A supercritical fluid-based coating technology. 3: preparation and characterization of bovine serum albumin particles coated with lipids

Solvent-free microparticles, loaded with bovine serum albumin as a model protein, were produced using a novel supercritical (SC) fluid-based coating technology. Coating material consists either of trimyristin (Dynasan 114) or of Gelucire 50-02. Microparticles obtained were characterized as regards their morphology, protein content and in vitro release profile. A discontinuous coating made of micro-needles of trimyristin led to an initial burst release of approximately 70% in 30 min. However, a prolonged release of the BSA could be achieved in a phosphate buffer solution at 37 degrees C over a 24 h period from particles coated with Gelucire 50-02. Furthermore, it was shown that BSA does not undergo any degradation after SC CO(2) treatment under the conditions used in the coating process.     

A supercritical fluid-based coating technology. 2: Solubility considerations

Solubility measurements of candidate coating materials have been performed in supercritical (SC) CO₂ so as to select appropriate coating materials for implementation of a solvent-free coating process previously described. Solubility of lipidic compounds such as waxes (paraffin, beeswax, Carnauba wax), pure triglycerides (tricaprin, trimyristin, tripalmitin, tristearin) and mixture of glycerides and fatty acid esters (Gelucire®) in SC CO₂ were evaluated in a static mode under different temperature and pressure conditions, ranging from 13–52°C and from 50–220 bar, whether the CO₂ was in its liquid or SC state. It was shown that the compounds which are mixtures of various components give rise to a selective extraction of the lower melting point components, as evidenced from thermal analysis of soluble and insoluble fractions of the coating materials.     

Microencapsulation of protein particles within lipids using a novel supercritical fluid process

Solvent-free microparticles, loaded with a model protein (bovine serum albumin: BSA) have been produced using a novel coating process based on supercritical (SC) fluid technology. Coating material consists of either trimyristin (Dynasan114) or Gelucire50-02, two lipidic compounds having a high melting point. Microparticles were characterized as regards their morphology, protein content and in vitro release profile. A discontinuous coating made of crystalline micro-needles was obtained using Dynasan114. It led to particles with an initial burst release of about 70% in 30 min at 37 degrees C. However, a prolonged release of the protein has been achieved over a 24 h period from particles coated with Gelucire50-02, which produces a more homogeneous, film-forming coating. Furthermore, it was shown that BSA does not undergo any degradation after SCCO2 treatment under the SC conditions used in the coating process.     

A supercritical fluid-based coating technology 1: Process considerations

A novel solvent-free particle coating process, based on the solvent properties of supercritical fluids (SCF) is described. It consists of dissolving one or more coating materials in supercritical CO₂ and then adjusting T/P conditions in the autoclave so that the coating material becomes insoluble in the CO₂. This insolubilization step causes a coating to deposit on the surface of suspended particles. This process has been applied to bovine serum albumin and sugar granules. Coating was effective, but different morphologies were obtained depending on the coating material used. A discontinuous coating has been made using trimyristin, since this material precipitates as micro-needles. Conversely, a smooth, regular coating has been obtained with a commercially available mixture of glycerides and glyceride esters of PEG (Gelucire® 50/02 acting like a film-forming agent.     

Release behaviour of diclofenac sodium dispersed in Gelucire® and encapsulated with alginate beads

Sustained release polymeric particles containing diclofenac sodium dispersed in Gelucire® matrix and encapsulated in calcium alginate shell were prepared with different drug-to-polymer ratios and also with different concentrations of sodium alginate for a fixed drug-to-polymer ratio in an aqueous environment. Spherical particles were formed by dropping an emulsion of diclofenac sodium in Gelucire® matrix, emulsified with sodium alginate, into calcium chloride solution. The gelled beads formed by ionotropic gelation of alginate with calcium ions showed sustained release of the water soluble drug in in-vitro release study. Drug release was a function of square-root of time, suggesting a matrix diffusion release pattern. The rate of release was significantly suppressed with increasing proportions of Gelucire® in the mixture. Sustained and complete release was achieved with Gelucire® of low melting point and low HLB value. No significant drug release occurred in a dissolution medium of pH 1.5, whereas complete release was observed at pH 6.8, consistent with considerable swelling of the alginate gel at this pH.     

