Influence of In Situ Calcium Pectinate Coating on Metoprolol Tartrate Pellets for Controlled Release and Colon-Specific Drug Delivery.

In situ calcium pectinate-coated pellets were proposed by applying an alternate coating method to drug-layered pellets to achieve colon-specific drug delivery. Solution layering of metroprolol tartrate, a water-soluble model drug, on inert core pellets was achieved using a centrifugal granulator followed by successive alternate coating with pectin and calcium chloride layers using a fluidized bed bottom spray coater. The effect of the coating sequence on the drug release was studied in phosphate buffer pH 7.4 and 6.0. These test conditions were used to mimic the physiological environments in the distal small intestine and proximal colon, respectively. The results showed that the in situ calcium pectinate layer was successfully generated from the alternate coating of pectin and calcium layers after hydration to form gelation, which was able to control the drug release. The coating sequence played an important role in the drug release. The outermost pectin layer tended to retard the drug release whilst the outermost calcium layer accelerated the release regardless of the number of coating layers. These findings indicate that the release behavior followed the Higuchi model, with the drug release from the coated pellets described by a diffusion control mechanism. It is concluded that the success of the in situ calcium pectinate-coated pellets in controlling the drug release is due to the coating of the outermost layer with pectin and the maintenance of the optimum ratio of calcium to pectin upon hydration.

Development of phyllanthin-loaded self-microemulsifying drug delivery system for oral bioavailability enhancement.

Phyllanthin, a poorly water-soluble herbal active component from Phyllanthus amarus, exhibited a low oral bioavailability. This study aims at formulating self-microemulsifying drug delivery systems (SMEDDS) containing phyllanthin and evaluating their in-vitro and in-vivo performances. Excipient screening was carried out to select oil, surfactant and co-surfactant. Formulation development was based on pseudo-ternary phase diagrams and characteristics of resultant microemulsions. Influences of dilution, pH of media and phyllanthin content on droplet size of the resultant emulsions were studied. The optimized phyllanthin-loaded SMEDDS formulation (phy-SMEDDS) and the resultant microemulsions were characterized by viscosity, self-emulsification performance, stability, morphology, droplet size, polydispersity index and zeta potential. In-vitro dissolution and oral bioavailability in rats of phy-SMEDDS were studied and compared with those of plain phyllanthin. Phy-SMEDDS consisted of phyllanthin/Capryol 90/Cremophor RH 40/Transcutol P (1.38:39.45:44.38:14.79) in % w/w. Phy-SMEDDS could be emulsified completely within 6 min and formed fine microemulsions, with average droplet range of 27-42 nm. Phy-SMEDDS was robust to dilution and pH of dilution media while the resultant emulsion showed no phase separation or drug precipitation after 8 h dilution. The release of phyllanthin from phy-SMEDDS capsule was significantly faster than that of plain phyllanthin capsule irrespective of pH of dissolution media. Phy-SMEDDS was found to be stable for at least 6 months under accelerated condition. Oral absorption of phyllanthin in rats was significantly enhanced by SMEDDS as compared with plain phyllanthin. Our study indicated that SMEDDS for oral delivery of phyllanthin could be an option to enhance its bioavailability.

Intranasal chitosan-DNA vaccines that protect across influenza virus subtypes.

An egg-free and broadly effective influenza vaccine that can be produced rapidly, adequately and cost-effectively is needed. In this study, chitosan-associated DNAs prepared at various nitrogen/phosphate charge (N/P) ratios were studied for their physicochemical properties, stability, cytotoxicity, and protein expression ability. The chitosan-DNA complexes (chitoplexes) of the N/P ratio 2 had the required characteristics including optimal size range, positive surface charge, high DNA association efficiency, tolerability to DNase digestion and mammalian cell viability compatibility. The N/P ratio 2-chitoplexes revealed the highest green fluorescent protein and luciferase expressions in the transfected mammalian cell cultures and in the mouse lungs. Mice immunized intranasally with the N/P ratio 2-chitoplex vaccines carrying DNAs coding for conserved proteins of influenza virus, i.e., ion channel protein (M2) and/or nucleoprotein (NP), had both mucosal and systemic humoral as well as cell mediated immune responses to the in vivo expressed antigens which conferred protection in mice against the lethal challenges not only with the homologous influenza virus subtype (H1N1), but also the heterologous subtype (H3N2). The chitoplexes should be considered as influenza vaccine candidates especially during the period of high vaccine demand. They are suitable for developing areas of the world where conventional vaccine production capacity is lacking.

