Biopharmaceutical characterization of a novel sustained-release formulation of allopurinol with reduced nephrotoxicity.

The present study was aimed to develop a novel sustained-release formulation for allopurinol (ALP/SR) with the use of a pH-sensitive polymer, hydroxypropyl methylcellulose acetate succinate, to reduce nephrotoxicity. ALP/SR was evaluated in terms of crystallinity, the dissolution profile, pharmacokinetic behavior, and nephrotoxicity in a rat model of nephropathy. Under acidic conditions (pH1.2), sustained release behavior was seen for ALP/SR, although both crystalline ALP and ALP/SR exhibited rapid dissolution at neutral condition. After multiple oral administrations of ALP samples (10 mg-ALP/kg) for 4 days in a rat model of nephropathy, ALP/SR led to a low and sustained plasma concentration of ALP, as evidenced by half the maximum concentration of ALP and a 2.5-fold increase in the half-life of ALP compared with crystalline ALP, possibly due to suppressed dissolution behavior under acidic conditions. Repeated-dosing of ALP/SR resulted in significant reductions in plasma creatinine and blood urea nitrogen levels by 73% and 69%, respectively, in comparison with crystalline ALP, suggesting the low nephrotoxic risk of ALP/SR. From these findings, a strategic SR formulation approach might be an efficacious dosage option for ALP to avoid severe nephrotoxicity in patients with nephropathy.

Impact of Green Tea Catechin Ingestion on the Pharmacokinetics of Lisinopril in Healthy Volunteers.

Lisinopril, a highly hydrophilic long-acting angiotensin-converting enzyme inhibitor, is frequently prescribed for the treatment of hypertension and congestive heart failure. Green tea consumption may reduce the risk of cardiovascular outcomes and total mortality, whereas green tea or its catechin components has been reported to decrease plasma concentrations of a hydrophilic ß blocker, nadolol, in humans. The aim of this study was to evaluate possible effects of green tea extract (GTE) on the lisinopril pharmacokinetics. In an open-label, randomized, single-center, 2-phase crossover study, 10 healthy subjects ingested 200 mL of an aqueous solution of GTE containing ~ 300 mg of (-)-epigallocatechin gallate, a major catechin component in green tea, or water (control) when receiving 10 mg of lisinopril after overnight fasting. The geometric mean ratio (GTE/control) for maximum plasma concentration and the area under the plasma concentration-time curve of lisinopril were 0.289 (90% confidence interval (CI) 0.226-0.352) and 0.337 (90% CI 0.269-0.405), respectively. In contrast, there were no significant differences in time to reach maximum lisinopril concentration (6 hours in both phases) and renal clearance of lisinopril (57.7 mL/minute in control vs. 56.9 mL/minute in GTE). These results suggest that the extent of intestinal absorption of lisinopril was significantly impaired in the presence of GTE, whereas it had no major effect on the absorption rate and renal excretion of lisinopril. Concomitant use of lisinopril and green tea may decrease oral exposure to lisinopril, and therefore result in reduced therapeutic efficacy.

Photochemically stabilized formulation of dacarbazine with reduced production of algogenic photodegradants.

The present study aimed to develop a photochemically stabilized formulation of dacarbazine [5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide; DTIC] for reducing the production of algogenic photodegradant (5-diazoimidazole-4-carboxamide; Diazo-IC). Photochemical properties of DTIC were characterized by UV-visible light spectral analysis, reactive oxygen species (ROS) assay, and photostability testing. A pharmacokinetic study was conducted after intravenous administration of DTIC formulations (1 mg-DTIC/kg) to rats. DTIC exhibited strong absorption in the UVA range, and photoirradiated DTIC exhibited marked ROS generation. Thus, DTIC had high photoreactive potential. After exposure of DTIC (1 mM) to simulated sunlight (250 W/m2) for 3 min, remaining DTIC and yielded Diazo-IC were estimated to be ca. 230 µM and 600 µM, respectively. The addition of radical scavenger (1 mM), including l-ascorbic acid, l-cysteine (Cys), l-histidine, D-mannitol, l-tryptophan, or l-tyrosine, to DTIC (1 mM) could attenuate DTIC photoreactions, and in particular, the addition of Cys to DTIC brought ca. 34% and 86% inhibition of DTIC photodegradation and Diazo-IC photogeneration, respectively. There were no significant differences in the calculated pharmacokinetic parameters of DTIC between DTIC and DTIC with Cys (0.67 mg/kg). From these findings, the supplementary use of Cys would be an effective approach to improve the photostability of DTIC with less production of Diazo-IC.

