Discovery of Orally Bioavailable and Liver-Targeted Hypoxia-Inducible Factor Prolyl Hydroxylase (HIF-PHD) Inhibitors for the Treatment of Anemia.

We report herein the design and synthesis of a series of orally active, liver-targeted hypoxia-inducible factor prolyl hydroxylase (HIF-PHD) inhibitors for the treatment of anemia. In order to mitigate the concerns for potential systemic side effects, we pursued liver-targeted HIF-PHD inhibitors relying on uptake via organic anion transporting polypeptides (OATPs). Starting from a systemic HIF-PHD inhibitor (1), medicinal chemistry efforts directed toward reducing permeability and, at the same time, maintaining oral absorption led to the synthesis of an array of structurally diverse hydroxypyridone analogues. Compound 28a was chosen for further profiling, because of its excellent in vitro profile and liver selectivity. This compound significantly increased hemoglobin levels in rats, following chronic QD oral administration, and displayed selectivity over systemic effects.

Evaluation of JAK3 Biology in Autoimmune Disease Using a Highly Selective, Irreversible JAK3 Inhibitor.

Reversible janus associated kinase (JAK) inhibitors such as tofacitinib and decernotinib block cytokine signaling and are efficacious in treating autoimmune diseases. However, therapeutic doses are limited due to inhibition of other JAK/signal transducer and activator of transcription pathways associated with hematopoiesis, lipid biogenesis, infection, and immune responses. A selective JAK3 inhibitor may have a better therapeutic index; however, until recently, no compounds have been described that maintain JAK3 selectivity in cells, as well as against the kinome, with good physicochemical properties to test the JAK3 hypothesis in vivo. To quantify the biochemical basis for JAK isozyme selectivity, we determined that the apparent Km value for each JAK isozyme ranged from 31.8 to 2.9 µM for JAK1 and JAK3, respectively. To confirm compound activity in cells, we developed a novel enzyme complementation assay that read activity of single JAK isozymes in a cellular context. Reversible JAK3 inhibitors cannot achieve sufficient selectivity against other isozymes in the cellular context due to inherent differences in enzyme ATP Km values. Therefore, we developed irreversible JAK3 compounds that are potent and highly selective in vitro in cells and against the kinome. Compound 2, a potent inhibitor of JAK3 (0.15 nM) was 4300-fold selective for JAK3 over JAK1 in enzyme assays, 67-fold [interleukin (IL)-2 versus IL-6] or 140-fold [IL-2 versus erythropoietin or granulocyte-macrophage colony-stimulating factor (GMCSF)] selective in cellular reporter assays and >35-fold selective in human peripheral blood mononuclear cell assays (IL-7 versus IL-6 or GMCSF). In vivo, selective JAK3 inhibition was sufficient to block the development of inflammation in a rat model of rheumatoid arthritis, while sparing hematopoiesis.

Development and optimization of a high-throughput micro-computed tomography imaging method incorporating a novel analysis technique to evaluate bone mineral density of arthritic joints in a rodent model of collagen induced arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune disease resulting in joint inflammation, pain, and eventual bone loss. Bone loss and remodeling caused by symmetric polyarthritis, the hallmark of RA, is readily detectable by bone mineral density (BMD) measurement using micro-CT. Abnormalities in these measurements over time reflect the underlying pathophysiology of the bone. To evaluate the efficacy of anti-rheumatic agents in animal models of arthritis, we developed a high throughput knee and ankle joint imaging assay to measure BMD as a translational biomarker. A bone sample holder was custom designed for micro-CT scanning, which significantly increased assay throughput. Batch processing 3-dimensional image reconstruction, followed by automated image cropping, significantly reduced image processing time. In addition, we developed a novel, automated image analysis method to measure BMD and bone volume of knee and ankle joints. These improvements significantly increased the throughput of ex vivo bone sample analysis, reducing data turnaround from 5 days to 24 hours for a study with 200 rat hind limbs. Taken together, our data demonstrate that BMD, as quantified by micro-CT, is a robust efficacy biomarker with a high degree of sensitivity. Our innovative approach toward evaluation of BMD using optimized image acquisition and novel image processing techniques in preclinical models of RA enables high throughput assessment of anti-rheumatic agents offering a powerful tool for drug discovery.

Etanercept ameliorates inflammation and pain in a novel mono-arthritic multi-flare model of streptococcal cell wall induced arthritis.

