Boram Ohk

Docosahexaenoic acid signaling attenuates the proliferation and differentiation of bone marrow-derived osteoclast precursors and promotes apoptosis in mature osteoclasts

Docosahexaenoic acid (DHA), a component of omega-3 fatty acids, has been reported to protect against inflammatory bone diseases such as osteoporosis and rheumatoid arthritis. However, its exact mechanism in bone resorbing cells has not been elucidated. In this study, we investigated the effects and the molecular mechanism of DHA on the proliferation, differentiation, and survival of osteoclast lineage cells using mouse bone marrow-derived macrophages (BMMs). DHA suppressed the macrophage colony-stimulating factor (M-CSF)-induced proliferation of osteoclast precursors, BMMs, in a dose-dependent manner. The attenuated proliferation of DHA-treated BMMs was related to M-CSF inhibition that selectively decreased Akt activation and downregulated cyclin D1 and cyclin D2 expression. DHA also blocked receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation from BMMs. At the molecular level, DHA inhibited JNK, ERK, and p38 MAPKs. In addition, it inhibited NF-κB signaling cascades, as demonstrated by the suppression of RANKL-mediated IκBα phosphorylation, NF-κB subunit p65 nuclear translocation, and NF-κB transcriptional activation. Accordingly, DHA attenuated the induction of c-Fos and nuclear factor of activated T cells c1 (NFATc1). Furthermore, DHA accelerated the apoptosis of mature osteoclasts by inducing Bim expression, a critical modulator of osteoclast apoptosis. Collectively, our data demonstrate that DHA exerts an anti-osteoclastogenic effect by suppressing the proliferation and differentiation of BMMs and enhancing the apoptosis of mature osteoclasts, thereby resulting in a diminished number of bone-resorptive cells.

Deficiency of Lipocalin-2 Promotes Proliferation and Differentiation of Osteoclast Precursors via Regulation of c-Fms Expression and Nuclear Factor-kappa B Activation.

Background Lipocalin-2 (LCN2), a small glycoprotein, has a pivotal role in diverse biological processes such as cellular proliferation and differentiation. We previously reported that LCN2 is implicated in osteoclast formation induced by receptor activator of nuclear factor-kappa B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). In the present study, we used a knockout mouse model to further investigate the role of LCN2 in osteoclast development. Methods Osteoclastogenesis was assessed using primary bone marrow-derived macrophages. RANKL and M-CSF signaling was determined by immunoblotting, cell proliferation by bromodeoxyuridine (BrdU) enzyme-linked immunosorbent assay (ELISA), and apoptosis by cell death detection ELISA. Bone morphometric parameters were determined using a micro-computed tomography system. Results Our results showed that LCN2 deficiency increases tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclast formation in vitro, a finding that reflects enhanced proliferation and differentiation of osteoclast lineage cells. LCN2 deficiency promotes M-CSF-induced proliferation of bone marrow macrophages (BMMs), osteoclast precursors, without altering their survival. The accelerated proliferation of LCN2-deficient precursors is associated with enhanced expression and activation of the M-CSF receptor, c-Fms. Furthermore, LCN2 deficiency stimulates the induction of c-Fos and nuclear factor of activated T cells c1 (NFATc1), key transcription factors for osteoclastogenesis, and promotes RANKL-induced inhibitor of kappa B (IκBα) phosphorylation. Interestingly, LCN2 deficiency does not affect basal osteoclast formation in vivo, suggesting that LCN2 might play a role in the enhanced osteoclast development that occurs under some pathological conditions. Conclusions Our study establishes LCN2 as a negative modulator of osteoclast formation, results that are in accordance with our previous findings.

