Osamu Sakata

Chromatographic and mass spectrometric analysis of glutathione in biological samples.

Biological thiol compounds are classified into high-molecular-mass protein thiols and low-molecular-mass free thiols. Endogenous low-molecular-mass thiol compounds, namely, reduced glutathione (GSH) and its corresponding disulfide, glutathione disulfide (GSSG), are very important molecules that participate in a variety of physiological and pathological processes. GSH plays an essential role in protecting cells from oxidative and nitrosative stress and GSSG can be converted into the reduced form by action of glutathione reductase. Measurement of GSH and GSSG is a useful indicator of oxidative stress and disease risk. Many publications have reported successful determination of GSH and GSSG in biological samples. In this article, we review newly developed techniques, such as liquid chromatography coupled with mass spectrometry and tandem mass spectrometry, for identifying GSH bound to proteins, or for localizing GSH in bound or free forms at specific sites in organs and in cellular locations.

Preparation of chitosan/alginate/calcium complex microparticles loaded with lactoferrin and their efficacy on carrageenan-induced edema in rats.

Background: Although lactoferrin (LF) possesses useful functions such as antitumor, antiviral, and anti-inflammatory acitivities, it is subject to gastric digestion, resulting in the reduction of efficacy. Therefore, it is important to develop a system delivering LF efficiently to intestinal mucosa or gut-associated lymphoid tissue. Method: Chitosan/alginate/calcium complex microparticles containing LF at a high loading were prepared using alginate, LF, and calcium chloride at the ratio of 6:3:8 (w/w). The release test was performed using Japanese Pharmacopoeia, Fifteenth Edition (JP15) first fluid (pH 1.2) for initial 2 hours, followed by JP15 second fluid (pH 6.8) for another 5 hours. Furthermore, the in vivo efficacy was evaluated from anti-inflammatory effect using rats with carrageenan-induced edema, in which dosing was performed intragastrically at 50 mg LF eq./kg 5, 3, and 1 days before carrageenan injection. Results: Microparticles have 20 – 30 % (w/w) LF content and 1 – 3 mm size. Nearly 60 % of LF was released at pH 1.2 at the first 1 hour, and then slowly released up to 80 % at 7 hours. Suppressive effect against the edema was greater in the order of microparticles LF solution control (saline). Initial burst of LF from microparticles was not associated with their promoted efficacy. Conclusion: Chitosan/alginate/calcium complex microparticles are suggested to be useful for promotion of efficacy of LF at oral administration

Preparation and evaluation of ritodrine buccal tablets for rational therapeutic use.

Ritodrine hydrochloride (RD-HCl) tablets containing alginate (AL) and lactose (LC) with or without microcrystalline cellulose (MC) as excipients were produced as a buccal dosage form. The RD-HCl (2 mg) tablets with AL/LC but no MC swelled and dissolved gradually in the in vitro dissolution test. The tablet showing the fastest dissolution and highest drug release rate, called Tablet A1, was selected as a tablet to show rapid and prolonged absorption. However, in the in vivo buccal absorption test using rats, it could not give a plasma concentration over the human minimal effective level (15 ng/mL). The modified tablet containing AL, LC, MC and RD-HCl (4 mg), named Tablet B/MC, showed better hardness and faster drug release. Tablet B/MC gave a plasma concentration over the human effective level within 15 min, and the plasma concentration was maintained at >15 ng/mL over 4 h. Moreover, the deconvolution analyses demonstrated that a prolonged high absorption rate could be achieved in vivo best with Tablet B/MC. Tablet B/MC improved the pharmacokinetic profile in comparison with Tablet A1 and the solution dosage form. The RD-HCl buccal tablets with AL, LC and MC as excipients are suggested to be possibly useful for the treatment of premature labor.

Clonazepam Oral Droplets for the Treatment of Acute Epileptic Seizures

Oral droplet formulations of clonazepam (CZ) were developed to examine their potentials as an alternative to i.v. administration for the treatment of acute epileptic seizures. Propylene glycol containing 2.5% (wt/wt) CZ with or without 5.0% (wt/wt) oleic acid (OA) was prepared as a solution by heating at 90°C and subsequently lowering the temperature to 30°C. The droplet (20 μL) was administered to the oral cavity between the lower gum and bottom lip before CZ precipitation started. With a droplet of propylene glycol loaded with 2.5% (wt/wt) CZ and 5.0% (wt/wt) OA, the plasma concentration reached 20 ng/mL (minimal effective concentration) within 10 min and was maintained between 20 and 60 ng/mL, less than a toxic level, for a period of 60 min. For a droplet of propylene glycol loaded only with CZ at 2.5% (wt/wt), it took more than 15 min for the plasma concentration to reach 20 ng/mL. It is suggested that a droplet of CZ/OA/propylene glycol (2.5:5.0:92.5, wt/wt) might be useful as an alternative to i.v. injection of CZ for the treatment of acute epileptic seizures.

