Utility of nano-sized, water-in-oil emulsion as a sustained release formulation of glycyrrhizin.

The aim of this study was to determine the efficiency of nano-sized water-in-oil (w/o) emulsions that encapsulate glycyrrhizin (GZ) (Rp-I) as a sustained release formulation for subcutaneous administration. Four formulations were assessed in rats for 8-72 h: nano-sized water-in-oil (w/o) emulsion encapsulating GZ (Rp-I), GZ aqueous solution (Rp-II), oil-in-water (o/w) emulsion containing GZ (Rp-III), and w/o emulsion containing solid GZ (Rp-IV). All had a GZ concentration of 150 mg/ml. Over an 8-h period, GZ elimination in bile after subcutaneous administration of Rp-I, Rp-II, Rp-III, and Rp-IV (50 mg/kg GZ) was 10.8%, 97.0%, 81.0%, and 7.1%, respectively. The elimination of GZ into bile after the administration of Rp-IV was the lowest (30.5%) at the 72-h endpoint, dropping significantly from 48 to 72 h. On the other hand, the elimination rate of GZ after the administration of Rp-I was sustained at a constant level (1.8-2.1 mg/24 h) over 72 h. GZ concentration in liver at 72 h in Rp-I was highest (19.9 µg/g tissue) among the four formulations, suggesting that the release of GZ from the Rp-I formulation is constant, at least up to 72 h after administration. These results suggest that a nano-sized w/o emulsion is useful as a sustained release formulation for long-term therapy of chronic hepatitis.

[Preparation of PLGA microspheres encapsulating highly concentrated glycyrrhizin and the bile elimination of glycyrrhizin after subcutaneous administration in rats].

The aim of this study was to prepare poly (lactide-co-glycolide) (PLGA) microspheres encapsulating highly concentrated glycyrrhizin (GZ), a hydrophilic drug, and to compare the release characteristics of GZ in in vitro experiments and GZ elimination into bile after subcutaneous administration in rats. The preparation was carried out based on water drying using a (w/o)/w emulsion. The encapsulation rate of GZ in microspheres was 76% when the GZ concentration in the outer water phase was equal to that in the inner water phase for the preparation of (w/o)/w emulsion. The release of GZ from the microspheres showed a biphasic zero-order profile, that is, the behavior boundary was approximately 12 h. The release of GZ from the microspheres at the periods of 0.5-8 h and 12-672 h was 0.18 mg/h and 0.0012 mg/h, respectively. On the other hand, 0.25% of GZ administered (5.0 mg) was eliminated into bile by 12 h, and the bile clearance rate (1.13 ml/h) of GZ after the subcutaneous administration of the microspheres was the same as that (1.13 ml/h) after the administration of GZ solution. From the results, it is suggested that the initial controlled release (0.18 mg/h) of GZ from microspheres may be beneficial for the hepatic bioavailability of GZ.

[Novel formulations of a liver protection drug glycyrrhizin].

In Japan, glycyrrhizin injections have been used as a therapeutic drug for allergy inflammation since 1948 and for chronic hepatitis since 1979. A 20 ml injection of glycyrrhizin contains 53 mg of monoammonium glycyrrhizinate (40 mg as glycyrrhizin acid), 400 mg of glycine, and 20 mg of L-cysteine. Patients receiving glycyrrhizin injections two or three times per week are forced to accept a decline in quality of life. Because administering glycyrrhizin by injection has some disadvantages, many researchers have systematically searched for novel glycyrrhizin formulations that can be administered through oral, rectal, intranasal, and subcutaneous routes. There are two problems, however, in developing new formulations: (1) glycyrrhizin has low membrane permeability and is thus poorly absorbed, and (2) highly concentrated glycyrrhizin readily forms gels in aqueous solutions. Here, we describe the utility of glycyrrhizin formulations prepared in safe solubility agents and absorption-enhancing agents, as assessed in animal experiments. We also discuss pharmaceutical issues in developing various glycyrrhizin formulations. In the near future, convenient pharmaceutical preparations of glycyrrhizin will be developed for chronic hepatitis patients who require glycyrrhizin therapy.

Enhancing mechanism of Labrasol on intestinal membrane permeability of the hydrophilic drug gentamicin sulfate.

