Population Pharmacokinetic and Pharmacodynamic Analysis of Polmacoxib in Healthy Volunteers and Patients With Osteoarthritis.

PURPOSE: Polmacoxib, a new coxib dually inhibiting cyclooxygenase-2 and carbonic anhydrase I/II, was recently approved for osteoarthritis treatment in South Korea. This study explored the population pharmacokinetic and pharmacodynamic characteristics of polmacoxib. METHODS: Nonlinear mixed-effects modeling was performed using pooled pharmacokinetic data from a Phase I study in healthy individuals and pharmacokinetic properties and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) data from a Phase IIb study in patients with osteoarthritis. Pharmacodynamic models for WOMAC were sequentially fit using individual pharmacokinetic parameter estimates. FINDINGS: Polmacoxib concentrations in whole blood were adequately described by the 2-compartment model, with mixed zero- and first-order absorption kinetics. Iron concentration was the significant covariate associated with clearance of polmacoxib. The relationship between the whole blood concentration of polmacoxib and WOMAC was best described by a 2-effect compartment model that consisted of central and peripheral compartments with the rate constant of 0.408 min-1 for distribution to the central effect compartment. A decrease in WOMAC was linked to the central effect site compartment concentration through an ordinary maximum effect model with an effect site concentration needed to achieve 50% of the maximum effect of 508 ng/mL. IMPLICATIONS: The current model accurately characterized the pharmacokinetic and pharmacodynamic properties of polmacoxib and could provide a basis for individualized drug therapy.

HDAC Inhibitor, CG-745, Enhances the Anti-Cancer Effect of Anti-PD-1 Immune Checkpoint Inhibitor by Modulation of the Immune Microenvironment.

Histone deacetylase inhibitors (HDACis) are well-known epigenetic regulators with therapeutic potential in various diseases. Recent studies have shown that HDACis are involved in immune-mediated anti-cancer effects and may modulate the activity of immunotherapy agents. CG-745, a histone deacetylase inhibitor, has shown anti-cancer effects in pancreatic cancer, colorectal cancer, and non-small cell lung cancer. However, the exact role of CG-745 within the immune system is largely unknown. In this study, we have shown that CG-745 induces microenvironment changes promoting anti-cancer effect of anti-PD-1 antibody in syngeneic mouse models. Specifically, CG-745 induces or extends IL-2 and IFN-γ expression with or without additional stimulation, and increases proliferation of cytotoxic T cells and NK cells, while inhibiting proliferation of regulatory T cells. The analysis of immune cell distribution in the tumor microenvironment and spleen reveals that CG-745 suppresses M2 macrophage polarization and decreases the myeloid-derived suppressor cells. Recent advances in immunotherapy highlight the anti-cancer effects of immune checkpoint inhibitor despite a relatively limited clinical benefit in the subset of patients. Our results indicate that CG-745 enables the synergistic effects of the immune checkpoint inhibitor combination therapy in various cancers by suppressing tumor microenvironment.

CG200745, a Novel HDAC Inhibitor, Attenuates Kidney Fibrosis in a Murine Model of Alport Syndrome.

Histone deacetylases have been a target of therapy for organ fibrosis. Here, we report the protective effect of CG200745 (CG), a novel histone deacetylase inhibitor, on tubulointerstitial fibrosis in Col4a3-/- mice, a murine model of Alport syndrome. Morphological analyses revealed CG treatment markedly alleviated kidney fibrosis in Col4a3-/- mice at the age of 7 weeks. CG prevented the activation of transforming growth factor ß (TGFß) and its downstream SMAD signaling in the kidney of Col4a3-/- mice. As critical upstream regulators of TGFß signaling, immunoblotting of whole kidney lysate of Col4a3-/- mice reveled that intra-renal renin-angiotensin system (RAS) was activated with concurrent upregulation of inflammation and apoptosis, which were effectively suppressed by CG treatment. CG suppressed both activation of RAS and up-regulation of TGFß signals in angiotensin II-stimulated HK-2 cells, a human kidney proximal tubular epithelial cell line. CG inhibited activation of TGFß-driven signals and fibrosis in NRK-49F cells, a rat kidney fibroblast cell line, under angiotensin II-rich conditions. Collectively, CG was found to be effective both in proximal tubular epithelial cells by inhibiting local RAS and TGFß signaling activation, as well as in fibroblasts by blocking their transition to myofibroblasts, attenuating renal fibrosis in a murine model of Alport syndrome.

