BRD2-specific inhibitor, BBC0403, inhibits the progression of osteoarthritis pathogenesis in osteoarthritis-induced C57BL/6 male mice.

BACKGROUND AND PURPOSE: The discovery of new bromo- and extra-terminal inhibitors presents new drugs to treat osteoarthritis (OA). EXPERIMENTAL APPROACH: The new drug, BBC0403, was identified in the DNA-encoded library screening system by searching for compounds that target BRD (bromodomain-containing) proteins. The binding force with BRD proteins was evaluated using time-resolved fluorescence energy transfer (TR-FRET) and binding kinetics assays. Subsequently, in vitro and ex vivo analyses demonstrated the effects of the BRD2 inhibitor, BBC0403, on OA. For animal experiments, medial meniscus destabilization was performed to create a 12-week-old male C57BL/6 mouse model, and intra-articular (i.a.) injections were administered. Histological and immunohistochemical analyses were then performed. The underlying mechanism was confirmed by gene set enrichment analysis (GSEA) using RNA-seq. KEY RESULTS: TR-FRET and binding kinetics assays revealed that BBC0403 exhibited higher binding specificity for BRD2 compared to BRD3 and BRD4. The anti-OA effects of BBC0403 were tested at concentrations of 5, 10 and 20 µM (no cell toxicity in the range tested). The expression of catabolic factors, prostaglandin E2 (PGE2) production and extracellular matrix (ECM) degradation was reduced. Additionally, the i.a. injection of BBC0403 prevented OA cartilage degradation in mice. Finally, BBC0403 was demonstrated to suppress NF-κB and MAPK signalling pathways. CONCLUSION AND IMPLICATIONS: This study demonstrated that BBC0403 is a novel BRD2-specific inhibitor and a potential i.a.-injectable therapeutic agent to treat OA.

Novel molecule BBC0901 inhibits BRD4 and acts as a catabolic regulator in the pathogenesis of osteoarthritis.

Osteoarthritis (OA) is induced by matrix degradation and inflammation mediated by bromo-domain-containing protein 4 (BRD4)-dependent catabolic factors. BRD4 acts as both a transcriptional regulator and an epigenetic reader. BBC0901 was identified as an inhibitor of BRD4 using a DNA-encoded library screening system. We aimed to demonstrate the effects of BBC0901 on OA pathogenesis by in vitro, ex vivo, and in vivo analyses. BBC0901 inhibited the expression of catabolic factors that degrade cartilage without significantly affecting the viability of mouse articular chondrocytes. Additionally, ex vivo experiments under conditions mimicking OA showed that BBC0901 suppressed extracellular matrix degradation. RNA sequencing analysis of gene expression patterns showed that BBC0901 inhibited the expression of catabolic factors, such as matrix metalloproteinases (MMPs) and cyclooxygenase (COX)2, along with reactive oxygen species (ROS) production. Furthermore, intra-articular (IA) injection of BBC0901 into the knee joint blocked osteoarthritic cartilage destruction by inhibition of MMP3, MMP13, COX2, interleukin (IL)6, and ROS production, thereby obstructing the nuclear factor kappa-light-chain-enhancer of activated B cell and mitogen activated protein kinase signaling. In conclusion, BBC0901-mediated BRD4 inhibition prevented OA development by attenuating catabolic signaling and hence, can be considered a promising IA therapeutic for OA.

Streamlined DNA-encoded small molecule library screening and validation for the discovery of novel chemotypes targeting BET proteins.

Targeting aberrant epigenetic programs that drive tumorigenesis is a promising approach to cancer therapy. DNA-encoded library (DEL) screening is a core platform technology increasingly used to identify drugs that bind to protein targets. Here, we use DEL screening against bromodomain and extra-terminal motif (BET) proteins to identify inhibitors with new chemotypes, and successfully identified BBC1115 as a selective BET inhibitor. While BBC1115 does not structurally resemble OTX-015, a clinically active pan-BET inhibitor, our intensive biological characterization revealed that BBC1115 binds to BET proteins, including BRD4, and suppresses aberrant cell fate programs. Phenotypically, BBC1115-mediated BET inhibition impaired proliferation in acute myeloid leukemia, pancreatic, colorectal, and ovarian cancer cells in vitro. Moreover, intravenous administration of BBC1115 inhibited subcutaneous tumor xenograft growth with minimal toxicity and favorable pharmacokinetic properties in vivo. Since epigenetic regulations are ubiquitously distributed across normal and malignant cells, it will be critical to evaluate if BBC1115 affects normal cell function. Nonetheless, our study shows integrating DEL-based small-molecule compound screening and multi-step biological validation represents a reliable strategy to discover new chemotypes with selectivity, efficacy, and safety profiles for targeting proteins involved in epigenetic regulation in human malignancies.