Physicochemical characterization of phyllanthin from Phyllanthus amarus Schum. et Thonn.

Phyllanthin is a major bioactive lignan component of Phyllanthus amarus, with several known biological activities. This study dealt with the isolation and physicochemical characterization of phyllanthin. Phyllanthin was isolated from P. amarus leaves by column chromatography and purified by recrystallization to obtain phyllanthin crystals with a purity of more than 98%. UV, IR, MS, (1)H NMR and (13)C NMR spectra were employed to identify phyllanthin. The physicochemical properties of phyllanthin were characterized using differential scanning calorimetry, thermogravimetric analysis, X-ray diffraction, pH-solubility, ionization property and lipophilicity. The results indicated that phyllanthin crystals had the melting point and melting enthalpy range of 96.67-97.03 °C and 109.61-116.34 J/g, respectively. Three kinds of phyllanthin crystals, recrystallized by petroleum ether, absolute ethanol and 25% ethanol solution, showed only one polymorph and no polymorphic impurity. Phyllanthin in a solid state was found to undergo significant thermal decomposition above 200 °C. The compound demonstrated good stability in aqueous solution over a pH range of 1.07-10.02 for at least 4 h. The solubility of phyllanthin appeared to be pH-independent of pH range from 1.07 to 10.26. Ionization property studied by absorbance spectroscopy method was in agreement with the result of pH-solubility study, showing that phyllanthin has no pKa over a pH range of 1.12-10.02. The log Pow value of phyllanthin was found to be 3.30 ± 0.05 at pH 7.48, suggesting that phyllanthin may have good permeability through biological membranes. The findings could be useful tools for the development of stable and bioavailable oral dosage forms of phyllanthin.

Chitosan nanoparticle encapsulated hemagglutinin-split influenza virus mucosal vaccine.

Subunit/split influenza vaccines are less reactogenic compared with the whole virus vaccines. However, their immunogenicity is relatively low and thus required proper adjuvant and/or delivery vehicle for immunogenicity enhancement. Influenza vaccines administered intramuscularly induce minimum, if any, mucosal immunity at the respiratory mucosa which is the prime site of the infection. In this study, chitosan (CS) nanoparticles were prepared by ionic cross-linking of the CS with sodium tripolyphosphate (TPP) at the CS/TPP ratio of 1:0.6 using 2 h mixing time. The CS/TPP nanoparticles were used as delivery vehicle of an intranasal influenza vaccine made of hemagglutinin (HA)-split influenza virus product. Innocuousness, immunogenicity, and protective efficacy of the CS/TPP-HA vaccine were tested in influenza mouse model in comparison with the antigen alone vaccine. The CS/TPP-HA nanoparticles had required characteristics including nano-sizes, positive charges, and high antigen encapsulation efficiency. Mice that received two doses of the CS/TPP-HA vaccine intranasally showed no adverse symptoms indicating the vaccine innocuousness. The animals developed higher systemic and mucosal antibody responses than vaccine made of the HA-split influenza virus alone. The CS/TPP-HA vaccine could induce also a cell-mediated immune response shown as high numbers of IFN-γ-secreting cells in spleens while the HA vaccine alone could not. Besides, the CS nanoparticle encapsulated HA-split vaccine reduced markedly the influenza morbidity and also conferred 100% protective rate to the vaccinated mice against lethal influenza virus challenge. Overall results indicated that the CS nanoparticles invented in this study is an effective and safe delivery vehicle/adjuvant for the influenza vaccine.

In vitro studies on the cytotoxicity, and elastase and tyrosinase inhibitory activities of marigold (Tagetes erecta L.) flower extracts.

