High-Throughput SFC-MS/MS Method to Measure EPSA and Predict Human Permeability.

Permeability is a key factor driving the absorption of orally administered drugs. In early discovery, the efficient evaluation of permeability, particularly for compounds violating Lipinski's Rule of 5, remains challenging. Addressing this, we established a high-throughput method to measure the experimental polar surface area (HT-EPSA) as an in vitro surrogate to measure permeability. Compared to earlier methods, HT-EPSA significantly reduces data acquisition time with enhanced sensitivity, selectivity, and data quality. In the effort of translating EPSA to human in vitro and in vivo passive permeability, we demonstrated the application of EPSA for predicting Caco-2 cell and human intestinal permeability, showing improvements over topological polar surface area and the parallel artificial membrane permeability assay for rank-ordering permeability in a proteolysis targeting chimera case study. The HT-EPSA method is expected to be highly beneficial in guiding early stage compound rank-ordering, faster decision-making, and in predicting in vitro and/or in vivo human intestinal permeability.

Discovery of Potent Azetidine-Benzoxazole MerTK Inhibitors with In Vivo Target Engagement.

Inhibition of the receptor tyrosine kinase MerTK by small molecules has the potential to augment the immune response to tumors. Potent, selective inhibitors with high levels of in vivo target engagement are needed to fully evaluate the potential use of MerTK inhibitors as cancer therapeutics. We report the discovery and optimization of a series of pyrazinamide-based type 1.5 MerTK inhibitors bearing an azetidine-benzoxazole substituent. Compound 31 potently engages the target in vivo and demonstrates single agent activity in the immune-driven MC-38 murine syngeneic tumor model.

Discovery of A-910, a Highly Potent and Orally Bioavailable Dual MerTK/Axl-Selective Tyrosine Kinase Inhibitor.

TAM receptor tyrosine kinases have emerged as promising therapeutic targets for cancer treatment due to their roles in both tumor intrinsic survival mechanisms and suppression of antitumor immunity within the tumor microenvironment. Inhibiting MerTK and Axl selectively is believed to hinder cancer cell survival, reverse the protumor myeloid phenotype, and suppress efferocytosis, thereby eliciting an antitumor immune response. In this study, we present the discovery of A-910, a highly potent and selective dual MerTK/Axl inhibitor, achieved through a structure-based medicinal chemistry campaign. The lead compound exhibits favorable oral bioavailability, exceptional kinome selectivity, and significantly improved in vivo target engagement. These findings support the use of A-910 as an orally bioavailable in vivo tool compound for investigating the immunotherapy potential of dual MerTK/Axl inhibition.

Integrity and efficiency: AbbVie's journey of building an integrated nonregulated bioanalytical laboratory.

While bioanalytical outsourcing is widely adopted in the pharmaceutical industry, AbbVie is one of the few large biopharmaceutical companies having an internal bioanalytical unit to support nearly all its drug metabolism and pharmacokinetic studies. This article highlights our experience and perspective in building an integrated and centralized laboratory to provide early discovery and preclinical-stage bioanalytical support with high operational efficiency, cost-effectiveness and data integrity. The advantages of in-house nonregulated bioanalytical support include better control of data quality, faster turnaround times, real-time knowledge sharing and troubleshooting, and lower near- and long-term costs. The success of an in-house model depends upon a comprehensively optimized and streamlined workflow, fueled by continuous improvements and implementation of innovative technologies.

Global Analysis of Models for Predicting Human Absorption: QSAR, In Vitro, and Preclinical Models.

Models intended to predict intestinal absorption are an essential part of the drug development process. Although many models exist for capturing intestinal absorption, many questions still exist around the applicability of these models to drug types like "beyond rule of 5" (bRo5) and low absorption compounds. This presents a challenge as current models have not been rigorously tested to understand intestinal absorption. Here, we assembled a large, structurally diverse dataset of ∼1000 compounds with known in vitro, preclinical, and human permeability and/or absorption data. In silico (quantitative structure-activity relationship), in vitro (Caco-2), and in vivo (rat) models were statistically evaluated for predictive performance against this human intestinal absorption dataset. We expect this evaluation to serve as a resource for DMPK scientists and medicinal/computational chemists to increase their understanding of permeability and absorption model utility and applications for academia and industry.

Determination of IL-23 Pharmacokinetics by Highly Sensitive Accelerator Mass Spectrometry and Subsequent Modeling to Project IL-23 Suppression in Psoriasis Patients Treated with Anti-IL-23 Antibodies.

