Correction: Sato et al. Design and Characterizations of Inhalable Poly(lactic-co-glycolic acid) Microspheres Prepared by the Fine Droplet Drying Process for a Sustained Effect of Salmon Calcitonin. Molecules 2020, 25, 1311.

The author wishes to make the following correction to this paper [...].

Design and Characterizations of Inhalable Poly(lactic-co-glycolic acid) Microspheres Prepared by the Fine Droplet Drying Process for a Sustained Effect of Salmon Calcitonin.

The present study aimed to develop inhalable poly (lactic-co-glycolic acid) (PLGA)-based microparticles of salmon calcitonin (sCT) for sustained pharmacological action by the fine droplet drying (FDD) process, a novel powderization technique employing printing technologies. PLGA was selected as a biodegradable carrier polymer for sustained-release particles of sCT (sCT/SR), and physicochemical characterizations of sCT/SR were conducted. To estimate the in vivo efficacy of the sCT/SR respirable powder (sCT/SR-RP), plasma calcium levels were measured after intratracheal administration in rats. The particle size of sCT/SR was 3.6 µm, and the SPAN factor, one of the parameters to present the uniformity of particle size distribution, was calculated to be 0.65. In the evaluation of the conformational structure of sCT, no significant changes were observed in sCT/SR even after the FDD process. The drug release from sCT/SR showed a biphasic pattern with an initial burst and slow diffusion in simulated lung fluid. sCT/SR-RP showed fine inhalation performance, as evidenced by a fine particle fraction value of 28% in the cascade impactor analysis. After the insufflation of sCT samples (40 µg-sCT/kg) in rats, sCT/SR-RP could enhance and prolong the hypocalcemic action of sCT possibly due to the sustained release and pulmonary absorption of sCT. From these observations, the strategic application of the FDD process could be efficacious to provide PLGA-based inhalable formulations of sCT, as well as other therapeutic peptides, to enhance their biopharmaceutical potentials.

Respirable powder formulation of a shortened vasoactive intestinal peptide analog for treatment of airway inflammatory diseases.

The aim of present study was to develop a respirable powder (RP) of a shortened vasoactive intestinal peptide (VIP) analog for inhalation. VIP and C-terminally truncated VIP analogs were synthesized with a solid-phase method. A structure-activity relationship (SAR) study was carried out in terms with binding and relaxant activities of the peptides. Prepared RP formulation of a shortened VIP analog was physicochemically characterized by morphological, in vitro aerodynamic, and pharmacological assessments. The SAR study demonstrated that the N-terminal 23 amino acid residues were required for biological activity of VIP. Upon chemical modification of VIP(1-23), [R15, 20, 21 , L17 ]-VIP(1-23) was newly developed, which had higher binding activity in rat lung and smooth muscle relaxant effect in mouse stomach than VIP(1-23). The [R15, 20, 21 , L17 ]-VIP(1-23)-based RP, [R15, 20, 21 , L17 ]-VIP(1-23)/RP, exhibited fine in vitro inhalation performance. Airway inflammation evoked by sensitization of antigen in rats was attenuated by pre-treatment with the [R15, 20, 21 , L17 ]-VIP(1-23)/RP at a dose of 50 µg-[R15, 20, 21 , L17 ]-VIP(1-23)/rat as evidenced by a 70% reduction of recruited inflammatory cells in bronchoalveolar lavage fluid. On the basis of these results, [R15, 20, 21 , L17 ]-VIP(1-23)/RP might be a promising agent for treatment of airway inflammatory diseases.

Self-micellizing solid dispersion of cyclosporine A for pulmonary delivery: Physicochemical, pharmacokinetic and safety assessments.

