Characterization of collagen gel solutions and collagen matrices for cell culture.

The influence of glutaraldehyde as a crosslinking agent to increase the strength of collagen matrices for cell culture was examined in this study. Collagen solutions of 1% were treated with different concentrations (0-0.2%) of glutaraldehyde for 24 h. The viscoelasticity of the resulting collagen gel solution was measured using dynamic mechanical analysis (DMA), which demonstrated that all collagen gel solutions examined followed the same model pattern. The creep compliance model of Voigt-Kelvin satisfactorily described the change of viscoelasticity expressed by these collagen gel solutions. These crosslinked collagen gel solutions were freeze-dried to form a matrix with a thickness of about 0.2-0.3 mm. The break modulus of these collagen matrices measured by DMA revealed that the higher the degree of crosslinking. the higher the break modulus. The compatibility of fibroblasts isolated from nude mouse skin with these collagen matrices was found to be acceptable at a cell density of 3 x 10(5) cells/cm2 with no contraction, even when using a concentration of glutaraldehyde of up to 0.2%.

Fungal mycelia as the source of chitin and polysaccharides and their applications as skin substitutes.

A wovenable skin substitute (Sacchachitin) made from the residue of the fruiting body of Ganoderma tsugae was developed in this study. Chemical analysis revealed that the treated residue was a copolymer of beta-1,3-glucan (ca 60%) and N-acetylglucosamine (ca 40%) with a filamental structure of mycelia form, as demonstrated by both optical and scanning electron microscopy. The pulp-like white residue was then woven into thin, porous sheets 7.0 cm in diameter and 0.1-0.2 mm in thickness by filtration and lyophilized for use as a skin substitute. The wound area produced by dissecting rat skin of full thickness was found to almost completely heal on the side covered with Sacchachitin, whereas the control side covered with cotton gauge was around 6.0 cm2 on the 28th day. Furthermore, the wound healing effects of the chitin sheet from crab shell (Beschitin) and Sacchachitin were not found to be significantly different.

Development of fungal mycelia as skin substitutes: effects on wound healing and fibroblast.

In this study, Sacchachitin membrane, prepared from the residue of the fruiting body of Ganoderma tsugae, was estimated for its effects on wound healing and the proliferation and migration of fibroblast cells. Two mirror-image wounds were made on the back of female guinea pigs by dissecting a 1.5 x 1.5 cm2 skin surface of full thickness. Sacchachitin membrane was placed randomly on one of the wounds and gauze or Beschitin on the other. Changes in the wound area were measured and photographed after a predetermined amount of time postoperatively. Histological examination of the wound and surrounding tissue was also performed to reveal any interaction of tissue with the dressing. The results showed that the wound area covered with Sacchachitin membrane was statistically smaller than that covering with gauze on day 10, whereas there was no significant difference in the wound size compared to that with Beschitin. Fibroblast cells from the dermis layer of guinea pigs were used. The number of fibroblast cells were counted on the predetermined days in the culture suspended with or without 0.01% w/v dressing materials. By layering on DMEM plates, the number of fibroblast cells migrating across the center line or outside of the central hole were counted after five days. All the results indicated that both 0.01% w/v of Sacchachitin and chitin significantly enhanced the proliferation and migration of fibroblast cells.

Diffusion characteristics of collagen film.

Collagen films prepared by treating collagen gel solutions with different concentrations of glutaraldehyde were evaluated as a biodegradable and biocompatible drug carrier for cosmetically effective agents in this study. The influences of concentration of glutaraldehyde (0, 0.05, 0.075, 0.1, 0.2, 0.25, and 0.3%, v/w) with a fixed concentration (1%, w/w) of collagen on the crosslinking rate of collagen gel solutions and on the crosslinking extent of the collagen contained within were examined by monitoring changes in viscosity. In addition, the influences of the addition of different model drugs (retinoic acid, retinol palmitate, ascorbic acid 6-palmitate, and tocopherol acetate) on viscosity changes of collagen gel solutions were compared. The results demonstrate that the maximal viscosity of collagen gel solutions increases with increasing concentrations of glutaraldehyde. When the concentration of glutaraldehyde exceeds 0.2%, the maximal viscosity of collagen gel solutions reaches a plateau. However, model drugs showed insignificant effects on viscosity changes of collagen gel solutions. The diffusion characteristics of collagen films prepared from those gel solutions crosslinked with different concentrations of glutaraldehyde were assessed using two different matrix forms of solution or gel for the model drugs in a flow-through diffusion system. The matrix effect on the flux of model drugs from both solution and gel matrix through collagen films was inconclusive. However, both fluxes show the same tendency to decrease when the concentration of glutaraldehyde used for crosslinking is increased. However, when the concentration of glutaraldehyde exceeds 0.2%, these model drugs, except retinoic acid, show similar diffusion characteristics across the collagen films.

