In Vitro Protein Release and Degradation of Poly-dl-lactide-poly(ethylene glycol) Microspheres with Entrapped Human Serum Albumin: Quantitative Evaluation of the Factors Involved in Protein Release Phases

Purpose. To quantitatively evaluate the correlations between the amount of initial burst release and the surface-associated protein, and between the onset time for the second burst release and the matrix polymer degradation.Methods. Human serum albumin (HSA) was microencapsulated in polylactide (PLA) and poly-dl-lactide-poly(ethylene glycol) (PELA) with PEG contents of 5, 10, 20, and 30%, respectively, using the solvent extraction procedure based on formation of double emulsion w/o/w. Microspheres with similar particle size (1.7-2.0m), similar protein entrapment (2.1-2.8%) but different surface-associated proteins (9.3-53.6%) were used to evaluate the in vitro matrix degradation and protein release profiles. Degradation was characterized by studying the intrinsic viscosity decrease, medium pH change, and weight loss of the microspheres.Results. The matrix degradation and protein release profiles were highly dependent on the polymer composition of the microspheres. Faster decreases in the intrinsic viscosity of recovered matrix polymer, the microspheres weight, and the pH of degradation medium, and earlier onsets for the break in intrinsic viscosity reduction and the mass loss were detected for PELA microspheres with higher PEG content. The hydration and swelling of microspheres matrix contributed greatly to the degradation of matrix polymer. The HSA release showed triphasic profile and involved two mechanisms for all the microsphere samples. Smaller amount of initial burst release, larger gradual release rate, and earlier onset for the second burst release were observed for HSA from matrix polymer with higher PEG content. The extent of the initial burst release was quantitatively related with the surface-associated protein. The second burst release of HSA was observed to occur within 1 week after the onset for mass loss, which was also the break in the intrinsic viscosity reduction rate.Conclusion. Protein release profiles could be rationalized by optimizing the matrix polymer degradation and microsphere characteristics.     

Biodegradable PLGA Microspheres Loaded with Ganciclovir for Intraocular Administration. Encapsulation Technique,in Vitro Release Profiles,and Sterilization Process

Purpose. The purpose of this work was to obtain a sterilized formulation consisting of biodegradable microspheres of poly (DL-lactide-co-glycolide) (PLGA) for intraocular sustained release of ganciclovir. Methods. Microspheres were prepared using a dispersion of ganciclovir in fluorosilicone oil (FSiO) that was further dispersed in an acetone solution of PLGA [50/50 and inherent viscosity 0.41 dl/g], and emulsified in silicone oil with a surfactant. Once prepared, the formulation was exposed with an effective radiation dose of 2.5 megarads. The release rate data of ganciclovir from the sterilized and nonsterilized batches were compared using the similarity factor (f₂). Results. The dispersion of the drug in FSiO contributed to achieving a drug payload of up to 95% of the theoretical in the 300-500 m microspheres. Ten mg released ganciclovir in vitroat 1.3 g/h for the first 21 days, but decreased to 0.2 g/h from day 25 until the end of the release study (42 days). No significant differences in the amounts of encapsulated drug (=0.05) were observed between the sterilized and nonsterilized microspheres. Furthermore, dissolution profiles of formulations behaved similarly before and after gamma radiation exposure. Conclusions. The technique of microsphere preparation described resulted in high ganciclovir loading (95%) and prolonged drug release. The ganciclovir formulation behaved similarly before and after the sterilization process.     

