Shingen Misaka

Structure-activity relationship of vasoactive intestinal peptide (VIP): potent agonists and potential clinical applications

Vasoactive intestinal peptide (VIP) has been identified as one of major peptide transmitters in the central and peripheral nervous systems, being involved in a wide range of biological functions. The general physiologic effects of VIP include vasodilation, anti-inflammatory actions, cell proliferation, hormonal secretion, regulation of gastric motility, and smooth muscle relaxation; therefore, VIP has emerged as a promising drug candidate for the treatment of several diseases. A number of clinical applications of VIP or its derivatives have been developed; however, VIP-based drugs are not yet in clinical use, possibly because of mainly two serious problems: (1) poor metabolic stability and (2) poor penetration to the desired site of action. To overcome these shortcomings, the development of efficacious VIP analogues and several drug delivery systems has been attempted on the basis of numerous structure–activity relationships (SAR) studies and pharmacological experiments. Combination of the use of potent VIP analogues and an appropriate drug delivery system might be advantageous for the VIP-based therapy. We review in this paper SAR studies of VIP for the identification of potent therapeutic agents, describe the development of selective and/or metabolically stable VIP receptor agonists/antagonists, and discuss the potential application for clinical treatment using drug delivery systems.

Novel dry powder formulation of ovalbumin for development of COPD-like animal model: Physicochemical characterization and biomarker profiling in rats

This study was directed toward the development of novel ovalbumin dry powder inhalation system (OVA-DPI) for preparing experimental animal models of chronic obstructive pulmonary disease, with the aim of aiding the drug discovery. OVA-DPI, prepared with jet mill, showed high dispersion and emission from capsule as evaluated by cascade impactor. Based on the results from long term stability studies employing scanning electron microscopy, UPLC/ESI-MS analysis, powder X-ray diffraction and TG/DTA analyses, the OVA-DPI, stored at room temperature, was found to be stable for more than 3 years as evidenced by no significant degradation and crystal polymorphism. Intratracheal administration of OVA-DPI in OVA-sensitized rats resulted in 11-fold increase of infiltrated granulocytes, especially neutrophil, which would be characteristics of severe asthma/COPD symptoms. Of all plasma biomarkers monitored, myeloperoxidase activity and lactate dehydrogenase leakage into blood seemed to be sensitive indicators of lung injury in this model. In addition, biphasic increase of LDH was observed with peak responses at 3 and 24h after antigen challenge, suggesting that OVA-DPI could cause both acute and delayed inflammatory reactions. Upon these findings, OVA-DPI can be useful and reproducible research tool for the development of experimental asthma/COPD model.

Inhalable powder formulation of a stabilized vasoactive intestinal peptide (VIP) derivative: Anti-inflammatory effect in experimental asthmatic rats

Vasoactive intestinal peptide (VIP) exerts immunomodulating and anti-inflammatory activities through its specific receptors, such as VPAC1 and 2 receptors. Previously, a stabilized VIP derivative, [R(15,20,21), L(17)]-VIP-GRR (IK312532), was proposed as a candidate of anti-asthma drug, and a dry powder inhaler system of IK312532 was also developed for inhalation therapy with minimal systemic side-effects. In the present study, the anti-inflammatory properties of IK312532 respirable powder (RP) were characterized in an asthma/COPD-like animal model, with the use of newly developed ovalbumin (OVA)-RP for lung inflammation. Marked inflammatory events in the lung were observed after OVA-RP challenge in rats as evidenced by significant increase of inflammatory biomarkers such as eosinophil peroxidase (EPO), myeloperoxidase (MPO) and lactate dehydrogenase (LDH). However, intratracheal administration of IK312532-RP led to significant attenuation of plasma EPO, MPO and LDH activities, as well as significant reduction of recruited inflammatory cells in BALF, especially macrophages and eosinophils. In the rats pretreated with IK312532-RP, histochemical examinations revealed that the inflammatory cells infiltrating to the lung and the epithelial wall thickness decreased significantly by 85% and 58%, respectively. Thus, inhalable powder formulation of IK312532 exerts its anti-inflammatory activity by suppressing granulocyte recruitment to the lung and epithelial hyperplasia, followed by the reduction of cytotoxic peroxidases.

