Suppression of transforming growth factor-ß effects in rabbit subconjunctival fibroblasts by activin receptor-like kinase 5 inhibitor.

PURPOSE: Transforming growth factor-ß (TGF-ß) activity has been implicated in subconjunctival scarring in eyes following glaucoma filtration surgery (GFS). The purpose of this study is to determine whether an inhibitor for activin receptor-like kinase (ALK) 5 (also known as TGF-ß receptor type I) could suppress TGF-ß activity and thereby promote filtering bleb survival after GFS in a rabbit model. METHODS: An ALK-5 inhibitor, SB-505124, was used. A docking study was performed to investigate the interaction between the inhibitor and the receptor. Immunofluorescence for connective tissue growth factor (CTGF) and α-smooth muscle actin (α-SMA) was performed in cultured rabbit subconjunctival fibroblasts. Immunoblotting for phosphorylated Smad2 (pSmad2), CTGF, and α-SMA was also performed. In an in vivo rabbit GFS model, SB-505124 was delivered in a lactose tablet during surgery. Eyes were examined by slit-lamp and intraocular pressure (IOP) was measured until the time of bleb failure or up to 28 days after surgery. Tissue sections on day 5 after surgery were histologically evaluated after staining with hematoxylin and eosin. The sections were also immunostained for CTGF and α-SMA. In addition, cell outgrowth from dissected subconjunctival tissues placed in a cell culture flask with media was investigated. RESULTS: The docking study indicated hydrogen bond interactions between SB-505124 and amino acids His-283 and Ser-280 of ALK-5. Suppression of pSmad2, CTGF, and α-SMA by SB-505124 was observed in cultured fibroblasts. Filtering blebs in the GFS with SB-505124 group were maintained for more than 10 days, and the period of bleb survival was significantly longer than that in controls. IOP levels after surgery seemed to be related to bleb survival. Histologically, subconjunctival cell infiltration and scarring at the surgical site in the GFS with SB-505124 and mitomycin C (MMC) groups were much subsided compared to controls. Suppression of CTGF and α-SMA by SB-505124 was also observed by immunofluorescence. Cell outgrowth from explants dissected from eyes to which SB-505124 was applied during GFS was robust while outgrowth was poor from those treated with MMC. CONCLUSIONS: The ALK-5 inhibitor SB-505124 was efficacious both in vitro and in vivo in suppressing the TGF-ß action. The inhibitor may provide a novel therapy for preventing ocular inflammation and scarring.

Investigations on factors affecting chitosan for dissolution enhancement of oxcarbazepine by spray dried microcrystal formulation with an experimental design approach.

In the present work effect of chitosan on microcrystal formulation for dissolution enhancement of oxcarbazepine using controlled crystallization technique coupled with spray drying was explored. The work was extended for exploration of simplified approach for stable particle size reduction. The study was performed with an experimental design approach i. e. a fractional factorial design of resolution 5 (with all 2 factor interaction) for the screening of predefined independent variables drug concentration, chitosan concentration, feed rate, inlet temperature and percent aspiration for spray drying. Whereas percent drug dissolved, wettability time, flowability in terms of angle of repose and particle size were designated as response variables. Resultant models were analyzed using multiple linear regression analysis, which generated equation to plot response surface curves along with desirability function. Results showed that chitosan concentration had significant effect on dissolution enhancement of oxcarbazepine at a level of 2% w/v. Increase in drug concentration showed decreased dissolution rate however on particle size it did not show statistically significant effect. Topographical characterization was carried out by SEM which showed that feed rate, percent aspiration and inlet temperature had significant effect on particle morphology. For deriving optimized formulation results were analyzed using desirability function for the maximum percent drug dissolved and least drug polymer matrix particle size. DSC studies showed that drug was molecularly associated with chitosan matrix or particles.

Nateglinide quantification in rabbit plasma by HPLC: optimization and application to pharmacokinetic study.