The pro-inflammatory cytokine interleukin (IL)-23 is a key modulator of the immune response, making it an attractive target for the treatment of autoimmune disease. Correspondingly, several monoclonal antibodies against IL-23 are either in development or approved for autoimmune indications such as psoriasis. Despite being a clinical validated target, IL-23 pharmacokinetics (e.g., IL-23 synthesis and elimination rates) and the degree of target suppression (i.e., decrease in free "active" IL-23) associated with clinical efficacy are not well understood, primarily due to its ultra-low circulating levels and the lack of sensitive and accurate measurement methods. In the current work, this issue was overcome by using accelerator mass spectrometry (AMS) to measure the concentration and pharmacokinetics of human recombinant [14C]-IL-23 following an intravenous trace-dose in cynomolgus monkeys. IL-23 pharmacokinetic parameters along with clinical drug exposure and IL-23 binding affinities from four different anti-IL-23 antibodies (ustekinumab, tildrakizumab, guselkumab, and risankizumab) were used to build a pharmacokinetics/pharmacodynamics (PK/PD) model to assess the time course of free IL-23 over one year in psoriasis patients following different dosing regimens. The predicted rank order of reduction of free IL-23 was consistent with their reported rank order of Psoriasis Area and Severity Index (PASI) 100 scores in clinical efficacy trials (ustekinumab < tildrakizumab < guselkumab < risankizumab), thus demonstrating the utility of highly sensitive AMS for determining target pharmacokinetics to inform PK/PD modeling and assessing target suppression associated with clinical efficacy.

Identification of aminopyrazolopyridine ureas as potent VEGFR/PDGFR multitargeted kinase inhibitors.

Tumor angiogenesis is mediated by KDR and other VEGFR and PDGFR kinases. Their inhibition presents an attractive approach for developing anticancer therapeutics. Here, we report a series of aminopyrazolopyridine ureas as potent VEGFR/PDGFR multitargeted kinase inhibitors. A number of compounds have been identified to be orally bioavailable and efficacious in the mouse edema model.

Community-based surveillance in the united states of macrolide-resistant pediatric pharyngeal group A streptococci during 3 respiratory disease seasons.

BACKGROUND: In 2001, a total of 48% of pharyngeal group A streptococci (GAS) from Pittsburgh children were macrolide resistant. We assessed macrolide resistance, resistance genes, and emm types among GAS in the United States. METHODS: In prospective, multicenter, community-based surveillance of pharyngeal GAS recovered from children 3-18 years old during 3 respiratory seasons (the 2000-2001 season, the 2001-2002 season, and the 2002-2003 season), GAS were tested for macrolide resistance and underwent emm gene sequencing. Macrolide-resistant GAS were tested for resistance to clindamycin, and resistance genes were determined. RESULTS: Erythromycin resistance was observed in 4.4% of isolates from the 2000-2001 season, 4.3% from the 2001-2002 season, and 3.8% from the 2002-2003 season (P=.80). Clindamycin resistance was found in 1.04% of isolates; annual rates of clindamycin resistance were stable (P=.75). The predominant resistance genotype each year was mef A (65%-76.9%; overall, 70.3%). Resistant isolates included strains representing 8-11 different emm types each year. Heterogeneity of emm subtypes, resistance genes, and clindamycin resistance was evident among resistant isolates within some emm types. Geographic variability in resistance rates was present each year. CONCLUSIONS: The macrolide resistance rate among pharyngeal GAS was <5% and was stable over the 3 seasons. However, rates varied among sites each year. There was no evidence of spread of a specific resistant clone, increasing clindamycin resistance, or escalation in median erythromycin MICs.

Epidemiology of macrolide and/or lincosamide resistant Streptococcus pneumoniae clinical isolates with ribosomal mutations.

Twenty macrolide and/or lincosamide resistant Streptococcus pneumoniae clinical isolates from various sources with 50S ribosomal mutations were identified. Mutations were identified in the 23S rDNA with substitutions at A2058, A2059, or C2611 and in L4 or L22 ribosomal protein genes. Fourteen were A2059G substitutions, one was A2058G, two were C2611T, two had an altered L4 and one isolate contained an altered L22 gene. Susceptibility testing with erythromycin, josamycin, clindamycin, and two ketolides including cethromycin was performed. The L4 mutants had the amino acid changes of (69)GTG(71) to (69)TPS(71). The isolate with the L22 mutation contained an 18 base pair tandem duplication/insertion at the 3' end of the gene. 50s ribosomal mutations are the least frequent mechanism of S. pneumoniae resistance, occurring at an extremely low frequency and are identified only by genome sequence data.