The aim of this study was to clarify the mechanism by which caprylocaproyl macrogol-8 glyceride (Labrasol) enhances the intestinal absorption of gentamicin sulfate (GM), a drug that has poor permeability but relatively high solubility. We studied the following characteristics: (i) the phase behavior of Labrasol in aqueous solution, (ii) the affinity of GM to Labrasol micelles, and (iii) the interaction between Labrasol and membrane lipids. We measured the critical micelle concentration of Labrasol in aqueous solution to be approximately 0.01%. The average diameters of Labrasol micelles in 2% and 25% solutions were approximately 10 nm and 20 nm, respectively, indicating that micelles increase in size with increasing Labrasol concentration. Although GM dissolved in 5% Labrasol solution was dialyzable, GM dissolved in either 25% or 50% Labrasol solutions was not, suggesting that GM exists in the hydrophilic region of the Labrasol micelle or in the high affinity region of the micelle surface where GM is retained. In membrane permeability experiments and electrophysiological studies conducted with rat ileum, only 25% Labrasol solution enhanced GM permeability, but did not remarkably affect membrane resistance. Furthermore, Labrasol increased membrane lipid fluidity as determined by fluorescence anisotropy in porcine intestinal brush border membrane liposomes. These results suggest that high concentrations of Labrasol solution enhance membrane permeability of GM via a transcellular rather than a paracellular route. We propose the following mechanism: Labrasol micelles grow when the concentration of Labrasol exceeds 20%, at which point GM shows high affinity for the hydrophilic region of the micelles. Since Labrasol micelles increase membrane lipid fluidity, the membrane permeability of GM is concomitantly enhanced.

Cytotoxic evaluation of cubic boron nitride in human origin cultured cells.

The aim of this study was to evaluate the cytotoxicity of cubic boron nitride (cBN), a component of surgical cutting tools. The small quantities of cBN that typically remain on implants as a result of the manufacturing process may act as abrasives, injuring tissues surrounding the implant. To determine how cBN affects cells, we treated human neuroblastoma cells (NB-1) and human articular chondrocytes (nHAC-kn) with different concentrations of cBN powder and assessed cell growth and cell survival using the methyl-thiazol-tetrazolium (MTT) assay and a fluorescence probe assay. We also assessed the effects of tungsten carbide (WC) and cobalt (Co), two common components of joint implants, on cell growth and cell survival. Both cBN and WC moderately inhibited NB-1 and nHAC-kn cell growth. However, cBN and WC did not affect cell survival, even at high concentrations (40 microg/ml). By contrast, Co affected cell survival, inducing cell death in both cell types at increasing concentrations. These results suggest that cBN may be less toxic than WC alloys containing Co.

In vitro and in situ evidence for the contribution of Labrasol and Gelucire 44/14 on transport of cephalexin and cefoperazone by rat intestine.

In vitro and in situ intestinal transport of beta-lactam antibiotics in the presence of two novel pharmaceutical excipients, caprylocaproyl and lauroyl macrogolglycerides (Labrasol and Gelucire 44/14), is described. The objective was to compare the effects of both macrogolglycerides on the intestinal transport of cephalexin, a substrate of oligopeptide transporters, and cefoperazone, a non-substrate of them. The in vitro transport studies were performed using a sheet of rat jejunum mounted in Ussing-type diffusion chambers. The in situ studies used an isolated internal loop model in the rat. Labrasol and Gelucire 44/14 were used as the excipients at low concentrations (0.01-0.5%, w/v). The membrane permeability of both drugs was compared by apparent permeability coefficients (P(app)) determined from changes in the amount of permeation vs. time in in vitro studies and by apparent absorptive clearance (CL(app)) determined from changes in the steady state drug concentration of perfusate in in situ studies. The P(app) value of cephalexin increased with an increase in the concentration of Labrasol (0.05-0.5%) compared to the value without Labrasol. The enhancing effect of Labrasol on cephalexin transport was similarly observed in in situ studies, and when 0.5% Labrasol was used in the presence of glycyl-L-leucine or L-alanyl-L-alanine, 60 or 46% enhancement of the active transport of cephalexin by Labrasol was obtained. On the other hand, Gelucire 44/14 did not affect the P(app) and CL(app) of either drug. The different effects of the excipients on cephalexin transport were thought to be due to the influences of size parameters such as a polydispersity index and particle size, and the change in the short-circuit current of jejunum by the addition of the excipient.

