prevalent in RLM. However, a subsequent rat mass balance study with [nitrile-14C]BIIB104 showed incomplete recovery of administered radioactivity (∼80%) from urine and feces over 7 days following an oral dose, and an exceptionally long plasma total radioactivity half-life. Radiochromatographic metabolite profiling and identification, including chemical derivation, revealed that [14C]cyanide was a major metabolite of [nitrile-14C]BIIB104 in RLM, but a minor and trace metabolite in DLM and HLM, respectively. Correspondingly in bile duct-cannulated rats, [14C]thiocyanate accounted for ∼53% of total radioactivity excreted over 48 hours postdose and it, as an endogenous substance, explained the exceptionally long plasma radioactivity half-life. The release of [14C]cyanide from the 2-cyanothiophene moiety is postulated to follow an epoxidation-initiated thiophene-opening based on the detection of non-radiolabeled counterpart metabolites in RLM. This unusual biotransformation serves as a lesson regarding placement of the radioactive label on an aryl nitrile when material will be used for evaluating the metabolism of a new drug candidate. Additionally, the potential cyanide metabolite of nitrile-containing drug molecules may be detected in liver microsomes with liquid chromatography-mass spectrometry following a chemical derivatization. SIGNIFICANCE STATEMENT: Using [nitrile-14C]BIIB104, non-intuitive metabolites of BIIB104 were discovered involving a novel cyanide release from the 2-cyanothiophene motif via a postulated epoxidation-initiated thiophene-opening. This unusual biotransformation serves as a lesson regarding placement of the radioactive label on an aryl nitrile when material will be used for evaluating the metabolism of a new drug candidate.

Preclinical safety, toxicokinetics and metabolism of BIIB131, a novel prothrombolytic agent for acute stroke.

BIIB131, a small molecule, is currently in Phase 2 for the treatment of acute ischemic stroke. Safety and metabolism of BIIB131 were evaluated following intravenous administration to rats and monkeys. Exposure increased dose-proportionally in rats up to 60 mg/kg and more than dose-proportionally in monkeys at greater than 10 mg/kg accompanied by prolonged half-life and safety findings. The BIIB131 was poorly metabolized in microsomes with no inhibition of CYPs. BIIB131-glucuronide, formed by UGT1A1, accounted for 21.5% metabolism in human hepatocytes and 28-40% in rat bile. In rats, excretion was primarily via the bile. BIIB131 inhibited the hERG and Nav1.5 cardiac channels by 39% but showed no effect on cardiovascular parameters in monkeys. Toxicology findings were limited to reversable hematuria, changes in urinary parameters and local effects. A MTD of 30 mg/kg was established in monkeys, the most sensitive species, at total plasma Cmax and AUC of 6- and 14-fold, respectively, greater than the NOAEL. The Phase 1 study started with intravenous 0.05 mg/kg and ascended to 6.0 mg/kg which corresponded to safety margins of 147- to 0.9-fold (for Cmax) within the linear drug exposure. Thus, the preclinical profile of BIIB131 has been appropriately characterized and supports its further clinical development.

The Importance of Tracking "Missing" Metabolites: How and Why?

Technologies currently employed to find and identify drug metabolites in complex biological matrices generally yield results that offer a comprehensive picture of the drug metabolite profile. However, drug metabolites can be missed or are captured only late in the drug development process. This could be due to a variety of factors, such as metabolism that results in partial loss of the molecule, covalent bonding to macromolecules, the drug being metabolized in specific human tissues, or poor ionization in a mass spectrometer. These scenarios often draw a great deal of attention from chemistry, safety assessment, and pharmacology. This review will summarize scenarios of missing metabolites, why they are missing, and associated uncovering strategies from deeper investigations. Uncovering previously missed metabolites can have ramifications in drug development with toxicological and pharmacological consequences, and knowledge of these can help in the design of new drugs.

Optimization of a novel piperazinone series as potent selective peripheral covalent BTK inhibitors.

BTK is a tyrosine kinase playing an important role in B cell and myeloid cell functions through B cell receptor (BCR) signaling and Fc receptor (FcR) signaling. Selective inhibition of BTK has the potential to provide therapeutical benefits to patients suffering from autoimmune diseases. Here we report the design, optimization, and characterization of novel potent and highly selective covalent BTK inhibitors. Starting from a piperazinone hit derived from a selective reversible inhibitor, we solved the whole blood cellular potency issue by introducing an electrophilic warhead to reach Cys481. This design led to a covalent irreversible BTK inhibitor series with excellent kinase selectivity as well as good whole blood CD69 cellular potency. Optimization of metabolic stability led to representative compounds like 42, which demonstrated strong cellular target occupancy and inhibition of B-cell proliferation measured by proximal and distal functional activity.