Marigold (Tagetes erecta L.) has long been used as a medicinal herb for a number of therapeutic activities. In the present study, the cytotoxicities of ethanol and ethyl acetate extracts of marigold flowers and their inhibitory effects on elastase and tyrosinase enzymes were investigated. An MTT assay was performed to measure the cytotoxicity of these two extracts on the H460 lung cancer and the Caco-2 colon cancer cell lines. An elastase assay kit, based on the digestion of a non-fluorescent elastin substrate to highly fluorescent fragments by elastase, was used for the elastase inhibition assay. Tyrosinase inhibition activity was investigated using the dopachrome method with L-3,4-dihydroxyphenylalanine (L-DOPA) as a substrate. The data obtained in this study demonstrated that the extracts were nontoxic to H460 and Caco-2 cell lines. The elastase inhibition activities of ethanol (250 µg/ml) and ethyl acetate (125 µg/ml) extracts were found to be significantly higher than that of the negative control. The tyrosinase inhibition activities of ethanol and ethyl acetate extracts, in terms of the mean inhibition concentration (IC50), were 1,078 and 1,467 µg/ml, respectively. To the best of our knowledge, the present study has demonstrated for the first time that marigold flower extracts possess tyrosinase inhibition activity. The activities of ethanol and ethyl acetate extracts of marigold flowers were investigated in vitro and indicated that these extracts possess useful properties that may be of interest for cosmetic development.

Fabrication of chitosan--polyacrylic acid complexes as polymeric osmogents for swellable micro/nanoporous osmotic pumps.

AIM: The aims of this study were to prepare and evaluate chitosan-polyacrylic acid complex (CS-PAA) as polymeric osmogents for swellable micro/nanoporous osmotic pump propranolol tablets. METHODS: The complexes were characterized and evaluated for their swelling characteristics. The selected complexes were incorporated into the core propranolol tablets as polymeric osmogents. The core tablets were formulated, compressed as monolithic and two-layered tablets, and finally coated with cellulose acetate containing PEG 400 and PVP K30 as plasticizers and pore formers, respectively. As a final point, the drug release was determined. RESULTS: A direct correlation was found between the CS content in the complex and the maximum swelling force and swelling ratio of the complex mixture. In vitro drug release revealed that the percent drug release increased with the amount of osmogent in the two-layered tablets. Drug release could be prolonged up to 12h and conformed to the USP 31 criteria. In contrast, percent release decreased with the increasing amount of complexes in monolithic tablets. It was postulated that two opposing mechanisms were involved. Following water uptake, the complexes of polymers swelled and pushed the drug out of the tablets, and the drug bound to the polymer network and remained in the tablets. CONCLUSIONS: The results indicated that the complex of CS-PAA at optimal proportion and amount was a promising polymeric osmogent for a zero-order controlled release from two-layered swellable micro/nanoporous osmotic pump tablets.

Development and evaluation of chitosan-coated liposomes for oral DNA vaccine: the improvement of Peyer's patch targeting using a polyplex-loaded liposomes.

The aim of this study was to develop chitosan-coated and polyplex-loaded liposomes (PLLs) containing DNA vaccine for Peyer's patch targeting. Plain liposomes carrying plasmid pRc/CMV-HBs were prepared by the reverse-phase evaporation method. Chitosan coating was carried out by incubation of the liposomal suspensions with chitosan solution. Main lipid components of liposomes were phosphatidylcholine/cholesterol. Sodium deoxycholate and dicetyl phosphate were used as negative charge inducers. The zeta potentials of plain liposomes were strongly affected by the pH of the medium. Coating with chitosan variably increased the surface charges of the liposomes. To increase the zeta potential and stability of the liposome, chitosan was also used as a DNA condensing agent to form a polyplex. The PLLs were coated with chitosan solution. In vivo study of PLLs was carried out in comparison with chitosan-coated liposomes using plasmid encoding green fluorescence protein as a reporter. A single dose of plasmid equal to 100 µg was intragastrically inoculated into BALB/c mice. The expression of green fluorescence protein (GFP) was detected after 24 h using a confocal laser scanning microscope. The signal of GFP was obtained from positively charged chitosan-coated liposomes but found only at the upper part of duodenum. With chitosan-coated PLL540, the signal of GFP was found throughout the intestine. Chitosan-coated PLL demonstrated a higher potential to deliver the DNA to the distal intestine than the chitosan-coated liposomes due to the increase in permanent positive surface charges and the decreased enzymatic degradation.

Development of push-pull osmotic tablets using chitosan-poly(acrylic acid) interpolymer complex as an osmopolymer.