A rapid, simple, and sensitive HPLC method with UV detection was developed and validated for the determination of nateglinide (NTG) from rabbit plasma. The retention behavior of NTG and gliclazide (GLZ, internal standard-IS) as a function of mobile phase pH, composition and flow rate was investigated. Separation was developed on a reverse-phase C(18) column (250 mm x 4.6mm i.d., 5 microm particle size), using a mixture of acetonitrile (ACN):10mM phosphate buffer (PBS, pH 3.0) in the ratio of 70:30(%v/v) at a flow rate of 1.0 ml/min with UV detection at 203 nm within 8 min, and quantified based on drug/IS peak area ratios. The plasma samples were prepared by a simple deproteinization with a mixture of methanol and acetonitrile, yielding more than 97.86% extraction efficiencies. The calibration curve was linear (correlation coefficient of 0.9984) in the concentration range of 10-2500 ng/ml. The limit of detection (LoD) and limit of quantitation (LoQ) were found to be 2.91 and 9.70 ng/ml, respectively. Both the intra-day and inter-day precisions at four tested concentrations were below 1.32% R.S.D. The present method was selective enough to analyze NTG in rabbit plasma without any tedious sample clean-up procedure and was successfully applied for estimating the pharmacokinetic parameters of NTG following oral administration of a single 15 mg NTG to white albino rabbits.

Reversed chitosan-alginate polyelectrolyte complex for stability improvement of alpha-amylase: optimization and physicochemical characterization.

The present work explores, using response surface methodology, the main and interaction effects of some process variables on the preparation of a reversed chitosan-alginate polyelectrolyte complex (PEC) with entrapped alpha-amylase for stability improvement. A 3(3) full factorial design was used to investigate the effect of the chitosan and alginate concentrations and hardening time on the percent entrapment, time required for 50% (T(50)) and 90% (T(90)) enzyme release, and particle size. The beads were prepared by dropping chitosan containing alpha-amylase into a sodium alginate solution without any salt. The in vitro enzyme release profile of the beads was fitted to various release kinetics models to study the release mechanism. A topographical characterization was carried out using scanning electron microscopy (SEM), and the entrapment was confirmed using Fourier transform infrared (FTIR) spectroscopy and differential scanning calorimetry (DSC). Stability testing was carried out according to the International Conference on Harmonization (ICH) guidelines for zones III and IV. Beads prepared using 2.5%w/v chitosan and 3%w/v sodium alginate with a hardening time of 60 min had more than 90% entrapment and a T(90) value greater than 48 min. Moreover, the shelf-life of the enzyme-loaded beads was found to increase to 3.68 years, compared with 0.99 years for the conventional formulation. It can be inferred that the proposed methodology can be used to prepare a reversed PEC of chitosan and alginate with good mechanical strength, provided both the reactants are in a completely ionized form at the time of the reaction. Proper selection of the reaction pH, polymer concentration and hence charge density, and hardening time is important and determines the characteristics of the PEC.

Physicochemical characterization of papain entrapped in ionotropically cross-linked kappa-carrageenan gel beads for stability improvement using Doehlert shell design.

This work examines the influence of various process parameters on papain entrapped in cross-linked kappa-carrageenan beads for improvement of its stability. A Doehlert shell design (DSD) was employed to investigate the effect of three process variables, namely kappa-carrageenan concentration, KCl concentration, and hardening time, on the entrapment, time required for 50% enzyme release (T50), time required for 90% enzyme release (T90), and particle size. The beads were prepared by dropping the kappa-carrageenan containing papain into a magnetically stirred KCl solution. Topographical characterization was carried out by scanning electron microscopy and entrapment was confirmed by Fourier transform infrared spectroscopy and differential scanning calorimetry. Stability testing was carried out according to the International Conference on Harmonization (ICH) guidelines for zone III and IV. A polymeric matrix was prepared with kappa-carrageenan (3.5% w/v) and potassium chloride (0.5 M) using the ionotropic gelation method, with a hardening time of 20 min. Beads characterized by a spherical disc shape with a collapsed center, an absence of aggregates, an entrapment of 82.75%, a T90 value of 55.36 min, and a composite index of 88.55 were produced. The shelf-life of the enzyme-loaded beads was found to increase to 3.63 years compared with 1.01 years for the conventional formulation. It can be inferred that the proposed methodology can be used to prepare papain-loaded kappa-carrageenan beads for stability improvement.