PLG Microsphere Size Controls Drug Release Rate Through Several Competing Factors

Purpose. Although the rate of drug release from poly(D,L-lactide-co-glycolide) (PLG) microspheres is often modulated by changing fabrication conditions or materials, the specific factors directly controlling the release profiles are often unclear. We have fabricated uniform rhodamine- and piroxicam-containing microspheres, 10 to 100 m in diameter, to better understand how microsphere size controls drug release. Methods. Drug distribution within the microspheres was examined using confocal fluorescence microscopy. The rate of polymer degradation was determined as the change in molecular weight, measured by gel permeation chromatography, during in vitro degradation experiments. Further, changes in the surface and interior morphology of the particles during in vitro degradation were investigated by scanning electron microscopy. Results. Microsphere size greatly affected drug distribution. Small (10-m) microspheres showed an essentially uniform drug distribution. Larger (100-m) microspheres showed redistribution of drug to specific regions of the microspheres. Rhodamine partitioned to the surface and piroxicam partitioned to the interior of large PLG microspheres. Further, the rate of polymer degradation increased with microsphere size, possibly the result of a more acidic interior caused by increased accumulation of hydrolyzed polymer products in larger particles. Finally, larger microspheres developed a more porous interior structure during the drug release. Conclusions. Microsphere size affects drug release not only through changes in diffusion rates but also through secondary effects including drug distribution in the particle, polymer degradation rate, and microsphere erosion rates.     

Salivary nystatin concentrations after administration of an osmotic controlled release tablet and a pastille

Mucosal oral therapeutic system (MOTS) is a controlled-release osmotic system for oral cavity therapy. MOTS (nystatin) is designed to deliver approximately 200,000 units of nystatin over several hours. A crossover study was conducted in five healthy volunteers to evaluate the amount of nystatin released (based on residual drug content) when the system is held in the mouth for 30 min, 1 h, and 2 h, and to compare these concentrations with those achieved with a Mycostatin (nystatin) pastille.An average of 37% of the nystatin content was released intra-orally from MOTS during 2 h in the mouth, which was very similar to the percentage delivered in vitro. Mean salivary drug concentrations were as follows: 279 g·ml^–1 at 30 min; 654 g·ml^–1 after 1 h; and 532 g·ml^–1 at 2 h. These concentrations consistently exceeded those produced by the pastille at the same time points. Fifteen minutes after placement of the pastille in the mouth (i.e., immediately after its dissolution) mean nystatin concentrations reached 746 g·ml^–1 but fell rapidly to 13.2 g·ml^–1 at 30 min, 7.2 g·ml^–1 at 1 h, and 5.6 g·ml^–1 at 2 h.The study demonstrates that MOTS maintains high salivary nystatin concentrations throughout a 2 h dosing interval.     

Preparation and Characterization of Poly (D,L-Lactide-Co-Glycolide) Microspheres for Controlled Release of Poly(L-Lysine) Complexed Plasmid DNA

Purpose. To produce and characterize controlled release formulations of plasmid DNA (pDNA) loaded in poly (D,L-lactide-co-glycolide) (PLGA) microspheres both in free form and as a complex with poly (L-lysine). Methods. Poly (L-lysine) (PLL) was used to form pDNA/PLL complexes with complexation ratio of 1:0.125 and 1:0.333 w/w to enhance the stability of pDNA during microsphere preparation and protect pDNA from nuclease attack. pDNA structure, particle size, zeta potential, drug loading, in vitro release properties, and protection from DNase I were studied. Results. The microspheres were found to be spherical with average particle size of 3.1-3.5 m. Drug loading of 0.6% was targeted. Incorporation efficiencies of 35.1% and 29.4-30.6% were obtained for pDNA and pDNA/PLL loaded microspheres respectively. Overall, pDNA release kinetics following the initial burst did not correlate with blank microsphere polymer degradation profile suggesting that pDNA release is convective diffusion controlled. The percentage of supercoiled pDNA in the pDNA and pDNA/PLL loaded microspheres was 16.6 % and 76.7-85.6% respectively. Unencapsulated pDNA and pDNA/PLL degraded completely within 30 minutes upon the addition of DNase I. Encapsulation of DNA/PLL in PLGA microspheres protected pDNA from enzymatic degradation. Conclusions. The results show that using a novel process, pDNA can be stabilized and encapsulated in PLGA microspheres to protect pDNA from enzymatic degradation.     

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.     