AhR activation underlies the CYP1A autoinduction by A-998679 in rats.

Xenobiotic-mediated induction of cytochrome P450 (CYP) drug metabolizing enzymes (DMEs) is frequently encountered in drug discovery and can influence disposition, pharmacokinetic, and toxicity profiles. The CYP1A subfamily of DMEs plays a central role in the biotransformation of several drugs and environmental chemicals. Autoinduction of drugs through CYP3A enzymes is a common mechanism for their enhanced clearance. However, autoinduction via CYP1A is encountered less frequently. In this report, an experimental compound, A-998679 [3-(5-pyridin-3-yl-1,2,4-oxadiazol-3-yl) benzonitrile], was shown to enhance its own clearance via induction of Cyp1a1 and Cyp1a2. Rats were dosed for 5 days with 30, 100, and 200 mg/kg/day A-998679. During the dosing period, the compound's plasma AUC decreased at 30 mg/kg (95%) and 100 mg/kg (80%). Gene expression analysis and immunohistochemistry of the livers showed a large increase in the mRNA and protein levels of Cyp1a, which was involved in the biotransformation of A-998679. Induction of CYP1A was confirmed in primary rat, human, and dog hepatocytes. The compound also weakly inhibited CYP1A2 in human liver microsomes. A-998679 activated the aryl hydrocarbon receptor (AhR) in a luciferase gene reporter assay in HepG2 cells, upregulated expression of genes associated with AhR activation in rat liver and enhanced nuclear migration of AhR in HepG2 cells. Collectively these results demonstrate that A-998679 is an AhR activator that induces Cyp1a1 and Cyp1a2 expression, resulting in an autoinduction phenomenon. The unique properties of A-998679, along with its novel structure distinct from classical polycyclic aromatic hydrocarbons (PAHs), may warrant its further evaluation as a tool compound for use in studies involving AhR biology and CYP1A-related mechanisms of drug metabolism and toxicity.

Liver-specific knockdown of JNK1 up-regulates proliferator-activated receptor gamma coactivator 1 beta and increases plasma triglyceride despite reduced glucose and insulin levels in diet-induced obese mice.

The c-Jun N-terminal kinases (JNKs) have been implicated in the development of insulin resistance, diabetes, and obesity. Genetic disruption of JNK1, but not JNK2, improves insulin sensitivity in diet-induced obese (DIO) mice. We applied RNA interference to investigate the specific role of hepatic JNK1 in contributing to insulin resistance in DIO mice. Adenovirus-mediated delivery of JNK1 short-hairpin RNA (Ad-shJNK1) resulted in almost complete knockdown of hepatic JNK1 protein without affecting JNK1 protein in other tissues. Liver-specific knockdown of JNK1 resulted in significant reductions in circulating insulin and glucose levels, by 57 and 16%, respectively. At the molecular level, JNK1 knockdown mice had sustained and significant increase of hepatic Akt phosphorylation. Furthermore, knockdown of JNK1 enhanced insulin signaling in vitro. Unexpectedly, plasma triglyceride levels were robustly elevated upon hepatic JNK1 knockdown. Concomitantly, expression of proliferator-activated receptor gamma coactivator 1 beta, glucokinase, and microsomal triacylglycerol transfer protein was increased. Further gene expression analysis demonstrated that knockdown of JNK1 up-regulates the hepatic expression of clusters of genes in glycolysis and several genes in triglyceride synthesis pathways. Our results demonstrate that liver-specific knockdown of JNK1 lowers circulating glucose and insulin levels but increases triglyceride levels in DIO mice.

Differences in the DNA sequences in the upstream attenuator region of erm(A) in clinical isolates of Streptococcus pyogenes and their correlation with macrolide/lincosamide resistance.

The regulatory regions of 52 erm(A) [formerly erm(TR)] clinical Streptococcus pyogenes isolates were studied. Differences in the upstream regulatory region of erm(A) correlated with macrolide/lincosamide resistance patterns. Nine macrolide/lincosamide/streptogramin B-resistant isolates had changes in the leader sequence of erm(A) including base changes, insertions, or deletions. Isolates were also emm typed.