Membrane permeability and antipyrine absorption in a rat model of ischemic colitis.

The aim of this study was to determine whether the duration of ischemia affects antipyrine absorption in the large intestine. This was carried out in a rat model of ischemic colitis in which ischemia and associated inflammation was induced by marginal vessel ligation. Blood flow was disrupted by positioning an o-ring around the distal rectum and ligating the marginal vessel at two locations in the hind-gut ligament artery region. Ligation was performed for 1, 2, 3, and 5h. We assessed large intestine damage by measuring key indicators of inflammation, myeloperoxidase (MPO) activity and thiobarbituric acid reactant substrates (TBARS) in the mucosa and by histological staining with hematoxylin-eosin stain. Antipyrine membrane permeability was assessed in Ussing-type diffusion chambers, and related pharmacokinetics were calculated from antipyrine plasma concentration measurements following colon administration of the drug. Vessel ligation caused some sloughing of epithelial cells and elevated the MPO and TBARS levels. Prolonged ligation failed to affect the apparent permeability coefficient (P(app)) of antipyrine. Prolonged ligation, however, gradually increased plasma antipyrine concentrations to near control levels. This increase was paralleled by increases in the absorption rate constant AUC and antipyrine bioavailability. Taken together, these results suggest that the absorption kinetics of antipyrine may depend on blood flow changes in the large intestine that occur with inflammation.

Physicochemical and structural properties of glycerin gel prepared using glycyrrhizic acid diethyl ester.

Glycyrrhizic acid diethyl ester (GZ-DE) was developed as a prodrug of glycyrrhizic acid (GZ), a hepatitis therapeutic drug. We fortuitously found that GZ-DE gels with glycerin selectively while searching for a safe solvent with which to dissolve GZ-DE. Based on this gelation, the aim of this study was to investigate the preparation of the gel and study the rheology, physicochemical and structural properties of the glycerin gel by differential scanning calorimeter (DSC), capillary electrophoresis (CEP), nuclear magnetic resonance (NMR), and small angle X-ray scattering (SAXS). The glycerin gel was prepared by the addition of at least 2.0% w/w GZ-DE. This gel did not flow at room temperature. After mixing glycerin and GZ-DE, a gel was formed after 2 days at 25°C or 3 h at 60°C. Glycerin gel containing 2.4% w/w GZ-DE provided the following results: 1) The glycerin gel exhibited creep at a constant stress of less than 10 Pa, but it is a fragile gel, showing Newtonian flow at 10 Pa stress. 2) Dynamic viscoelastic measurements showed that the elastic modulus (G') exceeds the viscous modulus (G''), indicating that glycerin gel has solid-like properties. 3) DSC showed a significant difference between the glass transition temperature of glycerin and glycerin gel. 4) CEP did not reveal a new compound in the glycerin gel. 5) NMR confirmed that glycerin gel is a physical gel. 6) SAXS measurements revealed that the glycerin gel has an oval-shaped basic frame (119 nm long and 65 nm wide).

Intestinal absorption and biliary elimination of glycyrrhizic acid diethyl ester in rats.

BACKGROUND: The purpose of this study was to evaluate absorption and elimination from the gastrointestinal tract of glycyrrhizic acid diethyl ester (GZ-DE) which was prepared as a prodrug of glycyrrhizic acid (a poorly absorbed compound) in rats. METHODS: After the GZ-DE solution was administered via the intravenous, intraduodenal, intraileal, and stomach routes, GZ-DE and GZ concentrations in bile were determined by high-performance liquid chromatography. The stability of GZ-DE was estimated from residual GZ-DE and GZ produced in GZ-DE solutions prepared with distilled water, a pH 1.2 solution, 0.9% NaCl solution, and phosphate-buffered solution (pH 7.4) at 37°C. RESULTS: GZ-DE was eliminated into bile by the pharmacokinetic parameters of apparent distribution rate constant (4.56 ± 0.36 per hour) and apparent elimination rate constant (0.245 ± 0.042 per hour). After intravenous and intraduodenal administration of GZ-DE, the concentration ratio of GZ-DE to GZ in bile was approximately 4:1, and the bioavailability of GZ containing GZ-DE was three-fold higher compared with the bioavailability of GZ after intraduodenal administration. GZ-DE was immediately precipitated in pH 1.2 solution and was converted to GZ by hydrolysis in pH 7.4 solution. CONCLUSION: Improvement of intestinal absorption of GZ was made possible by administration of GZ-DE into the intestine where absorption of GZ is lower than in the strong acidic environment of the stomach. However, because the elimination rate in bile simulated from kinetic parameters of GZ-DE was higher than the conversion rate from GZ-DE to GZ by hydrolysis, it is thought that the availability of GZ as a revolutionary prodrug was not high from the viewpoint of bioavailability of GZ in the liver by intestinal administration of GZ-DE.