Influence of pluronic F-68 on dissolution and bioavailability characteristics of multiple-layer pellets of nifedipine for controlled release delivery.

A multiple-layer design of pellets for nifedipine was developed using pluronic F-68 to enhance dissolution rate. The influence of ratios of nifedipine in the inner layer to that in the outer layer, the ratios of pluronic F-68 to nifedipine in the solid dispersion, and the thickness of the control membrane on dissolution characteristics were investigated. With an increasing ratio of pluronic F-68 to nifedipine, the dissolution rate of nifedipine was gradually promoted and the extent of release was enhanced as well. DSC thermograms illustrate the gradual disappearance or broadening of the nifedipine melting peak with the presence of pluronic F-68. The decrease of the nifedipine ratio in the inner layer and the increase of the ratio of pluronic F-68 to nifedipine in the outer layer can enhance the release of nifedipine. With a fixed nifedipine ratio of 1.5 between the inner layer and the outer layer, increasing the ratio of pluronic F-68 to nifedipine in the outer layer significantly increased the initial release rate of nifedipine. By increasing the nifedipine ratio of the inner layer to the outer layer to 1:1, the increase of coating percentage referenced to the total weight decreased the release rate of nifedipine from the inner layer. The pharmacokinetic bioequivalence between the test product (Cardilate, N-6) and Coracten was found with a multiple-dose oral administration of 20 mg in 12 healthy, normal Chinese male volunteers.

Investigation of the percutaneous penetration of prostaglandin E1 and its ethyl ester.

The optimization of percutaneous delivery of PGE1 and its ethyl ester in alcoholic buffer solution through hairless mouse skin was investigated. A reversed-phase HPLC system with a photodiode array detector was used to differentiate the UV spectra of the penetration products. By comparison of the UV spectrum for each chromatographic peak, the conversion of PGE1 ethyl ester to PGE1 by enzymatic hydrolysis was found to be the predominant degradation pathway during the in vitro penetration. The quantification of ethyl ester was developed based on the same principle as that for PGE1. It was then applied to monitor the penetration of prostaglandins through hairless mouse skin from the vehicles containing various fractions of alcohol. Results demonstrated that the alkyl group promoted the penetration mainly as a result of enhancing the drug partitioning into the stratum corneum at its maximal thermodynamic activity. The alcohol fraction around 20% seemed to be optimal for the percutaneous delivery of the ethyl ester. The use of collagen gel to carry PGE1 ethyl ester for percutaneous application was included for comparison. The effect of adding alcohol in the collagen gel on the penetration of PGE1 ethyl ester was found to be slightly lower than that from the same vehicle without collagen.

Penetration enhancement by menthol combined with a solubilization effect in a mixed solvent system.

The improvement in solubility of indomethacin due to the presence of menthol in various cosolvent systems consisting of water, alcohol and propylene glycol was examined by a mixture design in this study. A proper model to quantitatively describe the effect of menthol at different concentrations on the solubility of indomethacin was compared based on the statistical parameters provided by DESIGN-EXPERT. Then three cosolvent systems with the addition of menthol to solubilize indomethacin to extents of 1.0, 1.5 or 2.0% w/v were selected. The penetration of indomethacin through nude mouse skin from these three cosolvent systems with the addition of 0-12% menthol was investigated and followed by a discussion on the penetration mechanism. The results showed that menthol was able to improve drug solubility to different extents for different cosolvent systems. Optimally, a cosolvent system with an equal ratio of the three solvents, water, alcohol and propylene glycol, showed the highest extent of improvement in the solubility at all concentrations of menthol. The enhancement factors for indomethacin penetration due to menthol in different cosolvent systems were compared, based either on the permeation coefficient (Kp) or the separate overall effects on the skin (Flux). Both comparisons gave similar results. The influence of menthol was more significant compared to that of the cosolvent systems and the extent of this influence increased with an increase in the amount added, reaching a maximum at a specific amount of menthol for each different cosolvent system.

