Evaluation of residual dentin thickness using quantitative light-induced fluorescence technology.

This study aimed to determine whether a correlation exists between residual dentin thickness and quantitative light-induced fluorescence (QLF) values and, if so, to analyze its tendencies. Forty extracted sound human molars were assigned to filled and unfilled groups. The teeth were submerged in a mold with clear acrylic resin. Red utility wax was inserted into the pulp chamber space in the filled group to simulate vital pulp. The specimen was sectioned longitudinally to observe the inside of the pulp space. The samples were cut horizontally from the highest point of the pulp space 2 mm apart. QLF images were then taken of 2 mm, 1 mm, and 0.5 mm samples using the QLF-D Biluminator™ 2 + system. Three operators independently evaluated the QLF images, and the statistical analysis was conducted using one-way analysis of variance, Pearson correlation coefficients, and intraclass correlation coefficients. In the filled group, the mean ΔF values for residual dentin thicknesses of 2 mm, 1 mm, and 0.5 mm were - 3.22, - 7.84, and - 11.52, respectively. In the unfilled group, the mean ΔF values were 0, - 6.90, and - 10.14, respectively. A positive correlation was found between residual dentin thickness and ΔF values (P < 0.05). The intraclass correlation coefficients for observations made by the three operators for the filled and unfilled groups were 0.831 and 0.917, respectively (P < 0.05). In conclusion, residual dentin thickness and ΔF values were significantly correlated and had a highly positive correlation regardless of the QLF device operator.

Dexamethasone Degradation in Aqueous Medium and Implications for Correction of In Vitro Release from Sustained Release Delivery Systems.

The in vitro drug release in an aqueous medium is a critical performance metric for a sustained release drug product. During long-term release studies, drugs may degrade in the release medium, and such degradation can lead to errors in drug release quantitation. Using dexamethasone as a model drug and LC-MS/MS methods employing dexamethasone-d4 as an internal standard, this study identified that dexamethasone can degrade into 13 major degradation products in phosphate buffered saline (PBS) as a function of time, temperature (25, 37, and 45°C), and light exposure. A putative scheme for dexamethasone degradation pathways in PBS has been proposed. In proof-of-concept studies, the analytical method was used to quantitate dexamethasone and its degradation products during in vitro release studies with sustained release dexamethasone-poly(D,L-lactide-co-glycolide) (PLGA) implants incubated in phosphate buffer saline (PBS). Further, mathematical approaches were developed to estimate drug release from implants after accounting for drug degradation in PBS. The LC-MS/MS analytical method and the mathematical approaches developed could be used for assessing the stability and/or release of dexamethasone during manufacturing, storage, and use of various dosage forms.

FDA's Poly (Lactic-Co-Glycolic Acid) Research Program and Regulatory Outcomes.

Poly (lactic-co-glycolic acid) (PLGA) has been used in many long-acting drug formulations which have been approved by the US Food and Drug Administration (FDA). However, generic counterparts for PLGA products have yet to gain FDA approval due to many complexities in formulation, characterization, and evaluation of test products. To address the challenges of generic development of PLGA-based products, the FDA has established an extensive research program to investigate novel methods and tools to aid both product development and regulatory review. The research focus have been: (1) analytical tools for characterization of PLGA polymers; (2) impacts of PLGA characteristics and manufacturing conditions on product performance; (3) in vitro drug release testing and in vitro-in vivo correlation of PLGA-based products, and (4) modeling tools to facilitate formulation design and bioequivalence study design of PLGA-based drugs. This article provides an overview of FDA's PLGA research program and highlights scientific accomplishments as well as regulatory outcomes that have resulted from successful research investigations.

Effect of implant formation on drug release kinetics of in situ forming implants.