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.

Optimal pH 8.5 to 9 for the hydrolysis of vixotrigine and other basic substrates of carboxylesterase-1 in human liver microsomes.

Vixotrigine is a voltage- and use-dependent sodium channel blocker under investigation for the potential treatment of neuropathic pain. One of the major in vivo metabolic pathways of vixotrigine in humans is the hydrolysis of the carboxamide to form the carboxylic acid metabolite M14.The in vitro formation of M14 in human hepatocytes was inhibited by the carboxylesterase (CES) inhibitor Bis(4-nitrophenyl) phosphate in a concentration-dependent manner. The hydrolysis reaction was identified to be catalysed by recombinant human CES1b.Initial observation of only trace level formation of M14 in human liver microsomes at pH 7.4 caused us to doubt the involvement of CES1, an enzyme localised at the endoplasmic reticulum and the dominant carboxylesterase in human liver. Further investigation has revealed that optimal pH for the hydrolysis of vixotrigine and two other basic substrates of CES1, methylphenidate and oseltamivir, in human liver microsomes was pH 8.5-9 which is higher than their respective pKa(base), suggesting that neutral form of basic substrates is probably preferred for CES1 catalysis in liver microsomes.

BTK-inhibitor drug covalent binding to lysine in human serum albumin using LC-MS/MS.

Irreversible Bruton's tyrosine kinase (BTK) inhibitor drugs are designed to bind covalently to a free-thiol cysteine in the BTK protein active site. However, these reactive drugs bind to off-target proteins as well. In this study, seven BTK-inhibitor drugs containing acrylamide warheads were incubated with human serum albumin (HSA) and analyzed using an LC-MS/MS peptide mapping approach to determine the amino acid sites of drug covalent binding. Significant adduction at the free-thiol cysteine of HSA was only observed for two of the drugs. However, significant adduction was observed for at least four lysine residues. This is just a small percentage of the 59 total lysine residues in HSA. These four lysine residues are likely partially buried, accessible to the drugs, and exist at least partially in a neutral state. The levels of adduction observed in the in-vitro experimental conditions are only indicative of a relative propensity for adduction with the individual lysine residues of HSA, and are not in-vivo predictions. Widespread off-target lysine binding could impact clearance and bioavailability for irreversible inhibitor drugs. However, the extent of the impact on clearance may be limited in comparison to conjugation with glutathione.

Discovery of a Series of 7-Azaindoles as Potent and Highly Selective CDK9 Inhibitors for Transient Target Engagement.

Optimization of a series of azabenzimidazoles identified from screening hit 2 and the information gained from a co-crystal structure of the azabenzimidazole-based lead 6 bound to CDK9 led to the discovery of azaindoles as highly potent and selective CDK9 inhibitors. With the goal of discovering a highly selective and potent CDK9 inhibitor administrated intravenously that would enable transient target engagement of CDK9 for the treatment of hematological malignancies, further optimization focusing on physicochemical and pharmacokinetic properties led to azaindoles 38 and 39. These compounds are highly potent and selective CDK9 inhibitors having short half-lives in rodents, suitable physical properties for intravenous administration, and the potential to achieve profound but transient inhibition of CDK9 in vivo.

Clinical Parameters and Metabolomic Biomarkers That Predict Inhospital Outcomes in Patients With ST-Segment Elevated Myocardial Infarctions.

