Ripasudil hydrochloride hydrate (K-115), a specific Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor, was the first ophthalmic solution developed for the treatment of glaucoma ...and ocular hypertension in Japan. Topical administration of K-115 decreased intraocular pressure (IOP) and increased outflow facility in rabbits. This study evaluated the effect of K-115 on monkey trabecular meshwork (TM) cells and Schlemm's canal endothelial (SCE) cells. K-115 induced retraction and rounding of cell bodies as well as disruption of actin bundles in TM cells. In SCE-cell monolayer permeability studies, K-115 significantly decreased transendothelial electrical resistance (TEER) and increased the transendothelial flux of FITC-dextran. Further, K-115 disrupted cellular localization of ZO-1 expression in SCE-cell monolayers. These results indicate that K-115 decreases IOP by increasing outflow facility in association with the modulation of TM cell behavior and SCE cell permeability in association with disruption of tight junction.
Abstract
Purpose: To evaluate the topical instillation of K-115, a selective Rho-associated coiled coil-containing protein kinase (ROCK) inhibitor, on intraocular pressure (IOP), ocular distribution, ...and aqueous humor dynamics in experimental animals.
Methods: Kinase inhibition by K-115 was measured by biochemical assay. IOP was monitored using a pneumatonometer in albino rabbits and monkeys after topical instillation of K-115. The ocular distribution of 14CK-115 was determined by whole-head autoradiography. The aqueous flow rate was determined by fluorophotometry. The total outflow facility and uveoscleral outflow were measured by two-level constant pressure perfusion and perfusion technique using fluorescein isothiocyanate-dextran, respectively.
Results: Biochemical assay showed that K-115 had selective and potent inhibitory effects on ROCKs. In rabbits, topical instillation of K-115 significantly reduced IOP in a dose-dependent manner. Maximum IOP reduction was observed 1 h after topical instillation, which was 8.55 ± 1.09 mmHg (mean ± SE) from the baseline IOP at 0.5%. In monkeys, maximum IOP reduction was observed 2 h after topical instillation, which was 4.36 ± 0.32 mmHg from the baseline IOP at 0.4%, and was significantly stronger than that of 0.005% latanoprost. Whole-head autoradiography showed that the radioactivity level was maximum at 15 min after instillation of 14CK-115 in the ipsilateral eye. Single instillation of 0.4% K-115 showed no effect on aqueous flow rate or uveoscleral outflow, but significantly increased conventional outflow facility by 2.2-fold compared to vehicle-treated eyes in rabbits.
Conclusions: These results indicated that K-115 ophthalmic solution, a selective and potent ROCK inhibitor, is a novel and potent antiglaucoma agent.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Despite significant reduction of cardiovascular events by statin treatment, substantial residual risk persists, driving emerging needs for the development of new therapies. We identified a novel ...cholesteryl ester transfer protein (CETP) inhibitor, K-312, that raises HDL and lowers LDL cholesterol levels in animals. K-312 also suppresses hepatocyte expression of proprotein convertase subtilisin/kexin 9 (PCSK9), a molecule that increases LDL cholesterol. We explored the underlying mechanism for the reduction of PCSK9 expression by K-312. K-312 inhibited in vitro human plasma CETP activity (IC50; 0.06 μM). Administration of K-312 to cholesterol-fed New Zealand White rabbits for 18 wk raised HDL cholesterol, decreased LDL cholesterol, and attenuated aortic atherosclerosis. Our search for additional beneficial characteristics of this compound revealed that K-312 decreases PCSK9 expression in human primary hepatocytes and in the human hepatoma cell line HepG2. siRNA silencing of CETP in HepG2 did not compromise the suppression of PCSK9 by K-312, suggesting a mechanism independent of CETP. In HepG2 cells, K-312 treatment decreased the active forms of sterol regulatory element-binding proteins (SREBP-1 and -2) that regulate promoter activity of PCSK9. Chromatin immunoprecipitation assays demonstrated that K-312 decreased the occupancy of SREBP-1 and SREBP-2 on the sterol regulatory element of the PCSK9 promoter. PCSK9 protein levels decreased by K-312 treatment in the circulating blood of cholesterol-fed rabbits, as determined by two independent mass spectrometry approaches, including the recently developed, highly sensitive parallel reaction monitoring method. New CETP inhibitor K-312 decreases LDL cholesterol and PCSK9 levels, serving as a new therapy for dyslipidemia and cardiovascular disease.