Photochemical and Pharmacokinetic Characterization of Orally Administered Chemicals to Evaluate Phototoxic Risk.

This study aimed to verify the applicability of a proposed photosafety screening system based on a reactive oxygen species (ROS) assay and a cassette-dosing pharmacokinetic (PK) study to chemicals with wide structural diversity. The orally taken chemicals, erythromycin, gatifloxacin, 8-methoxypsoralen (MOP), pirfenidone (PFD), trifluoperazine (TFP), and voriconazole (VRZ), were selected as test compounds. The ROS assay was conducted to evaluate their photoreactivity, and all test compounds excluding erythromycin generated significant ROS under simulated sunlight exposure. According to the ROS data, TFP had potent photoreactivity, and the photoreactivity of 4 other compounds was judged to be moderate. Regarding the oral cassette-dosing PK test in rats, the skin deposition of MOP, PFD, and VRZ was relatively high, and gatifloxacin and TFP exhibited moderate skin deposition properties. Based on the ROS and PK data of test compounds, PFD and TFP were judged to be potent phototoxic compounds, and MOP and VRZ were deduced to have phototoxic risk. The predicted phototoxic risk of test compounds by proposed screening was mostly in agreement with observed in vivo phototoxicity in the rat skin. The proposed screening system could provide reliable photosafety information on orally administered compounds with wide structural diversity.

Supersaturable Self-Emulsifying Drug Delivery System of Krill Oil with Improved Oral Absorption and Hypotriglyceridemic Function.

This study aimed to develop a supersaturable self-emulsifying drug delivery system (S-SEDDS) of krill oil (KO), a rich source of docosahexaenoic acid and eicosapentaenoic acid (EPA), to improve its hypotriglyceridemic function. S-SEDDS of KO (KO/S-SEDDS) was prepared by the addition of lysolecithin, glycerin, and hydroxypropyl methylcellulose (HPMC). Self-emulsifying drug delivery system of KO (KO/SEDDS) and KO with HPMC (KO/HPMC) were also prepared for comparison purposes. The physicochemical and pharmacokinetic properties of KO samples were characterized, and the hypotriglyceridemic function of KO/S-SEDDS was evaluated. Micronized droplets in KO/SEDDS and KO/S-SEDDS with a mean diameter of ca. 270 nm could be observed in comparison to KO and KO/HPMC. Both KO/HPMC and KO/S-SEDDS tended to enhance the dissolution behavior of KO, and the S-SEDDS formulation improved the dissolution behavior of KO as a result of micronized droplets and the addition of HPMC. KO/S-SEDDS (60 mg of EPA/kg) improved the oral absorption of KO based on the pharmacokinetic profiling of EPA, and repeated oral administration of KO/S-SEDDS (250 mg of KO kg-1 day-1) for 7 days had a potent hypotriglyceridemic effect on rats with corn-oil-induced hypertriglyceridemia compared to orally administered KO. On the basis of these findings, the S-SEDDS approach might be an efficacious dosage option to enhance the nutraceutical properties of KO.

Role of (-)-epigallocatechin gallate in the pharmacokinetic interaction between nadolol and green tea in healthy volunteers.

PURPOSE: The aim of the present study is to investigate a possible role of a single dose of (-)-epigallocatechin gallate (EGCG), the major catechin in green tea, for the pharmacokinetic interaction between green tea and nadolol in humans. METHODS: In a randomized three-phase crossover study, 13 healthy volunteers received single doses of 30 mg nadolol orally with water (control), or an aqueous solution of EGCG-concentrated green tea extract (GTE) at low or high dose. Plasma concentrations and urinary excretion of nadolol were determined up to 48 h. In addition, blood pressure and pulse rate were monitored. In vitro transport kinetic experiments were performed using human embryonic kidney 293 cells stably expressing organic anion transporting polypeptide (OATP)1A2 to evaluate the inhibitory effect of EGCG on OATP1A2-mediated substrate transport. RESULTS: Single coadministration of low and high dose GTE significantly reduced the plasma concentrations of nadolol. The geometric mean ratios with 90% CI for area under the plasma concentration-time curves from 0 to infinity of nadolol were 0.72 (0.56-0.87) for the low and 0.60 (0.51-0.69) for the high dose. There were no significant differences in Tmax, elimination half-life, and renal clearance between GTE and water phases. No significant changes were observed for blood pressure and pulse rate between phases. EGCG competitively inhibited OATP1A2-mediated uptake of sulphobromophthalein and nadolol with Ki values of 21.6 and 19.4 µM, respectively. CONCLUSIONS: EGCG is suggested to be a key contributor to the interaction of green tea with nadolol. Moreover, even a single coadministration of green tea may significantly affect nadolol pharmacokinetics.