Local Stabilization of Hypoxia-Inducible Factor-1α Controls Intestinal Inflammation via Enhanced Gut Barrier Function and Immune Regulation.

Intestinal epithelial cells are adapted in mucosal hypoxia and hypoxia-inducible factors in these cells can fortify barrier integrity to support mucosal tissue healing. Here we investigated whether hypoxia-related pathways could be proposed as potential therapeutic targets for inflammatory bowel disease. We developed a novel hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitor, CG-598 which stabilized HIF-1α in the gut tissue. Treatment of CG-598 did not affect extra-intestinal organs or cause any significant adverse effects such as erythropoiesis. In the experimental murine colitis model, CG-598 ameliorated intestinal inflammation with reduction of inflammatory lesions and pro-inflammatory cytokines. CG-598 treatment fortified barrier function by increasing the expression of intestinal trefoil factor, CD73, E-cadherin and mucin. Also, IL-10 and IL-22 were induced from lamina propria CD4+ T-cells. The effectiveness of CG-598 was comparable to other immunosuppressive therapeutics such as TNF-blockers or JAK inhibitors. These results suggest that CG-598 could be a promising therapeutic candidate to treat inflammatory bowel disease.

The Anti-Fibrotic Effects of CG-745, an HDAC Inhibitor, in Bleomycin and PHMG-Induced Mouse Models.

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease with poor prognosis and progression to lung fibrosis related to genetic factors as well as environmental factors. In fact, it was discovered that in South Korea many people who used humidifier disinfectants containing polyhexamethylene guanidine (PHMG), died of lung fibrosis. Currently two anti-fibrotic drugs, pirfenidone and nintedanib, have been approved by the FDA, but unfortunately, do not cure the disease. Since the histone deacetylase (HDAC) activity is associated with progression to chronic diseases and with fibrotic phenomena in the kidney, heart and lung tissues, we investigated the anti-fibrotic effects of CG-745, an HDAC inhibitor. After lung fibrosis was induced in two animal models by bleomycin and PHMG instillation, the regulation of fibrosis and epithelial mesenchymal transition (EMT)-related markers was assessed. CG-745 exhibited potent prevention of collagen production, inflammatory cell accumulation, and cytokines release in both models. Additionally, N-cadherin and vimentin expression were lowered significantly by the treatment of CG-745. The anti-fibrotic effects of CG-745 proven by the EMT regulation may suggest a potential therapeutic effect of CG-745 on lung fibrosis.

Renoprotective Effect of the Histone Deacetylase Inhibitor CG200745 in DOCA-Salt Hypertensive Rats.

The novel histone deacetylase inhibitor CG200745 was initially developed to treat various hematological and solid cancers. We investigated the molecular mechanisms associated with the renoprotective effects of CG200745 using deoxycorticosterone acetate (DOCA)-salt hypertensive (DSH) rats. DOCA strips (200 mg/kg) were implanted into rats one week after unilateral nephrectomy. Two weeks after DOCA implantation, DSH rats were randomly divided into two groups that received either physiological saline or CG200745 (5 mg/kg/day) for another two weeks. The extent of glomerulosclerosis and tubulointerstitial fibrosis was determined by Masson's trichrome staining. The renal expression of fibrosis and inflammatory markers was detected by semiquantitative immunoblotting, a polymerase chain reaction, and immunohistochemistry. Pathological signs such as glomerulosclerosis, tubulointerstitial fibrosis, increased systolic blood pressure, decreased creatinine clearance, and increased albumin-to-creatinine ratios in DSH rats were alleviated by CG200745 treatment compared to those manifestations in positive control animals. Furthermore, this treatment counteracted the increased expression of αSMA, TGF-ß1, and Bax, and the decreased expression of Bcl-2 in the kidneys of DSH rats. It also attenuated the increase in the number of apoptotic cells in DSH rats. Thus, CG200745 can effectively prevent the progression of renal injury in DSH rats by exerting anti-inflammatory, anti-fibrotic, and anti-apoptotic effects.

