Yoon-Gyoon Kim

Antiplatelet effect of AMP-activated protein kinase activator and its potentiation by the phosphodiesterase inhibitor dipyridamole

AMP-activated protein kinase (AMPK) activates endothelial nitric oxide synthase (eNOS) via phosphorylation at the activating site. The eNOS-nitric oxide (NO)/soluble guanylate cyclase (sGC)-cGMP/cGMP-dependent protein kinase (PKG) signaling axis is a major antiaggregatory mechanism residing in platelets. Based on the hypothesis that direct activation of AMPK might be a potential strategy to inhibit platelet aggregation, the antiplatelet effect of AMPK activators was investigated. Treatment of isolated platelets with the AMPK activator, 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) resulted in AMPK activation and a decrease in aggregation, which was abolished by pretreatment with the AMPK inhibitors compound C (CC) and ara-A. Such an AMPK-dependent antiaggregatory effect was also observed with other AMPK activators such as A-769662 and PT1. AICAR induced eNOS activation was followed by NO synthesis, cGMP production, and subsequent phosphorylation of vasodilator-stimulated phosphoprotein (VASP), a PKG substrate. All these events were blocked by CC or ara-A pretreatment, and each event was inhibited by the eNOS inhibitor L-NAME, the sGC inhibitor ODQ, and the PKG inhibitor Rp-8-pCPT-cGMPS. Simultaneous treatment of dipyridamole, a phosphodiesterase (PDE) inhibitor, with AICAR potentiated the antiaggregatory effect by enhancing the cGMP elevation. Administration of AICAR increased platelet cGMP and prolonged FeCl3-induced arterial occlusion time in rats, which further increased in combination with dipyridamole. In conclusion, AMPK activators inhibited platelet aggregation by stimulating the eNOS-NO/sGC-cGMP/PKG signaling pathway. The antiplatelet effect of AMPK activators could be potentiated in combination with a PDE inhibitor through the common mechanism of elevating cGMP. Thus, AMPK may serve as a potential target for antiplatelet therapy.

Discovery of Novel 2,5-Dioxoimidazolidine-Based P2X7 Receptor Antagonists as Constrained Analogues of KN62

Novel 2,5-dioxoimidazolidine-based conformationally constrained analogues of KN62 (1) were developed as P2X7 receptor (P2X7R) antagonists using a rigidification strategy of the tyrosine backbone of 1. SAR analysis of the 2,5-dioxoimidazolidine scaffold indicated that piperidine substitution at the N3 position and no substitution at N1 position were preferable. Further optimization of the substituents at the piperidine nitrogen and the spacer around the skeleton resulted in several superior antagonists to 1, including 1-adamantanecarbonyl analogue 21i (IC50 = 23 nM in ethidium uptake assay; IC50 = 14 nM in IL-1β ELISA assay) and (3-CF3-4-Cl)benzoyl analogue (-)-21w (54 nM in ethidium uptake assay; 9 nM in IL-1β ELISA assay), which was more potent than the corresponding (+) isomer. Compound 21w displayed potent inhibitory activity in an ex vivo model of LTP-induced pain signaling in the spinal cord and significant anti-inflammatory activity in in vivo models of carrageenan-induced paw edema and type II collagen-induced joint arthritis.

Evaluation of the transfusion safety of blood products and determination of plasma concentrations of acitretin and etretinate in patients receiving transfusions

BACKGROUND: Acitretin and etretinate are potentially teratogenic. Many people taking acitretin for psoriasis have donated blood during the deferral period in Korea. Therefore, many of the blood products from these donors treated with acitretin have been circulated in Korea.

Discovery of Potent Antiallodynic Agents for Neuropathic Pain Targeting P2X3 Receptors

Antagonism of the P2X3 receptor is one of the potential therapeutic strategies for the management of neuropathic pain because P2X3 receptors are predominantly localized on small to medium diameter C- and Aδ-fiber primary afferent neurons, which are related to the pain-sensing system. In this study, 5-hydroxy pyridine derivatives were designed, synthesized, and evaluated for their in vitro biological activities by two-electrode voltage clamp assay at hP2X3 receptors. Among the novel hP2X3 receptor antagonists, intrathecal treatment of compound 29 showed parallel efficacy with pregabalin (calcium channel modulator) and higher efficacy than AF353 (P2X3 receptor antagonist) in the evaluation of its antiallodynic effects in spinal nerve ligation rats. However, because compound 29 was inactive by intraperitoneal administration in neuropathic pain animal models due to low cell permeability, the corresponding methyl ester analogue, 28, which could be converted to compound 29 in vivo, was investigated as a prodrug concept. Intravenous injection of compound 28 resulted in potent antiallodynic effects, with ED50 values of 2.62 and 2.93 mg/kg in spinal nerve ligation and chemotherapy-induced peripheral neuropathy rats, respectively, indicating that new drug development targeting the P2X3 receptor could be promising for neuropathic pain, a disease with high unmet medical needs.

Bioequivalence of Ramiprin ® tablet to Tritace Protect ® tablet (Ramipril 10 mg)

To evaluate the bioequivalence of two ramipril formulations, a standard 2-way randomized cross-over study was conducted in twenty-six healthy male Korean volunteers. A single oral dose of 10 mg of test formulation (tablet) or reference formulation Tritace (tablet) was administered with one-week washout period. Plasma concentrations of ramipril were assayed over a period of 12 hr with a well validated method using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The values of area under the plasma concentration-time curve, from time zero to last sampling time and from time zero to time infinity were for test, and for reference formulation, respectively. Similarly, maximum concentration and elimination half-life were and for test, and and for reference formulations, respectively. Time to reach maximum concentration for the test and the reference, were , respectively. The parametric 90% confidence intervals on the mean of the differences between the two formulations (test-reference) of the log-transformed values of were 1.03 to 1.19 and 0.98 to 1.17, respectively. The overall results indicate that the two formulations are bioequivalent and can be prescribed interchangeably.