BACKGROUND: The impact of anti-TNF, corticosteroid and analgesic therapy on inflammation and pain was evaluated in a novel mono-arthritic multi-flare rat Streptococcal Cell Wall (SCW) model using Etanercept, Dexamethasone and Buprenorphine. METHODS: Multiple flares of arthritis were induced with an intra-articular injection of SCW in the hind ankle on day 1, followed by intravenous challenges on days 21 and 42. Inflammation and pain were monitored in the hind paws. Cytokine profiling, cell phenotyping, bioluminescence imaging and histopathological evaluation were also performed. RESULTS: Local injection of SCW caused a rapid onset of inflammation and pain in the injected ankle which resolved within 4 days (Flare 1). Intravenous injection 20 days after sensitization resulted in an increase in ankle diameter and pain, which partially resolved in 8 days (Flare 2). The subsequent intra-venous injection in the same animals 14 days after resulted in a more chronic disease with inflammation and pain persisting over a period of 10 days (Flare 3). In Flare 2, therapeutic administration of Dexamethasone inhibited paw swelling (95%; P<0.001) and pain (55%; P<0.05). Therapeutic administration of Buprenorphine inhibited pain (80%; P<0.001) without affecting paw swelling (0%). Prophylactic administration of Etanercept in Flare 2 inhibited paw swelling (≥60%; P<0.001) and pain by ≥30%. Expression of IL-1ß, IL-6, MCP-1 and CINC was reduced by >50% (P<0.001). Treatment with Etanercept in Flare 3 inhibited paw swelling by 60% (P<0.001) and pain by 25%. Prior treatment with Etanercept in Flare 2 followed by re-administration in Flare 3 led to a complete loss in the efficacy of Etanercept. Systemic exposure of Etanercept corroborated with lack of efficacy. Dexamethasone inhibited inflammation and pain in both Flares 2 and 3 (P<0.001). CONCLUSIONS: We established a novel multi-flare SCW arthritis model enabling drug intervention in different stages of disease. We show for the first time the evaluation of inflammation and pain simultaneously in this model. Etanercept and Dexamethasone inhibited inflammation, pain and proinflammatory cytokines in this model. Taken together, this model facilitates the assessment of anti-rheumatic agents targeting inflammation and pain in the multiple flare paradigm and offers a powerful tool for drug discovery.

The preclinical efficacy, selectivity and pharmacologic profile of MK-5932, an insulin-sparing selective glucocorticoid receptor modulator.

Glucocorticoids are used widely in the treatment of inflammatory diseases, but use is accompanied by a significant burden of adverse effects. It has been hypothesized that gene- and cell-specific regulation of the glucocorticoid receptor by small molecule ligands could be translated into a therapeutic with an improved risk-benefit profile. MK-5932 is a highly selective glucocorticoid receptor modulator that is anti-inflammatory in vivo with an improved profile on glucose metabolism: Bungard et al. (2011). Bioorg. Med. Chem. 19, 7374-7386. Here we describe the full biological profile of MK-5932. Cytokine production following lipopolysaccharide (LPS) challenge was blocked by MK-5932 in both rat and human whole blood. Oral administration reduced inflammatory cytokine levels in the serum of rats challenged with LPS. MK-5932 was anti-inflammatory in a rat contact dermatitis model, but was differentiated from 6-methylprednisolone by a lack of elevation of fasting insulin or glucose levels after 7 days of dosing, even at high exposure levels. In fact, animals in the vehicle group were consistently hyperglycemic at the end of the study, and MK-5932 normalized glucose levels in a dose-dependent manner. MK-5932 was also anti-inflammatory in the rat collagen-induced arthritis and adjuvant-induced arthritis models. In healthy dogs, oral administration of MK-5932 exerted acute pharmacodynamic effects with potency comparable to prednisone, but with important differences on neutrophil counts, again suggestive of a dissociated profile. Important gaps in our understanding of mechanism of action remain, but MK-5932 will be a useful tool in dissecting the mechanisms of glucose dysregulation by therapeutic glucocortiocids.

Increased muscle force production and bone mineral density in ActRIIB-Fc-treated mature rodents.

Myostatin is a highly conserved member of the transforming growth factor-ß ligand family known to regulate muscle growth via activation of activin receptors. A fusion protein consisting of the extracellular ligand-binding domain of activin type IIB receptor with the Fc portion of human immunoglobulin G (ActRIIB-Fc) was used to inhibit signaling through this pathway. Here, we study the effects of this fusion protein in adult, 18-month-old, and orchidectomized mice. Significant muscle growth and enhanced muscle function were observed in adult mice treated for 3 days with ActRIIB-Fc. The ActRIIB-Fc-treated mice had enhanced fast fatigable muscle function, with only minor enhancement of fatigue-resistant fiber function. The ActRIIB-Fc-treated 18-month-old mice and orchidectomized mice showed significantly improved muscle function. Treatment with ActRIIB-Fc also increased bone mineral density and serum levels of a marker of bone formation. These observations highlight the potential of targeting ActRIIB receptor to treat age-related and hypogonadism-associated musculoskeletal degeneration.