The present study aimed to develop an inhalable self-micellizing solid dispersion of cyclosporine A (SMSD/CsA) for the direct delivery to the respiratory system with improved therapeutic efficacy and minimized systemic exposure. SMSD/CsA was obtained by wet-milling, and then jet-milled SMSD/CsA was blended with lactose carrier, producing a respirable powder of SMSD/CsA (SMSD/CsA-RP). The physicochemical, pharmacological, and pharmacokinetic properties of SMSD/CsA-RP were characterized, and the hepatotoxic and nephrotoxic potentials were investigated by biomarker analysis. Cascade impactor analysis demonstrated that SMSD/CsA-RP had high in vitro inhalation performance, with a fine particle fraction of 36%. In simulated lung fluid, the SMSD/CsA exhibited better dissolution behavior than amorphous CsA. Pretreatment with SMSD/CsA-RP resulted in significant suppression of antigen-evoked inflammatory events in rats. After intratracheal administration of SMSD/CsA-RP at a pharmacologically effective dose (100µg-CsA/rat), the AUC0-24 value was <1% of that after oral administration of Neoral® at a toxic dose (10mg-CsA/kg). Compared with oral Neoral®, insufflated SMSD/CsA-RP showed 99% reductions of CsA concentrations in both liver and kidney. No significant increases of biomarker levels in plasma were observed even after repeated intratracheal administration of SMSD/CsA-RP for 7days. From these findings, SMSD/CsA-RP might be a favorable dosage form for effective and safe inhalation therapy of CsA.

Physicochemical stability study on cyclosporine A loaded dry-emulsion formulation with enhanced solubility.

The main purpose of the present study was to evaluate the physicochemical stability of cyclosporine A (CsA)-loaded glycerol monooleate-based dry emulsion (DE). DE formulations containing 5-25% CsA (DE5-25) were stored at 25°C/60% relative humidity for 4 weeks, and freeze-dried solid dispersion formulations containing 5-30% CsA (FD5-30) were also prepared as reference formulations. Even after the storage, no significant changes were observed in the appearance of any formulations. In the dissolution study, both DE and FD exhibited marked enhancement of solubility and there was at least 2.0-fold improvement in the initial dissolution rate of DE formulations compared with that of FD formulations. After storage, DE5, DE15 and FD5 maintained relatively high solubility, with 10% reduction compared with the initial state. However, the solubility of DE25 gradually decreased during storage, as evidenced by 76% reduction of the dissolution amount. No significant changes were seen in DE5-25 using powder X-ray diffraction, although thermal analysis revealed moderate changes in crystallinity in DE25 after storage, possibly leading to the decreased dissolution. Furthermore, particle size distributions of micelles in DE5 and DE15 were almost unchanged after storage for 4 weeks. From these findings, it appears that the physicochemical stability of CsA-loaded DE might vary depending on the manufacturing method and that further optimization could improve physical properties and stability.

Self-micellizing solid dispersion of cyclosporine A with improved dissolution and oral bioavailability.

The present study aimed to develop a self-micellizing solid dispersion (SMSD) of cyclosporine A (CsA) using an amphiphilic block copolymer, poly[MPC-co-BMA], to improve the biopharmaceutical properties of CsA. The cytotoxicity of poly[MPC-co-BMA] was assessed in rat intestinal IEC-6 cells, and the pMB was less cytotoxic than polysorbate 80, a non-ionic surfactant with a wide safety margin. SMSD/CsA was prepared using a wet-milling system, and its physicochemical properties were characterized in terms of morphology, crystallinity, dissolution, particle size distribution, and stability. The SMSD/CsA exhibited immediate formation of fine micelles with a mean diameter of ca. 180 nm when introduced into aqueous media. There was marked improvement in the dissolution behavior of the SMSD/CsA compared with amorphous CsA. Even after storage at 40°C/75% relative humidity, the dissolution behavior of aged SMSD/CsA seemed to be almost identical to that of its freshly prepared equivalent, and CsA in aged SMSD/CsA was still in amorphous form. After oral administration of SMSD/CsA (10 mg CsA/kg) in rats, enhanced CsA exposure was observed with increases of Cmax and BA by ca. 11- and 42-fold, respectively, compared with those of amorphous CsA. The poly[MPC-co-BMA]-based SMSD formulation system might be an efficacious dosage option for CsA to achieve improvements in oral bioavailability.