Tissue pharmacokinetics of DHP107, a novel lipid-based oral formulation of paclitaxel, in mice and patients by positron emission tomography.

DHP107 is a newly developed lipid-based oral formulation of paclitaxel. We evaluated the in vivo tissue pharmacokinetics (PKs) of DHP107 in mice and patients using positron emission tomography (PET). Radioisotope-labeled [3 H]DHP107 and [18 F]DHP107 for oral administration were formulated in the same manner as the manufacturing process of DHP107. In vivo tissue PK were assessed in healthy ICR mice and breast cancer xenografted SCID mice. Two patients with metastatic breast cancer were clinically evaluated for absorption at the target lesion after internal absorbed dose estimation. Whole-body PET/computed tomography data were acquired in healthy and xenografted mice and in patients up to 10-24 h after administration. Tissue [18 F]DHP107 signals were plotted against time and PK parameters were determined. The amounts of radioactivity in various organs and excreta were determined using a beta-counter and are expressed as the percentage of injected dose (ID). Oral [18 F]DHP107 was well-absorbed and reached the target lesion in mice and patients with breast cancer. Significant amounts of radioactivity were found in the stomach, intestine, and liver after oral administration of [3 H]- and [18 F]DHP107 in healthy mice. The [18 F]DHP107 reached a peak distribution of 0.7-0.8%ID in the tumor at 5.6-7.3 h in the xenograft model. The [18 F]DHP107 distribution in patients with metastatic breast cancer was the highest at 3-4 h postadministration. Systemic exposures after administration of a DHP107 therapeutic dose were comparable with those in previous studies. PET using radioisotope-labeled drug candidates is useful for drug development and can provide valuable information that can complement plasma PK data, particularly in early phase clinical trials.

Use of oral paclitaxel for the treatment of bladder tumors in dogs.

An oral paclitaxel formulation that overcomes the hypersensitivity reaction of paclitaxel has been evaluated for safety and efficacy in humans, but not in dogs. We present the first case report on the use of oral paclitaxel in dogs. In this study, oral paclitaxel was well-tolerated in four dogs with either transitional cell carcinoma or prostate cancer; adverse effects were limited to mild neutropenia. Each of the dogs had progressive disease at the end, but clinical responses, including changes in mass size and improvement of clinical symptoms, were confirmed in some of the animals following oral paclitaxel chemotherapy. Although this study is somewhat limited by a small sample size, it suggests that oral paclitaxel may be a chemotherapeutic option for malignant tumors in dogs.

Antitumour effects of Liporaxel (oral paclitaxel) for canine melanoma in a mouse xenograft model.

Paclitaxel, a member of the taxane family, exhibits antitumour effects by targeting the microtubules in cancer cells. Recently, oral paclitaxel has been developed to overcome the side effects of intravenous paclitaxel administration in human patients. The objective of this study was to investigate the antitumour effects of oral paclitaxel in vitro and in vivo. Three weeks after inoculation, oral paclitaxel (25 and 50 mg/kg) or saline was administered every week for three consecutive weeks. To explore the underlying mechanism, tumour angiogenesis was examined by immunohistochemistry with an anti-CD31 antibody. Tumour cell apoptosis was detected by Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling assay, and cell cycle arrest was confirmed by western blot analysis. Oral paclitaxel treatment of canine melanoma cells exerted mediated antiproliferative effects and mediated cell cycle arrest in vitro. In animal experiments, after oral paclitaxel administration, the average tumour size decreased to approximately 30% of that in the control. Histologically, oral paclitaxel showed anti-angiogenic effects and induced the apoptosis in tumour tissues. Oral paclitaxel also downregulated the intratumoural expression of cyclin D1 and inhibited cell proliferation. The study findings support potential application of oral paclitaxel as a novel chemotherapeutic strategy to treat canine melanoma. This is the first study to investigate the potential of oral paclitaxel as a therapeutic drug against canine tumours.