Stabilization of Proteins Encapsulated in Cylindrical Poly(lactide-co-glycolide) Implants: Mechanism of Stabilization by Basic Additives

Purpose. A previous study from our group has shown that in theacidic microclimate of poly(lactide-co-glycolide) (PLGA) implants,encapsulated BSA forms insoluble noncovalent aggregates and ishydrolyzed during in vitro release. Incorporation of Mg(OH)₂ stronglyinhibits these mechanisms of instability and facilitates continuousprotein release. The purpose of this study was to determine the proteinstabilization mechanism in the presence of basic additives. Methods. BSA, as a model protein, was encapsulated in PLGAmillicylinders by a solvent extrusion method. The release of BSA fromthe PLGA millicylinders with and without basic additives (Mg(OH)₂,Ca(OH)₂, ZnCO₃ and Ca₃(PO₄)₂) in a physiological buffer was carriedout at 37°C and quantified by a modified Bradford assay. The insolubleaggregates extracted from the polymer with acetone were reconstitutedin a denaturing (6 M urea) or denaturing/reducing solvent (6 M urea/10 mM DTT) to determine the type of aggregation. Results. Aggregation of encapsulated BSA was inhibited withincreasing amount of base co-encapsulated in the polymer, irrespective of thetype of base used. The pH drop in the release medium and extent ofacid-catalyzed PLGA degradation were both inhibited in the presenceof base. The resultant effect was also reflected in an increase in wateruptake and porosity of the devices. The inhibition and mechanism ofBSA aggregation was correlated with the basicity of the additive.For Ca(OH)₂, at 3% loading, covalent BSA aggregation due tothiol-disulfide interchange was observed (indicative of ionization ofalbumin's free thiol at high pH), whereas at 3% ZnCO₃ or Ca₃(PO₄)₂, ahigher percentage of non-covalent aggregates was observed comparedto Mg(OH)₂. Decreasing the loading of BSA at constant Mg(OH)₂content caused an increase in BSA aggregation. Conclusions. The mechanism by which Mg(OH)₂ stabilizesencapsulated BSA in PLGA implants is through neutralizing the acidicmicroclimate pH in the polymer. The successful neutralization afforded by thebasic additives requires a percolating network of pores connecting bothbase and protein. The microclimate pH inside PLGA implants can becontrolled by selecting the type of basic salt, which suggests a potentialapproach to optimize the stability of encapsulated pharmaceuticals inPLGA including therapeutic proteins.     

A supercritical fluid-based coating technology. 2: solubility considerations

Solubility measurements of candidate coating materials have been performed in supercritical (SC) CO(2) so as to select appropriate coating materials for implementation of a solvent-free coating process previously described. Solubility of lipidic compounds such as waxes (paraffin, beeswax, Carnauba wax), pure triglycerides (tricaprin, trimyristin, tripalmitin, tristearin) and mixture of glycerides and fatty acid esters (Gelucire) in SC CO(2) were evaluated in a static mode under different temperature and pressure conditions, ranging from 13-52 degrees C and from 50-220 bar, whether the CO(2)was in its liquid or SC state. It was shown that the compounds which are mixtures of various components give rise to a selective extraction of the lower melting point components, as evidenced from thermal analysis of soluble and insoluble fractions of the coating materials.     

Zero-order release and bioavailability enhancement of poorly water soluble Vinpocetine from self-nanoemulsifying osmotic pump tablet

Abstract

Solid self-nanoemulsifying (S-SNEDDS) asymmetrically coated osmotic tablets of the poorly water-soluble drug Vinpocetine (VNP) were designed. The aim was to control the release of VNP by the osmotic technology taking advantage of the solubility and bioavailability-enhancing capacity of S-SNEDDS. Liquid SNEDDS loaded with 2.5?mg VNP composed of Maisine? 35-1, Transcutol^® HP, and Cremophor^® EL was adsorbed on the solid carrier Aeroperl^®. S-SNEDDS was mixed with the osmotic tablet excipients (sodium chloride, Avicel^®, HPMC-K4M, PVP-K30, and Lubripharm^®), then directly compressed to form the core tablet. The tablets were dip coated and mechanically drilled. A 3²*2¹ full factorial design was adopted. The independent variables were: type of coating material (X₁), concentration of coating solution (X₂), and number of drills (X₃). The dependent variables included % release at 2?h (Y₁), at 4?h (Y₂), and at 8?h (Y₃). The in vivo performance of the optimum formula was assessed in rabbits. Zero-order VNP release was obtained by the single drilled 1.5% Opadry^® CA coated osmotic tablets and twofold increase in VNP bioavailability was achieved. The combination of SNEDDS and osmotic pump tablet system was successful in enhancing the solubility and absorption of VNP as well as controlling its release.     