Upregulation of C/EBPß and TSC2 by an HDAC inhibitor CG200745 protects heart from DOCA-induced hypertrophy.

Histone deacetylases (HDACs) are a vast family divided into four major classes: class I (1, 2, 3, and 8), class II (4, 5, 6, 7, 9 and 10), class III (sirtuin family) and class IV (HDAC11). HDAC inhibition attenuates cardiac hypertrophy through suppression of the mechanistic target of rapamycin complex1 (mTORC1) signaling. HDAC inhibitors upregulate the expression of tuberous sclerosis complex 2 (TSC2), an mTORC1 inhibitor. However, the molecular mechanism underlying HDAC inhibitor-mediated upregulation of TSC2 is unclear. We hypothesized that an HDAC inhibitor, CG200745 (CG), ameliorates cardiac hypertrophy through the inhibition of mTORC1 signaling by upregulating of the CCAAT/enhancer-binding protein-ß (C/EBP-ß)/TSC2 pathway. To establish a cardiac hypertrophy model, deoxycorticosterone acetate (DOCA, 40 mg/kg/wk) was subcutaneously injected for 4 weeks into Sprague-Dawley rats. All rats were unilaterally nephrectomized and had free access to drinking water containing 1% NaCl with or without CG of different concentrations. The expression level of TSC2 and C/EBP-ß was measured by quantitative real-time PCR (qRT-PCR) and western blot analysis. Acetylation of C/EBP-ß was analyzed by immunoprecipitation. The recruitment of C/EBP-ß and polymerase II (Pol II) on TSC2 promoter region was analyzed by chromatin immunoprecipitation (ChIP). CG treatment increased the expression of TSC2. In addition, CG treated rats showed an increased in the expression and acetylation of C/EBP-ß, owing to the increase in the recruitment of C/EBP-ß and Pol II at Tsc2 gene promoter. Thus, CG ameliorates cardiac hypertrophy through the inhibition of mTORC1 signaling via upregulation of the C/EBP-ß/TSC2 pathway in DOCA-induced hypertensive rats.

Histone deacetylase inhibitor, CG200745 attenuates renal fibrosis in obstructive kidney disease.

Tubulointerstitial fibrosis is a common feature of kidney disease. Histone deacetylase (HDAC) inhibitors have been reported to attenuate renal fibrosis progression. Here, we investigated the effect of CG200745, a novel HDAC inhibitor, on renal fibrosis development in a mouse model of unilateral ureteral obstruction (UUO). To examine the effects of CG200745 on renal fibrosis in UUO, C57BL/6 J male mice were divided into three groups: control, UUO, and CG200745 (30 mg/kg/day)-treated UUO groups. CG 200745 was administered through drinking water for 1 week. Human proximal tubular epithelial (HK-2) cells were also treated with CG200745 (10 µM) with or without TGF-ß (2 ng/mL). Seven days after UUO, plasma creatinine did not differ among the groups. However, plasma neutrophil gelatinase-associated lipocalin (NGAL) levels were markedly increased in the UUO group, which were attenuated by CG200745 treatment. UUO kidneys developed marked fibrosis as indicated by collagen deposition and increased α-smooth muscle actin (SMA) and fibronectin expression. CG200745 treatment attenuated these fibrotic responses and suppressed UUO-induced production of transforming growth factor-beta1 (TGF-ß) and phosphorylation of Smad-2/3. CG200745 treatment also attenuated UUO-induced inflammation as indicated by the expression of inflammatory markers. Furthermore, CG200745 attenuated phosphorylation of p38 mitogen-activated protein kinase in UUO kidneys. In HK-2 cells, TGF-ß induced the expression of α-SMA and fibronectin, which were attenuated by CG200745 cotreatment. These results demonstrate that CG200745, a novel HDAC inhibitor, has a renoprotective effect by suppressing renal fibrosis and inflammation in a UUO mouse model.

Structural Basis for Inhibitor-Induced Hydrogen Peroxide Production by Kynurenine 3-Monooxygenase.