Beeinflussung der analgetischen Wirkung des Morphins durch Reserpin und Thiopropazat

Zusammenfassung Es wurde die Beeinflussung der Wirkungen von Morphin durch Thiopropazat und Reserpin an der Maus in verschiedenen Versuchsanordnungen verglichen.1. Thiopropazat verstärkte die analgetische Wirkung des Morphins an der Heißen Platte und bei mechanischer Reizung der Schwanzwurzel. Diese Wirkungskomponente war nach 30 min bereits optimal ausgeprägt und kann zu den Tests auf Sedation im Sinne von Courvoisier (1957) gerechnet werden.2. Thiopropazat antagonisierte die Hemmung eines polysynaptischen Reflexes (Wegziehen einer Hinterpfote bei Wärmereiz) durch Morphin. Diese Wirkungskomponente trat erst im Verlauf von 2 Std ein und ist ihrem Mechanismus nach der Katalepsie bei der Ratte vergleichbar.3. Reserpin schwächte die Wirkung von Morphin in allen Versuchsanordnungen ab. Seine sedierende Wirkung bei Mensch und Tier ist wahrscheinlich nicht mit der von Chlorpromazin zu vergleichen.

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Summary In mice, the influence of thiopropazate and reserpine upon the effects of morphine was compared in several tests.1. Thiopropazate enhanced the analgesic effect of morphine on the hot plate and on mechanical stimulation of the root of the tail. This component was optimally developped 30 min after the injection of thiopropazate and can be regarded as a test for sedation according to the definition of Courvoisier (1957).2. Thiopropazate antagonized the inhibition of a polysynaptic reflex (withdrawal of a hind foot upon irradiation) by morphine. This component could be demonstrated after 2 hours and is probably brought about by the same mechanism as the catatonia in rats.3. Reserpine inhibited the effects of morphine in all tests. Its sedative action in man and beast is probably not comparable to that of chlorpromazine.

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Herrn Prof. Dr. O. Schaumann, Innsbruck, zum 70. Geburtstag gewidmet.     

Viscoelastic Properties of Bioadhesive,Chlorhexidine-Containing Semi-Solids for Topical Application to the Oropharynx

Purpose. This study examined the viscoelastic properties of bioadhesive, chlorhexidine-containing semi-solid formulations, designed for topical application to the oropharynx. Methods. Oscillatory rheometry was performed using a Carri-Med CSL²-100 rheometer at 20.0 ± 0.1° C in conjunction with parallel plate geometry (2 cm diameter, 0.5 mm sample thickness). Samples were subjected to a constant strain (6.5 × 10^–3 rad) and defined viscoelastic parameters, namely storage modulus (G), loss modulus (G), loss tangent (tan ) and dynamic viscosity (), measured over a defined frequency range (0.01-1.0 Hz). Results. As the oscillatory frequency was increased, G G of all formulations increased, whereas both and tan significantly decreased. The magnitude of increase of G and G as a function of frequency was relatively small, indicating that, in general, the formulations were non-cross-linked elastic systems. Increasing concentrations of HEC, PVP and PC significantly increased G, G, yet decreased tan observations that may be attributed to the physical state of each polymer in the formulations. Formulation elasticity increased (i.e. tan decreased) as a result of increased entanglement of polymeric chains of dissolved components (i.e. HEC and PVP) and the restrained extension of swollen, cross-linked chains of PC. Additionally, in formulations where the saturation solubility of PVP was exceeded and/or insufficient 'free-water' was available for maximal swelling of PC, formulation elasticity increased as a result of the increasing mass of dispersed solid particles of PVP and/or PC. Formulation increased due to the attendent effects of polymer chain entanglement and polymer state on overall formulation viscosity. Conclusions. Following application to the oropharynx, the formulations will behave as elastic systems. Thus, these formulations would be expected to offer advantageous clinical properties, e.g., prolonged drug release, increased bioadhesion. However, it is noteworthy that the final choice of formulation for clinical evaluation will involve a compromise between viscoelastic characteristics and acceptable textural properties, e.g. ease of product application. This study has shown the applicability of oscillatory rheometry for both the characterisation and selection of candidate, topical bioadhesive formulations for clinical evaluation.     