Systems analysis of eleven rodent disease models reveals an inflammatome signature and key drivers.

Common inflammatome gene signatures as well as disease-specific signatures were identified by analyzing 12 expression profiling data sets derived from 9 different tissues isolated from 11 rodent inflammatory disease models. The inflammatome signature significantly overlaps with known drug targets and co-expressed gene modules linked to metabolic disorders and cancer. A large proportion of genes in this signature are tightly connected in tissue-specific Bayesian networks (BNs) built from multiple independent mouse and human cohorts. Both the inflammatome signature and the corresponding consensus BNs are highly enriched for immune response-related genes supported as causal for adiposity, adipokine, diabetes, aortic lesion, bone, muscle, and cholesterol traits, suggesting the causal nature of the inflammatome for a variety of diseases. Integration of this inflammatome signature with the BNs uncovered 151 key drivers that appeared to be more biologically important than the non-drivers in terms of their impact on disease phenotypes. The identification of this inflammatome signature, its network architecture, and key drivers not only highlights the shared etiology but also pinpoints potential targets for intervention of various common diseases.

Non-invasive muscle contraction assay to study rodent models of sarcopenia.

BACKGROUND: Age-related sarcopenia is a disease state of loss of muscle mass and strength that affects physical function and mobility leading to falls, fractures, and disability. The need for therapies to treat age-related sarcopenia has attracted intensive preclinical research. To facilitate the discovery of these therapies, we have developed a non-invasive rat muscle functional assay system to efficiently measure muscle force and evaluate the efficacy of drug candidates. METHODS: The lower leg muscles of anesthetized rats are artificially stimulated with surface electrodes on the knee holders and the heel support, causing the lower leg muscles to push isometric pedals that are attached to force transducers. We developed a stimulation protocol to perform a fatigability test that reveals functional muscle parameters like maximal force, the rate of fatigue, fatigue-resistant force, as well as a fatigable muscle force index. The system is evaluated in a rat aging model and a rat glucocorticoid-induced muscle loss model RESULTS: The aged rats were generally weaker than adult rats and showed a greater reduction in their fatigable force when compared to their fatigue-resistant force. Glucocorticoid treated rats mostly lost fatigable force and fatigued at a higher rate, indicating reduced force from glycolytic fibers with reduced energy reserves. CONCLUSIONS: The involuntary contraction assay is a reliable system to assess muscle function in rodents and can be applied in preclinical research, including age-related sarcopenia and other myopathy.

Microscopic image analysis for quantitative characterization of muscle fiber type composition.

Skeletal muscles consist of muscle fibers that are responsible for contracting and generating force. Skeletal muscle fibers are categorized into distinct subtypes based on several characteristics such as contraction time, force production and resistance to fatigue. The composition of distinct muscle fibers in terms of their number and cross-sectional areas is characterized by a histological examination. However, manual delineation of individual muscle fibers from digitized muscle histology tissue sections is extremely time-consuming. In this study, we propose an automated image analysis system for quantitative characterization of muscle fiber type composition. The proposed system operates on digitized histological muscle tissue slides and consists of the following steps: segmentation of muscle fibers, registration of successive slides with distinct stains, and classification of muscle fibers into distinct subtypes. The performance of the proposed approach was tested on a dataset consisting of 25 image pairs of successive muscle histological cross-sections with different ATPase stain. Experimental results demonstrate a promising overall segmentation and classification accuracy of 89.1% in identifying muscle fibers of distinct subtypes.

A cell-based ultra-high-throughput screening assay for identifying inhibitors of D-amino acid oxidase.