Stability improvement of alpha-amylase entrapped in kappa-carrageenan beads: physicochemical characterization and optimization using composite index.

PURPOSE: This work examines the influence of various process parameters on alpha-amylase entrapped in crosslinked kappa-carrageenan beads for stability improvement. A three level full factorial design was employed to investigate the effect of three process variables namely kappa-carrageenan concentration, potassium chloride concentration and hardening time on % entrapment, time required for 50% (T50) and 90% (T90) of enzyme release and particle size. METHODS: The beads were prepared by dropping the kappa-carrageenan-containing alpha-amylase to magnetically stirred potassium chloride solution. The composite index was applied to optimize the process under study. 'In vitro' enzyme release profile of the beads was fitted to various release kinetics models to understand the release mechanism. Topographical characterization was carried out by SEM and entrapment was confirmed by FTIR and DSC. Stability testing according to the ICH guidelines for zone III and IV was carried out. RESULTS: With the use of ionotropic gelation method, a polymeric matrix prepared by 3.5% (w/v) kappa-carrageenan, 0.7 M potassium chloride and hardening time of 30 min resulted in the production of beads characterized by disc shaped with collapsed center, absence of aggregates, % entrapment of 73.79, T90 of 74.4 min, and composite index of 83.01. Moreover, shelf-life of the enzyme loaded beads was found to increase up to 3.53 years compared to 0.99 year of the conventional formulation. CONCLUSIONS: It can be inferred that the proposed method can be used to prepare alpha-amylase loaded kappa-carrageenan beads for stability improvement. Also the proper selection of rate-controlling carrageenan concentration and its interactive potential for crosslinking is important and will determine the overall size and shape of beads, the duration and pattern of dissolution profiles and enzyme loading capacity.

Papain entrapment in alginate beads for stability improvement and site-specific delivery: physicochemical characterization and factorial optimization using neural network modeling.

This work examines the influence of various process parameters (like sodium alginate concentration, calcium chloride concentration, and hardening time) on papain entrapped in ionotropically cross-linked alginate beads for stability improvement and site-specific delivery to the small intestine using neural network modeling. A 3(3) full-factorial design and feed-forward neural network with multilayer perceptron was used to investigate the effect of process variables on percentage of entrapment, time required for 50% and 90% of the enzyme release, particle size, and angle of repose. Topographical characterization was conducted by scanning electron microscopy, and entrapment was confirmed by Fourier transform infrared spectroscopy and differential scanning calorimetry. Times required for 50% (T(50)) and 90% (T(90)) of enzyme release were increased in all 3 of the process variables. Percentage entrapment and particle size were found to be directly proportional to sodium alginate concentration and inversely proportional to calcium chloride concentration and hardening time, whereas angle of repose and degree of cross-linking showed exactly opposite proportionality. Beads with >90% entrapment and T(50) of <10 minutes could be obtained at the low levels of all 3 of the process variables. The inability of beads to dissolve in acidic environment, with complete dissolution in buffer of pH >or=6.8, showed the suitability of beads to release papain into the small intestine. The shelf-life of the capsules prepared using the papain-loaded alginate beads was found to be 3.60 years compared with 1.01 years of the marketed formulation. It can be inferred from the above results that the proposed methodology can be used to prepare papain-loaded alginate beads for stability improvement and site-specific delivery.

Effect of pH on in vitro permeation of ondansetron hydrochloride across porcine buccal mucosa.