Simultaneous Determination of Five Cytochrome P450 Probe Substrates and Their Metabolites and Organic Anion Transporting Polypeptide Probe Substrate in Human Plasma Using Liquid Chromatography-Tandem Mass Spectrometry

A rapid and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of organic anion transporting polypeptide 1B1 (OATP1B1) and cytochrome P450 (P450) probe substrates and their phase I metabolites in human plasma was developed. The OATP1B1 (pitavastatin) and five P450 probe substrates, caffeine (CYP1A2), losartan (CYP2C9), omeprazole (CYP2C19), dextromethorphan (CYP2D6), and midazolam (CYP3A) and their metabolites were extracted from human plasma (50 µL) using methanol. Analytes were separated on a C18 column followed by selected reaction monitoring detection using MS/MS. All analytes were separated simultaneously within a 9 min run time. The developed method was fully validated over the expected clinical concentration range for all analytes tested. The intra- and inter-day precisions for all analytes were lower than 11.3% and 8.82%, respectively, and accuracy was 88.5–117.3% and 96.1–109.2%, respectively. The lower limit of quantitation was 0.05 ng/mL for dextromethorphan, dextrorphan, midazolam, and 1′-hydroxymidazolam; 0.5 ng/mL for losartan, EXP-3174, omeprazole, 5′-hydroxyomeprazole, and pitavastatin; and 5 ng/mL for caffeine and paraxanthine. The method was successfully used in a pharmacokinetic study in healthy subjects after oral doses of five P450 and OATP1B1 probes. This analytical method provides a simple, sensitive, and accurate tool for the determination of OATP1B1 and five major P450 activities in vivo drug interaction studies.

Pterional versus superciliary keyhole approach: direct comparison of approach-related complaints and satisfaction in the same patient

OBJECTIVEThe objective of this study was to evaluate and compare the level of patient satisfaction and approach-related patient complaints between a superciliary keyhole approach and a pterional approach.METHODSPatients who underwent an ipsilateral superciliary keyhole approach and a contralateral pterional approach for bilateral intracranial aneurysms during an 11-year period were contacted and asked to complete a patient satisfaction questionnaire. The questionnaire covered 5 complaint areas related to the surgical approaches: craniotomy-related pain, sensory symptoms in the head, cosmetic complaints, palpable cranial irregularities, and limited mouth opening. The patients were asked to rate the 5 complaint areas on a scale from 0 (asymptomatic or very pleasant) to 4 (severely symptomatic or very unpleasant). Finally, the patients were asked to rate the level of overall satisfaction related to each surgical procedure on a visual analog scale (VAS) from 0 (most unsatisfactory) to 100 (most satisfactory).RESULTSA total of 21 patients completed the patient satisfaction questionnaire during a follow-up clinic visit. For the superciliary procedures, no craniotomy-related pain, palpable irregularities, or limited mouth opening was reported, and only minor sensory symptoms (numbness in the forehead) and cosmetic complaints (short linear operative scar) were reported (score = 1) by 1 (4.8%) and 3 patients (14.3%), respectively. Compared with the pterional approach, the superciliary approach showed better outcomes regarding the incidence of craniotomy-related pain, cosmetic complaints, and palpable irregularities, with a significant between-approach difference (p < 0.05). Furthermore, the VAS score for patient satisfaction was significantly higher for the superciliary approach (mean 95.2 ± 6.0 [SD], range 80-100) than for the pterional approach (mean 71.4 ± 10.6, range 50-90). Moreover, for the pterional approach, a multiple linear regression analysis indicated that the crucial factors decreasing the level of patient satisfaction were cosmetic complaints, craniotomy-related pain, and sensory symptoms, in order of importance (p < 0.05).CONCLUSIONSIn successful cases in which the primary surgical goal of complete aneurysm clipping without postoperative complications is achieved, a superciliary keyhole approach provides a much higher level of patient satisfaction than a pterional approach, despite a facial wound. For a pterional approach, the patient satisfaction level is affected by the cosmetic results, craniotomy-related pain, and numbness behind the hairline, in order of importance.