A Biodegradable Injectable Implant Sustains Systemic and Ocular Delivery of an Aldose Reductase Inhibitor and Ameliorates Biochemical Changes in a Galactose-Fed Rat Model for Diabetic Complications

Purpose. To fabricate and characterize in vitro and in vivo performance of a sustained release biodegradable implant for N-4-(benzoylaminophenylsulfonyl glycine) (BAPSG), a novel aldose reductase inhibitor. Methods. The ability of BAPSG to inhibit aldose reductase activity and glucose-induced vascular endothelial growth factor (VEGF) expression was assessed in a retinal pigment epithelial cell line (ARPE-19). A poly (DL-lactic-co-glycolic acid) implant containing 50% w/w BAPSG was fabricated and characterized for drug loading, in vitro drug release, and the thermal behavior of the drug and the polymer. Implants were injected subcutaneously into a galactose-fed diabetic rat model and cataract scores, plasma and tissue drug levels, galactitol levels in the lens and the retina, glutathione levels in the plasma, lens, cornea and retina and VEGF expression in the retina were determined on or until 18 days. Results. BAPSG inhibited aldose reductase activity and reduced VEGF expression in ARPE-19 cells. Implants (1 × 4 mm), with a loading efficiency of 106 7% for BAPSG, were fabricated. Upon implant fabrication, while the glass transition temperature of the polymer decreased, the melting point of the drug was not affected. In vivo drug release correlated well with in vitro release, with 44% drug release occurring in vivo by the end of 18 days. The implant reduced galactitol accumulation, glutathione depletion, cataract scores, and VEGF expression in galactose-fed rats. Conclusions. An injectable biodegradable implant of BAPSG sustained drug release in vitro and in vivo, and reduced galactitol accumulation, glutathione depletion, cataract scores, and VEGF expression in galactose-fed rats.     

Targeted delivery of diclofenac sodium via gelatin magnetic microspheres formulated for intra-arterial administration

In the present work, we have attempted to deliver diclofenac sodium to a target site by intra-arterial injection of gelatin magnetic microspheres and subsequent localization using an external magnet. Drug-loaded magnetic microspheres were prepared by emulsification/cross-linking method, characterized by drug loading, magnetite content, size distribution, optical microscopy, scanning electron microscopy (SEM), Fourier transform infrared (FT-IR) analysis, differential scanning calorimetry (DSC), X-ray diffraction (XRD), absence of glutaraldehyde by gas chromatography, and in vitro release studies. The targeting efficiency and the therapeutic efficacy of microspheres were studied in vivo in rabbits. The microspheres showed drug loading of 9.1, 18.7, 24.9% w/w, magnetite content of 27.8–28.9% w/w with an average size range of 25–30.6 μm, depending upon the drug–polymer ratio. They were spherical in nature as evidenced by optical microscopy and SEM. FT-IR, DSC, and XRD studies revealed the absence of drug–polymer interaction. Gas chromatography confirmed the absence of residual glutaraldehyde. The microspheres were able to prolong the drug release over 24–30 days and the application of sonication during in vitro release study has slightly increased the release rate. After intra-arterial administration of microspheres, 77.7% of injected dose was recovered at the target site which revealed good targeting efficiency. The microspheres effectively reduced joint swelling, but lesser extent than the oral diclofenac sodium in high dose, in antigen induced arthritic rabbits without producing gastric ulceration which was observed in rabbits treated with oral diclofenac sodium.     

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.     