Comparison of emm typing and ribotyping with three restriction enzymes to characterize clinical isolates of Streptococcus pyogenes.

A total of 336 Streptococcus pyogenes isolates recently recovered from patients with pharyngitis from 13 countries were characterized by emm typing and riboprinting using an automated Riboprinter (Dupont/Qualicon) based on the patterns produced by three restriction enzymes, EcoRI, PstI, and HindIII. Three enzymes were necessary to increase the discrimination of ribogroups formed by each enzyme. A total of 40 ribogroups and 38 emm sequences (not counting allelic variations) were identified. Multilocus sequence typing was performed on a sampling of the isolates, and those results were consistent with those of both emm typing and ribotyping. Correlations were observed among all three methods.

Characterization and prevalence of MefA, MefE, and the associated msr(D) gene in Streptococcus pneumoniae clinical isolates.

Recent work has shown that the efflux genes in Streptococcus pneumoniae that are responsible for acquired macrolide resistance can be distinguished as either mef(E) or mef(A). The genetic elements on which mef(A) and mef(E) are found also carry an open reading frame (ORF) that is 56% homologous to msr(A) in Staphylococcus. The prevalence of mef(A/E) and of the msr-like ORF [msr(D)] was evaluated in 153 mef(+) S. pneumoniae clinical isolates collected in North America, Europe, Africa, and Asia from 1997 to 2002. Clinical isolates were screened with PCR primers specific for either mef(A) or mef(E) and for msr(D). mef(A), mef(E), and msr(D) were cloned from mef(+) strains and transformed into a susceptible, competent strain of S. pneumoniae. The transformants were tested for antimicrobial susceptibilities and efflux pump induction. The results of this work demonstrated that mef(A) is more often isolated in parts of Europe, with some incidence in Canada, and that the msr-like gene alone can confer the efflux phenotype.

Comparison of in vitro activities of ABT-773 and telithromycin against macrolide-susceptible and -resistant streptococci and staphylococci.

The activity of a new ketolide, ABT-773, was compared to the activity of the ketolide telithromycin (HMR-3647) against over 600 gram-positive clinical isolates, including 356 Streptococcus pneumoniae, 167 Staphylococcus aureus, and 136 Streptococcus pyogenes isolates. Macrolide-susceptible isolates as well as macrolide-resistant isolates with ribosomal methylase (Erm), macrolide efflux (Mef), and ribosomal mutations were tested using the NCCLS reference broth microdilution method. Both compounds were extremely active against macrolide-susceptible isolates, with the minimum inhibitory concentrations at which 90% of the isolates tested were inhibited (MIC90s) for susceptible streptococci and staphylococci ranging from 0.002 to 0.03 microg/ml for ABT-773 and 0.008 to 0.06 microg/ml for telithromycin. ABT-773 had increased activities against macrolide-resistant S. pneumoniae (Erm MIC90, 0.015 microg/ml; Mef MIC90, 0.12 microg/ml) compared to those of telithromycin (Erm MIC90, 0.12 microg/ml; Mef MIC90, 1 microg/ml). Both compounds were active against strains with rRNA or ribosomal protein mutations (MIC90, 0.12 microg/ml). ABT-773 was also more active against macrolide-resistant S. pyogenes (ABT-773 Erm MIC90, 0.5 microg/ml; ABT-773 Mef MIC90, 0.12 microg/ml; telithromycin Erm MIC90, >8 microg/ml; telithromycin Mef MIC90, 1.0 microg/ml). Both compounds lacked activity against constitutive macrolide-resistant Staphylococcus aureus but had good activities against inducibly resistant Staphylococcus aureus (ABT-773 MIC90, 0.06 microg/ml; telithromycin MIC90, 0.5 microg/ml). ABT-773 has superior activity against macrolide-resistant streptococci compared to that of telithromycin.

Molecular epidemiology of macrolide resistance in neonatal bloodstream isolates of group B streptococci.

Pulsed-field gel electrophoresis (PFGE) was performed on 122 neonatal bloodstream isolates of group B streptococci (GBS) to further examine the relationship between macrolide resistance and serotype V GBS (GBS-V). Over one-third (35%) of macrolide-resistant GBS belonged to a single PFGE subtype of GBS-V, which was also the most common GBS-V subtype noted in previous Centers for Disease Control and Prevention surveillance studies. Erm methylase (ermA and ermB) was the most common resistance mechanism detected, present in 12 of 20 macrolide-resistant GBS.