Sensitive analysis and pharmacokinetic study of epalrestat in C57BL/6J mice.

Epalrestat is clinically applied for the management of diabetic peripheral neuropathy, yet its pharmacokinetic properties are not well understood. In this study, a rapid and sensitive LC-MS/MS method was established for assaying epalrestat in bio-samples of mice. The method was validated and it showed a good linearity over the range of 2-5000ng/mL, a precision of less than 12.3%, and recovery and matrix effects of 112.5-123.6% and 87.9-89.5%, respectively. After administration of a single dose of epalrestat administered, the exposure level of AUC0-∞ was positively dose-dependent and the mean Cmax, AUC0-12h, T1/2, and MRT were 36.23±7.39µg/mL, 29,086.5µg/Lh, 1.2h and 1.8h, respectively. Epalrestat was highly exposed in stomach, intestine, liver and kidney, and only a small amount was detected in brain, urine and feces. Multi-dose of epalrestat significantly increased MRT and apparent volume of distribution (Vd) relative to those of a single-dose.

Thiazolidine derivatives as potent and selective inhibitors of the PIM kinase family.

The PIM family of serine/threonine kinases have become an attractive target for anti-cancer drug development, particularly for certain hematological malignancies. Here, we describe the discovery of a series of inhibitors of the PIM kinase family using a high throughput screening strategy. Through a combination of molecular modeling and optimization studies, the intrinsic potencies and molecular properties of this series of compounds was significantly improved. An excellent pan-PIM isoform inhibition profile was observed across the series, while optimized examples show good selectivity over other kinases. Two PIM-expressing leukemic cancer cell lines, MV4-11 and K562, were employed to evaluate the in vitro anti-proliferative effects of selected inhibitors. Encouraging activities were observed for many examples, with the best example (44) giving an IC50 of 0.75µM against the K562 cell line. These data provide a promising starting point for further development of this series as a new cancer therapy through PIM kinase inhibition.

Inhibition of Tau Protein Aggregation by Rhodanine-based Compounds Solubilized Via Specific Formulation Additives to Improve Bioavailability and Cell Viability.

BACKGROUND: Anti-aggregation drugs play an important role in therapeutic approaches for Alzheimer's disease. We have previously developed a number of compounds that are able to inhibit the pathological aggregation of Tau protein. One common obstacle to application is the limited penetration across the plasma membranes into cells, where Tau aggregation occurs in the cytosol. We used an inducible N2a cell line which expresses the repeat domain of tau and develops tau aggregates. OBJECTIVE: Several peptide-polymer conjugates were synthesized to enhance the uptake of compounds into cells and thus to improve their biomedical application. The aim of this study was to test whether the peptide-inhibitor complexes still retain their inhibitory activity on Tau aggregation. METHOD: We screened peptide sequences with high binding capacity to a subset of aggregation inhibitors and identified them by fluorescence microscopy and MALDI MS/MS with regard to drug solubility and effective complexion. To explore whether the synthesized complexes can influence the aggregation propensity of Tau we performed in vitro and cellular assays. The effect on toxicity was investigated by measuring apoptosis markers. RESULTS/CONCLUSION: The tested peptide-compound complexes show no decrease in the total Tau levels but decreased ratios of soluble to pelletable Tau species. This indicates a conversion of insoluble Tau oligomers into soluble forms which appear to be less toxic than the insoluble ones, as seen by a decrease of apoptotic cells. Thus the peptide-compound complexes have a higher potency than the compounds alone due to improved bioavailability of the drug.

Simultaneous determination of epalrestat and puerarin in rat plasma by UHPLC-MS/MS: Application to their pharmacokinetic interaction study.