Kynurenine 3-monooxygenase (KMO) inhibitors have been developed for the treatment of neurodegenerative disorders. The mechanisms of flavin reduction and hydrogen peroxide production by KMO inhibitors are unknown. Herein, we report the structure of human KMO and crystal structures of Saccharomyces cerevisiae (sc) and Pseudomonas fluorescens (pf) KMO with Ro 61-8048. Proton transfer in the hydrogen bond network triggers flavin reduction in p-hydroxybenzoate hydroxylase, but the mechanism triggering flavin reduction in KMO is different. Conformational changes via π-π interactions between the loop above the flavin and substrate or non-substrate effectors lead to disorder of the C-terminal α helix in scKMO and shifts of domain III in pfKMO, stimulating flavin reduction. Interestingly, Ro 61-8048 has two different binding modes. It acts as a competitive inhibitor in scKMO and as a non-substrate effector in pfKMO. These findings provide understanding of the catalytic cycle of KMO and insight for structure-based drug design of KMO inhibitors.

A novel HDAC inhibitor, CG200745, inhibits pancreatic cancer cell growth and overcomes gemcitabine resistance.

Pancreatic cancer is predominantly lethal, and is primarily treated using gemcitabine, with increasing resistance. Therefore, novel agents that increase tumor sensitivity to gemcitabine are needed. Histone deacetylase (HDAC) inhibitors are emerging therapeutic agents, since HDAC plays an important role in cancer initiation and progression. We evaluated the antitumor effect of a novel HDAC inhibitor, CG200745, combined with gemcitabine/erlotinib on pancreatic cancer cells and gemcitabine-resistant pancreatic cancer cells. Three pancreatic cancer-cell lines were used to evaluate the antitumor effect of CG200745 combined with gemcitabine/erlotinib. CG200745 induced the expression of apoptotic proteins (PARP and caspase-3) and increased the levels of acetylated histone H3. CG200745 with gemcitabine/erlotinib showed significant growth inhibition and synergistic antitumor effects in vitro. In vivo, gemcitabine/erlotinib and CG200745 reduced tumor size up to 50%. CG200745 enhanced the sensitivity of gemcitabine-resistant pancreatic cancer cells to gemcitabine, and decreased the level of ATP-binding cassette-transporter genes, especially multidrug resistance protein 3 (MRP3) and MRP4. The novel HDAC inhibitor, CG200745, with gemcitabine/erlotinib had a synergistic anti-tumor effect on pancreatic cancer cells. CG200745 significantly improved pancreatic cancer sensitivity to gemcitabine, with a prominent antitumor effect on gemcitabine-resistant pancreatic cancer cells. Therefore, improved clinical outcome is expected in the future.

Histone deacetylase inhibitor, CG200745, attenuates cardiac hypertrophy and fibrosis in DOCA-induced hypertensive rats.

CG200745 is a novel inhibitor of histone deacetylases (HDACs), initially developed for treatment of various hematological and solid cancers. Because it is water-soluble, it can be administered orally. We hypothesized that the HDAC inhibitor, CG200745, attenuates cardiac hypertrophy and fibrosis in deoxycorticosterone acetate (DOCA)-induced hypertensive rats. For establishment of hypertension, 40 mg/kg of DOCA was subcutaneously injected four times weekly into Sprague-Dawley rats. All the rats used in this study including those in the sham group had been unilaterally nephrectomized and allowed free access to drinking water containing 1% NaCl. Systolic blood pressure was measured by the tail-cuff method. Blood chemistry including sodium, potassium, glucose, triglyceride, and cholesterol levels was analyzed. Sections of the heart were visualized after trichrome and hematoxylin and eosin stain. The expression of hypertrophic genes such as atrial natriuretic peptide A (Nppa) and atrial natriuretic peptide B (Nppb) in addition to fibrotic genes such as Collagen-1, Collagen-3, connective tissue growth factor (Ctgf), and Fibronectin were measured by quantitative real-time PCR (qRT-PCR). Injection of DOCA increased systolic blood pressure, heart weight, and cardiac fibrosis, which was attenuated by CG200745. Neither DOCA nor CG200745 affected body weight, vascular contraction and relaxation responses, and blood chemistry. Injection of DOCA increased expression of both hypertrophic and fibrotic genes, which was abrogated by CG200745. These results indicate that CG200745 attenuates cardiac hypertrophy and fibrosis in DOCA-induced hypertensive rats.