Sustained protein release from hydrogel microparticles using layer-by-layer (LbL) technology

Context and objectives: Since most of developed therapeutic proteins are intended to treat chronic diseases, patients are prescribed multiple injections for long time periods, and therefore, sustained release formulations are much needed. However, challenges facing these formulations are quite significant. In this context, a model protein, lysozyme (Lys), was loaded on hydrogel microparticles (beads) and the ability of layer-by-layer (LbL) coating to control Lys release and maintain its activity over a one-month period was investigated.

Methods: LbL coating was composed of chondroitin sulfate as a negatively charged polyelectrolyte and a biocompatible, hydrolytically degradable poly β-aminoester as a positively charged polyelectrolyte. Loading distribution was monitored by fluorescence imaging, and followed by depositing a series of LbL coatings of different thicknesses. Release of Lys from these formulations was studied and activity of released fraction was determined.

Results: Lys was loaded effectively on hydrogel beads achieving about 9?mg protein/100?mg wet spheres. LbL coating was proven successful by monitoring the zeta potential of the beads, which was reversed after the addition of each layer. In vitro release studies showed sustained release profiles that depend on the thickness of the deposited coat, with t₅₀ extended from 4.9 to 143.9?h. More importantly, released Lys possessed a high degree of biological activity during the course of release maintaining at least 72% of initial activity.

Conclusions: Successful loading of Lys and extension of its release while maintaining a considerable degree of activity might make this formulation suitable for use with other active therapeutic proteins.     

Supercritical Fluid Technology-Based Trans-Resveratrol SLN for Long Circulation and Improved Radioprotection

Purpose

To alleviate the harmful effects of radiations during occupational radiology, radiotherapy and diagnosis, radioprotective system with lower toxicity and extended activity is imperative. Trans-resveratrol (RVL) acts through free radical scavenging/antioxidant mechanism to mitigate the radiation-induced damage. But, its poor solubility and fast metabolism impede its efficacy. Thus, encapsulation of RVL into long circulating solid lipid nanoparticle (SLN) is aimed.

Method

Supercritical CO₂ solution of RVL, Gelucire®50/02 and Gelucire®50/13 SLN, was rapidly expanded into aqueous phase containing Tween 80, sonicated and lyophilized to get SLN. Particle size, polydispersity index (PDI), entrapment efficiency (%EE), scanning electron microscopy (SEM), transmission electron microscopy (TEM), differential scanning calorimetry (DSC), X-ray diffraction (XRD), drug release, in vivo pharmacokinetics, antioxidant assays, radiation-induced lipid peroxidation and plasmid DNA relaxation assays were studied. Stability studies were performed to analyze drug degradation and shelf life.

Results

Optimized formulation (F9) had %yield, particle size, PDI, %EE and %drug release (after 72 h) of 68.48?±?5.73 %, 276.7?±?5.33 nm, 0.18?±?0.032, 62.66?±?4.52 % and 70.05?±?3.003 %, respectively. Electron microscopy revealed nearly spherical particles, while DSC and XRD showed reduced crystalline peaks. F9 showed higher AUC and sustained release of RVL in rats (i.v. bolus) and increased antioxidant activities and radioprotection as compared to RVL solution. Shelf life of >2 years was predicted for F9 (at 8 °C).

Conclusion

Results are encouraging to use F9 for radiation exposure prophylaxis.