In the present study, a simple, rapid and reliable ultrahigh-performance liquid chromatography-tandem mass spectrometric (UHPLC-MS/MS) method was developed and validated to determine simultaneously epalrestat (EPA) and puerarin (PUE) in rat plasma for evaluation of the pharmacokinetic interaction of these two drugs. Both the analytes and glipizide (internal standard, IS) were extracted using a protein precipitation method. The separation was performed on a C18 reversed phase column using acetonitrile and 5 mmol/L ammonium acetate in water as the mobile phase with a gradient elution program. The analytes, including IS, were quantified with multiple reaction monitoring under negative ionization mode. The optimized mass transition ion pairs (m/z) were 318.1 → 274.0 for EPA, 415.1 → 266.9 for PUE and 444.2 → 166.9 for IS. The linear calibration curves for EPA and PUE were obtained in the concentration ranges of 10-4167 and 20-8333 ng/mL, respectively (r > 0.99). The current method was successfully applied for the pharmacokinetic interaction study in rats following administration of EPA and PUE alone or co-administration (EPA 15 mg/kg, oral; PUE 30 mg/kg, intravenous). The results showed that the combination of EPA and PUE could increase t1/2 of EPA and reduce Tmax of EPA. These changes indicated that EPA and PUE might cause drug-drug interactions when co-administrated.

Identification and characterization of potent, selective and metabolically stable IKKß inhibitor.

We have previously reported the identification of a rhodanine compound (1) with well-balanced inhibitory activity against IKKß and collagen-induced TNFα activated cells. However, we need more optimized compounds because of its instability over plasma and microsome. As part of a program directed toward the optimization of IKKß inhibitor, we modified a substituent of parent compound to a series of functional groups. Among substituted compounds, fluorine substituent (12) on the para position of phenyl ring restored the stability toward plasma and microsome while retaining inhibitory potency and selectivity against IKKß over other kinases. Also, we have demonstrated that compound 12 is an ATP non-competitive inhibitor and safe enough to apply to animal experiment from an acute toxicity test.

LC-MS/MS method for the quantification of aldose reductase inhibitor-epalrestat and application to pharmacokinetic study.

A simple and rapid LC-MS/MS method was developed and validated for the quantification of epalrestat, an aldose reductase inhibitor for the treatment of diabetic neuropathy. Following protein precipitation epalrestat and IS were eluted with 10mM ammonium acetate and acetonitrile using a rapid gradient program on reverse phase column. Multiple reaction monitoring mode was used to monitor the transitions of m/z 318→58 for epalrestat and m/z 410→348 for the IS. The assay exhibited a linear dynamic range of 2-5,000 ng/mL for epalrestat in rat plasma. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The within batch accuracy was in the range of 101.3-108.0% with precision in the range of 3.0-12.3%. All the other validation parameters were within the acceptable limits. Validated method was applied to analyze rat plasma samples obtained from a pharmacokinetic study. After oral administration of epalrestat at 10mg/kg to wistar rats (n=3) mean C(max), AUC(0-24) (ngh/mL) and t(1/2) were found to be 4077 ± 1327 ng/mL, 8989 ± 1590 ngh/mL and 2.9 ± 1.4h, respectively. Bioavailability was found to be 90 ± 14% for epalrestat in male wistar rats.

Tau protein and tau aggregation inhibitors.

Alzheimer disease is characterized by pathological aggregation of two proteins, tau and Abeta-amyloid, both of which are considered to be toxic to neurons. In this review we summarize recent advances on small molecule inhibitors of protein aggregation with emphasis on tau, with activities mediated by the direct interference of self-assembly. The inhibitors can be clustered in several compound classes according to their chemical structure, with subsequent description of the structure-activity relationships, showing that hydrophobic interactions are prevailing. The description is extended to the pharmacological profile of the compounds in order to evaluate their drug-likeness, with special attention to toxicity and bioavailability. The collected data indicate that following the improvements of the in vitro inhibitory potencies, the consideration of the in vivo pharmacokinetics is an absolute prerequisite for the development of compounds suitable for a transfer from bench to bedside.

Epalrestat: an aldose reductase inhibitor for the treatment of diabetic neuropathy.