Chemical synthesis and formulation design of a PEGylated vasoactive intestinal peptide derivative with improved metabolic stability.

The present study aimed to design a PEGylated VIP derivative, [Arg(15, 20, 21), Leu(17)]-VIP-GRR (IK312532), with improved metabolic stability, and develop its respirable powder (RP) formulation for inhalation therapy. IK312532 was chemically conjugated with PEG (5 kDa, P5K), the physicochemical and biochemical properties of which were characterized by CD spectral analysis, binding assays, and metabolic stability. CD spectral analysis demonstrated that PEG conjugation had no impact on the conformational structure of IK312532. Although the receptor-binding activity of IK312532/P5K (IC50: 82 nM) was estimated to be ca. 30-fold less than that of IK312532 (IC50: 2.8 nM), the metabolic stability of IK312532/P5K was highly improved. The IK312532/P5K was jet-milled and blended with lactose carrier particles to provide RP formulation of IK312532/P5K (IK312532/P5K-RP). In vitro inhalation performance and in vivo pharmacological effects of the IK312532/P5K-RP in antigen-sensitized rats were also evaluated. In cascade impactor analyses, fine particle fraction of IK312532/P5K-RP was calculated to be ca. 37%. Insufflation of IK312532/P5K-RP (150 µg of IK312532/P5K) in antigen-sensitized rats resulted in marked attenuation of inflammatory events, as evidenced by significant decreases in inflammatory biomarkers and granulocyte recruitment in pulmonary tissue 24h after the antigen challenge. From these findings, PEGylation of a VIP derivative, as well as its strategic application to the RP formulation, may be a viable approach to improve its therapeutic potential for the treatment of airway inflammatory diseases.

Development of cyclosporine A-loaded dry-emulsion formulation using highly purified glycerol monooleate for safe inhalation therapy.

The main objective of this study was to improve the safety and oxidative stability of glycerol monooleate (GMO)-based dry-emulsion (DE) formulation containing cyclosporine A (CsA) for inhalation therapy. GMO or highly purified GMO (hpGMO) was used as surfactant for the DE formulations (GMO/DE or hpGMO/DE), the toxicological and physicochemical properties of which were characterized with a focus on oxidative stability, in vitro/in vivo toxicity, and dissolution property. Incubation of GMO at oxidation accelerating conditions for 10 days at 60°C resulted in the formation of lipid peroxides as evidenced by increased malondialdehyde (111 µmol/mg); however, hpGMO samples exhibited increase of only 20.7 µmol/mg in malondialdehyde level. No significant acute cytotoxicity was observed in rat alveolar L2 cells exposed to hpGMO (0.28mM), and intratracheal administration of hpGMO powder in rats did not cause an increase of the plasma LDH level. The hpGMO/DE exhibited marked improvement in dissolution behavior of CsA, and stable fine micelles with a mean diameter of 320 nm were formed when suspended in water. A respirable powder formulation of hpGMO/DE (hpGMO/DE-RP) was newly prepared, and its in vitro inhalation property and in vivo efficacy were also evaluated. The hpGMO/DE-RP exhibited high dispersibility in laser diffraction analysis and significantly improved potency to attenuate recruitment of inflammatory cells into airway and thickening of airway wall in an animal model. Thus, the strategic use of hpGMO would improve oxidative stability and local toxicity compared with a GMO-based DE formulation, and its application to RP formulation could be a promising approach for effective inhalation therapy.

Inhalable sustained-release formulation of long-acting vasoactive intestinal peptide derivative alleviates acute airway inflammation.