A New Mathematical Model Quantifying Drug Release from Bioerodible Microparticles Using Monte Carlo Simulations

Purpose. The major objectives of this study were to 1) develop a new mathematical model describing all phases of drug release from bioerodible microparticles; 2) evaluate the validity of the theory with experimental data; and 3) use the model to elucidate the release mechanisms in poly(lactide-co-glycolide acid)-based microspheres. Methods. 5-Fluorouracil-loaded microparticles were prepared with an oil-in-water solvent extraction technique and characterized in vitro. Monte Carlo simulations and sets of partial differential equations were used to describe the occurring chemical reactions and physical mass transport phenomena during drug release. Results. The new mathematical model considers drug dissolution, diffusion with nonconstant diffusivities and moving boundary conditions, polymer degradation/erosion, time-dependent system porosities, and the three-dimensional geometry of the devices. In contrast with previous theories, this model is able to describe the observed drug release kinetics accurately over the entire period of time, including 1) initial burst effects; 2) subsequent, approximately zero-order drug release phases; and 3) second rapid drug release phases. Important information, such as the evolution of the drug concentration profiles within the microparticles, can be calculated. Conclusions. A new, mechanistic mathematical model was developed that allows further insight into the release mechanisms in bioerodible microparticles.     

Optimized Formulation of Magnetic Chitosan Microspheres Containing the Anticancer Agent,Oxantrazole

A combined emulsion/polymer cross-linking/solvent evaporation technique was used to prepare magnetic chitosan microspheres (MCM) containing the anticancer drug, oxantrazole. A central composite experimental design was used to simultaneously evaluate a variety of formulation factors on a number of response variables, such as the percentage of oxantrazole entrapped in the MCM. In association with the study design, statistical optimization procedures indicated the factors that significantly influence MCM preparation and what levels of the factors are needed to produce optimum MCM. Entrapment of anticancer agents into biodegradable microspheres is difficult because of low aqueous drug solubility and porosity of the particles. The latter effect was circumvented by a chitosan cross-linking step that resulted in 3% (w/w) oxantrazole entrapment in the MCM via the optimization procedures. The combined formulation and statistical optimization strategy provide a basis to develop other microparticulate systems and led to a dosage form that can be used for future in vivo investigations.     

Preparation of Three-Month Depot Injectable Microspheres of Leuprorelin Acetate Using Biodegradable Polymers

To obtain a three-month release injection of leuprorelin acetate, microspheres were prepared with copoly(DL-lactic/glycolic acid) or poly(DL-lactic acid) (PLA) using an in-water drying method, and drug release was evaluated. The content of water-soluble oligomers in the polymers was found to strongly affect the initial burst, and reducing the content to less than 0.1% was necessary to keep the first-day release below 10%. Drug loading of more than 15% also increased the initial drug release; the acceptable maximum loading was 12%. Elevation of the glass transition temperature of the microspheres was observed with an increase in drug loading. This suggests formation of a rigid structure, possibly with arrangement of the polymer around the drug cores like in a micelle. This structure provides a hydrophobic barrier against diffusion of the hydrophilic peptide, resulting in high trapping efficiency and long-term sustained release dependent on polymer erosion. The microspheres prepared with PLA having a m.w. of 12,000 to 18,000 provided linear sustained release and persistent serum levels of the drug in rats for over 3 months.     

顺铂聚乳酸微球的药物释放特性及肝动脉栓塞研究

对顺铂聚乳酸微球进行了体外药物释放和家犬肝动脉栓塞研究。该微球粒径范围为50～200μm,平均粒径为115.76±35.94μm,顺铂含量为37.16%(W/W);体外药物释放机制符合Higuchi方程;肝动脉栓塞后8h,肝组织顺铂浓度高达21.55±12.18μg/g,明显高于肝动脉灌注顺铂组:3.16±0.09μg/g(P<0.05);肝动脉栓塞组的顺铂血浓峰值、各取血点浓度及曲线下面积AUC皆低于肝动脉灌注顺铂组。可望达到提高栓塞部位的药物疗效,降低全身毒副反应的作用。     

Blockade of γ-aminobutyric acid-receptors of the B-subtype inhibits the dopamine-induced enhancement of the release of cholecystokinin-like immunoreactivity from slices of rat dorsal caudatoputamen