Pharmacokinetic and bioequivalence study of a telmisartan/S-amlodipine fixed-dose combination (CKD-828) formulation and coadministered telmisartan and S-amlodipine in healthy subjects

Purpose A new fixed-dose combination (FDC) formulation of telmisartan 80 mg and S-amlodipine 5 mg (CKD-828) has been developed to increase convenience (as only one tablet is required per day) and improve treatment compliance. Methods The pharmacokinetic characteristics and tolerability of an FDC of telmisartan and S-amlodipine were compared to those after coadministration of the individual agents in this randomized, open-label, single-dose, two-way, four-period, crossover study. To analyze the telmisartan and S-amlodipine plasma concentrations using a validated liquid chromatography–tandem mass spectrometry method, serial blood samples were collected up to 48 hours post-dose for telmisartan and 144 hours post-dose for S-amlodipine, in each period. Results Forty-eight healthy subjects were enrolled, and 43 completed the study. The mean peak plasma concentration (Cmax) and the area under the plasma concentration–time curve from time 0 to the last measurement (AUC0–t) values of telmisartan were 522.29 ng/mL and 2,475.16 ng·h/mL for the FDC, and 540.45 ng/mL and 2,559.57 ng·h/mL for the individual agents concomitantly administered, respectively. The mean Cmax and AUC0–t values of S-amlodipine were 2.71 ng/mL and 130.69 ng·h/mL for the FDC, and 2.74 ng/mL and 129.81 ng·h/mL for the individual agents concomitantly administered, respectively. The geometric mean ratio (GMR) and 90% confidence interval (CI) for the telmisartan Cmax and AUC0–t (FDC of telmisartan and S-amlodipine/concomitant administration) were 0.8509 (0.7353–0.9846) and 0.9431 (0.8698–1.0226), respectively. The GMR and 90% CI for the S-amlodipine Cmax and AUC0–t (FDC/concomitant administration) were 0.9829 (0.9143–1.0567) and 0.9632 (0.8798–1.0546), respectively. As the intrasubject variability of the Cmax for telmisartan administered individually was 42.94%, all 90% CIs of the GMRs fell within the predetermined acceptance range. Both treatments were well tolerated in this study. Conclusion CKD-828 FDC tablets were shown to be bioequivalent to coadministration of the individual agents with the respective strength, in healthy subjects under fasting conditions. There was no significant difference in safety profile between the two treatments.

A Comprehensive In Vivo and In Vitro Assessment of the Drug Interaction Potential of Red Ginseng

Purpose: Red ginseng is one of the worlds most popular herbal medicines; it exhibits a wide range of pharmacologic activities and is often co-ingested with other herbal and conventional medicines. This open-label, randomized, 3-period study investigated the in vivo herb-drug interaction potential for red ginseng extract with cytochrome P-450 (CYP) enzymes and organic anion-transporting polypeptide (OATP) 1B1. METHODS Fifteen healthy male volunteers (22-28 years; 57.1-80.8 kg) were administered a single dose of cocktail probe substrates (caffeine 100 mg, losartan 50 mg, omeprazole 20 mg, dextromethorphan 30 mg, midazolam 2 mg, and pitavastatin 2 mg) and single or multiple doses of red ginseng extract for 15 days. FINDINGS The pharmacokinetic profiles of the probe substrates and metabolites after single- or multiple-dose administration of red ginseng extracts were comparable to the corresponding profiles of the control group. The geometric mean ratio of AUC0-t and 90% CIs for the probe substrate drugs between the control and multiple doses of red ginseng for 15 days were within 0.8 to 1.25 (CYP2C9, CYP3A4, and OATP1B1 probe substrates) or slightly higher (CYP1A2, CYP2C19, and CYP2D6 probe substrates). Additional assessments of the in vitro drug interaction potential of red ginseng extracts and the ginsenoside Rb1 on drug-metabolizing enzymes and transporters using human liver microsomes, cryopreserved human hepatocytes, and transporter-overexpressed cells were negative. IMPLICATIONS Red ginseng poses minimal risks for clinically relevant CYP- or OATP-mediated drug interactions and is well tolerated. Clinical Research Information Service registry no.