In situ forming implants are attractive long-acting implant dosage forms due to their: i) ability to control drug release; ii) simple manufacturing process; and iii) minimally invasive administration. In situ forming implants are typically made of a drug, solvent, and a biocompatible polymer that controls drug release. Once injected in the subcutaneous tissue, they form solid depots through solvent/non-solvent exchange and phase separation of the biodegradable polymer (such as poly (lactic-co-glycolic acid), PLGA and poly (lactic acid), PLA). However, the mechanism of implant formation and the changes in their microstructure that determine drug release behavior are not fully understood. Furthermore, there is no standardized in vitro release testing method for in situ forming implants due to limitations in recreating bio-relevant and reproducible implant formation in vitro with controllable implant shape, dimensions and surface-to-volume ratio. In the present study, bio-relevant implant formation was recreated in vitro by testing five different methods to determine their effect on drug release kinetics, reproducibility, and internal microstructure formation. The leuprolide acetate formulation Eligard® was used as a model in situ-forming implant, consisting of lyophilized leuprolide acetate, and PLGA dissolved in N-methyl pyrrolidone. The results revealed that the in vitro implant formation method is a crucial step in the dissolution testing process that significantly impacts the release profile of in situ forming implants. An implant formation method that utilizes dissolvable polyvinyl alcohol (PVA) films allowed for initial drug burst release control by modulating implant dimensions (i.e. surface area) and resulted in reproducible in vitro release profiles. In addition, implant formation was shown to affect the internal microstructure of in situ forming implant and was the main factor controlling the release profile which consisted of an initial release phase followed by a release plateau (lag phase) and then a second erosion-controlled release phase.

Impact of product design parameters on in vitro release from intrauterine systems.

Polydimethylsiloxane (PDMS)-based levonorgestrel intrauterine systems (LNG-IUSs) contain a large amount of potent LNG, and therefore it is important to understand the impact of product design parameters on the in vitro and in vivo performance to ensure safety and efficacy, as well as to avoid serious side effects resulting from dose dumping. LNG-IUS is a complex drug-device combination product, and its formulation design, requires consideration of additional factors such as device configuration and dimensions, in addition to formulation and processing parameters. In this study, ten qualitatively (Q1) and quantitatively (Q2) equivalent LNG-IUSs were manufactured with differences in source (supplier) and dimensions (i.e., thickness) of the outer membrane, drug particle size, dimensions of the drug reservoir (i.e., inner diameter), as well as configuration of the entire IUS. A real-time in vitro release testing method was developed for the LNG-IUSs. In addition, an accelerated release testing method was developed using hydro-alcoholic media in order to reduce the time associated with formulation design. Source variations and thickness of their outer membranes had a great impact on the in vitro drug release from the LNG-IUSs. It was demonstrated that the thicker the outer membrane, the slower the drug release rate. The physicochemical properties of the outer membranes obtained from different sources were characterized to understand their impact on the in vitro drug release of the LNG-IUSs. The composition and mechanical strength may play a role in differences in drug release. The LNG-IUS formulation prepared with the larger drug particle size showed a slightly slower daily release rate. The drug release rates from the compositionally equivalent LNG-IUSs linearly correlated to the surface area of the corresponding drug reservoirs. Another factor that affected the drug release rate was the configuration of the entire IUS. It was shown that the placement of the outer membrane was significant, i.e. whether the ends of the drug reservoir were covered or not. It is important to note that real-time release showed zero-order release kinetics over the test period of approximately 900 days. The current study provides a comprehensive understanding of the impact of product design parameters on the in vitro drug release of LNG-IUSs. In addition, the developed real-time and accelerated release testing methods showed good discriminatory ability for compositionally equivalent LNG-IUSs prepared using different product design parameters.

Manufacturing of Dexamethasone-Poly(d,l-Lactide-co-Glycolide) Implants Using Hot-Melt Extrusion: Within- and Between-Batch Product Performance Comparisons.

Purpose: Reliable drug therapy with injectable intravitreal implants requires implants of consistent quality. The purpose of this study was to prepare dexamethasone-poly(d,l-lactide-co-glycolide) (PLGA) biodegradable implants and assess implant quality within and between batches for different polymer compositions. Methods: Implants containing 20% w/w dexamethasone with 3 theoretical rates of release (fast, intermediate, and slow) were manufactured with decreasing proportion of acid-terminated PLGA (50:50) and increasing proportion of ester-terminated PLGA (50:50) in a batch process using hot-melt extrusion. The implants were manufactured without and with in-process modification of extrusion/conveyor speed in the late phase of each batch. Implant samples collected at early, middle, and late phases of each batch were analyzed for diameter, drug loading, mechanical properties (strength and toughness), and drug release. Results: With a fixed process, unlike a modified process with an increase in extrusion speed and reduction of conveyor speed in the late phase, all implant formulations tended to decrease in diameter and mechanical properties in the late phase. Drug release profiles for the intermediate and slow release compositions were similar with or without process modification, unlike the fast release composition. Addition of ester-terminated PLGA resulted in a slower drug release. When all formulations are grouped together, the implant diameter exhibited a moderate correlation with mechanical properties, but no correlation was observed with drug release. Conclusions: Within a hot-melt extrusion batch process, the dexamethasone-PLGA implant diameter and hence toughness and strength tend to decline in the latter phase. In-process adjustment of extrusion and conveyor speeds can improve batch consistency and, potentially, implant integrity or performance during or after injection. Process changes did not affect drug release for 2 of the 3 implant compositions.