BACKGROUND: Postoperative risk stratification is challenging in patients with ST-segment elevation myocardial infarction (STEMI) who undergo percutaneous coronary intervention. This study aimed to characterize the metabolic fingerprints of patients with STEMI with different inhospital outcomes in the early stage of morbidity and to integrate the clinical baseline characteristics to develop a prognostic prediction model. METHODS: Plasma samples were collected retrospectively from two propensity score-matched STEMI cohorts from May 6, 2020 to April 20, 2021. Cohort 1 consisted of 48 survivors and 48 non-survivors. Cohort 2 included 48 patients with unstable angina pectoris, 48 patients with STEMI, and 48 age- and sex-matched healthy controls. Metabolic profiling was generated based on ultra-performance liquid chromatography and a mass spectrometry platform. The comprehensive metabolomic data analysis was performed using MetaboAnalyst version 5.0. The hub metabolite biomarkers integrated into the model were tested using multivariate linear support vector machine (SVM) algorithms and a generalized estimating equation (GEE) model. Their predictive capabilities were evaluated using areas under the curve (AUCs) of receiver operating characteristic curves. RESULTS: Metabonomic analysis from the two cohorts showed that patients with STEMI with different outcomes had significantly different clusters. Seven differentially expressed metabolites were identified as potential candidates for predicting inhospital outcomes based on the two cohorts, and their joint discriminative capabilities were robust using SVM (AUC = 0.998, 95% CI 0.983-1) and the univariate GEE model (AUC = 0.981, 95% CI 0.969-0.994). After integrating another six clinical variants, the predictive performance of the updated model improved further (AUC = 0.99, 95% CI 0.981-0.998). CONCLUSION: A survival prediction model integrating seven metabolites from non-targeted metabonomics and six clinical indicators may generate a powerful early survival prediction model for patients with STEMI. The validation of internal and external cohorts is required.

The impact of radiochemistry in drug projects: The use of C-14 label in the AZD8529, AZD7325, and AZD6280 projects.

Understanding the metabolic transformations of a potential drug molecule is important to understanding the safety profile of a drug candidate. Liquid chromatography-mass spectrometry is a standard method for detecting metabolites in the drug discovery stage, but this can lead to an incomplete understanding of the molecule's metabolism. In this manuscript, we highlight the role radiolabeling played in determining the metabolism and in quantifying the metabolites of AZD8529, AZD7325, and AZD6280. A quantitative whole-body autoradiography study can detect covalent adducts in vivo as was the case with AZD5248 in which the compound was bound to the aorta. Ultimately another compound free of aortic binding was developed, AZD7986.

Discovery of AZD4573, a Potent and Selective Inhibitor of CDK9 That Enables Short Duration of Target Engagement for the Treatment of Hematological Malignancies.

A CDK9 inhibitor having short target engagement would enable a reduction of Mcl-1 activity, resulting in apoptosis in cancer cells dependent on Mcl-1 for survival. We report the optimization of a series of amidopyridines (from compound 2), focusing on properties suitable for achieving short target engagement after intravenous administration. By increasing potency and human metabolic clearance, we identified compound 24, a potent and selective CDK9 inhibitor with suitable predicted human pharmacokinetic properties to deliver transient inhibition of CDK9. Furthermore, the solubility of 24 was considered adequate to allow i.v. formulation at the anticipated effective dose. Short-term treatment with compound 24 led to a rapid dose- and time-dependent decrease of pSer2-RNAP2 and Mcl-1, resulting in cell apoptosis in multiple hematological cancer cell lines. Intermittent dosing of compound 24 demonstrated efficacy in xenograft models derived from multiple hematological tumors. Compound 24 is currently in clinical trials for the treatment of hematological malignancies.

Elevated microRNA­145­5p increases matrix metalloproteinase­9 by activating the nuclear factor­κB pathway in rheumatoid arthritis.

The present study explored whether miR­145­5p can aggravate the development and progression of rheumatoid arthritis (RA) by regulating the expression of matrix metalloproteinases (MMPs). ELISAs, reverse transcription­quantitative polymerase chain reaction (RT­qPCR), and western blotting were used to examine the expression levels of MMP­1, MMP­3, MMP­9, and MMP­13 in fibroblast­like synoviocytes (FLS) from patients with RA. Levels of MMP­1, MMP­3, MMP­9, and MMP­13 were assessed in the right hind ankles of a murine collagen­induced arthritis (CIA) model by RT­qPCR and immunohistochemical (IHC) analysis. The effects of activation or inhibition of the nuclear factor­κB (NF­κB) pathway on MMPs were evaluated by RT­qPCR and western blotting. Subcellular localization of NF­κB p65 was visualized by confocal microscopy. Overexpression of miR­145­5p increased the expression of MMP­3, MMP­9, and MMP­13 in RA­FLS. Moreover, injection of a miR­145­5p agomir into mice increased MMP­3, MMP­9, and MMP­13, as demonstrated by RT­qPCR and IHC analysis. A chemical inhibitor that selectively targets NF­κB (BAY11­7082) significantly attenuated MMP­9 expression, while it did not influence the levels of MMP­3 and MMP­13. Immunofluorescence analysis revealed that nuclear localization of p65 was significantly enhanced, indicating that miR­145­5p enhances activation of the NF­κB pathway by promoting p65 nuclear translocation. miR­145­5p overexpression also significantly increased phosphorylated p65 levels; however, the levels of IkB­a were reduced in response to this miRNA. Moreover, our results indicated that miR­145­5p aggravated RA progression by activating the NF­κB pathway, which enhanced secretion of MMP­9. In conclusion, modulation of miR­145­5p expression is potentially useful for the treatment of RA inflammation, by regulating the expression of MMPs, and MMP­9 in particular, through inhibition of the NF­κB pathway.