     

Preparation and evaluation of tamsulosin hydrochloride sustained-release pellets modified by two-layered membrane techniques

The aim of the present study was to develop tamsulosin hydrochloride sustained-release pellets using two-layered membrane techniques. Centrifugal granulator and fluidized-bed coater were employed to prepare drug-loaded pellets and to employ two-layered membrane coating respectively. The prepared pellets were evaluated for physicochemical characterization, subjected to differential scanning calorimetry (DSC) and in vitro release of different pH. Different release models and scanning electron microscopy (SEM) were utilized to analyze the release mechanism of Harnual^® and home-made pellets. By comparing the dissolution profiles, the ratio and coating weight gain of Eudragit^® NE30D and Eudragit^® L30D55 which constitute the inside membrane were identified as 18:1 and 10%–11%. The coating amount of outside membrane containing Eudragit^® L30D55 was determined to be 0.8%. The similarity factors (f₂) of home-made capsule and commercially available product (Harnual^®) were above 50 in different dissolution media. DSC studies confirmed that drug and excipients had good compatibility and SEM photographs showed the similarities and differences of coating surface between Harnual^® and self-made pellets before and after dissolution. According to Ritger-Peppas model, the two dosage form had different release mechanism.     

Design,in vitro release characterization and pharmacokinetics of novel controlled release pellets containing levodropropizine

Abstract

This study was performed to investigate the in vitro release characteristics of levodropropizine (LDP) from novel dual-coated sustained release (SR) pellets, and evaluate the pharmacokinetics of a novel controlled release (CR) preparation composed of the dual-coated SR pellets and immediate release (IR) LDP pellets. The dual-coated SR pellets composed of a drug-loaded nonpareil core, a sub-coating layer (HPMC 6cps) and an SR-coating layer (Aquacoat® ECD, Eudragit® RS 30D or Kollicoat® SR 30D) were prepared by a bottom-spray fluidized bed-coating method. The drug release from the dual-coated SR pellets coated with Aquacoat® ECD followed a zero-order profile in water, and the drug release was not affected by the coating level of the sub-coating layer and stable under the accelerated storage condition (40?°C, 75% RH) for 6 months. The CR preparation showed significantly decreased values of maximum drug concentration (C_max) and elimination rate (K) than the reference product (LEVOTUS® SYR) but the similar bioavailability (F?=?95.43%). The novel CR preparation presents promising delivery of LDP with an immediate and sustained release manner, with similar clinical effect as the commercial IR product.     

Stabilization and Controlled Release of Bovine Serum Albumin Encapsulated in Poly(D,L-lactide) and Poly(ethylene glycol) Microsphere Blends

Purpose. The acidic microclimate in poly(D, L-lactide-co-glycolide) 50/50 microspheres has been previously demonstrated by our group as the primary instability source of encapsulated bovine serum albumin (BSA). The objectives of this study were to stabilize the encapsulated model protein, BSA, and to achieve continuous protein release by using a blend of: slowly degrading poly(D, L-lactide) (PLA), to reduce the production of acidic species during BSA release; and pore-forming poly(ethylene glycol) (PEG), to increase diffusion of BSA and polymer degradation products out of the polymer. Methods. Microspheres were formulated from blends of PLA (Mw 145,000) and PEG (Mw 10,000 or 35,000) by using an anhydrous oil-in-oil emulsion and solvent extraction (O/O) method. The polymer blend composition and phase miscibility were examined by FT-IR and DSC, respectively. Microsphere surface morphology, water uptake, and BSA release kinetics were also investigated. The stability of BSA encapsulated in microspheres was examined by losses in protein solubility, SDS-PAGE, IEF, CD, and fluorescence spectroscopy. Results. PEG was successfully incorporated in PLA microspheres and shown to possess partial miscibility with PLA. A protein loading level of 5% (w/w) was attained in PLA/PEG microspheres with a mean diameter of approximately 100 m. When PEG content was less than 20% in the blend, incomplete release of BSA was observed with the formation of insoluble, and primarily non-covalent aggregates. When 20%-30% PEG was incorporated in the blend formulation, in vitro continuous protein release over 29 days was exhibited. Unreleased BSA in these formulations was water-soluble and structurally intact. Conclusions. Stabilization and controlled relaease of BSA from PLA/PEG microspheres was achieved due to low acid and high water content in the blend formulation.     