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.

Lack of pharmacokinetic interaction between fluvastatin and green tea in healthy volunteers.

PURPOSE: The objective of this study is to assess the effects of green tea and its major catechin component, (-)-epigallocatechin gallate (EGCG), on CYP2C9-mediated substrate metabolism in vitro, and the pharmacokinetics of fluvastatin in healthy volunteers. METHODS: The metabolism of diclofenac and fluvastatin in human recombinant CYP2C9 was investigated in the presence of EGCG. In a randomized three-phase crossover study, 11 healthy volunteers ingested a single 20-mg dose of fluvastatin with green tea extract (GTE), containing 150 mg of EGCG, along with water (300 mL), brewed green tea (300 mL), or water (300 mL) after overnight fasting. Plasma concentrations of fluvastatin and EGCG were measured by ultra-performance liquid chromatography with fluorescence detection and a single mass spectrometer. RESULTS: EGCG inhibited diclofenac 4'-hydroxylation and fluvastatin degradation with IC50 of 2.23 and 48.04 µM, respectively. Brewed green tea used in the clinical study also dose-dependently inhibited the metabolism of diclofenac and fluvastatin in vitro. However, no significant effects of GTE and brewed green tea were observed in plasma concentrations of fluvastatin. The geometric mean ratios with 90% CI for area under the plasma concentration-time curve (AUC0-∞) of fluvastatin were 0.993 (0.963-1.024, vs. brewed green tea) and 0.977 (0.935-1.020, vs. GTE). CONCLUSIONS: Although in vitro studies indicated that EGCG and brewed green tea produce significant inhibitory effects on CYP2C9 activity, the concomitant administration of green tea and fluvastatin in healthy volunteers did not influence the pharmacokinetics of fluvastatin.

Hybridization of polyvinylpyrrolidone to a binary composite of curcumin/α-glucosyl stevia improves both oral absorption and photochemical stability of curcumin.

The tri-component system curcumin/α-glucosyl stevia (Stevia-G)/polyvinylpyrrolidone (PVP) was developed to improve the oral bioavailability and physicochemical properties of curcumin (CUR). The tri-component CUR formulation with Stevia-G and PVP was prepared with freeze-drying. The tri-component CUR system exhibited 13,000-fold higher solubility of CUR than the equilibrium solubility of CUR for 24h, indicating a stable tri-composite structure involving CUR. CUR could be converted into an amorphous form in the presence of Stevia-G and PVP by freeze-drying. The photo-degradation of CUR in the tri-component system was negligible even under an amorphous state of CUR. After oral administration in rats, the oral absorption of the tri-component CUR formulation (20mgCUR/kg) was 6.7-fold higher than that of crystalline CUR. The tri-component CUR formulation would therefore be a promising option to improve physicochemical properties and oral absorption of CUR.

Formulation Approaches to Overcome Biopharmaceutical Limitations of Inhaled Peptides/Proteins.