Late-occurring and Long-circulating Metabolites of GABAAα2,3 Receptor Modulator AZD7325 Involving Metabolic Cyclization and Aromatization: Relevance to MIST Analysis and Application for Patient Compliance.

AZD7325 [4-amino-8-(2-fluoro-6-methoxyphenyl)-N-propylcinnoline-3-carboxamide] is a selective GABAAα2,3 receptor modulator intended for the treatment of anxiety disorders through oral administration. An interesting metabolic cyclization and aromatization pathway led to the tricyclic core of M9, i.e., 2-ethyl-7-(2-fluoro-6-methoxyphenyl)pyrimido[5,4-c]cinnolin-4(3H)-one. Further oxidative metabolism generated M10 via O-demethylation and M42 via hydroxylation. An authentic standard of M9 was synthesized to confirm the novel structure of M9 and that of M10 and M42 by liver microsomal incubation of the M9 standard. Metabolites M9, M10, and M42 were either minor or absent in plasma samples after a single dose; however, all became major metabolites in human and preclinical animal plasma after repeated doses and circulated in humans longer than 48 hours after the end of seven repeated doses. The absence of these long circulating metabolites from selected patients' plasma samples was used to demonstrate patient noncompliance as the cause of unexpected lack of drug exposure in some patients during a Phase IIb outpatient clinical study. The observation of late-occurring and long-circulating metabolites demonstrates the need to collect plasma samples at steady state after repeated doses when conducting metabolite analysis for the safety testing of drug metabolites. All 12 major nonconjugate metabolites of AZD7325 observed in human plasma at steady state were also observed in dog, rat, and mouse plasma samples collected from 3-month safety studies and at higher exposures in the animals than humans. This eliminated concern about human specific or disproportional metabolites.

Synthesis of C-14 labeled GABAA α2/α3 selective partial agonists and the investigation of late-occurring and long-circulating metabolites of GABAA receptor modulator AZD7325.

Anxiolytic activity has been associated with GABAA α2 and α3 subunits. Several target compounds were identified and required in C-14 labeled form to enable a better understanding of their drug metabolism and pharmacokinetic properties. AZD7325 is a selective GABAA α2 and α3 receptor modulator intended for the treatment of anxiety through oral administration. A great number of AZD7325 metabolites were observed across species in vivo, whose identification was aided by [14 C]AZD7325. An interesting metabolic cyclization and aromatization pathway leading to the tricyclic core of M9 and the oxidative pathways to M10 and M42 are presented.

Mouse Red Blood Cell-Mediated Rare Xenobiotic Phosphorylation of a Drug Molecule Not Intended to Be a Kinase Substrate.

Phosphorylation of xenobiotics is rare, probably owing to a strong evolutionary pressure against it. This rarity may have attracted more attention recently as a result of intentionally designed kinase-substrate analogs that depend on kinase-catalyzed activation to form phosphorylated active drugs. We report a rare phosphorylated metabolite observed unexpectedly in mouse plasma samples after an oral dose of a Tankyrase inhibitor that was not intended to be a kinase substrate, i.e., (S)-2-(4-(6-(3,4-dimethylpiperazin-1-yl)-4-methylpyridin-3-yl)phenyl)-8-(hydroxymethyl)quinazolin-4(3H)-one (AZ2381). The phosphorylated metabolite was not generated in mouse hepatocytes. In vitro experiments showed that the phosphorylation of AZ2381 occurred in mouse whole blood with heparin as anticoagulant but not in mouse plasma. The phosphorylated metabolite was also produced in rat, dog, and human blood, albeit at lower yields than in mouse. Divalent metal ions are required for the phosphorylation since the reaction is inhibited by the metal chelator EDTA. Further investigations with different cellular fractions of mouse blood revealed that the phosphorylation of AZ2381 was mediated by erythrocytes but did not occur with leukocytes. The levels of 18O incorporation into the phosphorylated metabolite when inorganic 18O4-phosphate and γ-18O4-ATP were added to the mouse blood incubations separately suggested that the phosphoryl transfer was from inorganic phosphate rather than ATP. It remains unclear which enzyme present in red blood cells is responsible for this rare phosphorylation.