Novel mucoadhesive oral patch containing diazepam.

The oral patch of diazepam (DZ) was developed to achieve a rapid absorption of DZ for the emergency treatment of epileptic seizure or anxiety disorder. The patch was composed of the outer mucoadhesive Carbopol 934 region, central drug region, and Tegaderm backing film. DZ (3 mg) was dissolved in propylene glycol (PG) alone or PG containing oleic acid (OA) at 5.6% (w/w), and used as the drug region. The patches with and without OA were attached to the mucosa of cheek in rats. The patch with OA exhibited the plasma level of more than 200 ng/mL at 10 min after administration, then the plasma concentration decreased gradually. The patch without OA displayed a plasma level of less than 30 ng/mL during 40 min after administration. To the contrary, in the in vitro drug permeation using a cellulose membrane, the patch without OA showed a three times faster permeation rate than the patch with OA, suggesting that the direct action of OA to mucosa might be associated with absorption enhancement. It was demonstrated that the patch with OA showed a good adhesion to oral mucosa and worked efficiently for rapid absorption of DZ.

Semi-solid dosage form of clonazepam for rapid oral mucosal absorption

Background: In order to obtain an alternative to the intravenous (i.v.) dosage form of clonazepam (CZ), an oral droplet formulation of CZ was developed previously; however, the droplet was physically unstable. Therefore, in the present study, it was attempted to develop an easily-handled dosage form, which was more physically stable and allowed rapid drug absorption from oral mucosa. Method: A semi-solid dosage form, composed of polyethylene glycol 1500 (PEG), CZ, and oleic acid (OA) at 37/1/2 (w/w) and named PEG/CZ/OA, and a semi-solid dosage form containing PEG and CZ at 39/1 (w/w), called PEG/CZ, were prepared. Their physical stability in air at room temperature and oral mucosal absorption in rats were investigated. Results: The semi-solid dosage forms were much more stable physically than the droplet, that is, no recrystallization of CZ was observed for at least 8 days. The effective concentration for humans and rats (20u2009ng/mL or more) was achieved within 30u2009min after buccal administration for both PEG/CZ/OA and PEG/CZ. The plasma concentration increased gradually and less varied at each time point for PEG/CZ/OA. PEG/CZ/OA was found to show more rapid and higher absorption of CZ in buccal administration than in sublingual administration. Conclusion: Buccal administration with the semi-solid dosage PEG/CZ with or without OA was suggested to be a possibly useful novel dosage form as an alternative to i.v. injection.

In vivo absorption study of ritodrine hydrochloride in the buccal administration to rats.

Background: Although ritodrine (RD)-hydrochloride (HCl), named RD-HCl, is widely used in the treatment of premature labor by intravenous prolonged infusion or frequent oral dosing of tablets, those administrations often lower patients’ quality of life (QOL) or cause undesirable side effects, such as tachycardia; therefore, in this study, the potential usefulness of buccal administration as a novel administration method was examined in vivo. Method: First, the HPLC method was assessed for the determination of plasma RD concentration. Then, after RD-HCl solution in saline was administered intravenously (1u2009mg/kg), intragastrically (10u2009mg/kg) or buccally (10u2009mg/kg) in rats, the plasma concentration–time profiles were investigated, and the absorption extent and rate compared. Results: The present modified determination method by HPLC with fluorescence detection (Ex. 278u2009nm, Em. 306u2009nm) was suitable to analyze the plasma level at 8–200u2009ng/mL. Buccal administration gave the best plasma concentration–time profile for maintenance of an effective plasma level and fewer side effects. Absorption rates calculated by deconvolution also supported better sustained absorption in buccal dosing. Conclusion: Buccal application of RD-HCl was demonstrated to be a potentially useful dosing method in the treatment of premature labor with RD-HCl.

