Correlation Between In Vitro Release and In Vivo Immune Response from Biodegradable Polymer Particles Entrapping Tetanus Toxoid

Release of immunoreactive antigen from biodegradable polymer particles in a manner mimicking the normal vaccination schedule is one of the most important requirements for the successful development of single dose vaccine formulation. Tetanus toxoid (TT) was used as a model antigen to develop single dose vaccine formulations using poly(lactide-co-glycolide) and poly(lactide)polymers. An amphiphilic stabilizer such as RSA or PVA was used at an optimal concentration in the internal aqueous phase during preparation of immunoreactive TT particles using the multiple emulsion solvent evaporation method. Particles made from different polymers released immunoreactive TT continuously for more than 4 months in vitro and release profiles were in accordance with the degradation characteristics of the polymer. Initial burst release of antigen from the particles was controlled by incorporation of different concentrations of PVA in the internal aqueous phase during particle formulation. The extent of antigen release from the particles was varied by changing the aqueous to organic phase volume during the primary emulsification stage of particles formulation. Use of above formulation variables resulted in the formulation of polymer particles having different in vitro release characteristics. Anti-TT antibody titers in vivo were also in accordance with the in vitro release characteristics of immunoreactive TT from the particles. Anti-TT antibody titers from the stabilized particles were much better than that observed from particles made without stabilizers. These results indicate the importance of stabilizers and different formulation variables for the preparation of polymer particles having desired in vitro release characteristics.     

In Vivo Evaluation of Biodegradable Progesterone Microspheres in Mares

Pharmaceutical Research -     

Comparative Assessment of In Vitro Release Kinetics of Calcitonin Polypeptide from Biodegradable Microspheres

The objective of our study was to compare the in vitro release kinetics of a sustained-release injectable microsphere formulation of the polypeptide drug, calcitonin (CT), to optimize the characteristics of drug release from poly-(lactide-co-glycolide) (PLGA) copolymer biodegradable microspheres. A modified solvent evaporation and double emulsion technique was used to prepare the microspheres. Release kinetic studies were carried out in silanized tubes and dialysis bags, whereby microspheres were suspended and incubated in phosphate buffered saline, sampled at fixed intervals, and analyzed for drug content using a modified Lowry protein assay procedure. An initial burst was observed whereby about 50% of the total dose of the drug was released from the microspheres within 24 hr and 75% within 3 days. This was followed by a period of slow release over a period of 3 weeks in which another 10-15% of drug was released. Drug release from the dialysis bags was more gradual, and 50% CT was released only after 4 days and 75% after 12 days of release. Scanning electron micrographs revealed spherical particles with channel-like structures and a porous surface after being suspended in an aqueous solution for 5 days. Differential scanning calorimetric studies revealed that CT was present as a mix of amorphous and crystalline forms within the microspheres. Overall, these studies demonstrated that sustained release of CT from PLGA microspheres over a 3-week period is feasible and that release of drug from dialysis bags was more predictable than from tubes.     

卡莫司汀聚乳酸微球的体外释放及其在大鼠脑组织中的分布研究

目的:探讨卡莫司汀聚乳酸微球(BCNU-PLA-MS)的体外释药过程及其在大鼠脑组织中的分布。方法:应用高效液相色谱法检测BCNU在磷酸盐缓冲溶液(PBS)和大鼠脑组织内自PLA-MS中释放的药物含量;应用3H标记BCNU,检测3H-BCNU-PLA-MS在正常大鼠脑组织及血清中的分布。结果:BCNU-PLA-MS在PBS和大鼠脑组织中均可持续释药2wk以上。在PBS中,其1、3、15d时药物释放率分别约为15%、50%、90%;在大鼠脑组织内,其4h、1d、3d时药物释放率分别约为8%、16%、60%,并可持续释药15d。大鼠药物植入处药物浓度是其它检测点的6～70倍。结论:BCNU-PLA-MS具有良好的缓释功能,而且安全性、生物相容性较好。     

Controlled Release of Contraceptive Steroids from Biodegradable and Injectable Gel Formulations: In Vivo Evaluation