Diabetic neuropathy is one of the most common long-term complications in patients with diabetes mellitus, with a prevalence of 60-70% in the United States. Treatment options include antidepressants, anticonvulsants, tramadol, and capsaicin. These agents are modestly effective for symptomatic relief, but they do not affect the underlying pathology nor do they slow progression of the disease. Epalrestat is an aldose reductase inhibitor that is approved in Japan for the improvement of subjective neuropathy symptoms, abnormality of vibration sense, and abnormal changes in heart beat associated with diabetic peripheral neuropathy. Unlike the current treatment options for diabetic neuropathy, epalrestat may affect or delay progression of the underlying disease process. Data from experimental studies indicate that epalrestat reduces sorbitol accumulation in the sciatic nerve, erythrocytes, and ocular tissues in animals, and in erythrocytes in humans. Data from six clinical trials were evaluated, and it was determined that epalrestat 50 mg 3 times/day may improve motor and sensory nerve conduction velocity and subjective neuropathy symptoms as compared with baseline and placebo. Epalrestat is well tolerated, and the most frequently reported adverse effects include elevations in liver enzyme levels and gastrointestinal-related events such as nausea and vomiting. Epalrestat may serve as a new therapeutic option to prevent or slow the progression of diabetic neuropathy. Long-term, comparative studies in diverse patient populations are needed for clinical application.

Optical recording of vagal pathway formation in the embryonic brainstem.

Multiple-site optical recording with a fast voltage-sensitive dye, absorption dye NK2761, was used to study the developmental organization of functional synaptic networks in the vagal pathway. Glutamatergic excitatory postsynaptic potentials (EPSPs) evoked by vagus nerve stimulation was first detected from the nucleus of the tractus solitarius (NTS) at embryonic day 7 (E7) in chick embryos and E15 in rat embryos, when morphological differentiation of pre- and postsynaptic neurons is incomplete. When extracellular Mg2+ was removed, small EPSPs were elicited at E6 in chick embryos and E14 in rat embryos. These results suggest that synaptic function mediated by N-methyl-D-aspartate (NMDA) receptors is latently generated 1 day before the expression of glutamatergic EPSP. Functional synapses related to the glossophyaryngeal nerve appear to be generated at the same time as the vagus nerve, but their spatial distribution was different from that of the vagus nerve. We further investigated the development of second synaptic pathways from the NTS to higher centers, and found that neuronal circuits from the NTS are already generated when the primary afferents form functional synapses with NTS neurons.

Permeability characteristics of novel aldose reductase inhibitors using rat jejunum in vitro.

The aim of this study was to estimate in vivo permeability and bioavailability of epalrestat and newly synthesized compounds with possible therapeutic activity as aldose enzyme inhibitors (ARIs). For this purpose permeability in vitro using rat jejunum mounted in side-by-side diffusion cells was determined. Tested substances were found to be low and moderately permeable and some of them were also substrates for efflux transporters. It was shown, that the higher efflux for some derivatives was due to MRP-2, but not Pgp involvement. Tested ARIs do not share the same efflux transporter with epalrestat, the only ARI currently on the market in Japan. The most permeable compound, a 2,6-difluoro-4-pyrrol-1ylphenol derivative, is not a substrate for efflux transporters and would therefore be the most promising lead compound for further investigation of potent ARIs.

Age-dependent emergence of a parieto-insular corticocortical signal flow in developing rats.

By using the procedure that we developed for inducing population oscillation, it was previously demonstrated that insular cortex stimulation can evoke insulo-parietal field potential propagation and synchronized population oscillation in the parietal cortex in slices obtained from mature rats (27-35 days old). By using the same procedure, we have now studied the reciprocal parieto-insular projection. Parietal cortex stimulation elicited synchronized population oscillation in the parietal--but not insular--cortex in mature tissues. In the insular cortex, the initial wavelet of the oscillation generated by parietal cortex stimulation propagated, but the entire oscillation did not. A prior induction--but not simultaneous occurrence--of oscillation in the parietal cortex sufficed to have this initial wavelet propagate. In immature tissue (9-10 days old), both the parietal cortex oscillation and the parieto-insular propagation were induced only with low [Mg2+]o. This age dependence is exactly the same as we previously observed for the reciprocal insulo-parietal propagation. Given that the parietal cortex receives somatosensory inputs from the oral cavity and the insular cortex receives primarily chemosensory inputs from the same source, the age-dependent changes in the availability of bidirectional signal traffic between these cortices might contribute to the development of multimodal responsiveness of taste neurons.