The objectives of this study were to prepare push-pull osmotic tablets (PPOT) of felodipine using an interpolymer complex of chitosan (CS) and poly(acrylic acid) (PAA) as an osmopolymer, and to study the mechanisms of drug release from these tablets. The interpolymer complexes were prepared with different weight ratios of CS to PAA. Preparation of PPOT involved the fabrication of bilayered tablets with the drug layer, containing felodipine, polyethylene oxide, and the polymeric expansion layer, containing the CS-PAA complex. The effects of polymer ratios, type of plasticizers, and compression forces on release characteristics were investigated. It was found that drug release from PPOT exhibited zero-order kinetics and could be prolonged up to 12 or 24 h depending on the plasticizer used. PPOT using dibutyl sebacate showed a longer lag time and slower drug release than that using polyethylene glycol 400. In the case of polyethylene glycol 400, an increase in the CS proportion resulted in an increase in the drug release rate. The compression force had no effect on drug release from PPOT. Drug release was controlled by two consecutive mechanisms: an osmotic pump effect resulting in the extrusion of the drug layer from the tablet and subsequent erosion and dissolution of the extruded drug layer in the dissolution medium. The mathematical model (zero-order) related to extrusion and erosion rates for describing the mechanism of drug release showed a good correlation between predicted and observed values.

Development and optimization of micro/nanoporous osmotic pump tablets.

Micro/nanoporous osmotic pump tablets coated with cellulose acetate containing polyvinylpyrolidone (PVP) as pore formers were fabricated. Propranolol hydrochloride was used as a model drug in this study. Formulation optimization based on USP 31 requirements was conducted following a central composite design using a two-level factorial plan involving two membrane variables (pore former and coating levels). Effect of molecular weight of pore former (PVP K30 and PVP K90) was also evaluated. Responses of drug release to the variables were analyzed using statistical software (MINITAB 14). Scanning electron microscopy and atomic force microscopy showed that the pores formed by PVP. The drug release was dependent on the molecular weight and concentration of PVP and the level of coating. The results showed that acceptable 12-h profile could be achieved with only specific range of PVP K30-containing membrane at the defined membrane thickness. However, satisfactory 24-h profile could be accomplished by both PVP K30 and PVP K90-containing membrane at the range and membrane thickness tested. Preparation and testing of the optimized formulation showed a good correlation between predicted and observed values.

Characterization of drug-chitosan interaction by 1H NMR, FTIR and isothermal titration calorimetry.

Electrostatic interaction between opposite charge of drugs (insulin and benzoic acid) and chitosan was studied by 1H NMR, FTIR and isothermal titration calorimetry (ITC). No ionic interaction between the carboxyl group of benzoic acid and the amine group of chitosan could be detected. There was a minor change in the FTIR spectra of insulin-chitosan microparticles made of different concentrations of insulin. Exothermic heat of reaction between insulin and chitosan was obtained by ITC. However, the measured interaction enthalpy change (delta H) was possibly due to the conformational changes and the adsorption phenomena of insulin onto the surfaces of the particles but not to a binding interaction. The binding of tripolyphosphate, a widely used cross-linking agent, to pH 3.3 and pH 5 chitosan was also studied by ITC. The interaction enthalpy change of the binding between tripolyphosphate and chitosan indicated that tripolyphosphate provided a stronger interaction to pH 5 chitosan than to pH 3.3 chitosan. However, it can be stated that the electrostatical interaction forces between the tested molecules insulin, benzoic acid, and tripolyphosphate and chitosan are found to be very weak.

Cloning, protein expression and immunogenicity of HBs-murine IL-18 fusion DNA vaccine.