Evaluation of preventive and therapeutic activity of novel non-steroidal anti-inflammatory drug, CG100649, in colon cancer: Increased expression of TNF-related apoptosis-inducing ligand receptors enhance the apoptotic response to combination treatment with TRAIL.

Non-steroidal anti-inflammatory drugs (NSAIDs) have been suggested as the potential new class of preventive or therapeutic antitumor agents. The aim of the present study was to evaluate the antitumor activity of the novel NSAID, CG100649. CG100649 is a novel NSAID dual inhibitor for COX-2 and carbonic anhydrase (CA)-I/-II. In the present study, we investigated the alternative mechanism by which CG100649 mediated suppression of the colon cancer growth and development. The anchorage­dependent and -independent clonogenic assay showed that CG100649 inhibited the clonogenicity of human colon cancer cells. The flow cytometric analysis showed that CG100649 induced the G2/M cell cycle arrest in colon cancer cells. Animal studies showed that CG100649 inhibited the tumor growth in colon cancer xenograft in nude mice. Furthermore, quantitative PCR and FACS analysis demonstrated that CG100649 upregulated the expression of TNF-related apoptosis-inducing ligand (TRAIL) receptors (DR4 and DR5) but decreased the expression of decoy receptors (DcR1 and DcR2) in colon cancer cells. The results showed that CG100649 treatment sensitized TRAIL­mediated growth suppression and apoptotic cell death. The combination treatment resulted in significant repression of the intestinal polyp formation in APCmin/+ mice. Our data clearly demonstrated that CG100649 contains preventive and therapeutic activity for colon cancer. The present study may be useful for identification of the potential benefit of the NSAID CG100649, for the achievement of a better treatment response in colon cancer.

Growth arrest and forced differentiation of human primary glioblastoma multiforme by a novel small molecule.

Glioblastoma multiforme is the most common malignant brain tumor in adults, with an average survival of less than one year due to its resistance to therapy. Recent studies reported that GBM initiates from CD133-expressing cancer stem cells (CSC). However, the efficacy of CSC targeting is limited. A newly developed approach in cancer treatment is the forced differentiation of cancer cells. Here, we show that the treatment of the novel small molecule, CG500354, into CD133-expressing human primary GBM cells induces growth arrest by cell cycle regulators, p53, p21, p27 and phase-specific cyclins, and neural differentiation, as confirmed by neural progenitor/precursor markers, nestin, GFAP and Tuj1. When GBM-derived cells caused the tumors in NOD/SCID mice, CG500354 induced GBM-derived cells differentiation into Tuj1 and GFAP expressing cells. We next demonstrated that CG500354 plays a tumor-suppressive role via cAMP/CREB signaling pathway. CG500354 increases not only the extracellular cAMP level but also the protein level of PKA and CREB. Additionally, both mimetic substances, Forskolin and Rolipram, revealed comparable results with CG500354. Our findings indicate that induction of growth arrest and neural differentiation via cAMP/CREB signaling pathway by CG500354 treatment suggests the novel targeting of PDE4D in the development of new drugs for brain tumor therapy.

Discovery of novel 2-[2-(3-hydroxy-pyridin-2-yl)-thiazol-4-yl]-acetamide derivatives as HIF prolyl 4-hydroxylase inhibitors; SAR, synthesis and modeling evaluation.

The design, synthesis, and capacity to inhibit HIF prolyl 4-hydroxylases (PHDs) are described for 2-[2-(3-hydroxy-pyridin-2-yl)-thiazol-4-yl]-acetamide analogs. These analogs revealed two kinds of novel scaffolds as PHD2 inhibitors. Synthetic routes were developed for the preparation of their analogs containing the new scaffolds. In addition, the structure-activity relationship (SAR) of the 2-[2-(3-hydroxy-pyridin-2-yl)-thiazol-4-yl]-acetamide derivatives and their biological activities were reported. The complex structure of compound 18 with PHD2 was also obtained for the purpose of more efficient lead optimization.