Aims/Introduction
Dipeptidyl peptidase‐4 inhibitors are used for treatment of patients with type 2 diabetes. In addition to glycemic control, these agents showed beneficial effects on lipid ...metabolism in clinical trials. However, the mechanism underlying the lipid‐lowering effect of dipeptidyl peptidase‐4 inhibitors remains unclear. Here, we investigated the lipid‐lowering efficacy of anagliptin in a hyperlipidemic animal model, and examined the mechanism of action.
Materials and Methods
Male low‐density lipoprotein receptor‐deficient mice were administered 0.3% anagliptin in their diet. Plasma lipid levels were assayed and lipoprotein profile was analyzed using high‐performance liquid chromatography. Hepatic gene expression was examined by deoxyribonucleic acid microarray and quantitative polymerase chain reaction analyses. Sterol regulatory element‐binding protein transactivation assay was carried out in vitro.
Results
Anagliptin treatment significantly decreased the plasma total cholesterol (14% reduction, P < 0.01) and triglyceride levels (27% reduction, P < 0.01). Both low‐density lipoprotein cholesterol and very low‐density lipoprotein cholesterol were also decreased significantly by anagliptin treatment. Sterol regulatory element‐binding protein‐2 messenger ribonucleic acid expression level was significantly decreased at night in anagliptin‐treated mice (15% reduction, P < 0.05). Anagliptin significantly suppressed sterol regulatory element‐binding protein activity in HepG2 cells (21% decrease, P < 0.001).
Conclusions
The results presented here showed that the dipeptidyl peptidase‐4 inhibitor, anagliptin, exhibited a lipid‐lowering effect in a hyperlipidemic animal model, and suggested that the downregulation of hepatic lipid synthesis was involved in the effect. Anagliptin might have beneficial effects on lipid metabolism in addition to a glucose‐lowering effect.
The results presented here indicated that the DPP‐4 inhibitor, anagliptin, exhibited a lipid‐lowering effect in LDLR‐deficient mice as a hyperlipidemic animal model, and suggested that the downregulation of hepatic lipid synthesis was involved in the effect.
Hepatic ATP-binding cassette transporter A1 (ABCA1) plays a key role in high-density lipoprotein (HDL) production by apolipoprotein A-I (ApoA-I) lipidation. 3-Hydroxy-3-methylglutaryl coenzyme A ...(HMG-CoA) reductase inhibitors, statins, increase ABCA1 mRNA levels in hepatoma cell lines, but their mechanism of action is not yet clear. We investigated how statins increase ABCA1 in rat hepatoma McARH7777 cells. Pitavastatin, atorvastatin, and simvastatin increased total ABCA1 mRNA levels, whereas pravastatin had no effect. Pitavastatin also increased ABCA1 protein. Hepatic ABCA1 expression in rats is regulated by both liver X receptor (LXR) and sterol regulatory element–binding protein (SREBP2) pathways. Pitavastatin repressed peripheral type ABCA1 mRNA levels and its LXR-driven promoter, but activated the liver-type SREBP-driven promoter, and eventually increased total ABCA1 mRNA expression. Furthermore, pitavastatin increased peroxisome proliferator–activated receptor α (PPARα) and its downstream gene expression. Knockdown of PPARα attenuated the increase in ABCA1 protein, indicating that pitavastatin increased ABCA1 protein via PPARα activation, although it repressed LXR activation. Furthermore, the degradation of ABCA1 protein was retarded in pitavastatin-treated cells. These data suggest that pitavastatin increases ABCA1 protein expression by dual mechanisms: SREBP2-mediated mRNA transcription and PPARα-mediated ABCA1 protein stabilization, but not by the PPAR–LXR–ABCA1 pathway.