The pulmonary dosing route has been advocated as an attractive alternative to injection and oral administration for the systemic delivery of therapeutic peptides and proteins. The lung possesses many favorable physiological characteristics for systemic absorption of inhaled peptides/proteins, so inhalable formulation systems of these drugs have generated considerable interest as a valid and non-invasive dosing approach. A major obstacle to the widespread use of inhalation therapies for many peptides/proteins is the limited bioavailability and thereby insufficient therapeutic outcomes because of biopharmaceutical challenges such as rapid pulmonary clearance, limited pulmonary deposition, delayed dissolution in lung environment, poor membrane permeability, and low metabolic stability. A better understanding of the biopharmaceutical properties of inhaled peptides/proteins would be indispensable to overcome these drawbacks with the aid of strategic drug delivery systems and chemical synthesis of new derivatives based on structure-activity relationship information, possibly leading to improved therapeutic potential of pharmaceutical products. The present paper reviews biopharmaceutical properties of inhaled peptides/proteins, with a focus on the pharmacokinetic fate of inhaled peptides/proteins and critical determinants of therapeutic potential. The emphasis in this mini-review will also be on viable formulation approaches for breakthroughs beyond the biopharmaceutical limitations of inhalation therapy with peptides/proteins.

Development of nanocrystal formulation of meloxicam with improved dissolution and pharmacokinetic behaviors.

The present study aimed to develop nanocrystal formulations of meloxicam (MEL) in order to enhance its biopharmaceutical properties and provide a rapid onset of action. Nanocrystal formulations were prepared by wet-milling and lyophilization with hydrophilic polymers used as aggregation inhibitors. Aggregation inhibitors were selected based on high-throughput screening of crystal growth inhibition in supersaturated MEL solution. Supersaturation of MEL was observed in PVP K-30, HPC-SSL, and POVACOAT Type F solution. Although the particle size distributions of pulverized MEL with PVP K-30 (MEL/PVP), HPC-SSL (MEL/HPC), and POVACOAT Type F (MEL/POVA) were in the nanometer range following lyophilization, increases in micron-sized aggregates were observed after storage at 60°C for 21 days. The order of increased amount of aggregates was MEL/POVA≫MEL/HPC>MEL/PVP. These findings showed that hydrophilic polymers that inhibited crystal growth in supersaturated MEL solutions tended to prevent aggregation. The dissolution behavior of all nanocrystal formulations tested was markedly enhanced compared with that of unpulverized MEL. Oral administration of MEL/PVP showed a 2.0h shortened Tmax and a 5.0-fold increase in bioavailability compared with unpulverized MEL. These findings showed that the MEL/PVP mixture was physicochemically stable and provided a rapid onset of action and enhanced bioavailability after oral administration.

Biopharmaceutical characterization of nanocrystalline solid dispersion of coenzyme Q10 prepared with cold wet-milling system.

The present study aimed to develop a nano-crystalline solid dispersion (CSD) of coenzyme Q10 (CoQ10) using a newly developed cold wet-milling (CWM) system to enhance the dissolution and biopharmaceutical properties of CoQ10. CSD formulations of CoQ10 were prepared by the CWM system, and their physicochemical properties were characterized in terms of morphology, crystallinity, particle size distribution, dissolution, and photostability. Application of the CWM system to CoQ10 led to successful development of a CSD formulation (CoQ10/CWM) with a mean CoQ10 diameter of ca. 129 nm, although a conventional wet-milling system failed due to evident formation of large particles. In comparison with crystalline CoQ10, marked improvement in the aqueous dissolution was seen for the CoQ10/CWM, with no significant decrease of photostability. Oral bioavailability and hepatoprotective effects of orally dosed CoQ10 samples were also evaluated in rats. After oral administration of CoQ10/CWM (100 mg CoQ10/kg) in rats, there appeared to be a similar Tmax value and 13-fold increase of bioavailability compared with crystalline CoQ10. In a rat model of acute liver injury, pretreatment with CoQ10/CWM (100 mg CoQ10/kg, twice) led to marked attenuation of hepatic damage as evidenced by decreased ALT and AST, surrogate biomarkers for hepatic injury, whereas crystalline CoQ10 was less effective. The CSD approach with the new CWM system might be a promising dosage option for improving the nutraceutical values of CoQ10.

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.

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.

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.

Up-regulation of nicotinic and muscarinic receptor mRNA in rat bladder by repeated administration of nicotine in relation to the pharmacokinetics.