Purpose. The purpose of this study was to investigate in vivo biocompatibility, biodegradability and biological effects of contraceptive steroids, such as levonorgestrel and ethinyl estradiol, released from gels prepared with a combination of derivatized vegetable oil (Labrafil 1944 CS) and glyceryl ester of fatty acids (Precirol ATO 5). Methods. Biocompatibility, biodegradability, and in vivo effects of levonorgestrel and ethinyl estradiol were studied by histologic evaluation of rat tissue, visual estimate of changes in gel size, and assessment of drug effects on reproductive cyclicity of female rats, respectively, following subcutaneous injection of gel formulations. Results. Histological evaluation of the tissue samples following an injection of the gel revealed an inflammatory reaction for about 7 days, after which the tissues did not show any inflammatory response. Complete degradation of the gels containing 10% wax was observed between 5 and 6 weeks. Normal rat estrous cycles were completely blocked by the contraceptive steroids released from the gels. Gel formulations containing 0.25% w/w levonorgestrel were more effective in blocking the estrous cycle of female rats compared to the oil formulations containing an identical drug loading. The duration of the biological effect induced by levonorgestrel appears to be dose-related. The gel formulation containing 2.00% ethinyl estradiol was superior to oil formulation containing an identical drug loading in terms of controlling drug release and toxicity. Conclusions. These observations suggest that Labrafil-Precirol gels are biocompatible and biodegradable. Moreover, controlled release of steroids is possible in vivo for a prolonged period of time.     

In Vitro Release Profile of Mitomycin C from Albumin Microspheres: Extrapolation from Macrospheres to Microspheres

To analyze the in vitro release profiles of mitomycin C from albumin microspheres prepared by chemical denaturation in a multiparticulate system, a method to calculate the total cumulative amount of mitomycin C released from a batch of microspheres was developed. Mitomycin C-loaded albumin macrospheres (diameter in mm range) were prepared, and the in vitro release kinetics of mitomycin C from individual macrospheres were determined. Then the relationship between the kinetic parameters and the physical parameters (e.g., diameter, weight) was investigated under the assumption that macrospheres and microspheres behave identically. Further, the size distribution of microspheres was measured, and the total cumulative amount of mitomycin C released from albumin microspheres was calculated. The release profiles of mitomycin C from individual macrospheres fitted first-order release kinetics better than spherical matrix kinetics. The calculated initial mitomycin C contents and first-order release rate constants for individual macrospheres were correlated with the weight and reciprocal of surface area of the macrospheres, respectively. The observed in vitro release profile for the microspheres agreed with the calculated values. These results suggest that this method is valid for calculating drug release from albumin microspheres.     

In Vitro/in Vivo Correlation for 14C-Methylated Lysozyme Release from Poly(Ether-Ester) Microspheres

PURPOSE: The purpose of this study was to obtain an in vitro/in vivo correlation for the sustained release of a protein from poly(ethylene glycol) terephthalate (PEGT)/poly(butylene terephthalate) (PBT) microspheres. METHODS: Radiolabeled lysozyme was encapsulated in PEGT/PBT microspheres via a water-in-oil-in-water emulsion. Three microsphere formulations varying in copolymer composition were administered subcutaneously to rats. The blood plasma was analyzed for radioactivity content representing released lysozyme at various time points post-dose. The in vitro release was studied in phosphate-buffered saline. RESULTS: The encapsulation efficiency, calculated from the radioactivity in the outer water phase of the emulsion, varied from 60-87%. Depending on the PEG segment length and wt% PEGT, the lysozyme was released completely in vitro within 14 to 28 days without initial burst. 14C-methylated lysozyme could be detected in the plasma over the same time courses. The in vitro/in vivo correlation coefficients obtained from point-to-point analysis were greater than 0.96 for all microsphere formulations. In addition, less then 10% of administered radioactivity remained at dose site at 28 days for the microsphere formulations, indicating no notable retention of the protein at the injection site. CONCLUSION: The in vitro release in phosphate-buffered saline and the in vivo release in rats showed an excellent congruence independent of the release rate of 14C-methylated lysozyme from PEGT/PBT microspheres.     

Controlled Release of Liposomes from Biodegradable Dextran Microspheres: A Novel Delivery Concept

