[Evaluation of Photostability of Small Molecular Compounds with Solid-state UV Spectra].

It is crucial to evaluate the photostability of drugs. However, it requires a longer period of time to evaluate the photodegradation of compounds because extended light exposure to the compound is required to detect photodegradation products with the help of the commonly utilized technique of chromatography. Therefore, a simple and easy approach to estimate the photostability of the compound is required particularly for the initial screening of the drug candidates. It was reported in our previous manuscript that, focusing on ultraviolet-visible (UV/vis) spectrometry, the area under the spectrum curve in the ultraviolet-A (UVA) range (AUSCUVA) are closely related to the photodegradation of indomethacin polymorphs. In this study, the solid-state UV/vis absorption spectra of compound A polymorphs, indomethacin complexes and some small molecule compounds were determined and analyzed to check the applicability of this method. AUSCUVA are closely related to the photodegradation of compound A polymorphs as well as indomethacin. On the contrary, no close relation was observed between AUSCUVA of indomethacin complexes and their photodegradation. Additionally, the result indicated that the differences in their solid-state UV/vis absorption spectra were observed between photosensitive and photostable compounds. Photosensitive compounds show absorption in UVA range, while photostable compounds exhibit less absorption. In conclusion, the solid-state UV/vis absorption spectra of small molecular compounds might provide the key information on the photosensitivity.

Improving the Solid-State Photostability of Furosemide by Its Cocrystal Formation.

The photostability of three types of furosemide (FUR) cocrystal (FUR-caffeine, FUR-urea, and FUR-nicotinamide cocrystals) was studied under irradiation with a D65 fluorescent lamp. The coloration of the FUR-urea pellets was significantly faster than that of the intact FUR, whereas the coloration of FUR-nicotinamide was suppressed compared with that of intact FUR and the other cocrystals. In the case of FUR-urea, the chemical degradation of FUR increased by approximately 6.6% after irradiation for 90 d. On the other hand, FUR-nicotinamide showed better chemical stability, with only 1.3% of FUR degraded, which was significantly lower than the other cocrystals. The FUR-urea pellets showed a UV-Visible absorption spectrum similar to that of intact FUR, while the absorption range of FUR-nicotinamide shifted to a shorter wavelength. The light sensitivity of FUR-nicotinamide was improved because of the much lower emission of the D65 fluorescent lamp in the absorption range of the cocrystal.

A New Method for Classification of Salts and Cocrystals Using Solid-State UV Spectrophotometry.

Salt and cocrystal formulations are widely used as techniques to improve physicochemical properties of compounds. Some spectrometric techniques to distinguish cocrystals from salts have been reported; however, it has not been possible to adapt these formulations with many compounds, because of limitations, high difficulty, and exceptions. Therefore, we focused on the possibility of UV spectrometry, which had not been reported as a classification technique for salts and cocrystals. The integration values of solid-state UV/visible (Vis) spectra of indomethacin salts were larger than those of physical mixtures of indomethacin and counter molecules, while that of indomethacin cocrystal was not large compared with that of the physical mixture. From these results, differences between a salt and a cocrystal were observed in their solid-state UV/Vis absorption spectra for indomethacin complexes. Therefore, it is suggested that solid-state UV/Vis absorption spectra can be used as a new technique to classify salts and cocrystals.

Utility of Solid-State UV/Vis Spectra on the Evaluation of Photostability of Indomethacin Crystals.

Evaluating the stability of drugs over ranges of various environmental conditions is necessary to ensure the quality of the drug throughout the shelf life. In this study, we used indomethacin, which is well known as a photosensitive drug, and evaluated the photostability of prepared crystals. HPLC analysis revealed that all the indomethacin crystals were degraded by light exposure. However, the indomethacin crystals involving solvates had different degrees of photodegradation and different solid-state UV/Vis spectra. The value of the absorption integral in the UVA range related closely to the photodegradation of indomethacin crystals involving solvates. Therefore, it is easy to compare the photosensitivity among the crystals without actual analytical data, by use of a suitable analytical method and using light exposure samples. Moreover, it is possible to predict the value of photodegradation ratio from the solid-state UV/Vis spectra of indomethacin crystals. Therefore, this method may provide key information for selecting the most appropriate crystal form of photosensitive drugs.

Novel strategy for improving the bioavailability of curcumin based on a new membrane transport mechanism that directly involves solid particles.