Supplementary Figures: available only at http://dx.doi.org/10.1254/jphs.10241FP
Abstract Acyl-coenzyme A:cholesterol O-acyltransferase-1 (ACAT-1) plays an essential role in macrophage foam cell formation and progression of atherosclerosis. We developed a potent and selective ...ACAT-1 inhibitor, K-604, and tested its effects in apoE-knockout mice. Administration of K-604 to 8-week-old apoE-knockout mice for 12 weeks at a dose of 60 mg/kg/day significantly reduced macrophage-positive area and increased collagen-positive area in atherosclerotic plaques in the aorta without affecting plasma cholesterol levels or lesion areas, indicating direct plaque-modulating effects of K-604 on vascular walls independent of plasma cholesterol levels. Pactimibe, a nonselective inhibitor of ACAT-1 and ACAT-2, reduced plasma cholesterol levels but did not affect macrophage- or collagen-positive areas. The size of macrophages and cholesteryl ester contents in the aorta were reduced by K-604. Exposure of cultured human aortic smooth muscle cells to K-604 resulted in increased procollagen type 1 contents in the culture supernatant and increased procollagen type 1 mRNA levels. Procollagen production was unaffected by pactimibe even at a concentration that inhibited cholesterol esterification to the basal level. Thus, the plaque-modulating effects of K-604 can be explained by stimulation of procollagen production independent of ACAT inhibition in addition to potent inhibition of macrophage ACAT-1.
K-134 is a more potent antiplatelet drug with a selective inhibitory effect on phosphodiesterase 3 (PDE3) compared with its analogue, cilostazol.
This study was performed to compare the ameliorating ...effects of K-134 and cilostazol on brain damage in an experimental photothrombotic cerebral infarction model.
We investigated the effects of oral preadministration of PDE3 inhibitors in a rat stroke model established by photothrombotic middle cerebral artery (MCA) occlusion. K-134 significantly prolonged MCA occlusion time at doses >10 mg/kg, and reduced cerebral infarct size at 30 mg/kg in the stroke model (n = 12, 87.5±5.6 vs. 126.8±7.5 mm(3), P<0.01), indicating its potent antithrombotic effect. On the other hand, the effects of cilostazol on MCA occlusion time and cerebral infarct size are relatively weak even at the high dosage of 300 mg/kg. Furthermore, K-134 blocked rat platelet aggregation more potently than cilostazol in vitro. Also in an arteriovenous shunt thrombosis model, K-134 showed an antithrombotic effect greater than cilostazol.
These findings suggest that K-134, which has strong antithrombotic activity, is a promising drug for prevention of cerebral infarction associated with platelet hyperaggregability.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Rationale
Osilodrostat is an inhibitor of 11‐beta‐hydroxylase (CYP11B) and is used for the treatment of Cushing's disease but also categorized as an anabolic agent. The use of osilodrostat is ...prohibited in horseracing and equestrian sports. To the best of our knowledge, this is the first metabolic study of osilodrostat in equine plasma.
Methods
Potential metabolites of osilodrostat were identified by differential analysis using data acquired from pre‐ and post‐administration plasma samples after protein precipitation with liquid chromatography electrospray ionization high‐resolution mass spectrometry (LC/ESI–HRMS). Correction added on 27 January 2023, after first online publication: In the preceding sentence, “C–HRMS” was changed to “LC/ESI–HRMS” in this version. For quantification of osilodrostat, a strong cation exchange solid‐phase extraction was employed, and the extracts were analyzed using LC/ESI–triple quadrupole tandem mass spectrometry (LC/ESI–QqQ‐MS/MS) to establish its elimination profile. Such extracts were further analyzed using LC/ESI–HRMS to investigate the detectability of osilodrostat and its identified mono‐hydroxylated metabolite over a 2‐week sampling period.
Results
Mono‐hydroxylated osilodrostat was identified based on the differential analysis and mass spectrometric interpretations, and it was found to be the most abundant metabolite in plasma. Elimination profile of osilodrostat in plasma was successfully established over the 24‐h post‐administration period. Both osilodrostat and its mono‐hydroxylated metabolite were detected up to the last sampling point at 2 weeks using HRMS, and osilodrostat could be confirmed up to 8‐day post‐administration with its reference material using HRMS as well.