Purpose. Previous experimental work suggests that convection maybe important in determining the biodistribution of drugs implanted orinjected in the vitreous humor. To develop accurate biodistributionmodels, the relative importance of diffusion and convection inintravitreal transport must be assessed. This requires knowledge of both thediffusivity of candidate drugs and the hydraulic conductivity of thevitreous humor. Methods. Hydraulic conductivity of cadaveric bovine vitreous humorwas measured by confined compression tests at constant loads of 0.15and 0.2 N and analyzed numerically using a two-phase model. Diffusioncoefficient of acid orange 8, a model compound, in the same mediumwas measured in a custom-built diffusion cell. Results. Acid orange 8 diffusivity within vitreous humor is about halfthat in free solution. When viscous effects are properly accounted for,the hydraulic conductivity of bovine vitreous humor is 8.4 ± 4.5 ×10^–7 cm²/Pa s. Conclusions. We predict that convection does not contributesignificantly to transport in the mouse eye, particularly forlow-molecular-weight compounds. For delivery to larger animals, such as humanswe conclude that convection accounts for roughly 30% of the totalintravitreal drug transport. This effect should be magnified forhigher-molecular-weight compounds, which diffuse more slowly, and inglaucoma, which involves higher intraocular pressure and thus potentiallyfaster convective flow. Thus, caution should be exercised in theextrapolation of small-animal-model biodistribution data to human scale.     

石杉碱甲缓释微球的体外释放度及其体内外相关性研究

目的建立体内外相关性良好的石杉碱甲缓释微球的体外释放度测定方法。方法采用直接释药法和透析释药法测定石杉碱甲缓释微球的体外释放度，考察加入吐温的量和透析袋内递质的体积对释药速度的影响;采用高效液相色谱法测定石杉碱甲缓释微球在大鼠注射部位的残留量，计算微球在体内的释药速度。通过相关性评价确定最佳的体外释放度测定方法。结果透析释药法可获得良好的体内外相关性(r＝0.990 2)。结论透析释药法可用于测定石杉碱甲缓释微球的体外释放度。     

In Vitro Evaluation of Sustained Drug Release from Biodegradable Elastomer

Poly(DL-lactic acid) (PLA), poly(-caprolactone) (PCL), and their copolymers (PLA-CL) with various monomer compositions were synthesized, and their properties as matrix for the sustained release of drugs were evaluated. The copolymerization technique produced very soft films which incorporated the drugs without deterioration of the elastic properties. Cisplatin and MD-805 were loaded in the films by casting the polymer solution containing the drugs. Fractions of the drugs released from the PLA-CL films were governed by the initial loading, the film thickness, and the polymer molecular weight. The drug release profiles obeyed the classical Fickian diffusion equation at least in the early stage, but significant hydrolytic degradation of the matrix polymers occurred in the later stage, influencing the kinetics of drug release. The monomer composition of copolymer affected the release profile more strongly than the initial molecular weight of the copolymer.     

Kinetics of a Model Nucleoside (Guanosine) Release from Biodegradable Poly(dl-lactide-co-glycolide) Microspheres: A Delivery System for Long-Term Intraocular Delivery

The objective of this study was to prepare poly(dl-lactide-co-glycolide)(PLGA) microspheres containing guanosine as a model drug for intraocular administration. Microspheres were prepared by solvent evaporation technique using o/w emulsion system. The influence of composition and molecular weight of PLGA, drug loading efficiency, microsphere size, and in vitro and in vivo release rates were determined. Differential scanning calorimetry (DSC) and FTIR studies were conducted to examine the guanosine–polymer interaction. In vitro release studies indicated that the permeant release from microspheres exhibits an initial burst followed by slow first-order kinetics. Ascending molecular weights of the polymers generated progressively slower release rates. Three different sizes of microspheres were prepared. The release continued for 7 days with a maximum of 70% of the content released within that time period. DSC and FTIR studies showed no polymer–guanosine interaction. A novel microdialysis technique was used to examine the initial release kinetics from microspheres in isolated vitreous humor. This technique was also employed to observe in vivo intravitreal release in albino rabbits. A good correlation exists between in vitro and in vivo release rates from both 75 and 140 kDa PLGA microspheres. Guanosine-loaded microspheres could be prepared for once-a-week intravitreal injection with minimum required concentration maintained throughout the dosing interval. Because the structural and solubility characteristics of guanosine are similar to those of acyclovir and ganciclovir (two acycloguanosine analogues effective against herpes simplex virus [HSV-1] and cytomegalovirus [CMV], respectively), similar biodegradable polymer-based microsphere technology can be employed for the long-term intraocular delivery of these two drugs.     