Theophylline colon specific tablets for chronotherapeutic treatment of nocturnal asthma

The ultimate goal is to design a new chronotherapeutic system for theophylline (TPH) with high potential benefits in treating nocturnal asthma. TPH core tablets were prepared by wet granulation using a developed formula. Compression coating over core tablets containing 200?mg TPH was done using granulated chitosan with 10% PVP K30. Different formulae F1, F2 and F3 were prepared using coat weights 260, 300 and 360?mg, respectively. The in vitro release characteristics in both variant pH media mimicking the gastrointestinal media and in media containing rat cecal content were monitored. The in vivo performance of the optimum formula was compared with Avolen^® SR in Beagle dogs. F3 with high coat thickness exhibited a minimal release after 5-h release study. Both F2 and F3 showed more than 50% drug release after 4?h in the rat cecal medium. This reflects the colon selectivity of the system. The C_max values were found to be 5.49?±?0.46 and 5.12?±?0.85?μg/mL for F3 and Avolen^® SR, respectively, F3 showed higher mean plasma concentration than Avolen^® SR from the beginning and continued till 7?h post administration indicating high potential as chronotherapeutic treatment of nocturnal asthma.     

Multiple response optimisation of processing and formulation parameters of pH sensitive sustained release pellets of capecitabine for targeting colon

Purpose: To optimise the Eudragit/Surelease^®-coated pH-sensitive pellets for controlled and target drug delivery to the colon tissue and to avoid frequent high dosing and associated side effects which restrict its use in the colorectal-cancer therapy.

Methods: The pellets were prepared using extrusion-spheronisation technique. Box–Behnken and 3² full factorial designs were applied to optimise the process parameters [extruder sieve size, spheroniser-speed, and spheroniser-time] and the coating levels [%w/v of Eudragit S100/Eudragit-L100 and Surelease^®], respectively, to achieve the smooth optimised size pellets with sustained drug delivery without prior drug release in upper gastrointestinal tract (GIT).

Results: The design proposed the optimised batch by selecting independent variables at; extruder sieve size (X₁?=?1?mm), spheroniser speed (X₂?=?900 revolutions per minute, rpm), and spheroniser time (X₃?=?15?min) to achieve pellet size of 0.96?mm, aspect ratio of 0.98, and roundness 97.42%. The 16%w/v coating strength of Surelease^® and 13%w/v coating strength of Eudragit showed pH-dependent sustained release up to 22.35?h (t_99%). The organ distribution study showed the absence of the drug in the upper part of GIT tissue and the presence of high level of capecitabine in the caecum and colon tissue. Thus, the presence of Eudragit coat prevent the release of drug in stomach and the inner Surelease^® coat showed sustained drug release in the colon tissue.

Conclusion: The study demonstrates the potential of optimised Eudragit/Surelease^®-coated capecitabine-pellets for effective colon-targeted delivery system to avoid frequent high dosing and associated systemic side effects of drug.     

Protein Spray-Freeze Drying. Effect of Atomization Conditions on Particle Size and Stability

Purpose. To investigate the effect of atomization conditions on particle size and stability of spray-freeze dried protein. Methods. Atomization variables were explored for excipient-free (no zinc added) and zinc-complexed bovine serum albumin (BSA). Particle size was measured by laser diffraction light scattering following sonication in organic solvent containing poly(lactide-co-glycolide) (PLG). Powder surface area was determined from the N₂ vapor sorption isotherm. Size-exclusion chromatography (SEC) was used to assess decrease in percent protein monomer. Fourier-transform infrared (FTIR) spectroscopy was employed to estimate protein secondary structure. PLG microspheres were made using a non-aqueous, cryogenic process and release of spray-freeze dried BSA was assessed in vitro. Results. The most significant atomization parameter affecting particle size was the mass flow ratio (mass of atomization N₂ relative to that for liquid feed). Particle size was inversely related to specific surface area and the amount of protein aggregates formed. Zinc-complexation reduced the specific surface area and stabilized the protein against aggregation. FTIR data indicated perturbations in secondary structure upon spray-freeze drying for both excipient-free and zinc-complexed protein. Conclusions. Upon sonication, spray-freeze dried protein powders exhibited friability, or susceptibility towards disintegration. For excipient-free protein, conditions where the mass flow ratio was > 0.3 yielded sub-micron powders with relatively large specific surface areas. Reduced particle size was also linked to a decrease in the percentage of protein monomer upon drying. This effect was ameliorated by zinc-complexation, via a mechanism involving reduction in specific surface area of the powder rather than stabilization of secondary structure. Reduction of protein particle size was beneficial in reducing the initial release (burst) of the protein encapsulated in PLG microspheres.     