Comparative study for pharmaceutical quality among bland-name drug and generic drugs of compound glycyrrhizin injections in China.

The physicochemical properties (pH and osmolarity), ingredients, and impurities containing in compound glycyrrhizin injections (eight items) marketed in China were compared with those in bland-name drug (Stronger Neo-Minophagen C injection). Glycyrrhizin (GZ), glycine (Gly), and l-cysteine (CysH) as the ingredients, moreover, glycyrrhetinic acid (GA), 3-monoglucuronyl-glycyrrhetinic acid (MGGA), and l-cystine (CysS) as the impurity were determined by HPLC. The pH and osmolarity were different every each pharmaceutical product, but the variation between batch was very small. On the other hand, although the contents of GZ, Gly, and CysH in bland-name drug were approximately 100% of the label claim, the contents of GZ in generic drugs were the range of 91.8-100.9%, indicating the GZ contents in four products were clearly less than value indicated in label (<97%). The remarkable difference was not accepted by impurities content such as GA and MGGA. The contents of CysH in generic drugs were the range of 79.9-100.4%, and CysS was determined in all generic drugs, suggesting that CysH may decompose to be CysS depending on the pH of injections in generic drug only. Because the variation of the ingredient content was big and products with a little quantity for the ingredients were recognized, establishment of the preparation that can maintain the prescribed ingredient content and the severity of the assay will be required.

Nano-sized water-in-oil-in-water emulsion enhances intestinal absorption of calcein, a high solubility and low permeability compound.

Our goal was to develop safe and stable multilayer emulsions capable of enhancing intestinal absorption of biopharmaceutics classification system (BCS) class III drugs. First, w/o emulsions were prepared using calcein as a model BCS class III compound and condensed ricinoleic acid tetraglycerin ester as a hydrophobic emulsifier. Then water-in-oil-in-water (w/o/w) emulsions were prepared with shirasu porous glass (SPG) membranes. Particle size analyses and calcein leakage from oil droplets in w/o/w emulsions led us to select stearic acid hexaglycerin esters (HS-11) and Gelucire 44/14 as hydrophilic emulsifiers. Analyses of the absorption-enhancing effects of w/o/w emulsions on intestinal calcein absorption in rats showed that calcein bioavailability after intraduodenal (i.d.) administration of HS-11 or Gelucire 44/14+polyvinyl alcohol (PVA) w/o/w emulsions prepared with 0.1-microm pore-sized SPGs was significantly higher than that of the calcein control. However, serum calcein concentration vs. time profiles after i.d. administration of w/o/w emulsions prepared with 1.1-microm and 30-microm pore-sized SPGs and an emulsion prepared with a calcein-containing outer water phase were comparable to control profiles. These results suggested that HS-11 or Gelucire 44/14+PVA are safe outer water phase additives and that 0.1-microm pore-sized SPGs are important for preparing w/o/w emulsions that enhanced intestinal calcein absorption.

Pharmacokinetics of glycyrrhizin in normal and albumin-deficient rats.