Oral Biodisposition Study of Ritodrine after Its Buccal Administration in Rats

BACKGROUNDnIn the previous study, buccal absorption of ritodrine (RD) hydrochloride was reported in vivo. As a result, buccal dosing of RD solution was found to be useful for the maintenance of effective plasma concentration. However, in order to find out the dosing schedule more clearly, it is important to clarify the in vivo drug behavior.nnnOBJECTIVEnThe biodistributions of RD in oral cavity and buccal mucosal were investigated in order to understand the in vivo drug behavior after the buccal application.nnnMETHODnThe pharmacokinetic parameters for 0 - infinite time and the absorption rate were calculated based on the plasma level-time profiles in the intravenous (1 mg/kg), buccal (10 mg/kg) and intragastric (10 mg/kg) dosings using rats. The drug concentrations in buccal mucosa and the remaining drug amounts in the oral cavity were examined over time after the buccal administration. From those drug distributions and drug absorption rates, the kinetic aspects were discussed.nnnRESULTSnThe absolute bioavailabilities of RD were 14.2% and 4.3% in buccal and intragastric (0.043) administrations, respectively. The oral cavity concentration was quickly eliminated within 0.5 h, and then decreased slowly. In both the administration site and distant region, the mucosal RD concentrations were observed at several dozen to approximately 100 μg/g during 0.5-4 h, indicating the rapid diffusion in the oral cavity. For both the mucosal parts, the buccal mucosal level reached a maximal level at 1 h, and then was slowly eliminated. The absorption rates were not related linearly to the buccal mucosal level, suggesting that the other mucosal parts such as sublingual mucosa and tongue ventral surface should be involved considerably in the absorption.nnnCONCLUSIONnThe changes in RD concentration in oral cavity and oral mucosa showed the drug behavior in vivo. The present study revealed that RD is not accumulated in the buccal mucosa and transfers relatively fast from the oral mucosa to systemic circulation. It was suggested that the buccal dosing of RD should be acceptable even in the repeated manner as an alternative to the intravenous or oral administration of RD.

In Vitro and in Vivo Evaluations of Buccal Tablet Formulations of Ritodrine Hydrochloride

Buccal tablets of ritodrine (RD) hydrochloride (HCl), called RD-HCl, were prepared using the direct compression method with alginate (AL), lactose (LC), magnesium stearate (ST), and microcrystalline cellulose (MC) as excipients. The tablets were evaluated based on hardness, and tablets weighing 80u2009mg and with hardness of greater than 30u2009N were chosen as appropriate ones. As a result, tablets composed of RD-HCl (4u2009mg)/LC (38.5u2009mg)/ST (0.5u2009mg)/MC (37u2009mg) and RD-HCl (4u2009mg)/AL(7u2009mg)/LC (28.5u2009mg)/ST (0.5u2009mg)/MC (37u2009mg), called D9 and D10, respectively, were selected. These tablets were further evaluated based on in vitro dissolution and in vivo absorption studies in rats. D9 rapidly released RD, achieved an effective plasma concentration from 15u2009min to 7u2009h after its buccal administration, and did not exceed the toxic plasma level of 80u2009ng/mL. D10 gradually released RD, and maintained an effective concentration from 1u2009h to 7u2009h after its buccal administration, without exceeding the toxic plasma level. The absorption was more prolonged in D10 than D9. Their in vivo release was considered to be caused gradually from the amount of RD remaining in the oral cavity at 7u2009h, in particular D10. The superior retention of D10 in plasma and oral cavity appeared to be related to its higher mucoadhesive properties. Although these results were obtained using rats, they suggest that the chosen tablets should have adequate characteristics from the viewpoints of plasma levels.

Absorption behavior of etilefrine after buccal administration in rats

Graphical abstract Figure. No Caption available. Abstract Etilefrine hydrochloride (ET‐HCl) is used in the treatment of hypotension. Dosage forms of orally administered tablets and parenteral injections are clinically available, but exhibit unfavorable characteristics, including cardiac toxicity, headaches, and damage at the injection site for the parenteral dosage form, and initially high plasma levels, fast elimination, and first‐pass effects for its oral administration. Therefore, the buccal application of ET‐HCl was herein investigated as an alternative to conventional administration routes. I.v., intragastric, and buccal administration were performed using rats, and absorption features were compared. Buccal application at open conditions for 1 h exhibited absolute bioavailability of more than 20%, while the intragastric administration gave much lower bioavailability (<10%). The drug residue and drug distribution in the oral mucosa were investigated in order to clarify drug transfer behaviors. In the application of ET‐HCl solution using a cotton ball, higher plasma concentrations and their maintenance at higher levels were achieved at 10 mg/kg than at 2.5 mg/kg. In addition, absorption was greater with a longer application (4 h) than with a shorter application (1 h). Etilefrine (ET) was rapidly absorbed using aqueous buffer of pH 9.5 as the solvent. Open application was appropriate for achieving and maintaining higher plasma levels. Thus, in the buccal application of ET‐HCl aqueous droplets, a wide distribution throughout the mucosal surface is important for achieving rapid absorption and the maintenance of plasma levels. These findings suggested that the buccal application should be feasible administration of ET‐HCl.