Characterization of drug release from diltiazem-loaded polylactide microspheres prepared using sodium caseinate and whey protein as emulsifying agents

The influence of milk protein emulsifying agents on the characteristics, particularly drug release, of polylactide microspheres was investigated. Diltiazem loaded polylactide (PL) microspheres were successfully prepared using the dairy proteins, sodium casinate (SC) and whey protein isolate (WPI) as the emulsifying agents. Microspheres were characerized in terms of microsphere yield, electron microscopy, particle size, drug loading, DSC and XRD analysis and drug release. The yields of microspheres obtained were 53-63% and were independent of the emulsifying agent used. SEM revealed that, regardless of the emulsifying agent employed, the microspheres were of good sphericity, but the surface appearance of the microspheres was not the same in all cases. The milk proteins resulted in microspheres approximately half the size of those obtained with methylcellulose (MC). Significant differences in drug loading were observed between the three emulgents, the MC systems giving the highest values. Release profiles were sigmoidal in shape and were well fitted to the equation ln (x/1-x) = k·t - k·t_max, reflecting degradation controlled drug release. The parameter k increased with drug loading, while t_max decreased. The relationships between the release parameters [P(k and t_max)] and loading (L) could be quantified by equations of the form P = a·L^N, N being negative in the case of t_max. Apart from the effect on loading efficiency, neither SC nor WPI appeared to significantly alter drug release. The quantitative relationships observed in this study may have more general application in quantifying drug release from drug polymer composites at low loadings where polymer degradation controls drug release.     

A Heterogeneously Structured Composite Based on Poly(lactic-co-glycolic acid) Microspheres and Poly(vinyl alcohol) Hydrogel Nanoparticles for Long-Term Protein Drug Delivery

Purpose. To prepare a heterogeneously structured composite based on poly (lactic-co-glycolic acid) (PLGA) microspheres and poly(vinyl alcohol) (PVA) hydrogel nanoparticles for long-term protein drug delivery. Methods. A heterogeneously structured composite in the form of PLGA microspheres containing PVA nanoparticles was prepared and named as PLGA-PVA composite microspheres. A model protein drug, bovine serum albumin (BSA), was encapsulated in the PVA nanoparticles first. The BSA-containing PVA nanoparticles was then loaded in the PLGA microspheres by using a phase separation method. The protein-containing PLGA-PVA composite microspheres were characterized with regard to morphology, size and size distribution, BSA loading efficiency, in vitroBSA release, and BSA stability. Results. The protein-containing PLGA-PVA composite microspheres possessed spherical shape and nonporous surface. The PLGA-PVA composite microspheres had normal or Gaussian size distribution. The particle size ranged from 71.5 m to 282.7 m. The average diameter of the composite microspheres was 180 m. The PLGA-PVA composite microspheres could release the protein (BSA) for two months. The protein stability study showed that BSA was protected during the composite microsphere preparation and stabilized inside the PLGA-PVA composite microspheres. Conclusions. The protein-containing PLGA-PVA composite may be suitable for long-term protein drug delivery.     

Functional expression of human 5-HT1A receptors and differential coupling to second messengers in CHO cells