Hepatitis B is a global serious disease caused by hepatitis B virus (HBV). There is no known cure for hepatitis B. The best way to deal with the disease is by preventing with hepatitis B vaccine. However, the current protein-based vaccines made up of recombinant hepatitis B surface antigen (HBsAg) are ineffective in chronic HBV carriers and a significant number of the vaccinees do not mount the protective immune response. Novel DNA-based immunization may overcome the deficits of the protein-based immunization and may provide more effective prophylactic and therapeutic outcomes. In this study, we constructed a recombinant plasmid carrying gene encoding the HBV surface antigen (HBs) linked to DNA segment encoding full-length murine interleukin-18, i.e. pcDNA-HBs-IL-18. Immunogenicity of the DNA construct was carried out in BALB/c mice in comparison with mock, i.e. pcDNA3.1+ and vaccines comprised of pRc/CMV-HBs and pRc/CMV-HBs plus pcDNA-IL-18. All vaccinated mice revealed significant serum anti-HBs IgG response after two intramuscular injections of the vaccines at 28 day interval as compared to the level of mock. Co-administration of pRc/CMV-HBs and pcDNA-IL-18 elicited arbitrarily higher levels of anti-HBs IgG than the levels in mice immunized with pRc/CMV-HBs alone and mice that received pcDNA-HBs-IL-18 although not statistically different. Further experiments are needed to investigate the subisotypes of the IgG antibody, the kinetics of cytokine and the cell-mediated immune response. For this communication, the prototype HBs-IL-18 DNA vaccine was successfully constructed and the gene encoding murine IL-18 was successfully cloned. The latter can be co-injected with the antigen coding DNA or used as a fusion partner to the DNA for priming the immune response. The recombinant HBs and full-length IL-18 proteins have potential for other research purposes. They may be used also as standard proteins in the protein quantification assay.

Chitosan drug binding by ionic interaction.

Three model drugs (insulin, diclofenac sodium, and salicylic acid) with different pI or pKa were used to prepare drug-chitosan micro/nanoparticles by ionic interaction. Physicochemical properties and entrapment efficiencies were determined. The amount of drug entrapped in the formulation influences zeta potential and surface charge of the micro/nanoparticles. A high entrapment efficiency of the micro/nanoparticles could be obtained by careful control of formulation pH. The maximum entrapment efficiency did not occur in the highest ionization range of the model drugs. The high burst release of drugs from chitosan micro/nanoparticles was observed regardless of the pH of dissolution media. It can be concluded that the ionic interaction between drug and chitosan is low and too weak to control the drug release.

Influences of layering on theophylline pellet characteristics.

This study evaluated and compared theophylline pellets prepared by both suspension and powder layering processes using the bottom spray coater and the tangential rotary granulator, respectively. Hydroxypropyl methylcellulose (HPMC) and hydroxypropyl cellulose (HPC) were employed as binders at various concentrations. The pellets were coated with Eudragit RS and RL to various levels. It was found that pellet sizes, true densities, and drug contents were comparable and independent of processes and binder levels. However, the increase in binder resulted in lower porosity and pore size, as well as smoother pellet surface. The powder layered pellets possessed higher pellet density and smoother surface than did the suspension layered pellets due to the greater consolidation resulted from tumbling and colliding of pellets. Powder x-ray diffraction pattern revealed that theophylline present in the suspension layered pellets was a mixture of anhydrous form II and hydrate, indicating that transformation could occur in aqueous medium. Drug release from uncoated pellets was found to be complete within 20 min. For coated pellets, the release was markedly decreased with the increase in Eudragit level. Both film thickness and anhydrous/hydrate form influenced the release of drug from the pellets. In general, two methods of layering produced the pellets of slightly differences in pellet properties; however, changes of drug characteristics could occur in suspension.

Design of salbutamol EOP tablets from pharmacokinetics parameters.

Salbutamol elementary osmotic pump (EOP) tablets were developed, and fundamental variables affecting their release characteristics were evaluated. The effects of film thickness and compression force on drug release from the tablets containing fixed amount of sodium chloride used as osmogent were evaluated. The core tablets were directly compressed at four compression forces and coated with 3% wt/vol cellulose acetate in acetone to levels of 2%, 3%, and 4% wt/wt. Coated tablets were drilled with CO2 laser beam to form drug delivery orifice of approximately 400 microm in diameter. The drug release was found to follow zero order fashion. The release rate decreased with the increased film thickness and was not affected by the compression force or porosity. The tablets coated to 3% and 4% levels exhibited the release rates within the range calculated from pharmacokinetic data. To illustrate the effect of osmogent content, the tablets were prepared at four osmogent levels and compressed at a constant compression force. The core tablets were coated to a level of 3% wt/wt. The release rate was initially increased with osmogent content and then decreased. At higher osmogent contents, the drug fraction in soluble component was decreased and resulted in the reduction of drug release. In conclusion, film thickness and osmogents played important roles in drug release from EOP tablets.