A型肉毒杆菌毒素的壳聚糖／海藻酸钠缓释微囊研制和药物的体外释放速率研究

目的：研制包裹A型肉毒杆菌毒素的壳聚糖/海藻酸钠微囊,并测定其体外药物释放动力学。方法：采用注射器滴注法制成包裹A型肉毒杆菌毒素的壳聚糖/海藻酸钠微囊混悬液。采用高效液相色谱法测定A型肉毒杆菌毒素的标准曲线,每隔2周测定微囊所包裹的A型肉毒杆菌毒素体外释放浓度。结果：微囊形态为圆形透明颗粒状,粒径在1mm左右。用SPSS统计软件进行分析微囊中A型肉毒杆菌毒素的体外每2周释放量,显示药物累积释放量与时间之间为直线关系,基本符合药物零级释放动力学。结论：滴注法制备包裹A型肉毒杆菌毒素的壳聚糖/海藻酸钠缓释微囊制作方法简便,其药物体外释放稳定。     

Hyaluronic Acid and Poly-l-Lysine Layers on Calcium Alginate Microspheres to Modulate the Release of Encapsulated FITC-Dextran

Alginate solutions crosslink into microspheres in calcium alginate, enabling the encapsulation and subsequent release of biological macromolecules and drugs. However, release from calcium alginate into PBS is relatively fast because it will decrosslink the gel relatively quickly. In this research, FITC-dextran (MW 10 kDa) was encapsulated in 2% (w/v) calcium alginate microspheres by electrospraying. The resulting microspheres (diameter = 247 ± 13 μm) were then layered with thin polyelectrolyte films of hyaluronic acid (HA) and poly-l-lysine (PLL) to attempt to slow the diffusion of FITC-dextran out of the microspheres and the coating parameters were modified to modulate diffusion and release. Increasing the concentration of FITC-dextran encapsulated in the microspheres resulted in an increase in its release over time into PBS. Crosslinking PLL/HA layers on the microspheres did not decrease the in vitro release rates of encapsulated FITC-dextran into PBS. Increasing the number of layers on the microspheres from 3 to 5 layers significantly decreased the amount of encapsulated FITC-dextran released. However, increasing the number of layers to 7 did not further sustain the release of FITC-dextran, likely because these microspheres collapsed to a smaller size during the coating procedure, resulting in release controlled by both diffusion and swelling. Multiple layers of PLL and HA provided a robust mechanism to sustain and control release of large molecules from calcium alginate.     

Utilization of an Amorphous Form of a Water-Soluble GPIIb/IIIa Antagonist for Controlled Release from Biodegradable Microspheres

Purpose. We prepared injectable microspheres for controlled release of TAK-029, a water-soluble GPIIb/IIIa antagonist and discussed the characteristics of controlled release from microspheres. Methods. Copoly(dl-lactic/glycolic)acid (PLGA) microspheres were used for controlled release of TAK-029 [4-(4-amidinobenzoylglycyl)-3-methoxycarbonyl-2-oxopiperazine-l-acetic acid]. They were prepared with a solid-in-oil-in-water (S/O/W) emulsion solvent evaporation technique using either a crystalline form or an amorphous form of the drug. Results. An amorphous form of TAK-029 gave more homogeneous S/O dispersion and higher viscosity than its crystalline form when added to dichloromethane solution of PLGA, resulting in a high drug entrapment into microspheres and a well-controlled release of the drug. Additions of sodium chloride into an external aqueous phase and L-arginine into an oil phase also increased entrapment of the drug, and reduced initial burst of the drug from the microspheres. The micro-spheres demonstrated a desirable plasma level profile in therapeutic range (20–100 ng/ml) for 3 weeks in rats after single subcutaneous injection. Conclusions. A well-controlled release of TAK-029, a water-soluble neutral drug, with small initial burst was achieved by utilizing its amorphous form as a result of possible interaction with PLGA and L-arginine.     

In Vitro Characterization and in Vivo Testosterone Suppression of 6-Month Release Poly(D,L-Lactide) Leuprolide Microspheres

Pharmaceutical Research -     

Osmotic-Driven Release Kinetics of Bioactive Therapeutic Proteins from a Biodegradable Elastomer are Linear, Constant, Similar, and Adjustable