Conclusions
For doping control, screening of both the parent drug osilodrostat and its mono‐hydroxylated metabolite in equine plasma would be recommended due to their extended detection windows of up to 2 weeks. Given the availability of reference material for potential confirmation in forensic samples, osilodrostat is considered the most appropriate monitoring target.
Abstract only
Background:
Despite potent LDL-cholesterol (LDL-C) therapies, substantial residual cardiovascular risk remains. Proprotein convertase subtilisin/kexin 9 (PCSK9) is a promising target ...for lowering LDL-C. We recently demonstrated that K-312, originally found as a cholesteryl ester transfer protein (CETP) inhibitor, decreased PCSK9 expression in vitro through the reduction of its promoter activity. The present study has further examined the underlying molecular mechanisms and in vivo effects of K-312 on PCSK9 levels using mass spectrometry.
Methods and Results:
CETP silencing by siRNA in the human hepatocyte cell line HepG2 did not compromise suppression of PCSK9 expression by K-312, indicating a mechanism independent of CETP inhibition. Transcription factors sterol-regulatory element binding protein (SREBP)-1 and SREBP-2 bind to the sterol regulatory element (SRE) of PCSK9 promoter when their precursor forms are cleaved into active forms. K-312 treatment decreased binding of SREBP-1 and SREBP-2 to SRE of the PCSK9 promoter (chromatin immunoprecipitation) and active forms of SREBP-1 and SREBP-2 (western blotting), suggesting the suppression of PCSK9 expression by K-312 involves SREBP-1 and SREBP-2. We then examined the effects of K-312 in cholesterol-fed New Zealand White rabbits administered either vehicle (N=7) or K-312 30 mg/kg (N=7) for 2 weeks. K-312 treatment decreased LDL-C (vehicle: 272.5 ± 42.2 mg/dl; K-312: 193.0 ± 36.5 mg/dl), increased HDL cholesterol (vehicle: 29.9 ± 3.7 mg/dl; K-312: 93.3 ± 14.7 mg/dl) and decreased PCSK9 mRNA levels in the liver by 63%. We further used two mass spectrometry-based approaches to measure PCSK9 levels in rabbit plasma: spectral counting and a novel high resolution (HR)-MS/MS quantification method. Immunoprecipitation of PCSK9 successfully enriched PCSK9 signal. Spectral counting and HR-MS/MS of PCSK9 peptides both demonstrated that K-312 treatment significantly decreased plasma PCSK9 levels by 76% (p<0.01) and 60% (p<0.05), respectively.
Conclusion:
K-312 may lower LDL cholesterol levels by suppressing CETP activity and PCSK9 expression, serving as a novel therapy for dyslipidemia and cardiovascular disease.
Statins exert atheroprotective effects through the induction of specific transcriptional factors in multiple organs. In endothelial cells, statin-dependent atheroprotective gene up-regulation is ...mediated by Kruppel-like factor (KLF) family transcription factors. To dissect the mechanism of gene regulation, we sought to determine molecular targets by performing microarray analyses of human umbilical vein endothelial cells (HUVECs) treated with pitavastatin, and KLF4 was determined to be the most highly induced gene. In addition, it was revealed that the atheroprotective genes induced with pitavastatin, such as nitric oxide synthase 3 (NOS3) and thrombomodulin (THBD), were suppressed by KLF4 knockdown. Myocyte enhancer factor-2 (MEF2) family activation is reported to be involved in pitavastatin-dependent KLF4 induction. We focused on MEF2C among the MEF2 family members and identified a novel functional MEF2C binding site 148 kb upstream of the KLF4 gene by chromatin immunoprecipitation along with deep sequencing (ChIP-seq) followed by luciferase assay. By applying whole genome and quantitative chromatin conformation analysis {chromatin interaction analysis with paired end tag sequencing (ChIA-PET), and real time chromosome conformation capture (3C) assay}, we observed that the MEF2C-bound enhancer and transcription start site (TSS) of KLF4 came into closer spatial proximity by pitavastatin treatment. 3D-Fluorescence in situ hybridization (FISH) imaging supported the conformational change in individual cells. Taken together, dynamic chromatin conformation change was shown to mediate pitavastatin-responsive gene induction in endothelial cells.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
PDF