Enzymes are often considered less "druggable" targets than ligand-regulated proteins such as G-protein-coupled receptors, ion channels, or other hormone receptors. Reasons for this include cellular location (intracellular vs. cell surface), typically lower affinities for the binding of small molecules compared to ligand-specific receptors, and binding (catalytic) sites that are often charged or highly polar. A practical drawback to the discovery of compounds targeting enzymes is that screening of compound libraries is typically carried out in cell-free activity assays using purified protein in an inherently artificial environment. Cell-based assays, although often arduous to design for enzyme targets, are the preferred discovery tool for the screening of large compound libraries. The authors have recently described a novel cell-based approach to screening for inhibitors of a phosphatase enzyme and now report on the development and implementation of a homogeneous 3456-well plate assay for D-amino acid oxidase (DAO). Human DAO was stably expressed in Chinese hamster ovary (CHO) cells, and its activity was measured as the amount of hydrogen peroxide detected in the growth medium following feeding the cells with D-serine. In less than 12 weeks, the authors proved the concept in 96-and then 384-well formats, miniaturized the assay to the 3456-well (nanoplate) scale, and screened a library containing more than 1 million compounds. They have identified several cell-permeable inhibitors of DAO from this cell-based high-throughput screening, which provided the discovery program with a few novel and attractive lead structures.

GABA transporter deficiency causes tremor, ataxia, nervousness, and increased GABA-induced tonic conductance in cerebellum.

GABA transporter subtype 1 (GAT1) knock-out (KO) mice display normal reproduction and life span but have reduced body weight (female, -10%; male, -20%) and higher body temperature fluctuations in the 0.2-1.5/h frequency range. Mouse GAT1 (mGAT1) KO mice exhibit motor disorders, including gait abnormality, constant 25-32 Hz tremor, which is aggravated by flunitrazepam, reduced rotarod performance, and reduced locomotor activity in the home cage. Open-field tests show delayed exploratory activity, reduced rearing, and reduced visits to the central area, with no change in the total distance traveled. The mGAT1 KO mice display no difference in acoustic startle response but exhibit a deficiency in prepulse inhibition. These open-field and prepulse inhibition results suggest that the mGAT1 KO mice display mild anxiety or nervousness. The compromised GABA uptake in mGAT1 KO mice results in an increased GABA(A) receptor-mediated tonic conductance in both cerebellar granule and Purkinje cells. The reduced rate of GABA clearance from the synaptic cleft is probably responsible for the slower decay of spontaneous IPSCs in cerebellar granule cells. There is little or no compensatory change in other proteins or structures related to GABA transmission in the mGAT1 KO mice, including GAT1-independent GABA uptake, number of GABAergic interneurons, and GABA(A)-, vesicular GABA transporter-, GAD65-, and GAT3-immunoreactive structures in cerebellum or hippocampus. Therefore, the excessive extracellular GABA present in mGAT1 KO mice results in behaviors that partially phenocopy the clinical side effects of tiagabine, suggesting that these side effects are inherent to a therapeutic strategy that targets the widely expressed GAT1 transporter system.

Characterization and localization of a human serine racemase.

D-serine is present in the mammalian central nervous system, where it acts as one of the co-activators of N-methyl-D aspartate receptors. Synthesis of D-serine is catalyzed by the serine racemase enzyme. The current studies report on the isolation of a cDNA encoding a human serine racemase (SRR) from the human neuronal like cell line, NT2N. The SRR gene was localized on chromosome 17q13. The full-length cDNA has 1020 nucleotides which encode for a protein of 340 amino acids. The human protein shares 89% sequence identity with the mouse serine racemase. Human embryonic kidney 293 cells transiently transfected with this SRR gene were able to produce d-serine, indicating that the sequence encodes for an active enzyme. In Northern blot analysis the SRR mRNA was expressed in human brain, heart, skeletal muscle, kidney and liver tissues. Different splice forms of SRR were present in the peripheral tissues. Transcripts of at least three different sizes were present in heart and kidney, while in Western blot analysis multiple bands of different sizes were observed. Immunohistochemical studies, using a polyclonal anti-human serine racemase antibody, revealed a peripheral expression of serine racemase protein in human cardiac myocytes and convoluted tubules of the kidney. Experiments in non-human primate brain demonstrated the localization of SRR in amygdala nuclei, cortex, thalamus and hippocampus. Co-localization studies in the hippocampus demonstrated the exclusive expression of serine racemase in glial cells. The cloning of a functional human serine racemase and its expression in central nervous system of primates support a role for D-serine in neuronal activity. Furthermore, its presence in human periphery such as in heart and kidney suggest a potential biological role for D-serine in the regulation of N-methyl-D-aspartate (NMDA) receptor activity in these peripheral organs as well.

GABA transporter-1 (GAT1)-deficient mice: differential tonic activation of GABAA versus GABAB receptors in the hippocampus.