The present study was undertaken to develop a respirable sustained-release powder (RP) formulation of long-acting VIP derivative, [Arg(15, 20, 21), Leu(17)]-VIP-GRR (IK312532), using PLGA nanospheres (NS) with the aim of improving the duration of action. NS formulation of IK312532 (IK312532/NS) was prepared by an emulsion solvent diffusion method in oil, and a mixture of the IK312532/NS and erythritol was jet-milled and mixed with lactose carrier to obtain the IK312532/NS-RP. Physicochemical properties were characterized focusing on appearance, particle size, and drug release, and in vivo pharmacological effects were assessed in antigen-sensitized rats. The IK312532/NS with a diameter of 140 nm showed a biphasic release pattern in distilled water with ca. 20% initial burst for 30 min and a sustained slow release up to ca. 55% for 24h. Laser diffraction analysis demonstrated that IK312532/NS-RP had fine dispersibility and suitable particle size for inhalation. In antigen-sensitized rats, insufflated IK312532/NS-RP (10 µg of IK312532/rat) could suppress increases of granulocyte recruitment and myeloperoxidase in pulmonary tissue for up to 24h after antigen challenge, although IK312532-RP at the same dose was less effective with limited duration of action. From these findings, newly prepared IK312532/NS-RP might be of clinical importance in improving duration of action and medication compliance for treatment of airway inflammatory diseases.

Comparative studies on physicochemical stability of cyclosporine A-loaded amorphous solid dispersions.

The present study aimed to evaluate the physical stability on amorphous solid dispersion (SD) of cyclosporine A (CsA) employing hydroxypropyl cellulose (HPC). SD formulations (5-30% CsA) of CsA such wet-milled SD (WM/SD) and freeze-dried SD (FD/SD) were prepared, and both SD formulations were stored at 40 °C/75% relative humidity for 8 weeks. Transitions in morphology, dissolution behavior, crystallinity and thermal behavior of CsA were evaluated. There was at least 84-fold improvement in initial dissolution rate of SD formulations compared with that of amorphous CsA powder, although their dissolution rate was gradually decreased under accelerated conditions. In particular, aged FD/SD with a drug load of 30% exhibited highly limited dissolution as evidenced by 40% reduction of solubility after 8 weeks of storage. In contrast, aged WM/SD exhibited less reduction in dissolution rate compared with FD/SD. No significant changes were seen in crystallinity and thermal behavior after aging of SD formulations for 8 weeks; however, electron microscopic observations revealed aggregation of drug molecules/particles in the aged FD/SD, possibly leading to the reduced dissolution. From these findings, stability on CsA-loaded SD might be variable depending on the preparation methodology, and the wet-milling approach could be a viable option for preparing efficacious SD formulations with improved stability.

Inhalable dry-emulsion formulation of cyclosporine A with improved anti-inflammatory effects in experimental asthma/COPD-model rats.

The main purpose of the present study was to develop a novel respirable powder (RP) formulation of cyclosporine A (CsA) using a spray-dried O/W-emulsion (DE) system. DE formulation of CsA (DE/CsA) was prepared by spray-drying a mixture of erythritol and liquid O/W emulsion containing CsA, polyvinylpyrrolidone, and glyceryl monooleate as emulsifying agent. The DE/CsA powders were mixed with lactose carriers to obtain an RP formulation of DE/CsA (DE/CsA-RP), and its physicochemical, pharmacological, and pharmacokinetic properties were evaluated. Spray-dried DE/CsA exhibited significant improvement in dissolution behavior with ca. 4500-fold increase of dissolution rate, and then, nanoemulsified particles were reconstituted with a mean diameter of 317 nm. Laser diffraction analysis on the DE/CsA-RP suggested high dispersion of DE/CsA on the surface of the lactose carrier. Anti-inflammatory properties of the inhaled DE/CsA-RP were characterized in antigen-sensitized asthma/COPD-model rats, in which the DE/CsA-RP was more potent than the RP formulation of physical mixture containing CsA and erythritol in inhibiting inflammatory responses, possibly due to the improved dissolution behavior. Pharmacokinetic studies demonstrated that systemic exposure of CsA after intratracheal administration of the DE/CsA-RP at a pharmacologically effective dose (100 µg-CsA/rat) was 50-fold less than that of the oral CsA dosage form at a toxic dose (10 mg/kg). From these findings, use of inhalable DE formulation of CsA might be a promising approach for the treatment of airway inflammatory diseases with improved pharmacodynamics and lower systemic exposure.

