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Two new bladder instillations of the investigational anticancer agent EO-9 containing 2-hydroxypropyl-beta-cyclodextrin (HP beta CD) and the alkalizers sodium bicarbonate (NaHCO3) and tri(hydroxymethyl)aminomethane (Tris) were developed. During the stability study of these freeze-dried products, formation of new degradation products was seen. We have characterized these products by high performance liquid chromatography in combination with photodiode array detection and mass spectrometry. In total, five new degradation products were identified of which three were detected in both freeze-dried products and two only in the freeze-dried product composed of EO-9/HP beta CD/NaHCO3. Furthermore, the purity profile of two lots of EO-9 drug substance was investigated. Five, probably synthetic intermediates were found. However, the amount of total impurities was very small for both lots of drug substance and below acceptable international limits for pharmaceutical use.

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For the development of a bladder instillation of the indoloquinone agent EO-9, use of the complexing agent 2-hydroxypropyl-beta-cyclodextrin (HPbetaCD) was considered. Therefore, a complexation study of EO-9 with HPbetaCD was performed. Complexation was studied in aqueous solution and in solid freeze-dried products. A phase solubility study, UV-visible spectroscopy (UV/VIS), and analysis of the effect of HPbetaD on the stability of EO-9 were performed. With the phase solubility study, a complexation constant (K1:1) of 32.9, a complexation efficiency (CE) of 0.0457, and a utility number (UCD) of 38.3 were calculated. These K1:1 and CE values indicate a weak complex, but the UCD shows that HPbetaCD can be very useful as solubilizer in the desired formulation. Furthermore, a positive effect of HPbetaCD on the chemical stability of EO-9 in solution was seen. Subsequently, complexation in the freeze-dried products was studied more thoroughly using Fourier transform infrared (FTIR), differential scanning calorimetry (DSC), X-ray diffraction (XRD), and scanning electron microscopy (SEM) analyses. HPbetaCD was found to be an excellent pharmaceutical complexing agent for application in formulations for EO-9 bladder instillations. Reconstitution before use of the developed freeze-dried products can be simply accomplished with water for injection.

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A bladder instillation of EO-9 (EOquin) is currently used in phase II clinical trials for the treatment of superficial bladder cancer. Three alternative formulations were developed to improve its pharmaceutical properties and clinical acceptability. Freeze-dried products composed of EO-9, 2-hydroxypropyl-beta-cyclodextrin (HPbetaCD), tri(hydroxymethyl) aminomethane (Tris), and sodium bicarbonate (NaHCO(3)) were tested. Selection of one formulation for further development was based on stability studies. These studies comprised stability of the freeze-dried products, stability after reconstitution and dilution and stability during bladder instillation in an experimental set-up. The stability study of the freeze-dried products showed that the formulation composed of EO-9/HPbetaCD/Tris (4/600/1mg/vial) was most stable. After reconstitution and dilution all products were stable for at least 8h. The product composed of EO9/HPbetaCD/NaHCO(3) (4/600/20mg/vial) was the least stable product both as freeze-dried formulation and after reconstitution and dilution. The bladder instillation simulation experiment showed that all products were stable when mixed with urine of pH 8 and unstable in urine of pH 4 and 6. The degradation products formed in urine were EO-5a and EO-9-Cl. Based on these results, the product composed of EO-9/HPbetaCD/Tris (4/600/1mg/vial) was selected for further pharmaceutical development.

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A series of 2,3-dialkyloxypropyl quaternary ammonium lipids containing hydroxyalkyl chains on the quaternary amine were synthesized, formulated with dioleoylphosphatidylethanolamine (DOPE) and assayed for their ability to enhance the activity of an intercellular adhesion molecule 1 (ICAM-1) antisense oligonucleotide, ISIS 1570. Cationic liposomes prepared with hydroxyethyl, hydroxypropyl and hydroxybutyl substituted cationic lipid all enhanced the activity of the ICAM-1 antisense oligonucleotide. Cationic lipids containing hydroxypentyl quaternary amines only marginally enhanced the activity of ISIS 1570. Hydroxyethyl cationic lipids synthesized with dimyristyl (Cl4:0) and dioleyl (C18:1) alkyl chains were equally effective. Activity of cationic lipids containing saturated alkyl groups decreased as the chain length increased, i.e. the dimyristyl (C14:0) was more effective than dipalmityl (C16:0) lipid, which was more effective than distearyl (C18:0). The phase transition temperature of cationic lipids containing saturated aliphatic chains was 56 degrees C for the distearyl lipid, 42 degrees C for the dipalmityl lipid and 24 degrees C for the dimyristyl lipid. Cationic lipids with dioleyl alkyl chains required DOPE for activity, with optimal activity occurring at 50 mole%. In contrast, a dimyristyl containing cationic lipid did not require DOPE to enhance the activity of ISIS 1570. Formulation with different phosphatidylethanolamine derivatives, revealed that optimal activity was obtained with DOPE. These studies demonstrate that several cationic lipid species enhance the activity of phosphorothioate antisense oligonucleotides and provide further information on the mechanism by which cationic lipids enhance the activity of phosphorothioate oligodeoxynucleotides.

