Reiko Teraoka

Pharmaceutical evaluation of carbamazepine modifications: comparative study for photostability of carbamazepine polymorphs by using Fourier-transformed reflection-absorption infrared spectroscopy and colorimetric measurement.

Abstract— The tablet surface was evaluated without physical damage by means of Fourier‐transform infrared reflection‐absorption spectroscopy (FT‐IR‐RAS) and colorimetric measurement (colour difference, AE) of the carbamazepine polymorphs I, II and III, after photodegradation at two irradiation intensities (30 and 120 J cm−2 s−1) under a near‐UV fluorescent lamp. The surface of sample pellets of all crystalline forms turned gradually from white to yellow‐orange upon exposure to light, and the discoloration rate of form II was faster than that of forms I and III, indicating that form II was the most unstable of the three. The major photoproducts were identified by HPLC, NMR and MS analyses. The carbamazepine content on the surface of the tablet was determined based on the absorption at 1685 cm−1 atributable to C = O stretch vibration in the FT‐IR‐RAS spectra before and after irradiation by a near‐UV fluorescent lamp. The semilogarithmic plots of the photodegradation profiles of the various polymorphs were straight lines, including the induction period, indicating that degradation of the drug on the surface followed first‐order kinetics. The induction periods of all forms were not significantly different. However, the degradation rate constant of form II at 12.0 Jcm−2 s−1 was 5·1 and 1·5 times larger than those of forms I and III, respectively.

Evaluation of photostability of solid-state dimethyl 1,4-dihydro-2, 6-dimethyl-4-(2-nitro-phenyl)-3,5-pyridinedicarboxylate by using Fourier-transformed reflection-absorption infrared spectroscopy.

Effect of particle size on the photostability of dimethyl 1, 4-dihydro-2,6-dimethyl-4-(2-nitro-phenyl)-3,5-pyridinedicarboxylate (nifedipine) powder and its tablet was investigated using high-pressure liquid chromatography (HPLC) method and Fourier-transformed infrared reflection-absorption spectroscopy (FT-IR-RAS) under the non-destructive condition. The nifedipine content on the surface of the tablet was determined based on the absorbance at 1682 cm(-1) attributable to the C=O stretch vibration in FT-IR-RAS spectra before and after irradiation by fluorescent lamp. The photodegradation followed apparently the first-order kinetics for any sample. The apparent photodegradation rate constant of nifedipine powder increased with decrease of the particle size, while that of its tablet was approximately constant irrespective of particle size. Semilogarithmic plots of the apparent degradation rate constant for nifedipine tablet against the reciprocal of illuminance demonstrated a linear relationship similar to that of the Arrhenius-type behavior.

Enzymatic measurement of phosphatidylserine in cultured cells

Phosphatidylserine (PS) is a quantitatively minor membrane phospholipid involved in diverse cellular functions. In this study, we developed a new fluorometric method for measuring PS using combinations of specific enzymes and Amplex Red. The calibration curve for PS measurement was linear and hyperbolic at low (0–50 µM) and high (50–1000 µM) concentrations, respectively, and the detection limit was 5 µM (50 pmol in the reaction mixture). This assay quantified PS regardless of the chain length and the number of double bonds. We applied this new method to the determination of PS content in HEK293 cells, which was validated by a recovery study and comparison with TLC-phosphorus assay. We showed that the PS content was high in sparse cells. The overexpression of PS synthase 1 elevated not only the cellular PS content but also the phosphatidylcholine (PC) and phosphatidylethanolamine (PE) contents, suggesting the conversion of PS into PE and the enhancement of PC production. This new assay for PS measurement is simple, specific, sensitive, and high throughput, and it will be useful to clarify the metabolism and biological functions of PS.