The influence of drug concentration, pH in donor chamber, and 1-octanol/buffer partition coefficient on transbuccal permeation of ondansetron hydrochloride (pKa, 7.4) across porcine buccal mucosa was studied by using an in-line Franz type diffusion cell at 37 degrees C. The pH was adjusted to several values and the solubility of the drug in different pH was measured. Solubility of ondansetron hydrochloride decreases with increasing pH. The permeability of the drug was evaluated at different donor pH and drug concentrations. Permeability of un-ionized (Pu) and ionized (Pi) species of drug was calculated by fitting the data to a mathematical model. The steady state flux increased linearly with the donor concentration (r2 = 0.9843) at pH 7.4. The permeability coefficient and the partition coefficient of the drug increased with increasing pH. The values of Pu and Pi were 4.86 x 10(-6) cm/sec and 7.18 x 10(-7) (c)m/sec, respectively. The observed permeability coefficients and the permeability coefficients calculated from the mathematical model at various pH showed good linearity (r2 = 0.9799). The total permeability coefficient increased with increasing the fraction of un-ionized form of the drug. The drug permeated through buccal mucosa by a passive diffusion process. The non-ionized species of drug penetrated well through buccal mucosa and the permeation was a function of pH. Transbuccal delivery is a potential route for the administration of ondansetron hydrochloride.

Transbuccal delivery of lamotrigine across porcine buccal mucosa: in vitro determination of routes of buccal transport.

PURPOSE: To determine the major routes of buccal transport of lamotrigine and to examine the effects of pH on drug permeation. METHODS: Transbuccal permeation of lamotrigine across porcine buccal mucosa was studied by using in-line Franz type diffusion cell at 37 degrees C. The permeability of lamotrigine was determined at pH 4.0 to 9.0. The permeability of unionized (Pu) and ionized (Pi) species of drug were calculated by fitting the data to a mathematical model. Lamotrigine was quantified by using the HPLC method. RESULTS: The steady state flux increased linearly with increasing the donor concentration (r2 = 0.9639) at pH 7.4. The permeability coefficient and the partition coefficient of the drug increased with increasing the pH. The values of Pu and Pi were 0.7291 x 10(-5) cm/sec and 0.2500 x 10(-5) cm/sec, respectively. The observed permeability coefficients and the permeability coefficients calculated from mathematical model at various pH showed good linearity (r(2) = 0.9267). The total permeability coefficient increased with increasing the fraction of unionized form of the drug. CONCLUSION. Lamotrigine permeated through buccal mucosa by a passive diffusion process. The partition coefficient and pH dependency of drug permeability indicated that lamotrigine was transported mainly via the transcellular route by a partition mechanism.

Estimation of trace amounts of chromium(III) in multi-vitamin with multi-mineral formulations.

Two new specific, selective, simple and inexpensive spectroscopic methods for estimating a trace amount of chromium (Cr3+) from a multi-vitamin with multi-mineral pharmaceutical formulations were developed. The proposed methods are based on the conversion of Cr3+ to Cr6+ either by oxidation with a nitric acid-perchloric acid mixture (method I) or by fusion with an excess amount of sodium carbonate (method II), followed by the complexation of Cr6+ with 1,5-diphenylcarbazide (DPC) in a mineral acidic solution of pH 1.0 +/- 0.5. The pink-colored complex was estimated at 544 nm. Both methods were found to be linear in the range of 0.1 - 0.8 microg/ml with a limit of detection in the range of 0.0123 - 0.0157 microg/ml and a limit of quantitation in the range of 0.0419 - 0.0525 microg/ml. Method I was found to be suitable for estimating Cr3+ species in various formulations, like tablets, capsules and syrups, while method II was found to be suitable for tablets and capsules. Satisfactory recovery from spiked samples of standard Cr3+ suggests no interference of any excipients and diverse ions present in the formulations. The developed methods were compared with AAS by ANOVA, and no significant difference was observed.