Factors affecting the particle size distribution and rheology of brinzolamide ophthalmic suspensions.

Drug particle size distribution (PSD) and dispersion viscosity are two critical quality attributes that govern the performance of topical ophthalmic suspensions, such as suspension physical stability, ocular retention, and drug release characteristics.. An in-depth knowledge of the effects of formulation and manufacturing process on these critical quality attributes may facilitate the product and process development, quality control, as well as support regulatory policy and approval. The current study has investigated the effect of formulation and process parameters on the quality attributes of brinzolamide ophthalmic suspensions. In the first step, three milling techniques (probe sonication, microfluidization, and media milling with a planetary centrifugal mixer) were evaluated for manufacturing of brinzolamide suspension. Out of the three techniques, the planetary centrifugal media milling yielded the narrowest PSD and thus was considered the most viable lab-scale technique for this purpose. In the next step, various process parameters of media milling were evaluated using a central-composite experimental design. The independent variables included bead size, agitating intensity, and process time while the PSD of drug particles (D50) was the response variable. The effect of shear rate and shear time of the homogenization process and the concentration of carbomer on the rheological properties of the suspension were studied using a Box-Behnken design. Additionally, effects of sodium chloride and mannitol concentration on the rheological properties of the suspension was also investigated. Sodium chloride was found to exert a pronounced effect on rheology of the suspension. Despite variations in the carbomer concentration, a suspension of comparable rheology could be prepared by controlling the process parameters namely the shear rate and process time.

Effect of Particle Size and Viscosity of Suspensions on Topical Ocular Bioavailability of Budesonide, a Corticosteroid.

Purpose: To determine the effect of particle size and viscosity of suspensions on topical ocular bioavailability of budesonide, a corticosteroid drug. Methods: Budesonide microparticle and nanoparticle (MP and NP) suspensions were prepared with or without homogenization and microfluidization. Using different grades of hydroxyl propyl methyl cellulose, low viscosity NP (NP-LV) and low and high viscosity MP (MP-LV and MP-HV) were prepared. Suspensions were characterized for particle size, viscosity, and osmolality. Budesonide suspensions were administered topically to rabbits and aqueous humor was collected and analyzed for budesonide. Budesonide Cmax, tmax, and the area under the concentration time curve (AUC (0-6h)) values were determined. The geometric mean ratio of AUC and bioequivalence was evaluated using a bootstrap method. Results: The particle sizes for NP and MP were ∼700 and 2,000 nm. The viscosities for low and HV formulations were ∼5 and 50 cP. The geometric mean budesonide Cmax values for the suspensions NP-LV, MP-LV, and MP-HV were 0.22, 0.22, and 0.31 µg/g, tmax values were 0.67, 0.60 and 0.53 h, and AUC0-6h values were 0.72, 0.53, and 0.95 µg h/g, respectively. Bootstrap analysis indicated that the 90% confidence intervals of the geometric mean ratio of AUC0-6h values were 1.00-1.74 (MP-HV vs. NP-LV), 0.57-0.96 (MP-LV vs. NP-LV), and 0.45-0.70 (MP-LV vs. MP-HV). Conclusions: The 3 budesonide suspensions assessed in this study were not bioequivalent. Results suggested that an increase in viscosity improves the bioavailability of budesonide from the microsuspension formulation.

Effect of minor manufacturing changes on stability of compositionally equivalent PLGA microspheres.