Microemulsion Using Polyoxyethylene Sorbitan Trioleate and Its Usage for Skin Delivery of Resveratrol to Protect Skin against UV-Induced Damage

We examined the phase behavior of various polyoxyethylene sorbitan fatty acid ester (polysorbates)/ethanol/isopropyl myristate (IPM)/150 mM NaCl solution (NaClaq) systems in order to prepare a microemulsion containing a low ratio of ethanol, which is more suitable for in vivo application. Using polyoxyethylene sorbitan trioleate (Tween 85), which has a large lipophilic moiety, as a surfactant component, single-phase domain of the phase diagram was the largest of all the polysorbates examined, and in particular a large oil-rich single-phase domain was obtained. When the ratio of Tween 85 to ethanol was changed from 1 : 1 to 3 : 1, the oil-rich single-phase domain further expanded, which led to a reduced ethanol concentration in the preparation. Thus, we determined the composition of the microemulsion to be Tween 85 : ethanol : IPM : NaClaq=30 : 10 : 53 : 7, and used it for skin delivery of resveratrol. Microemulsion gel was also prepared by adding 6.5% Aerosil) 200 into the microemulsion for ease of topical application. When applied with each vehicle, delivery of resveratrol into guinea pig skin in vitro was significantly enhanced compared with that by IPM, and resveratrol incorporated into the skin by microemulsion gel decreased lipid peroxidation to 29.5% compared with that of the control. Pretreatment of guinea pig dorsal skin with the microemulsion gel containing resveratrol almost completely prevented UV-B-induced erythema formation in vivo. These findings demonstrate that the microemulsion using Tween 85 containing a minimal concentration of ethanol enhanced the skin delivery of resveratrol and the incorporated resveratrol exhibited a protective effect against UV-induced oxidative damage.

Bile salt-stimulated phospholipid efflux mediated by ABCB4 localized in nonraft membranes.

ABCB4 is necessary for the secretion of phospholipids from hepatocytes into bile and for the protection of cell membranes against bile salts. Lipid rafts are plasma membrane microdomains containing high contents of cholesterol and sphingolipids, which are separated by Triton X-100 extraction or OptiPrep gradient centrifugation. In this study, we investigated the relationship between the function of ABCB4 and lipid rafts using mouse canalicular membranes and HEK293 cells stably expressing ABCB4. ABCB4 and ABCB1 were mainly distributed in nonraft membranes. The expression of ABCB4, but not ABCB1, led to significant increases in the phosphatidylcholine (PC), phosphatidylethanolamine (PE), and sphingomyelin (SM) contents in nonraft membranes and further enrichment of SM and cholesterol in raft membranes. The ABCB4-mediated efflux of PC, PE, and SM was significantly stimulated by taurocholate, while the efflux of PE and SM was much less than that of PC. This ABCB4-mediated efflux was completely abolished by BODIPY-verapamil, which hardly partitioned into raft membranes. In addition, ABCB1 and ABCB4 mediated the efflux of rhodamine 123 and rhodamine 6G from nonraft membranes, which was not affected by taurocholate. We conclude that ABCB4 located in nonrafts, but not in rafts, is predominantly involved in the efflux of phospholipids and other substrates.

Specific and sensitive enzymatic measurement of sphingomyelin in cultured cells.

Sphingomyelin (SM) is the most abundant sphingolipid in mammalian cell membranes and plays multiple physiological roles. In this study, we improved the sensitivity of the enzymatic measurement of SM and validated its specificity and accuracy. The enzymatic reaction sequence of the method involves the hydrolysis of SM by sphingomyelinase, dephosphorylation of phosphorylcholine, oxidation of choline, and reaction of hydrogen peroxide with Amplex Red. The calibration curve was shown to be quadratic and linear at low (0-10 μM) and high (10-100 μM) concentrations, respectively, and the detection limit was 0.5 μM (5 pmol in the reaction mixture), which was more sensitive than all other SM assays reported previously. This SM measurement using Triton X-100 detected only SM, but not other choline-containing phospholipids, sphingosylphosphocholine, phosphatidylcholine, and lysophosphatidylcholine, and quantified SM regardless of the length and double bonds of the acyl chain. By using this method, we demonstrated that an increase in the density of HEK293 cells was accompanied by an elevation in the cellular content of SM, and that the treatment of HEK293 cells with tumor necrosis factor α significantly decreased the SM content. This specific and sensitive method for measuring SM will be helpful in studying various cellular processes.

Prominent efficiency in skin delivery of resveratrol by novel sucrose oleate microemulsion.

To achieve an efficient skin delivery of resveratrol using sucrose fatty acid ester microemulsions and to clarify the mechanism of enhanced penetration.