Summary When slices of rat dorsal caudatoputamen (= neostriatum) are incubated in vitro, Choecystokinin-like immunoreactivity (CCK-LI) is released upon addition of veratridine (3.75 mol/l). This release is affected by dopamine and by -aminobutyric acid (GABA)-receptor agonists. Dopamine enhances the release by stimulating dopamine D₂-receptors and decreases it via D₁-receptors. GABA_A-receptor agonists enhance the veratridine-induced release of CCK-LI, while GABA_B-receptor agonists decrease it. In the present investigation, it was examined whether GABA-receptors are involved in the effect which dopamine exerts via D₂-receptors. The GABA_A-receptor antagonist bicuculline (10 mol/l)and the blocker of the GABA_A-receptor ionophore picrotoxin (1 mol/l) did not affect the dopamine (0.1 mol/1)-induced increase in the release of CCK-LI. However, the GABA_A-receptor agonist muscimol (1 mol/l) not only enhanced the release of CCK-LI, but also prevented a further enhancement by dopamine (0.1 mol/l). This effect of muscimol was blocked by bicuculline (10 mol/l). In the presence of -amino-n-valeric acid (0.1 mmol/l), which has been described to block GABA_B-receptors, dopamine no longer enhanced the veratridine-induced release of CCK-LI. -Amino-n-valeric acid also inhibited the pronounced enhancement of the release of CCK-LI caused by dopamine (0.1 mol/l) and 1 mol/l in the presence of the preferential D₁-receptor antagonist SCH 23390. The effect of -amino-n-valeric acid persisted in the presence of bicuculline (10 mol/l and 100 mol/l). (+)-Baclofen, a partial agonist at GABA_B-receptors, and the stereoisomer (–)-baclofen, a full agonist, also prevented the effect of dopamine on the veratridine-induced release of CCK-LI. The effects of both drugs may be due to desensitization of GABA_B-receptors, which has been described to develop quite rapidly. It is concluded that -amino-n-valeric acid blocks GABA_B-receptors and in this way prevents the enhancement of the veratridine-induced release of CCK-LI caused by dopamine via D₂-receptors. These data are interpreted as evidence that dopamine and GABA-neurons can directly or indirectly interact in the rat neostriatum. Send offprint requests to D. K. Meyer at the above address     

Preparation and Characterization of a Composite PLGA and Poly(Acryloyl Hydroxyethyl Starch) Microsphere System for Protein Delivery

Purpose. To prepare and characterize a novel composite microsphere system based on poly(D,L-lactide-co-glycolide) (PLGA) and poly(acryloyl hydroxyethyl starch) (acHES) hydrogel for controlled protein delivery. Methods. Model proteins, bovine serum albumin, and horseradish peroxidase were encapsulated in the acHES hydrogel, and then the protein-containing acHES hydrogel particles were fabricated in the PLGA matrix by a solvent extraction or evaporation method. The protein-loaded PLGA-acHES composite microspheres were characterized for protein loading efficiency, particle size, and in vitro protein release. Protein stability was examined by size-exclusion chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and monitoring the enzymatic activity. Results. Scanning electron microscopy showed discrete PLGA microspheres containing many acHES particles. The composite microspheres were spherical and smooth in size range of 39-93 m. The drug loading efficiency ranged from 51 to 101%. The composite microspheres showed more favorable in vitro release than conventional PLGA microspheres. The composite microspheres showed 20% less initial with a gradual sustained release compared to high burst (60%) followed by a very slow release with the conventional PLGA microspheres. The composite microspheres also stabilized encapsulated proteins from the loss of activity during the microsphere preparation and release. Proteins extracted from the composite microspheres showed good stability without protein degradation products and structural integrity changes in the size-exclusion chromatography and SDS-PAGE analyses. Horseradish peroxidase extracted from microspheres retained more than 81% enzymatic activity. Conclusion. The PLGA-acHES composite microsphere system could be useful for the controlled delivery of protein drugs.     