Film-forming polymer-granulated excipients as the matrix materials for controlled release dosage forms.

Lactose and dibasic calcium phosphate (DCP) were granulated with various concentrations of film-forming polymers by a stepwise spraying method to prepare a directly compressible matrix excipient. The film-forming polymeric latex of Eudragit RS-30D, Eudragit RL-30D, and Surelease (ethylcellulose) were used in this study as the source of the granulating materials. Better flowability and compressibility were observed for all the granulated particles than the polymer-free granules. Most tablets prepared from the polymer-granulated particles exhibited satisfactory friability of less than 1% except for those prepared from lactose particles granulated with low concentrations of ethylcellulose and from plain lactose granules. Change in tensile strength and tablet thickness were in good agreement with the plasticity of the granulating polymer. Polymer-granulated lactose and DCP provided for controlled release of captopril from matrix tablets. This investigation suggests that conventional excipients can be modified by a simple granulating procedure to provide better physical properties for being used as a matrix material.

The percutaneous penetration of prostaglandin E1 and its alkyl esters.

The percutaneous delivery of PGE1 and its alkyl esters in alcoholic saline solution through hairless mouse skin was compared. The quantification of alkyl esters was based on the same principle as that for PGE1, which was converted to PGB1 to enhance the sensitivity and minimize the interference. Results showed that it was PGE1 that appeared in the receiver compartment for all alkyl esters examined. The flux of all alkyl esters of PGE1 in the same concentration was higher than PGE1 itself at most of saline vehicle with various fractions of alcohol. The maximal flux for a fixed concentration of each alkyl ester appeared at different fractions of alcohol. When the fractions of alcohol was kept constant, the alkyl ester that showed the maximal flux at this concentration appeared to have a longer chain length with increasing the fraction of alcohol. But isopropyl ester deviated from this order. It was concluded that the alkyl ester derivatives promoted the penetration of PGE1 mainly as a result of enhancing the drug partitioning into the stratum corneum. The alcohol fraction that needed to achieve the maximal flux at the same concentration increased with the increase of alkyl chain length, which resulted in the decrease of solubility parameter. It is necessary to optimize the fraction of alcohol in the saline solution in order to achieve the maximal flux at a fixed concentration for these alkyl esters with different alkyl chain length.

Modification of physical characteristics of microcrystalline cellulose by codrying with beta-cyclodextrins.

In an attempt to modify the physical properties of microcrystalline cellulose (MCC), the slurry form of this material was codried with beta-cyclodextrin (beta-CD). MCC slurry was blended with beta-CD at concentrations of 10%-50% w/w as a dried mass relative to MCC. The mixtures were then granulated with water and codried at 60 degrees C for 12 h or until a constant weight was reached. Codried granules were pulverized, and the fraction between 61 and 150 microns in size was reserved. The powder and tableting properties of the codried products were compared to those of various grades of MCC and the corresponding components and physical mixtures. The results showed that the products of MCC codried with beta-CD significantly improved the flowability of MCC powder. It is probable that the improved flowability was due to the more rounded shape of particles formed with this codried process, which was confirmed by SEM photographs. Moreover, the compactibility and disintegration properties of tablets produced from the codried products were even better than those using MCC alone, physical mixtures, or various grades of MCC. MCC in a slurry form was more efficient than the existing MCC products in achieving these results, which is postulated to be due to the greater amount of water required and the higher solubility of beta-CD in water promoting the interaction between beta-CD and MCC during granulation. In conclusion, MCC codried with beta-CD provides a useful excipient for direct compression.

Preparation of microemulsions using polyglycerol fatty acid esters as surfactant for the delivery of protein drugs.

Phase diagrams containing the microemulsion region were constructed for pseudo-ternary systems composed for polyglycerol fatty acid ester/cosurfactant/Captex 300/water. It was found necessary to add ethanol, 1-propanol, 1-butanol as cosurfactant to produce microemulsions. The results also demonstrated microemulsions were only able to form when employing polyglycerol fatty acid esters with hydrophile-lipophile balances (HLBs) between 8 and 13, such as MO500, MO750, SO750, and ML310. Most microemulsions were determined to be Winsor type IV by dilution and dye solubility tests. Microemulsions stored at ambient temperature maintained constant viscosity, indicating that the system was thermodynamically stable for long periods. Further, several microemulsion formulations were demonstrated to be promising for oral delivery of insulin based on the results of stability tests and acid-protection efficiency.