AIMS: The current study aimed to investigate the effects of repeated treatment with nicotine on nicotinic and muscarinic receptors in the rat bladder. MAIN METHODS: Rats were administered nicotine (4.3 µmol/kg) subcutaneously (sc) twice a day for 10 days. The messenger RNA (mRNA) expression of nicotinic and muscarinic receptors in the bladder was examined by reverse transcription polymerase chain reaction (RT-PCR) and by a radioligand binding assay using [N-methyl-(3)H]scopolamine methyl chloride ([(3)H]NMS). The concentrations of nicotine and cotinine in plasma, tissues, and urine were estimated by ultra-performance liquid chromatography/electrospray ionization-mass spectrometry (UPLC/ESI-MS). KEY FINDINGS: mRNA of nicotinic receptor subunits (α1-α7, ß1-ß4, ε) and muscarinic receptor subtypes (M(1)-M(5)) was detected in the rat bladder. There was a significant increase in the mRNA expression of α3, ß1, ß2 and ß4 in the nicotine-treated rats (4.3 µmol/kg, s.c., b.i.d.) compared to the saline-treated group. Repeated administration of nicotine significantly increased the mRNA expression of the M(2) and M(3) muscarinic receptor subtypes in the rat bladder with a significant enhancement of [(3)H]NMS binding sites, compared to the saline-treated tissue. Nicotine was distributed at higher concentrations in the bladder than the heart and cerebral cortex. Furthermore, nicotine was excreted in rat urine at high concentrations. SIGNIFICANCE: The present study is the first to show that repeated treatment with nicotine affects pharmacologically-relevant nicotinic and muscarinic receptors in the bladder. Such changes may contribute to the etiology of lower urinary tract symptoms (LUTS) due to cigarette smoking.

Pharmacokinetic study of nicotine and its metabolite cotinine to clarify possible association between smoking and voiding dysfunction in rats using UPLC/ESI-MS.

The present study was undertaken to clarify the possible association between nicotine intake/cigarette smoking and detrusor instability. For pharmacokinetic characterization of nicotine and cotinine (a major and pharmacologically less active metabolite of nicotine), a rapid ultra-performance liquid chromatography/electrospray ionization-mass spectrometry (UPLC/ESI-MS) method was developed that requires only a small amount of sample and simple pretreatment. The UPLC/ESI-MS method was validated with a focus on specificity, sensitivity (limit of detection, 2.5 ng/mL; limit of quantification, 5 ng/mL), linearity (r > 0.998), accuracy (97.2-102.8%), precision (relative standard deviation <8%) and robustness in accordance with ICH guidelines (Q2B Validation of Analytical Procedures: Methodology). The developed method was successfully applied to determine nicotine and cotinine levels in rat biological samples such as plasma, urine and several tissues. After subcutaneous administration of nicotine ditartrate (2 mg/kg of body weight) in rats, the absorbed nicotine was rapidly and extensively metabolized into cotinine. However, nicotine was found to be predominant in cortex and bladder, where nicotinic acetylcholine receptors were expressed for neuronal control of voiding function. Repeated administration of nicotine led to a ca. 3-fold higher accumulation of nicotine than that of cotinine in rat urine. The results of the pharmacokinetic study using the UPLC/ESI-MS method further support the possible involvement of nicotine in increased risk of urinary dysfunction in smokers.

Development of high-energy amorphous solid dispersion of nanosized nobiletin, a citrus polymethoxylated flavone, with improved oral bioavailability.

Nobiletin (NOB), a citrus polymethoxylated flavone, attracts attention because of a wide range of pharmacological activities such as anti-inflammation, anticancer, and most notably ameliorative actions on memory impairment and ß-amyloid pathology. However, clinical use of NOB could be partly limited due to its poor solubility and bioavailability, which might necessitate high doses in order to reach therapeutic plasma concentrations in the central nervous system (CNS) after oral administration. In the present study, amorphous solid dispersion (SD) of nanosized NOB (NOB/SD) was prepared by wet-milling technique with the aim of improving dissolution behavior and pharmacokinetic properties of NOB. Physicochemical properties of the NOB/SD were characterized with focus on surface morphology, particle size distribution, dissolution, and crystallinity assessment. Wet-milled NOB particles in NOB/SD appeared to be amorphous with a diameter of approximately 270 nm, and there was marked improvement in the dissolution behavior compared with that of crystalline NOB. After oral administration of NOB/SD, higher exposure of NOB was observed with increases of bioavailability and CNS distribution by 13- and sevenfold, respectively, compared with those of crystalline NOB. These findings suggest that an amorphous, nanosized SD could be a viable option for enhancing the bioavailability and CNS delivery of NOB.

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.