Analysis of ethyl glucuronide and ethyl sulfate in blood to determine the absorption or elimination phase of alcohol for Korean.

This study was conducted to understand alcohol kinetics for Koreans and to determine whether an individual is in absorption phase or elimination phase at the time of blood collection by analyzing of ethyl glucuronide and ethyl sulfate in blood. A total of 50 healthy adults was selected and assigned to drink 1g of ethanol per kg body weight of individual within 1h. Blood samples were then collected every 15min for the first 3h, 30min next 3h, and 1h last 9h. Urine samples were also collected from the individual, but not under the controlled environment. All samples were then analyzed by gas chromatography with a flame ionization detector (GC-FID) for alcohol and liquid chromatography-mass/mass spectrometry (LC-MS/MS) for EtG and EtS. The maximum BAC (Cmax) was 0.138% (g/100mL) in average under the controlled experimental condition. Alcohol elimination rates (ß) in average were 0.020% for male and 0.024% for female, respectively. It was found that the ratio of UAC and BAC was less than 1 in the absorption phase and the average ratio of UAC and BAC was 1.47 in the elimination phase. The comparison of BAC (g/L) and EtG (mg/L) absorption and elimination curves showed that the intersection time was 3.9h in average. It is shown that the ratio of EtG (mg/L)/BAC (g/L) is higher than 1, the individual would be in elimination phase of BAC. At the time of Cmax, the ratio of EtG (mg/L)/BAC (g/L) was 0.255±0.132 (SD) in average.

Subacute toxicity and toxicokinetics study of DHP107, an oral paclitaxel formulation with once-weekly dosing in mice.

DHP107, an oral formulation of paclitaxel, is effectively and systemically absorbed in intestinal endothelial cells. Although the in vivo efficacy of DHP107 has been reported, the potential toxicity of DHP107 has not been evaluated. Therefore, this study was conducted to evaluate the toxicity and toxicokinetics of DHP107 orally administered to ICR mice at 25, 50, and 100 mg/kg via once-weekly dosing for six weeks. DHP107-related clinical signs were observed in both sexes at 100 mg/kg. There were significant increases in the number of platelets and percentages of reticulocytes and basophils in male mice. Also in males, there was a significant decrease in the absolute and relative weights of testes, epididymides, kidneys, and heart. Relative spleen weights were significantly increased in males treated with doses ≥50 mg/kg which had histopathological correlates. These changes were reversible after a two-week recovery period with the exception of the findings in the reproductive organs. Systemic exposure to paclitaxel increased with DHP107 doses in single and multiple dosing with no marked differences between sexes. In conclusion, the target organs were determined to be the reproductive and hematopoietic organs in male mice, suggesting of sex difference and the NOAEL of DHP107 was established to be < 25 mg/kg for males and 50 mg/kg for females.

Deposition of organochlorine pesticides into the surface snow of East Antarctica.

Organochlorine pesticides (OCPs) were measured in surface snow collected on a ~1400-km inland traverse beginning from the coastal regions of East Antarctica during the Japanese Antarctic Research Expedition (JARE) of 2007/2008. Of the 22 OCPs, α-hexachlorocyclohexane (HCH), γ-HCH, and hexachlorobenzene (HCB) were frequently detected in the snow with concentration ranges of 17.5-83.2, 33-137, and ND-182 pg L(-1), respectively. The most abundant pesticide was γ-HCH, with a mean concentration of 69.9 pg L(-1), followed by α-HCH, with an average concentration of 44.5 pg L(-1). The spatial variability of α-HCH and γ-HCH was narrow, and the concentrations of α-HCH and γ-HCH increased slightly with increasing altitude along the traverse route. Dome Fuji, the highest altitude sampling point, had the highest γ-HCH concentrations in the snow. Backward air trajectory analysis showed that the air masses at the sampling sites came mainly from the Indian and Atlantic Oceans and over the Antarctic continent, indicating that the OCPs were subjected to long-range atmospheric transport and were deposited in the surface snow. Our data suggest that the snow of Antarctica contains low levels of OCPs.