Amorphization has been widely recognized as a useful solubilization technique for poorly water-soluble drugs, such as curcumin. We have recently reported the novel finding that the membrane transport of curcumin was markedly enhanced when amorphous solid particles of curcumin came into direct contact with the lipid membrane surface, but this was not true for crystalline solid particles. The increase in the permeation of curcumin was found to be independent of the improvements in aqueous solubility brought about by amorphization. Thus, we have identified a novel membrane transport mechanism that directly involves solid particles. In addition, it might represent a novel strategy for improving the bioavailability of curcumin that does not focus on the aqueous solubility of the drug. In this study, the direct effects of the administration of amorphous nanoparticles of curcumin (ANC) on the in vivo intestinal absorption of curcumin were investigated. After the intraduodenal administration of a curcumin suspension, the area under the curve of the plasma concentration of curcumin increased in a manner that was dependent on the curcumin concentration of the suspension, while no significant absorption was observed from a saturated solution. This finding is consistent with the results from our in vitro transepithelial transport study. In the latter experiment, the bioavailability of curcumin was found to be 1-2%. The intrapulmonary insufflation of ANC powder resulted in a significant increase in the bioavailability of curcumin (it was two orders of magnitude higher than that seen after the application of a crystalline suspension). This was due to the ANC particles coming into contact with epithelial cells in a more efficient manner after the pulmonary application of the ANC powder than after the intestinal application of the ANC suspension. Therefore, the pulmonary insufflation of amorphous powder is a novel approach to improving the bioavailability of curcumin and might be a useful way of increasing the bioavailability of poorly water-soluble drugs, such as curcumin.

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.

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

OBJECTIVES: To achieve an efficient skin delivery of resveratrol using sucrose fatty acid ester microemulsions and to clarify the mechanism of enhanced penetration. METHODS: Skin delivery of resveratrol using different sucrose fatty acid ester microemulsions was examined in vitro. Vehicle-skin interaction was assessed by applying blank microemulsions to skin. Skin incorporation of microemulsion components was also assessed. KEY FINDINGS: The microemulsion consisting of sucrose oleate (SO), ethanol, isopropyl myristate (IPM) and water (MESO-E) showed a prominent increase in the amount of skin incorporation of resveratrol, which was more than 5-fold higher than those of all microemulsions we previously examined. Using MESO-E, resveratrol was rapidly incorporated into skin and mainly located in the dermis. When applied in the concentration range of 5-55 mm, the amount of skin incorporation of resveratrol increased with the applied concentration up to 30 mm, whereas skin incorporation efficiency was inversely proportional to the concentration. The microemulsion-skin interaction seemed to be involved in the enhanced skin delivery process of resveratrol by MESO-E. Stratum corneum modification due to the penetration of IPM, ethanol and SO is also involved in this interaction. CONCLUSIONS: MESO-E would be a promising vehicle for the efficient skin delivery of resveratrol, especially when applied at a low concentration.

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.

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.

Efficient delivery and distribution in skin of chlorogenic acid and resveratrol induced by microemulsion using sucrose laurate.

To achieve efficient skin delivery of polyphenols, we prepared a novel oil-in-water (o/w)-type microemulsion (MESL) using sucrose laurate as a surfactant and ethanol, isopropyl myristate and water as other components. We examined its usefulness by in vitro studies on skin delivery of chlorogenic acid and resveratrol as hydrophilic and hydrophobic polyphenols using Yucatan micropig skin, and also examined the difference in the distribution of these polyphenols in skin. MESL significantly improved skin incorporation of these polyphenols at all time points examined (6, 20, 40 h) in the epidermis and at 20 and 40 h in the dermis, compared with the microemulsion using Tween 80 as a surfactant component (MEK), although the solubilization capacity of MESL was lower than that of MEK. Using MESL, the incorporation amount in the dermis of each polyphenol increased with time, while the amount in the epidermis was almost constant during the time examined. Incorporation efficiencies into skin of chlorogenic acid and resveratrol induced by MESL at 40 h after application were about 6-fold and 19-fold higher in the epidermis and 3.5-fold and 15-fold higher in the dermis, respectively, than those by MEK. The increase was more prominent for resveratrol. Hydrophilic chlorogenic acid was distributed slightly more in the epidermis, while hydrophobic and smaller-molecular-weight resveratrol was mainly distributed in the dermis. These findings suggest that MESL could be a promising vehicle for the efficient skin delivery of chlorogenic acid and resveratrol, especially for resveratrol to the dermis.

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.

Distribution of polyphenols and a surfactant component in skin during Aerosol OT microemulsion-enhanced intradermal delivery.