Development of rapid and simultaneous quantitative method for green tea catechins on the bioanalytical study using UPLC/ESI-MS.

A rapid and quantitative analytical method for the simultaneous determination of green tea catechins using ultra-performance liquid chromatography/electrospray ionization-mass spectrometry was developed. Total analytical run time was 3.5 min for the detection of (-)-epicatechin (EC), (-)-epicatechin-3-O-gallate (ECG), (-)-epigallocatechin (EGC), (-)-epigallocatechin-3-O-gallate (EGCG) and myricetin as the internal standard (IS) in rat plasma. The calibration curves were linear over the range of 10-5000 ng/mL for all the catechins. The inter- and intra-day precision (relative standard deviation) and accuracy (percentage deviation) of the method were both lower than 10%. The average extraction recoveries in plasma ranged from 68.5 to 86.5%, and the lower limits of quantification of EC, EGC, ECG and EGCG were 10 ng/mL with a signal-to-noise ratio of >10. The assay developed was successfully applied to a pharmacokinetic study of catechins following intravenous and intragastric administrations of green tea extract in rats. Plasma concentrations of four catechins were detected up to 5-24 h after administration, and the pharmacokinetic parameters of catechins were in agreement with previous studies. From these findings, taken together with the high productivity and precision, the developed method could be a reliable and reproducible tool for the evaluation of pharmacokinetic properties of catechins.

Physicochemical and pharmacological characterization of novel vasoactive intestinal peptide derivatives with improved stability

Previously, [R(15,20,21), L(17)]-VIP-GRR (IK312532), a long-acting VIP derivative, was proposed as potential drug candidate for the treatment of asthma/COPD. The present work is aimed to elucidate solution-state stability of IK312532 and to develop further stabilized derivative with equipotent or higher biological functions. A stability study on IK312532 was carried out in solution state, and degradation mechanism was deduced by UPLC-MS and amino acid analyses. Three novel VIP derivatives were designed and chemically synthesized on the basis of stability data, being subjected to physicochemical and pharmacological characterization. Solution-state stability studies revealed the gradual degradation of IK312532, following pseudo-first-order kinetics. Chemical modification of IK312532, mainly position at 24, resulted in marked improvement of stability, although the chemical modification had no influence on the secondary structure, receptor binding, and activation of adenylate cyclase in rat lung cells. Novel derivatives also exhibited more potent neurite outgrowth in rat pheochromocytoma PC12 cells when compared to VIP and IK312532, possibly due to improved stability. Deamination of Asn at position 24 might be responsible for degradation of VIP derivative, and stability and chemical modification studies led us to the successful development of novel VIP derivatives with higher stability and biological functions.

Formulation design and in vivo evaluation of dry powder inhalation system of new vasoactive intestinal peptide derivative ([R15, 20, 21, L17, A24,25, des-N28]-VIP-GRR) in experimental asthma/COPD model rats

Vasoactive intestinal peptide (VIP) has been considered as a promising drug candidate for asthma and COPD because of its potent immunomodulating and anti-inflammatory activities. Recently, our group developed a new VIP derivative, [R(15, 20, 21), L(17), A(24,25), des-N(28)]-VIP-GRR (IK312548), with improved chemical and metabolic stability. In the present study, a dry powder inhaler system of IK312548 was designed for inhalation therapy with minimal systemic side effects, the physicochemical properties of which were also evaluated with a focus on morphology, particle size distribution, inhalation performance, and peptide stability. Laser diffraction and cascade impactor analysis suggested high dispersion and deposition in the respiratory organs with a fine particle fraction of 31.2%. According to UPLC/ESI-MS and circular dichroic spectral analyses, no significant changes in the purity and structure of VIP derivative were observed during preparation of respirable formulation. Anti-inflammatory properties of IK312548 respirable powder (RP) were characterized in antigen-sensitized asthma/COPD-model rats. There were marked inflammatory cells infiltrated into the lung tissues of experimental asthma/COPD-model rats; however, intratracheal administration of IK312548-RP led to significant reductions of recruited inflammatory cells in lung tissues and BALF by 72 and 78%, respectively. Thus, respirable powder formulation of IK312548 might be a promising medication for asthma, COPD, and other airway inflammatory diseases.