A statistical design to evaluate the influence of manufacturing factors on the material properties and functionalities of microcrystalline cellulose.

The aim of this study is to statistically evaluate the effects of manufacturing factors on the material properties and functionalities of microcrystalline cellulose (MCC) products. How the material properties of MCC products dominate their functionalities was further explored. Results demonstrate that the desired material properties and functionalities of MCC products can be obtained by manipulation of the manufacturing factors using proper polynomial equations, and the key manufacturing factor is temperature. On the other hand, the functionalities can be quantitatively predicted by material properties. Meanwhile, the key material property is molecular mass in controlling MCC functionalities. The particle morphologies may also serve as important material properties. In conclusion, the careful control of temperature during the manufacture of MCC might minimize inter-batch variation. The correlation of the material properties of MCC products with their functionalities might help the formulation designer rationally select proper MCC products. The universal harmonization of MCC products might be achieved by the regulation of their molecular mass, surface roughness, and roundness.

In-vitro and in-vivo studies of the diclofenac sodium controlled-release matrix tablets.

Controlled release matrix tablets for diclofenac sodium were developed in this study. Five matrix-tablet formulations were prepared by granulating two viscosity grades of HPMC (hydroxylpropylmethylcellulose) in varying ratios with water in the planetary mixer. The in-vitro dissolution tests indicate that all five matrix formulations prolong the release of diclofenac sodium. The main factors controlling drug release were the HPMC viscosity grade and the amount of HPMC used. The larger the amount of high viscosity grade HPMC used, the slower the resultant release rate of diclofenac sodium. There was no significant degradation of diclofenac sodium or change in drug release rate in any of the five formulations during a three-month period of stability testing. The sustained release ability of four formulations was further demonstrated in an in-vivo study in six healthy subjects. There were in-vitro/in-vivo correlations between Cmax, AUC0-14, and the time for 50 or 80% drug to be released.

Studies on the in-vitro percutaneous penetration of indomethacin from gel systems in hairless mice.

The influence of co-solvents on the in-vitro percutaneous penetration of indomethacin from gel systems was studied using a simplex lattice experimental design. Gel formulations were prepared by gelling the vehicle mixture of water, either alcohol or isopropanol and either propylene glycol or PEG 400 with 1% w/w Carbomer 940. Hairless mouse skin was employed as the barrier in a Franz-type diffusion cell. The penetration rates at steady state for seven formulations were fitted to a polynomial equation based on this simple lattice method and a three-dimensional plot was constructed. The formulation having the maximal penetration rate was determined to be the vehicle with a solvent ratio of water: alcohol: propylene glycol equal to 15:33:52, and which possessed a solubility parameter of 15 and a drug solubility of around 10 mg mL-1. When the solubility parameter of the vehicle was > 15, the drug solubility increased. However, the penetration rate decreased with an increasing solubility parameter. For those vehicles with a solubility parameter < 15, both the drug solubility and the penetration rate decreased with a decrease in the solubility parameter. There was shown to be an approximately 20-fold increase in the relative enhancement factor when using both alcohol and isopropanol, but only a threefold increase for both propylene glycol and PEG 400, when compared with water.

The influence of cosolvents on the in-vitro percutaneous penetration of diclofenac sodium from a gel system.

The influence of cosolovents on the in-vitro percutaneous penetration of diclofenac sodium from a gel system was studied using a simplex lattice experimental design. Gel formulations were prepared by gelling the vehicle mixture of water, alcohol and propylene glycol with Carbomer 940. The synthetic membrane Durapore and hairless mouse skin were employed as barriers in a Franz-type diffusion cell. It was found that the penetration through the synthetic membrane was well described by the Higuchi model. There existed a better inverse relationship between the penetration rate and the drug solubility in the respective vehicle. It appeared to be a membrane-controlled mechanism when using hairless mouse skin as the barrier. The penetration rates in steady-state for nine formulations were fitted to a polynomial equation based on this simplex lattice method. A three-dimensional plot was constructed in this simplex surface studied. The maximal penetration rate was found to be from the vehicle containing water and ethanol in an exact volume ratio of 3:1 and the minimal penetration rate was observed from the vehicle containing water only.