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A set of properties of the water contained within hydrogel contact lens materials was determined with the aim of developing a model which would predict the propensity of a hydrogel contact lens material to induce corneal desiccation staining. We postulated that materials containing a larger proportion of water with the properties of bulk water would tend to induce corneal desiccation more readily than materials with the same overall water content but containing a larger proportion of water that interacts strongly with the polymer. The water structure [as measured by differential scanning calorimetry (DSC)] and the permeabilities of water and glucose were determined for a series of commercial hydrogel lenses. Both glucose permeability and DSC measurements are sensitive indicators of water structure and able to distinguish between various materials. To illustrate the potential of our model, the results of a short-term clinical study are presented. Lower levels of staining were noted for a material with a lower glucose permeability and a larger amount of water melting below 0 degrees C than for a control lens, even though both materials were similar in water content and water permeability. Further clinical studies are needed to validate this model.

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An artificial tear solution containing the major types of proteins, glycoproteins, and lipids represented in human tears has been developed. The adsorption of lipids onto various hydrogel lens materials (polymacon, lidofilcon A, phemfilcon A, etafilcon A) was examined by exposing the lenses to our artificial tear solution for 18 h. The adsorbed lipids were detected using Nile red stain. The patterns of deposits obtained in vitro were similar to those obtained with human worn lenses. The Nile red stain appeared far more sensitive in detecting lipids adsorbed to hydrogel lenses than the oil red O stain. It was found that lipids adsorb to hydrogel materials quite readily either in a pure state or combined with mucin or other proteins. In view of this work more attention should be given to the role of lipids in the etiology of various contact lens wear complications.

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The effect of adsorbed substances on the properties of the water in various hydrogel contact lens materials was examined by exposing contact lenses (Hydron Zero 4, B&L 70, DuraSoft 3, Vistamarc, and Acuvue) to an artificial tear solution for various periods up to 14 days. The only materials affected were the high-water/ionic lenses which adsorbed a large amount of protein, predominantly lysozyme. In the DuraSoft 3 lenses the equilibrium water content (EWC) dropped from 49% to 46% and the freezing water from 28% to 21%. Similar changes were seen with the Vistamarc lenses. After a 10-day exposure of the Acuvue lens to artificial tears, the EWC decreased from 53% to 47% and the amount of freezing water from 33% to 23%. The decrease in the permeability of water seen with these materials was consistent with the decrease of the freezing water, i.e., the water able to participate in diffusion. Since the content of freezing water determines the transport through hydrogels it can be expected that any lens characteristics that depend upon the amount of this portion of water would be affected by the presence of proteins inside the polymer matrix. We extrapolated that an absolute change of 10% in the amount of freezing water could lead to a decrease in oxygen permeability of as much as 7 Dk units. In view of this work more attention should be given to changes in the properties of lenses during wear, in particular, in the high-water/ionic lenses.

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The lipid of the outermost layer of the skin is confined largely to the extracellular spaces surrounding the corneocytes of the stratum corneum where it forms a multilamellar adhesive matrix to act as the major permeability barrier of the skin. Knowledge of the molecular architecture of these intercellular domains is important for understanding various skin pathologies and their treatment, percutaneous drug delivery, and the cosmetic maintenance of the skin. We have surveyed by X-ray diffraction the structure of the intercellular domains and the extracted lipids of murine stratum corneum (SC) at 25, 45, and 70 degrees C which are temperatures in the vicinity of known thermal phase transitions [Rehfeld, S. J., & Elias, P. M. (1982) J. Invest. Dermatol. 79, 1-3]. The intercellular domains produce lamellar diffraction patterns with a Bragg spacing of 131 +/- 2 A. Lipid extracted from the SC and dispersed in excess water does not produce a simple lamellar diffraction pattern at any temperature studied, however. This and other facts suggest that another component, probably a protein, must be present to control the architecture of the intercellular lipid domains. We have also obtained diffraction patterns attributable to the protein envelopes of the corneocytes. The patterns suggest a beta-pleated sheet organizational scheme. No diffraction patterns were observed that could be attributed to keratin.

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Several amides of cyclic amines were prepared and tested as penetration enhancers in the diffusion of various drugs through hairless mouse skin in vitro. Hexamethylenelauramide (hexahydro-1-lauroyl-1H-azepine) was selected as a broad spectrum penetration enhancer worthy of further study. Later, the duration of the effect of various enhancers on the penetration barrier in vivo was determined by evaluating the in vitro diffusion of hydrocortisone through skins that had been pretreated in vivo. We found that the longer the pretreatment, the smaller the amount of penetrated hydrocortisone. Furthermore, our results suggested that differences exist in the retention of various enhancers in living mouse skin. The in vitro pretreatment experiments revealed that the penetration through dead skin is slow compared with the penetration through living skin. Neither the nature of the receptor phase, nor the increased temperature of the in vitro experiments, explain the striking differences between the in vivo and the in vitro experiments. Finally, the penetration of hydrocortisone through the stratum corneum in the presence of enhancers, as well as the penetration of 1-dodecylhexahydro-2H-azepin-2-one (laurocapram), hexamethylenelauramide, and oleic acid, were determined using a stratum corneum stripping technique. More hydrocortisone penetrated through the stratum corneum during the first 3 h in the presence of hexamethylenelauramide than in the presence of laurocapram or oleic acid.

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