Bioequivalence of Efexor ® XR capsule to Venfaxine ® OR tablet (Venlafaxine 75 mg)

To evaluate the bioequivalence of two venlafaxine formulations, a standard 2-way randomized cross-over study was conducted in twenty-four healthy male Korean volunteers. A single oral dose of 75 mg of test formulation Venfaxine (tablet) or reference formulation Efexor (capsule) was administered with one-week washout period. Plasma concentrations of venlafaxine were assayed for over a period of 72 hours with a well validated method using liquid chromatography coupled to tandem mass spectrometry (LC-MS-MS). The . of maximum concentration and elimination half-life were ng/mL, h, and ng/mL, h for test and reference formulations, respectively. Time to reach maximum concentration expressed in median value (range), for the test and the reference, were 10 h (6-14) and 8h (4-12), respectively. Similarly, area under the plasma concentration-time curve, from time zero to last sampling time and from time zero to time infinity , for test and reference formulations were , and , , respectively. The parametric 90% confidence intervals on the mean of the differences between the two formulations (test-reference) of the log transformed values of , and were 0.9630 to 1.1383 and 0.8650 to 1.0446, respectively. The overall results indicate that the two formulations are bioequivalent and can be prescribe interchangeably.

Validation of an LC/MS/MS Method for the Pharmacokinetic Study of Lercanidipine

A rapid, simple and sensitive LC/MS/MS method for the determination of lercanidipine in human serum was validated and applied to the pharmacokinetic study of lercanidipine. Lercanidipine and internal standard, amlodipine, were extracted from human serum by liquid-liquid extraction with hexan-isoamyl alcohol (100: 1, v/v) and analyzed on a MS column with the mobile phase of acetonitrile-0.2% aqueous formic acid (70: 30, v/v). Using MS/MS with multiple reaction monitoring (MRM) mode, lercanidipine and amlodipine were detected without severe interferences from human serum matrix. Lercanidipine produced a protonated precursor ion () at m/z 612.3 and a corresponding product ion at m/z 280.0. Internal standard produced a protonated precursor ion (]) at m/z 409.0 and a corresponding product ion at m/z 238.0. The ruggedness of this method was investigated using quality control (QC) samples. This method showed linear response over the concentration range of 0.05-20 ng/mL with correlation coefficient greater than 0.999. The lower limit of quantitation using 0.5 mL of serum was 0.05 ng/mL, which was sensitive enough for pharmacokinetic studies. The overall accuracy of the developed method ranged from 85.51 to 112.2% for lercanidipine with overall precision (% C.V.) being 3.56-13.1%. This method showed good ruggedness (within 15% C.V.) and was successfully applied for the analysis of lercanidipine in human serum samples for the pharmacokinetic studies, demonstrating the suitability of the method.

Bioequivalence of Lisihexal ® tablet to Zestril ® tablet(Lisinopril 10 mg)

Lisinopril is one of the angiotensin-converting enzyme inhibitors, which have been used for treatment of hypertension and heart failure. The aim of this study was to evaluate the bioequivalence of two lisinopril tablet, and as a test and reference, respectively. The study was came out on 28 healthy male Korean volunteers in crossover design. An analytical method with LC-MS-MS was developed for the quantification of lisinopril and enalapril(IS) using SPE method. The condition was selective, sensitive and precise in human plasma, that was enough for the pharmacokinetic study of lisinopril. The pharmacokinetic parameters such as were calculated and ANOVA test was used for the statistical analysis of the parameters using log transformed . of test and reference drugs were calculated , respectively. The 90% confidence intervals of were log 0.9245log 1.0603, log 0.9270log 1.0601 and log 0.9548log 1.1009, within the acceptable range of log 0.8 to log 1.25 by KFDA bioequivalence criteria. Two medications of lisinopril were evaluated bioequivalent and thus may be prescribed interchangeably.

Sensitive Determination of Pinaverium Bromide in Human Plasma by LC-ESI-MS/MS : Applicability to Oral Bioavailability Determination

A sensitive method for quantification of pinaverium bromide in human plasma was established using liquid chromatography-electrospray ionization tandem mass spectrometry(LC-ESI-MS/MS). Glimepiride was used as internal standard. Pinaverium bromide and internal standard in plasma sample were extracted using tert-butylmethylether(TBME). A centrifuged upper layer was then evaporated and reconstituted with mobile phase of acetonitrile-5 mM ammonium formate (80/20, pH 3.0). The reconstituted samples were injected into a reversed-phase column. Using MS/MS with multiple reaction monitoring (MRM) mode, pinaverium and glimepirde were detected without severe interference from human plasma matrix. Pinaverium produced a protonated precursor ion at m/z 510.3 and a corresponding product ion at m/z 228.9. Internal standard produced a protonated precursor ion at m/z 491.5 and a corresponding product ion at m/z 352.0. Detection of pinaverium bromide in human plasma was accurate and precise, with limit of quantitation at 0.5 ng/ml. The method has been successfully applied to bioavailability study of pinaverium bromide tablet in Korean healthy male volunteers. Pharmacokinetic parameters such as were calculated.