Inhalable powder formulation of vasoactive intestinal peptide derivative, [R15,20,21, L17]-VIP-GRR, attenuated neutrophilic airway inflammation in cigarette smoke-exposed rats

Cigarette smoke (CS) has been identified as a predominant causative factor for chronic obstructive pulmonary disease (COPD), so CS-exposed rodent model of COPD has drawn considerable interest and attention for fundamental study and drug discovery. In the present study, using experimental COPD model rats, the therapeutic potential of a newly prepared respirable powder (RP) formulation of a long-acting VIP derivative, [Arg(15,20,21), Leu(17)]-VIP-GRR (IK312532), was assessed with a focus on pro-inflammatory biomarkers, morphological and histochemical changes, and infiltrated cells in the respiratory system. CS exposure of rats for 11 days led to the marked infiltration of inflammatory cells, except for eosinophils, in bronchiolar epithelium, followed by goblet cell metaplasia and hyperplasia. However, inhalation of IK312532-RP (50μg/rat) in the CS-exposed rats resulted in 74 and 71% reductions of granulocyte recruitment in bronchoalveolar lavage fluids and lung tissues, respectively, with 68% decrease of goblet cells. Biomarker study demonstrated that the inhaled IK312532-RP could suppress the CS-evoked increase of myeloperoxidase in both plasma and lung by 87 and 70%, respectively, possibly leading to potent suppression of neutrophilic inflammatory symptoms. The results from TUNEL staining were indicative of apoptotic damage in respiratory tissues of the CS-exposed rats, and there appeared to be marked decrease of TUNEL-positive cells in the CS-exposed rat with inhaled IK312532-RP. The present findings suggest that an inhalable formulation of IK312532 might be efficacious as a therapy for COPD or other airway inflammatory diseases because of its potent immunomodulating activities.

Pharmacokinetics and pharmacodynamics of low doses of midazolam administered intravenously and orally to healthy volunteers

1. Midazolam, a short‐acting benzodiazepine, has been considered a probe for estimating hepatic and intestinal cytochrome P450 (CYP) 3A activity in humans. The aim of the present study was to evaluate the pharmacokinetics and pharmacodynamics of midazolam administered intravenously (i.v.) and orally (p.o.) at relatively low doses to healthy volunteers.

Novel vasoactive intestinal peptide derivatives with improved stability protect rat alveolar L2 cells from cigarette smoke-induced cytotoxicity and apoptosis

Vasoactive intestinal peptide (VIP) has been thought to be a promising candidate for asthma/chronic obstructive pulmonary disease (COPD), and our group previously developed several long-lasting VIP derivatives. The objective of the present study was to clarify the therapeutic potential of new VIP derivatives with improved chemical and metabolic stability. Exposure of rat alveolar L2 cells to cigarette smoke extract (CSE) for 1h led to release of lactate dehydrogenase (LDH) and decreased viability in a CSE concentration-dependent manner. There appeared to be marked induction of apoptosis after CSE exposure, as demonstrated by 59% elevation of caspase-3 activity and TUNEL staining. In contrast, a stabilized VIP derivative, [R(15,20,21), L(17)]-VIP-GRR (IK312532), at a concentration of 10(-7)M, exhibited 71% attenuation of LDH release and 85% decrease of the number of apoptotic cells. In addition to IK312532, new VIP derivatives also showed anti-apoptotic effects against CSE toxicity and marked reduction of nitric oxide production. In terms of cytoprotective effects, [R(15,20,21), L(17), A(24,25), des-N(28)]-VIP-GRR was more effective than VIP and IK312532, possibly due to the improved stability. Thus, the present study is the first to demonstrate that novel stabilized VIP derivatives exert anti-apoptotic and cytoprotective effects on CSE-induced cytotoxicity.

Evaluation of the pharmacokinetic interaction of midazolam with ursodeoxycholic acid, ketoconazole and dexamethasone by brain benzodiazepine receptor occupancy

Objectives  To clarify whether alterations in midazolam pharmacokinetics resulting from changes in cytochrome P450 3A (CYP3A) activity lead to changes in its pharmacodynamic effects, benzodiazepine receptor occupancy was measured in the brain of rats after oral administration of midazolam.