Purpose The aim of the study is to determine whether a biodegradable elastomeric device that uses an osmotic pressure delivery mechanism can release different therapeutic proteins at a nearly constant rate in nanomolar concentrations with high bioactivity, given the same formulation conditions. Vascular endothelial growth factor (VEGF) and interleukin-2 (IL-2) were embedded in the device as sample therapeutic proteins, and their release and bioactivity were compared to that achieved previously with interferon-γ (IFN-γ). Methods A photo-cross-linkable biodegradable macromer consisting of acrylated star(ɛ-caprolactone-co-d,l-lactide) was prepared. VEGF, IL-2, and IFN-γ were co-lyophilized with serum albumin and trehalose at different ratios and were then embedded into the elastomer by photo-cross-linking the lyophilized particles in a macromer solution. The protein mass and the bioactivity in the release supernatant were measured by enzyme-linked immunosorbent and cell-based assays. Results VEGF, IL-2, and IFN-γ were released at the same, nearly constant rate of 25.4 ng/day for over 18 days. Using the optimum elastomer formulation, the release profiles of the proteins were essentially identical, and their rates were linear and constant. Cell-based bioactivity assays showed that 70 and 88% of the released VEGF and IL-2, respectively, were bioactive. The rate of protein release can be adjusted by changing the trehalose loading concentration in the elastomer matrix without altering the linear nature of the protein release kinetics. The elastomeric device degraded in PBS buffer within 85 days. Conclusions The elastomer formulation shows promising potential as a sustained protein drug delivery vehicle for local delivery applications.     

Controlled Release of Paracetamol from Amylodextrin Tablets: In Vitro and in Vivo Results

Amylodextrin is a suitable excipient for the design of solid controlled-release systems. The release of paracetamol from tablets containing 30% drug and 70% amylodextrin was studied in vitro and in vivo. In vitro dissolution profiles showed almost-constant drug release rates during 8 hr, when measured in 0.05 M buffer, pH 6.8. Peroral administration of the tablets to man showed almost-constant paracetamol plasma levels up to 14 hr, as compared to fast absorption and fast elimination of a reference paracetamol solution. The plasma profiles of eight volunteers demonstrated a small intersubject variability during the first day after tablet administration. Increasing variability and decreasing plasma levels during the second day were caused by excretion of tablets from the bodies. Cumulative input as a function of time showed near-zero-order drug release during the first day. The in vivo results indicate that amylodextrin tablets are not hydrolyzed by -amylase, present in the gastrointestinal tract.     

Controlled Release of a Contraceptive Steroid from Biodegradable and Injectable Gel Formulations: In Vitro Evaluation

Purpose. The purpose of this study was to investigate the effects of formulation factors including varying wax concentration, drug loading and drug particle size, on drug release characteristics from both pure oil and gel formulations prepared with a combination of derivatized vegetable oil (Labrafil 1944 CS) and glyceryl palmitostearate (Precirol ATO 5), using levonorgestrel as a model drug. Methods. The effects of varying drug loadings, different drug particle sizes, and wax (Precirol) concentrations on in-vitro drug release rates were evaluated, and the mechanisms of drug release from the gels were determined. Results. Zero-order drug release rates from the 10% Precirol gel formulations containing 0.25, 0.50 and 2.00% w/v drug loadings were lower than those observed for oil formulations containing identical drug loadings. Higher zero-order release rates were observed from formulations containing smaller drug particles suspended in both oil and gel formulations. The mechanism of drug release from gels containing less than 0.25% w/w drug was diffusion-controlled. Increasing the wax concentrations in the gels from 5% w/w to 20% w/w significantly decreased the diffusivity of the drug through the gel formulations and markedly increased the force required to inject the gels from two different sizes of needles. Conclusions. This study shows how modification of the physicochemical properties of the gel formulations by changing the drug particle size, wax concentration and drug loading, affects drug release characteristics from the system.     

Controlled In Vivo Release of Nicorandil from a Carvone-Based Transdermal Therapeutic System in Human Volunteers

The aim of our present study was to prepare and evaluate a carvone-based transdermal therapeutic system (TTS) of nicorandil to find its ability in providing the desired in vivo controlled release profile on dermal application to human volunteers. The effect of EVA 2825, and adhesive-coated EVA 2825, and adhesive-coated EVA 2825-rat skin composite on the in vitro permeation of nicorandil from a carvone-based HPMC gel drug reservoir was studied against a control (rat abdominal skin alone). The carvone-based drug reservoir system was sandwiched between adhesive-coated EVA 2825-release liner composite and a backing membrane. The resultant drug reservoir sandwich was heat-sealed to produce a circle-shaped TTS (20 cm²) that was subjected to in vivo evaluation on dermal application to human volunteers against oral administration of immediate-release tablets of nicorandil. The carvone-based TTS provided a steady-state plasma concentration of 20.5 ng/ml for ～24 hr in human volunteers. We concluded that the carvone-based TTS of nicorandil provided the desired in vivo controlled-release profile of the drug for the predetermined period of time.