Formulation design and in vivo evaluation of dry powder inhalation system of new vasoactive intestinal peptide derivative ([R(15, 20, 21), L(17), A(24,25), des-N(28)]-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 sustained-release formulation of glucagon: in vitro amyloidogenic and inhalation properties, and in vivo absorption and bioactivity.

PURPOSE: The present study aimed to develop novel glucagon-loaded PLGA nanospheres without cytotoxic fibril formation for chronic glucagon replacement therapy. METHODS: Glucagon-loaded nanospheres (GLG/NS) were prepared by an emulsion solvent diffusion method in oil, and a respirable powder formulation (GLG/NS-RP) was prepared with a jet mill. Physicochemical and inhalation properties of GLG/NS-RP were characterized, and pharmacokinetic behavior and hyperglycemic effect of intratracheally instilled GLG/NS-RP were evaluated in rats. RESULTS: Although preparation of GLG/NS using glucagon solution at concentrations over 10 mg/mL led to significant formation of cytotoxic glucagon aggregates, glucagon solution at less than 5 mg/mL did not cause structural changes. Drug release behavior of GLG/NS showed a biphasic pattern with an initial burst and slow diffusion. Laser diffraction and cascade impactor analyses of GLG/NS-RP suggested high dispersion and deposition in the respiratory organs with a fine particle fraction of 20.5%. After the intratracheal administration of the GLG/NS-RP (200 µg glucagon/kg) in rats, glucagon was released in a sustained manner, leading to sustained hyperglycemic effects compared with those of normal glucagon powder. CONCLUSION: These data would suggest a therapeutic benefit of the newly developed GLG/NS-RP as an alternative to the injection form of glucagon currently used.

Development of PACAP38 analogue with improved stability: physicochemical and in vitro/in vivo pharmacological characterization.

Pituitary adenylate cyclase activating polypeptide 38 (PACAP38), one of the major peptide transmitters, has emerged as a promising drug candidate for the treatment of type 2 diabetes. In the present study, on the basis of previous structure-activity relationships, a new PACAP38 derivative, [R(15, 20, 21), L(17)]-PACAP38, was chemically synthesized with the aim of enhancing the therapeutic potential of PACAP38. The solution structure of the new derivative was almost identical to that of PACAP38 as evaluated by circular dichroic spectroscopy, and both PACAP38 and the new derivative stimulated adenylate cyclase in rat insulinoma RIN-m5F cells with EC(50) values of 4.6 and 5.5 nM, respectively. Stability studies revealed the gradual degradation of PACAPs in rat serum, although there appeared to be a 42% reduction in degradation kinetics for [R(15, 20, 21), L(17)]-PACAP38 compared with that of PACAP38. The novel derivative also exhibited more potent protective effects against streptozotocin (STZ)-induced apoptotic death of RIN-m5F cells, possibly due to the enhanced stability. The n0-STZ model, in which neonatal rats were injected with STZ at birth, developed a typical diabetic condition; however, chronic administration of [R(15, 20, 21), L(17)]-PACAP38 resulted in protection of pancreatic islets, followed by the improvement of glycemic control. Thus, the chemical modification of PACAP38 led to the development of a new promising derivative with enhanced stability and biological activity, and early administration of [R(15, 20, 21), L(17)]-PACAP38 might be of help for preventing the development of diabetes in type 2 diabetic model rats.

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.

Improved dissolution and pharmacokinetic behavior of cyclosporine A using high-energy amorphous solid dispersion approach.