[(4-Hydroxyl-benzo[4,5]thieno[3,2-c]pyridine-3-carbonyl)-amino]-acetic acid derivatives; HIF prolyl 4-hydroxylase inhibitors as oral erythropoietin secretagogues.

A new series of PHD (HIF prolyl 4-hydroxylase) inhibitors was designed based on the X-ray co-crystal structure of FG-2216. Using a lead generation process, a series of [(4-Hydroxyl-benzo[4,5]thieno[3,2-c]pyridine-3-carbonyl)-amino]-acetic acid derivatives was developed as potent PHD2 inhibitors. This class of compounds also showed the ability to stabilize HIF-α, to stimulate EPO secretion in in vitro studies, and to increase hematocrit, red blood cell count, and hemoglobin levels in an animal efficacy study.

Design, synthesis and biological evaluation of novel imidazopyridines as potential antidiabetic GSK3ß inhibitors.

Design, synthesis and biological evaluation of the imidazopyridine analogs as novel GSK3ß inhibitors for treatment of type 2 diabetes mellitus are described. Most of the analogs exhibited excellent inhibitory activities (IC50<44 nM) against glycogen synthase kinase 3ß (GSK3ß). The structure-activity relationship (SAR) of the imidazopyridine analogs and the binding mode of analog 23 in the catalytic domain of GSK3ß, based on our X-ray crystallography study, are described. In particular, analog 28, which was selected as a potential drug candidate for treatment of type 2 diabetes mellitus, exhibited excellent GSK3ß inhibition, pharmacokinetic profiles and blood glucose lowering effect in mouse.

Structure-activity relationship of the 7-hydroxy benzimidazole analogs as glycogen synthase kinase 3ß inhibitor.

Design, synthesis and the GSK3ß inhibitory activities of the 7-hydroxy benzimidazole analogs are described. The solid-phase synthetic route was also developed for preparation of the analogs consisting of the novel ATP competitive scaffold. In addition, the structure-activity relationship of the 7-hydroxy benzimidazole analogs and their biological activities are reported.

Structural basis of triclosan resistance.

Triclosan (5-chloro-2-(2,4-dichloro-phenoxy)-phenol, TCL) is a well known inhibitor against enoyl-acyl carrier protein reductase (ENR), an enzyme critical for cell-wall synthesis of bacteria. The inhibitory concentration at 50% inhibition (IC(50)) of TCL against the Escherichia coli ENR is 150nM for wild type (WT), 380, 470 and 68,500nM for Ala, Ser and Val mutants, respectively. To understand this high TCL resistance in the G93V mutant, we obtained the crystal structures of mutated ENRs complexed with TCL and NAD(+). The X-ray structural analysis along with the ab initio calculations and molecular dynamics simulations explains the serious consequence in the G93V mutant complex. The major interactions around TCL due to the aromatic(cation)-aromatic and hydrogen bonding interactions are found to be conserved both in WT and mutant complexes. Thus, the overall structural change of protein is minimal except that a flexible α-helical turn around TCL is slightly pushed away due to the presence of the bulky valine group. However, TCL shows substantial edge-to-face aromatic (π)-interactions with both the flexible R192-F203 region and the residues in the close vicinity of G93. The weakening of some edge-to-face aromatic interactions around TCL in the G93V mutant results in serious resistance to TCL. This understanding is beneficial to design new generation of antibiotics which will effectively act on the mutant ENRs.

Design and synthesis of 7-hydroxy-1H-benzoimidazole derivatives as novel inhibitors of glycogen synthase kinase-3beta.

A hydroxy functional group was introduced as the hydrogen bond donor and acceptor at the hinge region of protein kinase in order to develop novel ATP-competitive inhibitors. Several derivatives of 7-hydroxyl-1H-benzoimidazole were designed as inhibitors of glycogen synthase kinase-3beta with the help of ab initio calculations and a docking study. Enzymatic assay and an X-ray complex study showed that these designed compounds were highly potent ATP-competitive inhibitors.