CYP-Mediated Sulfoximine Deimination of AZD6738.

In hepatic S9 and human liver microsomes (HLMs) the sulfoximine moiety of the ATR inhibitor AZD6738 is metabolized to its corresponding sulfoxide (AZ8982) and sulfone (AZ0002). The initial deimination to AZ8982 is nominally a reductive reaction, but in HLMs it required both NADPH and oxygen and also was inhibited by 1-aminobenzotriazole at a concentration of 1 mM. Studies conducted in a panel of 11 members of the cytochrome P450 (P450) family (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 CYP2J2, CYP3A4, and CYP3A5) confirmed that deimination was an oxidative process that was mediated largely by CYP2C8 with some CYP2J2 involvement, whereas the subsequent oxidation to sulfone was carried out largely by CYP2J2, CYP3A4, and CYP3A5. There was no measureable metabolism in flavin-containing monooxygenase (FMO) enzymes FMO3, FMO5 or NADPH cytochrome C reductase. Studies using Silensomes, a commercially available HLM in which specific members of the P450 family have been inhibited by selective mechanism-based inhibitors, showed that when CYP2C8 was inhibited, the rate of deimination was reduced by 95%, suggesting that CYP2J2 is only playing a minor role in HLMs. When CYP3A4 was inhibited, the rate increased by 58% due to the inhibition of the subsequent sulfone formation. Correlation studies conducted in HLM samples from different individuals confirmed the role of CYP2C8 in the deimination over CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A. Hence, although nominally a reduction, the deimination of AZD6738 to its sulfoxide metabolite AZ8982 is an oxidation mediated by CYP2C8, and this metabolite is subsequently oxidized to the sulfone (AZ0002) largely by CYP3A.

Identification of Protonated Sulfone and Aromatic Carboxylic Acid Functionalities in Organic Molecules by Using Ion-Molecule Reactions Followed by Collisionally Activated Dissociation in a Linear Quadrupole Ion Trap Mass Spectrometer.

Gas-phase reactivity of protonated model compounds with different functional groups toward trimethoxymethylsilane (TMMS) was studied to explore the utility of this reagent in mass spectrometric identification of specific functionalities for potentially rapid characterization of drug metabolites. Only protonated analytes with a carboxylic acid, a sulfone, or a sulfonamide functionality formed diagnostic adducts that had lost a methanol molecule upon reactions with TMMS. Collisionally activated dissociation (CAD) of these methanol-eliminated adduct ions (MS3 experiments) produced characteristic fragment ions of m/z 75, 105, and 123 for sulfones, while an additional methanol elimination was observed for carboxylic acids and sulfonamides. CAD of latter products (MS4 experiments) resulted in elimination of diagnostic neutral molecules CO2 (44 Da) and C2H6O2Si (90 Da) for aromatic carboxylic acids. Both aliphatic carboxylic acids and sulfonamides yield several fragment ions in these MS4 experiments that are different from those observed for sulfones or aromatic carboxylic acids. Potential energy surfaces were calculated (at the M06-2X/6-311++G(d,p) level of theory) to explore the mechanisms of various reactions. In summary, sulfones and aromatic carboxylic acids can be differentiated from each other and also from sulfonamides and aliphatic carboxylic acids based on reactions with TMMS and one or two CAD experiments. Aliphatic carboxylic acids and sulfonamides could not be differentiated from each other.

Modulating the strength of hydrogen bond acceptors to achieve low Caco2 efflux for oral bioavailability of PARP inhibitors blocking centrosome clustering.

During the lead generation and optimization of PARP inhibitors blocking centrosome clustering, it was discovered that increasing hydrogen bond acceptor (HBA) strength improved cellular potency but led to elevated Caco2 and MDR1 efflux and thus poor oral bioavailability. Conversely, compounds with lower efflux had reduced potency. The project team was able to improve the bioavailability by reducing efflux through systematic modifications to the strength of the HBA by changing the electronic properties of neighboring groups, whilst maintaining sufficient acceptor strength for potency. Additionally, it was observed that enantiomers with different potency showed similar efflux, which is consistent with the promiscuity of efflux transporters. Eventually, a balance between potency and low efflux was achieved for a set of lead compounds with good bioavailability which allowed the project to progress towards establishing in vivo pharmacokinetic/pharmacodynamic relationships.