The aim of the present investigation is to develop solid dispersion (SD) formulations of cyclosporine A (CsA) for improving the oral bioavailability of CsA. Amorphous SDs of CsA with eight hydrophilic polymers were prepared with wet-mill employing zirconia beads. The physicochemical properties were characterized with a focus on morphology, crystallinity, thermal behavior, dissolution, and interaction of CsA with co-existing polymer. Although CsA molecules were found to be amorphous in all wet-milled formulations, some SD formulations failed to improve the dissolution. Of all CsA formulations, SD using polymer with HPC(SSL) exhibited the largest improvement in dissolution behavior. Pharmacokinetic profiling of orally dosed CsA in rats was carried out using UPLC/ESI-MS. After the oral administration of HPC(SSL)-based SD, enhanced CsA exposure was observed with increases in C(max) and AUC of ca. 5-fold, and the variation in AUC was ca. 40% less than that of amorphous CsA. Infrared spectroscopic studies suggested an interaction between CsA and HPC(SSL), as evidenced by the conformational transition of CsA. From the improved dissolution and pharmacokinetic data, the amorphous SD approach using wet-milling technology should lead to consistent and enhanced bioavailability, leading to an improved therapeutic potential of CsA.

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.

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.

Liposomal formulations of glucagon-like peptide-1: improved bioavailability and anti-diabetic effect.

Glucagon-like peptide-1 (GLP-1), an incretin hormone, is recognized to be potent drug candidate for treatment of diabetes, however its clinical application has been highly limited, because of rapid enzymatic degradation by dipeptidyl-peptidase IV. To protect GLP-1 from enzymatic degradation and improve pharmacological effects, liposomal formulations of GLP-1 were prepared using three types of lyophilized empty liposomes such as anionic, neutral and cationic liposomes. Electron microscopic and dynamic light scattering experiments indicated the uniform size distribution of GLP-1-loaded liposomes with mean diameter of 130-210 nm, and inclusion of GLP-1 did not affect the dispersibility and morphology of each liposome. Of all liposomal formulations tested, anionic liposomal formulation exhibited the highest encapsulation efficiency of GLP-1 (ca. 80%). In intraperitoneal glucose tolerance testing in rats, marked improvement of hypoglycemic effects were observed in anionic liposomal formulation of GLP-1 (100 nmol/kg) with 1.7-fold higher increase of insulin secretion, as compared to GLP-1 solution. In pharmacokinetic studies, intravenous administration of anionic liposomal formulation of GLP-1 (100 nmol/kg) resulted in 3.6-fold higher elevation of serum GLP-1 level as compared to GLP-1 injection. Upon these findings, anionic liposomal formulation of GLP-1 would provide the improved pharmacokinetics and insulinotropic action, possibly leading to efficacious anti-diabetic medication.

Novel dry powder inhaler formulation of glucagon with addition of citric acid for enhanced pulmonary delivery.

Glucagon, a gut hormone, is one of the key regulatory elements in glucose homeostasis, and is clinically used for treatment of hypoglycemia and premedication in peroral endoscopy. Dry powder inhaler (DPI) form of glucagon is believed to be a promising new dosage form, and the present study aimed to develop a novel glucagon-DPI using absorption enhancer for improved pharmacological effects. The cytotoxicity of citric and capric acids, the potential absorption enhancers, at 1 and 10 mM was assessed by monitoring extracellular LDH levels in rat alveolar L2 cells, and a concentration- and time-dependent release of LDH was observed in capric acid, but not in citric acid-treated cells. DPI form of glucagon containing citric acid was prepared with a jet mill, and laser diffraction and cascade impactor analyses of the newly developed glucagon-DPI suggested high dispersion and deposition in the respiratory organs with an emitted dose and fine particle fraction of 99.5 and 25%, respectively. Addition of citric acid in glucagon-DPI improved the dissolution behavior, and did not impair the solid-state stability of glucagon-DPI. Intratracheal administration of glucagon-DPI (50 microg-glucagon/kg body weight of rat) containing citric acid led to 2.9-fold more potent hyperglycemic effect in rats, as compared to inhaled glucagon-DPI without citric acid. Based on these physicochemical and pharmacological characterization, the dry powder inhaler of glucagon with addition of citric acid would be of use as an alternative to injection form.