The pharmacokinetics of glycyrrhizin (GZ) was compared in albumin-deficient rats (NAR) and normal rats (SDR) after intravenous administration. The study sought to clarify the relationship between GZ concentration and its elimination rate in serum, liver and bile when the serum protein binding of GZ decreased. Serum protein binding in SDR and NAR, respectively, was 99.7% and 68.2% for a GZ concentration of 2.5 microg/ml. At steady-state conditions after i.v. infusion of GZ (0.5-2.0 mg/h), the relationship between the GZ concentration in serum and liver was linear in the SDR but nonlinear in the NAR. For both NAR and SDR, the GZ liver level and the elimination rate was nonlinear, indicating that the elimination of GZ from liver into bile was the rate-limiting step regardless of serum protein binding, and that the liver GZ level was extremely high when serum protein binding was decreased. It is concluded that a typical dose of GZ in chronic hepatitis patients whose serum albumin level is low will not cause a decrease of therapeutic effect compared with patients with a normal serum albumin level.

Clove oil prevents glycyrrhizin gel formation in aqueous solution.

One persistent problem with using therapeutic concentrations of glycyrrhizin (GZ) is that, at these high concentrations, it forms a gel in an aqueous solution. We previously solved this problem by dissolving GZ in a highly concentrated phosphate buffer. Unfortunately, the resulting GZ solution has a hyperosmotic pressure that renders it unsuitable for use in patients. The aim of this study was to prepare a highly concentrated GZ solution having an osmotic pressure ratio of 1 and a pH of 7.4. By adding small amounts of oil and using a 100 mM phosphate buffer, we achieved an emulsified GZ solution that is stable at room temperature and has a physiological osmotic pressure and pH. When clove oil was used as an emulsifier, the gel formation temperature of GZ solution decreased appreciably compared to that of GZ solution without clove oil. Using scanning electron microscopy (SEM), we examined the detailed characteristics of GZ gels prepared from solutions with or without clove oil. SEM of cross sections of GZ gels revealed an irregular structure in gels prepared with clove oil, indicating that clove oil prevented the formation of the intermolecular GZ networks typically characterized by gels derived from pure GZ solutions.

Preparation and rectal absorption of highly concentrated glycyrrhizin solution.

We developed a simple method for preparing a highly concentrated solution of glycyrrhizin monoammonium salt (GZ) at low viscosity with no surfactants nor organic solvents and investigated the absorption profile after rectal administration to rats. GZ (200 mg/ml) was dissolved in phosphate buffered solution, pH 7.0; over 350 mM concentration was maintained for the aqueous solution without gel-formation. When glycerin was used as a non-aqueous formulation, GZ did not form gel. Apparent permeability coefficients of GZ obtained from 350 mM phosphate buffered solution (pH 7.0) and glycerin solution through rat rectal mucosa estimated by in vitro parallel diffusion chamber technique were 0.686 x 10(-6) and 0.379 x 10(-6) cm/s, respectively. On the other hand, the area under plasma concentration-time curves of GZ in 400 mM phosphate buffer (pH 7.0) and glycerin formulations after rectal administration to the rat were significantly higher than that in polyethylene glycol 400/propylene glycol (55 : 5) formulation. Maximum plasma concentrations of these formulations were dependent on the apparent permeability coefficients of GZ. Increased absorption observed by phosphate buffered formulation accompanied no pronounced histological damage in mucosa. These results demonstrate that addition of a highly concentrated phosphate salts is effective not only for lowering the viscosity of a highly concentration of GZ solution, but also for improving the mucosal GZ absorption.

A simple method for the selection of a suitable emulsifier based on color difference.

The purpose of this study was to establish a simple and rapid method for selecting a nonionic surfactant to prepare an emulsion that can maintain a stable emulsification phase. As an index of the degree of emulsification, the white chromaticity of the prepared sample was measured using a color difference meter. When liquid paraffin was used as an oil, the color difference (dE(H)) was shown as a change in a V-shaped curve depending on changes in the hydrophile-lipophile balance (HLB) number of sorbitan ester, polyoxyethylene sorbitan ester, or polyethyleneglycol ester. Although there was a difference in the minimal value of dE(H) in these experiments, the dE(H) was similar to the required HLB of liquid paraffin (HLB=10). On the other hand, no relationship was found between the HLB number and color difference when polyglycerol ester was used. It seems that there was possibility of the effect by the lowering of apparent HLB with a collapse of the hydration. These results indicate that color difference could be utilized for the selection of surfactants, except for polyglycerol ester.