After its release from interneurons in the CNS, the major inhibitory neurotransmitter GABA is taken up by GABA transporters (GATs). The predominant neuronal GABA transporter GAT1 is localized in GABAergic axons and nerve terminals, where it is thought to influence GABAergic synaptic transmission, but the details of this regulation are unclear. To address this issue, we have generated a strain of GAT1-deficient mice. We observed a large increase in a tonic postsynaptic hippocampal GABAA receptor-mediated conductance. There was little or no change in the waveform or amplitude of spontaneous inhibitory postsynaptic currents (IPSCs) or miniature IPSCs. In contrast, the frequency of quantal GABA release was one-third of wild type (WT), although the densities of GABAA receptors, GABAB receptors, glutamic acid decarboxylase 65 kDa, and vesicular GAT were unaltered. The GAT1-deficient mice lacked a presynaptic GABAB receptor tone, present in WT mice, which reduces the frequency of spontaneous IPSCs. We conclude that GAT1 deficiency leads to enhanced extracellular GABA levels resulting in an overactivation of GABAA receptors responsible for a postsynaptic tonic conductance. Chronically elevated GABA levels also downregulate phasic GABA release and reduce presynaptic signaling via GABAB receptors thus causing an enhanced tonic and a diminished phasic inhibition.

Time course of inner ear degeneration and deafness in mice lacking the Kir4.1 potassium channel subunit.

The Kir4.1 gene (KCNJ10) encodes an inwardly rectifying K(+) channel subunit abundantly expressed in the CNS. Its expression in the mammalian inner ear has been suggested but its function in vivo in the inner ear is unknown. Because diverse human hereditary deafness syndromes are associated with mutations in K(+) channels, we examined auditory function and inner ear structure in mice with a genetically inactivated Kir4.1 K(+) channel subunit. Startle response experiments suggest that Kir4.1-/- mice are profoundly deaf, whereas Kir4.1+/- mice react like wild-type mice to acoustic stimuli. In Kir4.1-/- mice, the Reissner membrane is collapsed, the tectorial membrane is swollen, and type I hair cells and spiral ganglion neurons as well as their central processes degenerate over the first postnatal weeks. In the vestibular ganglia, neuronal cell death with apoptotic features is also observed. Immunostaining reveals that Kir4.1 is strongly expressed in stria vascularis of wild-type but not Kir4.1-/- mice. Within the spiral ganglion, Kir4.1 labeling was detected on satellite cells surrounding spiral ganglion neurons and axons. We conclude that Kir4.1 is crucial for normal development of the cochlea and hearing, via two distinct aspects of extracellular K(+) homeostasis: (1). in stria vascularis, Kir4.1 helps to generate the cochlear endolymph; and (2). in spiral and vestibular ganglia, Kir4.1 in surrounding glial cells helps to support the spiral and vestibular ganglion neurons and their projecting axons.

Number, density, and surface/cytoplasmic distribution of GABA transporters at presynaptic structures of knock-in mice carrying GABA transporter subtype 1-green fluorescent protein fusions.

GABA transporter subtype 1 (GAT1) molecules were counted near GABAergic synapses, to a resolution of approximately 0.5 microm. Fusions between GAT1 and green fluorescent protein (GFP) were tested in heterologous expression systems, and a construct was selected that shows function, expression level, and trafficking similar to that of wild-type (WT) GAT1. A strain of knock-in mice was constructed that expresses this mGAT1-GFP fusion in place of the WT GAT1 gene. The pattern of fluorescence in brain slices agreed with previous immunocytochemical observations. [3H]GABA uptake, synaptic electrophysiology, and subcellular localization of the mGAT1-GFP construct were also compared with WT mice. Quantitative fluorescence microscopy was used to measure the density of mGAT1-GFP at presynaptic structures in CNS preparations from the knock-in mice. Fluorescence measurements were calibrated with transparent beads and gels that have known GFP densities. Surface biotinylation defined the fraction of transporters on the surface versus those in the nearby cytoplasm. The data show that the presynaptic boutons of GABAergic interneurons in cerebellum and hippocampus have a membrane density of 800-1300 GAT1 molecules per square micrometer, and the axons that connect boutons have a linear density of 640 GAT1 molecules per micrometer. A cerebellar basket cell bouton, a pinceau surrounding a Purkinje cell axon, and a cortical chandelier cell cartridge carry 9000, 7.8 million, and 430,000 GAT1 molecules, respectively; 61-63% of these molecules are on the surface membrane. In cultures from hippocampus, the set of fluorescent cells equals the set of GABAergic interneurons. Knock-in mice carrying GFP fusions of membrane proteins provide quantitative data required for understanding the details of synaptic transmission in living neurons.