As for most other polyphenols, intradermal delivery of curcumin and resveratrol is limited; however, it was significantly improved by a microemulsion using Aerosol OT (Aerosol OT microemulsion) and Tween 80 (Tween 80 microemulsion) as surfactants. Aerosol OT microemulsion was more effective and the incorporation ratio of these polyphenols into skin by Aerosol OT microemulsion was five-fold or ten-fold that by Tween 80 microemulsion. To clarify the mechanism of the enhancement we examined the distribution of these polyphenols and the surfactant component, Aerosol OT, using excised guinea pig skin and Yucatan micropig (YMP) skin. During permeation, polyphenols distributed deep in the skin. In particular, a small molecule, resveratrol, was mainly present in the dermis in YMP skin. Aerosol OT also distributed deep in the skin. These findings suggest the possible involvement of the interaction of surfactant molecules with skin components in the enhanced delivery process of polyphenols. The distribution ratio between the dermis and epidermis of the polyphenols, including quercetin, in the presence of Aerosol OT microemulsion decreased with the increase of molecular weight in YMP skin, suggesting the possibility that distribution to the dermis is regulated by the molecular size.

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.

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.

Effects of phosphatidylethanolamine N-methyltransferase on phospholipid composition, microvillus formation and bile salt resistance in LLC-PK1 cells.

Bile salts are potent detergents and can disrupt cellular membranes, which causes cholestasis and hepatocellular injury. However, the mechanism for the resistance of the canalicular membrane against bile salts is not clear. Phosphatidylethanolamine (PE) is converted to phosphatidylcholine (PC) in the liver by phosphatidylethanolamine N-methyltransferase (PEMT). In this study, to investigate the effect of PEMT expression on the resistance to bile salts, we established an LLC-PK1 cell line stably expressing PEMT. By using enzymatic assays, we showed that the expression of PEMT increased the cellular PC content, lowered the PE content, but had no effect on the sphingomyelin content. Consequently, PEMT expression led to reductions in PE/PC and sphingomyelin/PC ratios. Mass spectrometry demonstrated that PEMT expression increased the levels of PC species containing longer acyl chains and almost all ether-linked PC species. PEMT expression enhanced the resistance to duramycin and lysenin, suggesting decreased ratios of PE and sphingomyelin in the apical membrane, respectively. In addition, SEM revealed that PEMT expression increased the diameter of microvilli. The expression of PEMT resulted in reduced resistance to unconjugated bile salts, but surprisingly in increased resistance to conjugated bile salts, which might be attributable to modifications of the phospholipid composition and/or structure in the apical membrane. Because most bile salts exist as conjugated forms in the bile canaliculi, PEMT may be important in the protection of hepatocytes from bile salts and in cholestatic liver injury.

Efficient topical delivery of chlorogenic acid by an oil-in-water microemulsion to protect skin against UV-induced damage.

We examined the intradermal delivery of a hydrophilic polyphenol chlorogenic acid by in vitro study using excised guinea pig dorsal skin and Yucatan micropig skin. Skin accumulation as well as the solubility of chlorogenic acid in aqueous vehicles was much greater than for other polyphenols such as quercetin and genistein. However, since enhancement of skin delivery seemed to be necessary to exhibit its protective effects against oxidative damage of skin, we examined the effects of microemulsions as vehicles. Using microemulsions consisting of 150 mM NaCl solution, isopropyl myristate, polyoxyethylene sorbitan monooleate (Tween 80) and ethanol, skin accumulation as well as solubility of chlorogenic acid further increased. Enhancement effect of an oil-in-water (o/w-type) microemulsion was greater than that of a water-in-oil (w/o-type) microemulsion possibly due to the greater increase in solubility. This finding was quite different from previous findings on relatively hydrophobic polyphenols such as quercetin and genistein. Pretreatment of guinea pig dorsal skin with chlorogenic acid containing microemulsion gel prevented erythema formation induced by UV irradiation. These findings indicate the potential use of hydrophilic chlorogenic acid with o/w-type microemulsion as a vehicle to protect skin against UV-induced oxidative damage.

Enhanced skin delivery of genistein and other two isoflavones by microemulsion and prevention against UV irradiation-induced erythema formation.