High-performance liquid chromatographic analysis for the characterization of triterpenoids from Ganoderma.

A high-performance liquid chromatographic (HPLC) analysis of triterpenoids from Ganoderma is developed and validated in an attempt to explore a way to differentiate a number of species of the genus Ganoderma. Results show that 64 samples examined in this study could be divided into 18 groups based on characteristics of the HPLC pattern of triterpenoids. This result also conforms with those of the morphological examination and the interfertility test by di-monokaryotic mating. The HPLC analysis of triterpenoids further reveals that differentiation among samples from three different regions each of the two species G. lucidum and G. tsugae is workable. Even then, an incorrect designation is found for two of the groups of samples that were originally classified as G. resinaceum but showed different morphological characteristics and mating incompatibility. In conclusion, an HPLC analysis of triterpenoids is a simple and easy way to differentiate among different species of the genus Ganoderma.

Papaverine and prostaglandin E1 gel applications for impotence.

The transdermal permeability of papaverine and prostaglandin E1 (PGE1) could be determined in vitro. A chemical scheme of passive delivery or with enhancer, as well as a physical iontophoretic transdermal delivery system were designed in the laboratory. Clinically, up to 20% papaverine base gel and 500 micrograms PGE1 were applied onto the genital area without any side effect. Only vascular response could be detected by ultrasound. However, immediate erection could not be obtained at the present dosage. Further research on the combination of skin absorption enhancers, the different chemical derivatives of PGE1 or a different administration route is warranted.

Prolongation of drug release by covalent bonding of drugs to serum albumin microbeads.

Reaction conditions for the covalent bonding of 5'-deoxy-5-fluorouridine to serum albumin microbeads by means of a water-soluble carbodiimide were studied. Optimum coupling of dFUR to the microbeads occurred when pure water was used as solvent. There was no significant difference in the bonding efficiency for microbeads prepared at different stirring speeds, and there was a limit to the amount of dFUR that could be bound with increasing reaction time. Yields were low possibly because of competing coupling reactions involving carbodiimide and other reactive groups in the protein. The release of dFUR from dFUR-bound microbeads was slow and biexponential. The fraction of dFUR bound in the interior of the microbeads increased with increasing reaction time.

Fibrin based drug delivery systems.

Fibrinogen is the natural substrate for thrombin, the final proteolytic enzyme of the blood coagulation cascade. Following reaction with thrombin, fibrinogen is converted into fibrin monomers which then spontaneously polymerize into fibrin threads forming the fibrin matrix. This biochemical reaction between fibrinogen and thrombin has been used to produce natural, biodegradable, biocompatible systems for sustained drug delivery. Three potential delivery systems have been explored: 1) fibrin microparticles with drug entrapped are produced by an emulsion process, 2) reaction between fibrinogen adsorbed on suspended drug and thrombin in solution results in the formation of fibrin-coated drug particulates, and 3) fibrin sheets suitable as implants have been cast with drug entrapped. The drug release behavior of both small and large molecular weight substances from these systems was evaluated.

Development of a high-performance liquid chromatographic method for bioequivalence study of flavoxate tablets.

An improved HPLC method was developed for the concentration determination of the metabolite of flavoxate, 3-methyl-flavone-8-carboxylic acid (MFCA), in plasma in an attempt to compare two flavoxate tablet formulations. This HPLC method was validated by examining the precision and the accuracy for inter-day and intra-day runs in a linear concentration range of 0.1-24 microg/ml. The coefficients of variation (C.V.) of inter-day and intra-day assays were 0.24-7.18% and 0.06-5.70%, respectively. The standard errors of mean (S.E.M.) were -0.004-8.68% and -2.52-4.86% for inter-day and intra-day assays, respectively. Bioequivalence of the two formulations was determined on 12 normal healthy male volunteers in a single-dose, two-period, two-sequence, two-treatment crossover study. MFCA plasma concentrations were analyzed with this validated HPLC method. The normal pivotal parameters, AUC(0-last), AUC(0-inf) and Cmax, were calculated and compared using the SAS General Linear Model computer program. The two one-sided t distribution test was also performed, as well as the 90% confidence-interval method, for the mean difference of the three pivotal parameters. The results suggest that these two flavoxate tablet formulations are non-bioequivalent when orally administered in a 400-mg dose of two tablets. This result was consistent with the in vitro dissolution of these two formulations.