Determinants of Release Rate of Tetanus Vaccine from Polyester Microspheres

Controlled-release formulations based on poly(lactic) (PLA) and poly(lactic/glycolic) acid (PLGA) microspheres containing tetanus vaccine were designed. The polymers forming the microspheres were L-PLA of different molecular weights and DL-PLGA, 50:50. These microspheres were prepared by two solvent elimination procedures, both using a double emulsion, and were characterized for size, morphology, and toxoid release kinetics. The influence of formulation variables such as polymer type, vaccine composition, and vaccine/polymer ratio was also investigated. Both techniques yielded microspheres with similar size, morphology, and release properties. Microsphere size was dependent on the type of polymer and the presence of the surfactant L--phosphatidylcholine, which led to a reduction in microsphere size. On the other hand, the release kinetics of encapsulated protein were affected by the polymer properties (ratio lactic/glycolic acid and molecular weight) as well as by the vaccine composition, vaccine loading, and microsphere size. Moreover, for some formulations, a decrease in microsphere size occurred simultaneously, with an increase in porosity leading to an augmentation of release rate. The changes in the PLA molecular weight during in vitro release studies indicated that release profiles of tetanus toxoid from these microspheres were only marginally influenced by polymer degradation. A significant fraction of protein (between 15 and 35%) was initially released by diffusion through water-filled channels. In contrast, the decrease in the PLGA molecular weight over the first 10 days of incubation suggested that erosion of the polymer matrix substantially affects protein release from these microspheres. Among all formulations developed, two differing in microsphere size, polymer hydrophobicity, and release profile were selected for in vivo administration to mice. Administration of both formulations resulted in tetanus neutralizing antibody levels that were higher than those obtained after administration of the fluid toxoid.     

Viscosity of polymer solution phase and other factors controlling the dissolution of theophylline microspheres prepared by the emulsion solvent evaporation method

The objectives of this investigation are to evaluate the effect of the viscosity of polymer solution phase on microsphere properties, especially the drug release characteristics since no studies on this formulation variable have been reported. Also, since it is known that polymer molecular weight affects both the viscosity of the polymer solution and the release properties of microspheres, the interaction of these factors was studied. Microspheres with 33% theoretical drug loading of anhydrous theophylline core material were prepared by the emulsion solvent evaporation method. Two cellulose acetate butyrate polymers, (CAB381-2, CAB381-20), chemically similar but having different molecular weights, were used to prepare different polymer solutions having different apparent viscosities in acetone. A Brookfield viscometer was used to evaluate the viscosities of polymer solutions. Dissolution rates of microspheres prepared from the polymer solutions were inversely related to the initial polymer solution viscosities for both CAB381-2 and CAB381-20. The times for the release of 30 and 50% of the drug from the microspheres have a linear relationship with initial polymer solution viscosity. Initial release was significantly decreased with increasing polymer solution viscosity. Unlike CAB381-2 microspheres which follow Higuchi spherical matrix release kinetics, microspheres prepared from the higher molecular weight polymer (CAB381-20) showed extended release dissolution profiles with near zero order kinetics. It is evident that both the polymer solution viscosity and the molecular weight have an effect on the drug release from microspheres. These results suggest that release rates of matrix microspheres could be predictably optimized by adjusting the viscosity of polymer solutions.     

Pharmacological characterization of central α-adrenoceptors which mediate clonidine-induced locomotor hypoactivity in the developing rat

Summary The present experiment was designed to pharmacologically characterize receptors which mediate the clonidine-induced locomotor change in the developing rat. A subcutaneous injection of clonidine (0.78 mol/kg) produced locomotor hyperactivity in 7-day-old rats but hypoactivity in 20-day-old rats. Phenoxybenzamine (1.5 mol/kg, 5.9 mol/kg and 15 mol/kg) decreased spontaneous activity in a dosedependent manner but did not antagonize clonidineinduced hypoactivity in 20-day-old rats. By contrast, the significant reversal of the clonidine-induced hypoactivity by pretreatment with phentolamine (1.6 mol/kg and 6.3 mol/kg), yohimbine (1.3 mol/kg and 5.1 mol/kg) and piperoxan (7.4 mol/kg) was observed at such doses when the blockers did not cause and hypoactivity by themselves. It is suggested that clonidine could induce locomotor hypoactivity by activating presynaptic (₁-type) -adrenoceptors in the CNS of 20-day-old rat.