To improve the efficiency of the intradermal delivery of genistein and other two isoflavones (daidzein and biochanin A), we tried to clarify the usefulness of microemulsion by in vitro study on excised guinea pig dorsal skin and Yucatan micropig skin. Using microemulsion consisting of isopropyl myristate (IPM), 150 mM NaCl solution, Tween 80 and ethanol as a vehicle, the solubility of all the isoflavones markedly increased and significant amounts of isoflavones were delivered to the skin. The effect of water-in-oil (w/o)-type microemulsion D was larger than that of oil-in-water (o/w)-type microemulsion A. Among three isoflavones tested, the increase of genistein was most marked on both solubility and skin accumulation. Genistein retained in the skin significantly inhibited lipid peroxidation in vitro dose-dependently. Furthermore, pretreatment of guinea pig dorsal skin with genistein containing gel-like microemulsion D prevented UV irradiation-induced erythema formation. These findings indicate the potential use of w/o-type microemulsion for the delivery of genistein to protect skin against UV-induced oxidative damage.

Effect of pharmaceutical excipients on the stability of trichlormethiazide tablets under humid conditions.

The stability of trichlormethiazide (TCM) and the drug in the nine products available on the market (the original tablet (B) and 8 generic tablets (G1-G8)) were investigated under humid conditions. TCM was non-hygroscopic and was not degraded under humid conditions. Drug degradation in aqueous ethanol was accelerated with increased water concentration, and the drug stability in buffer solution was improved with decreased pH. TCM decomposition was not detected in each unwrapped tablet at low relative humidity. However, rapid degradation was observed for products G1 and G2, while product B and G7 showed higher stability at high relative humidity. The stability of products G1 and G2 decreased with increasing humidity. The same results were observed for the tablets in press-through packages (PTP), but the degradation rate was much slower than tablets without PTP packages. These results suggested that the adsorbed moisture by excipients cause TCM degradation. Various pharmaceutical excipients are added to TCM tablets and these vary between different pharmaceutical companies. Intact drug and pharmaceutical excipients, including lactose, microcrystalline cellulose, corn starch, hydroxypropylcellulose (HPC), low substituted HPC (L-HPC), calcium stearate, and light anhydrous silicic acid, were mixed, and the sample mixtures were stored in humid conditions. It was found that the TCM content decreased significantly in a binary mixture of TCM/HPC 1 : 1.

Physicochemical properties of tamoxifen hemicitrate sesquihydrate.

A novel modification of tamoxifen [(Z)-2-[4-(1,2-diphenyl-1-butenyl) phenoxy]-N,N-dimethylethylamine] citrate, tamoxifen hemicitrate hydrate was prepared. The crystalline form was identified and characterized by powder and single crystal X-ray diffractometries, differential scanning calorimetry (DSC), thermal gravimetric analysis (TGA), and hot-stage microscopy, and its physicochemical stability was also evaluated. The results of an elemental analysis, a single crystal X-ray analysis, and the TGA suggested that the molar ratio of tamoxifen:citric acid:water was 2:1:3 indicating it to be tamoxifen hemicitrate sesquihydrate. Simultaneous XRD-DSC measurements also indicated that two hydrates, sesquihydrate and hemihydrate, and an anhydrous form would exist during heating. The physicochemical stability of tamoxifen citrate forms A and B suspended in water and of form A during kneading and drying suggested that tamoxifen citrate was transformed into tamoxifen hemicitrate hydrate in water within 24 h, whereas tamoxifen citrate in a mixture with microcrystalline cellulose was quite stable during kneading. These results suggested that water and a mixture of water and organic solvent should be used for the manufacturing process with special attention paid to the transformation to tamoxifen hemicitrate sesquihydrate, because it showed a different stoichiometry from the active ingredient, tamoxifen citrate.

Physicochemical characterization of tamoxifen citrate pseudopolymorphs, methanolate and ethanolate.

Two novel pseudopolymorphs, methanolate and ethanolate of tamoxifen [(Z)-2-[4-(1,2-diphenyl-1-butenyl)phenoxy]-N,N-dimethylethylamine]citrate, were prepared in addition to forms A and B reported previously. Their crystalline forms were identified and characterized by powder and single crystal X-ray diffractometry, differential scanning calorimetry, thermogravimetric analysis, hot-stage microscopy, scanning electron microscopy and diffuse reflectance infrared Fourier-transform spectroscopy, and their physicochemical stability was also evaluated. The results of single crystal X-ray analysis and thermogravimetric analysis of methanolate and ethanolate suggested that the stoichiometry of tamoxifen citrate : methanol and tamoxifen citrate : ethanol could be composed of a 1 : 1 molecular ratio for both solvates. The results of physicochemical stability evaluations at 75 and 97% RH at 40 and 60 degrees C indicated that the metastable form A was quite stable for at least 2 months even under severe storage conditions, whereas methanolate immediately transformed to a crystalline mixture of forms A and B, and subsequently changed to the stable form B.