Summary The signal transduction linkages of the cloned human 5-HT_1A receptor as expressed stably in CHO cells were studied. A transfected clonal cell line which expresses 900 ± 36 fmol 5-HT_1A receptor/mg protein (designated CHO-5-HT_1A/WT-27) responded to 5-HT and/or 8-OH-DPAT by coupling to several second messenger pathways. The 5-HT_1A receptor inhibited, but did not stimulate, membrane adenylyl cyclase activity and whole cell cAMP accumulation in a dose-dependent manner (for 5-HT, IC₅₀ = 146 ± 27 and 55 ± 12 nM, respectively). Activation of the receptor was associated with other signal transduction linkages: (i) a 40–50% increase in hydrolysis of inositol phosphates (for 5-HT, EC₅₀ = 1.33 ± 0.15 M for 5-HT), (ii) a transient elevation of cytosolic Ca²⁺ levels (apparent at 1–100 M 5-HT) which was not affected by chelation of extracellular Ca²⁺ by EGTA, and (iii) an augmentation of [³H]-arachidonic acid release pharmacologically with the calcium ionophore A23187 or by activation of endogenous thrombin or P₂ purinergic receptors (for 5-HT, EC₅₀ = 1.22 ± 0.17 M). This pathway may be an amplification mechanism for signaling in anatomic regions with high concentrations of several neuro-transmitters, hormones or autacoids, such as at neuronal junctions or near areas of platelet aggregation. All linkages were sensitive to pertussistoxinpre-treatment (IC₅₀0.5–0.6 ng/ml × 4.5 h for all pathways), suggesting the involvement of G_i protein(s) in these signal transduction pathways. Coupling to varied signal transduction pathways in a single cell system may be a common feature of receptors which classically inhibit adenylyl cyclase such as the 5-HT_1A receptor.Abbreviations cAMP adenosine 3,5-cyclic monophosphate - PKC protein kinase C (calcium and phospholipid-dependent kinase) - 5-HT serotonin - 8-OH-DPAT 8-OH-2-(di-n-propylamino)1,2,3,4-tetrahydronaphthalene - PBS phosphate buffered saline - EDTA ethylenediaminetetraacetic acid - BSA bovine serum albumin - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - CHAPS 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfate - [¹²⁵I]-N₃-NAPS N-(p-azido-m-[¹²⁵I]iodophenethyl)-spiperone - G protein (guanine nucleotide binding protein) - mCPP 1-(3-chlorophenyl)piperazine - TFMPP m-trifluoromethylphenyl-piperazine - PAPP (LY-165,163) p-aminophenylethyl-m-trifluoromethylphenyl piperazine - mCPP 1-(3-chlorophenyl)piperazinedihydrochloride - WB 4101 2-(2,6-dimethoxyphenoxyethyl)aminoethyl1,4-benzodioxane hydrochloride - NAN-190 1-(2-methoxyphenyl)-4-(2-phthalimido)butyl]piperazine - H-7 1-(5-isoquinolinesulfonyl)-2-methylpiperazine - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis Send offprint requests to J. R. Raymond     

Development of Enteric-coated Pectin-based Matrix Tablets for Colonic Delivery of Theophylline

The present work was aimed at developing a new colonic drug delivery system which takes advantage of the combined approaches of a specifically colon-biodegradable pectin matrix with a pH-sensitive Eudragit_® S100 polymeric coating. The developed system was able to suitably retard the onset of drug release and to provide a colon-specific delivery, thus overcoming the problems of pectin solubility in the upper gastrointestinal tract and low site-specificity of simple pH-dependent systems. Due to the poor compactability properties of pectin, it was used in mixture with Emdex_®, a hydrophilic directly-compressible material, in order to make it possible to prepare tablets by direct compression. Theophylline (TP) was used as model drug due to its suitable pharmacokinetic properties for colonic delivery and good absorption in the large intestine. The effects of varying the type of pectin (low and high methoxylated, or amidated), the pectin:Emdex_® ratio and the level of the pH-dependent polymeric coating on drug release behavior were investigated. Release tests were performed using sequential liquids simulating the physiological variation of pH and the effect of the presence or not of pectinolytic enzymes into the simulated colonic medium was evaluated. Thirty percent (w/w) was the the minimum content of Emdex_® for obtaining directly compressible tablets with sufficient hardness to withstand the coating process and 27% (w/w) was the minimum coating amount for obtaining an adequate lag time before the onset of drug release. After lag time, linear nearly zero-order profiles were obtained whose slope (i.e. the drug release rate) depended on both the Emdex_® content and the pectin type. Comparison of the results obtained in the presence or not of pectynolitic enzymes allowed selection of the high methoxylated pectin as the most interesting candidate for specific colonic delivery since it was the least water-soluble and the most susceptible to enzymatic degradation, thus assuring a greater site-specificity of drug release. Finally, the importance of using appropriate dissolution test conditions to adequately characterize the drug release profiles from delivery systems endowed with a microflora-activated drug release triggering mechanism has been demonstrated.