Determination of sumatriptan in human plasma using liquid chromatography-mass spectrometry for pharmacokinetic study in healthy Korean volunteers

This study describes the development of an analytical method to determine sumatriptan levels in human plasma using high performance liquid chromatography (HPLC) coupled with triple quadrupole tandem mass spectrometry (MS/MS) and its application to a pharmacokinetic study in healthy Korean volunteers. A single 50 mg dose of sumatriptan was orally administered to twelve healthy volunteers (nine women and three men). The HPLC-MS/MS analytical method was validated with respect to its specificity, linearity, sensitivity, accuracy, precision, recovery, and stability. The calibration curve was linear over a concentration range of 0.3–100 ng/mL (r > 0.999). The lower limit of quantitation for sumatriptan in plasma was 0.3 ng/mL. The accuracy and precision of the analytical method were acceptable within 15% at all quality control levels. We compared plasma concentration-time curves as well as pharmacokinetic parameters such as the area under the curve (AUC) and maximum plasma concentration (Cmax). Both the mean AUC and Cmax of sumatriptan were 1.56 times higher in women than in men. These differences could be largely explained by the difference in body weight (44%) between women and men. The outcomes may provide insights into developing appropriate individualized treatment strategies.

Metabolomic analysis of healthy human urine following administration of glimepiride using a liquid chromatography-tandem mass spectrometry

Glimepiride, a third generation sulfonylurea, is an antihyperglycemic agent widely used to treat type 2 diabetes mellitus. In this study, an untargeted urinary metabolomic analysis was performed to identify endogenous metabolites affected by glimepiride administration. Urine samples of twelve healthy male volunteers were collected before and after administration of 2 mg glimepiride. These samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and then subjected to multivariate data analysis including principal component analysis and orthogonal partial least squares discriminant analysis. Through this metabolomic profiling, we identified several endogenous metabolites such as adenosine 3′, 5′-cyclic monophosphate (cAMP), quercetin, tyramine, and urocanic acid, which exhibit significant metabolomic changes between pre- and posturine samples. Among these, cAMP, which is known to be related to insulin secretion, was the most significantly altered metabolite following glimepiride administration. In addition, the pathway analysis showed that purine, tyrosine, and histidine metabolism was affected by pharmacological responses to glimepiride. Together, the results suggest that the pharmacometabolomic approach, based on LC-MS/MS, is useful in understanding the alterations in biochemical pathways associated with glimepiride action.

Pharmacokinetic and safety evaluation of MB12066, an NQO1 substrate

Objective This study evaluated the pharmacokinetics (PKs) and safety of a newly developed β-lapachone (MB12066) tablet, a natural NAD(P)H:quinone oxidoreductase 1 (NQO1) substrate, in healthy male volunteers. Methods In a randomized, double-blind, multiple-dose, two-treatment study, 100 mg MB12066 or placebo was given twice daily for 8 days to groups of eight or three fasted healthy male subjects, respectively, followed by serial blood sampling. Plasma concentrations for β-lapachone were determined using liquid chromatography–tandem mass spectrometry. PK parameters were obtained with non-compartmental analysis. Tolerability was assessed based on physical examinations, vital signs, clinical laboratory tests, and electrocardiograms. Results Following a single 100 mg MB12066 oral dose, maximum plasma concentration (Cmax) of β-lapachone was 3.56±1.55 ng/mL, and the median (range) time to reach Cmax was 3 h (2–5 h). After the 8 days of 100 mg twice daily repeated dosing was completed, mean terminal half-life was determined to be 18.16±3.14 h, and the mean area under the plasma concentration vs time curve at steady state was 50.44±29.68 ng·h/mL. Accumulation index was 2.72±0.37. No serious adverse events (AEs) were reported, and all reported intensities of AEs were mild. Conclusion The results demonstrated that MB12066 was safe and well tolerated in healthy volunteers and that there were no serious AEs. Accumulation in plasma with twice-daily administration was associated with a 2.72 accumulation ratio.