Effects of Mixing Procedure Itself on the Structure, Viscosity, and Spreadability of White Petrolatum and Salicylic Acid Ointment and the Skin Permeation of Salicylic Acid

White petrolatum is a mixture of solid and liquid hydrocarbons and its structure can be affected by shear stress. Thus, it might also induce changes in its rheological properties. In this study, we used polarization microscopy to investigate how different mixing methods affect the structure of white petrolatum. We used two different mixing methods, mixing using a rotation/revolution mixer and mixing using an ointment slab and an ointment spatula. The extent of the fragmentation and dispersal of the solid portion of white petrolatum depended on the mixing conditions. Next, we examined the changes in the structure of a salicylic acid ointment, in which white petrolatum was used as a base, induced by mixing and found that the salicylic acid solids within the ointment were also dispersed. In addition to these structural changes, the viscosity and thixotropic behavior of both test substances also decreased in a mixing condition-dependent manner. The reductions in these parameters were most marked after mixing with a rotation/revolution mixer, and similar results were obtained for spreadability. We also investigated the effects of mixing procedure on the skin accumulation and permeation of salicylic acid. They were increased by approximately three-fold after mixing. Little difference in skin accumulation or permeation was detected between the two mixing methods. These findings indicate that mixing procedures themselves affect the utility and physiological effects of white petrolatum-based ointments. Therefore, these effects should be considered when mixing is required for the clinical use of petrolatum-based ointments.

Chemical Stability of Ethyl Icosapentate Against Autoxidation. II. Effect of Photoirradiation on Oxidation Kinetics

The effect of ultraviolet (UV) or visible light (VIS) irradiation on the chemical stability of ethyl icosapentate [ethyl-(all-cis)-5,8,l l,14,17-icosapentaenoate] (EPA) was investigated at 45°C by means of HPLC and by measuring the peroxide value (POV). EPA was oxidized to peroxides after an induction period by photoirradiation, and the peroxide subsequently degraded to secondary products. The autoxidation of EPA followed consecutive reaction kinetics including an induction period, and the kinetic parameters of the oxidation were calculated based upon the consecutive reaction model by computer curve fitting. The results of the degradation rate constant, k, and the induction period obtained by HPLC showed that the radical and the peroxide formation rates are affected by UV, but not by VIS light irradiation. The formation rate constant of peroxide, kl, and its degradation rate constant to secondary products, k2, obtained from the POV under UV light irradiation, increased with irradiation intensity, during which the induction period decreased. On the other hand, kl, k2 and the induction period by VIS light irradiation did not change significantly. The relationship between the induction periods obtained by HPLC and POV and the UV light irradiation energy were superimposed in the plots, indicating that these parameters depended on the UV irradiation energy. The relationship between kl/k2 ratio and the UV irradiation energy suggested that the formation of secondary products was more remarkably accelerated by UV energy than that of peroxide.

Differences in the rheological properties and mixing compatibility with heparinoid cream of brand name and generic steroidal ointments: The effects of their surfactants

Most steroidal ointments contain propylene glycol (PG) and surfactants, which improve the solubility of corticosteroids in white petrolatum. Surfactants aid the uniform dispersal of PG within white petrolatum. Since the surfactants used in generic ointments are usually different from those used in brand name ointments, we investigated the effects of surfactants on the rheological properties of three brand name ointments and six equivalent generic ointments. We detected marked differences in hardness, adhesiveness, and spreadability among the ointments. Further examinations of model ointments consisting of white petrolatum, PG, and surfactants revealed that the abovementioned properties, especially hardness and adhesiveness, were markedly affected by the surfactants. Since steroidal ointments are often admixed with moisturizing creams prior to use, we investigated the mixing compatibility of the ointments with heparinoid cream and how this was affected by their surfactants. We found that the ointments containing glyceryl monostearate demonstrated good mixing compatibility, whereas those containing non-ionic surfactants with polyoxyethylene chains exhibited phase separation. These results were also consistent with the findings for the model ointments, which indicates that the mixing compatibility of steroidal ointments with heparinoid cream is determined by the emulsifying capacity of the surfactants in their oily bases.