The physicochemical properties and drug release characteristics of Q1/Q2 equivalent microspheres are sensitive to minor manufacturing changes, which may alter their stability under different storage-conditions. This may be undesirable due to the presence of a substantial amount of drug in microsphere products. Hence, the objective of the present work was to investigate the impact of minor manufacturing changes on the stability of Q1/Q2 equivalent microspheres under various storage conditions. Two Q1/Q2 equivalent risperidone microsphere formulations prepared with minor manufacturing changes (solvent system etc.) showed differences in their physicochemical properties (size, morphology, porosity etc.), drug release characteristics and hence, storage stability. Overall, both formulations were stable under long-term storage conditions (4 °C/ambient humidity). However, under the intermediate storage conditions (25 °C/ambient humidity), only formulation 1 was stable while formulation 2 showed significant polymer degradation, particle aggregation and alteration in the drug release characteristics. Lastly, under accelerated storage conditions (40 °C/ambient humidity vs 75% RH), the extent of polymer degradation, morphological changes and alteration of drug release characteristics of formulation 2 was significantly higher compared to that of formulation 1. Thus, minor manufacturing changes have the potential to significantly alter the storage stability and, hence, the quality and performance of complex drug products such as microspheres.

Drug release testing of long-acting intrauterine systems.

Performance evaluation of polydimethylsiloxane (PDMS) based long-acting (e.g. 3-5 years) levonorgestrel (LNG) intrauterine systems (IUSs), such as Mirena®, is challenging due to their complex formulation, locally-acting feature, and extremely long duration of drug release. To achieve such long-term release, a large amount of drug (up to 52 mg in Mirena®) must be incorporated as a drug reservoir in the IUS. Consequently, dose dumping or unanticipated changes in the LNG-IUS in vivo release characteristics may give rise to adverse product safety and efficacy. Therefore, it is crucial to understand, and have appropriate control over, the physicochemical properties and in vitro release characteristics of these products. This requires an understanding of the LNG-IUSs drug release mechanism and the development of a sensitive yet robust in vitro release testing method. There have been no previous reports on in vitro drug release and the release mechanism from LNG-IUSs. This is probably a consequence of the extremely slow drug release rate of LNG-IUSs under real-time in-use conditions (e.g., 3-5 years) and therefore it is impractical to obtain complete release profiles (e.g. there is only 60% release in 5 years for Mirena®). Therefore, the development of appropriate accelerated in vitro release methods is imperative. Following preparation of LNG-IUSs, similar to Mirena®, real-time release was tested in (0.9% w/v NaCl) media in a water shaker bath at 37 °C for over 2 years. Addition of surfactant (sodium dodecyl sulfate (SDS)), elevation of temperature, addition of organic solvents (ethanol (EtOH), isopropanol (IPA), tert-butanol (TBA) and tetrahydrofuran (THF)) and a combination thereof were utilized as release media to accelerate drug release for LNG-IUSs. Complete drug release was achieved in 32 and 672 days in THF and TBA hydro-organic media, respectively. The release profile in THF was considered too fast as it may result in change of release mechanism, whereas the release profile in TBA was deemed suitable following model fitting. Model fitting was performed to understand the release characteristics as well as the release mechanisms. The release rate in the hydro-alcoholic media was linearly proportional to the swelling ratio of the PDMS in the corresponding organic solvents. Zero-order, first-order and two-phase models were utilized to fit the release profiles obtained under the different release conditions. The data analysis was comparable using the parameters from different models given the high R2 values. However, the two-phase model was better in terms of the release mechanism of the LNG-IUSs considering the full drug release profile. The present study will facilitate the process of granting of biowaivers through an in vitro approach, thus reducing the necessity for clinical studies. In addition, it will help reduce the regulatory burden without sacrificing product quality of LNG-IUS products.

Development of Level A in vitro-in vivo correlations for peptide loaded PLGA microspheres.

Peptide loaded PLGA microsphere products are more complex in terms of manufacturing, drug release characteristics as well as release mechanism compared to small molecule loaded PLGA microsphere products. This is due to the complex structure of peptides, their hydrophilicity, charged state, large size and potential for instability. Moreover, therapeutic peptides are highly potent and therefore, any unintended change in the microsphere release profile may lead to undesirable side effects and toxicity. Accordingly, the objectives of the present work were: 1) to evaluate the impact of minor manufacturing changes on the quality and performance of peptide microspheres; and 2) to investigate the feasibility of developing Level A in vitro-in vivo correlations (IVIVCs) for peptide microspheres. Compositionally equivalent leuprolide acetate (LA) microspheres prepared with minor manufacturing changes (solvent system/homogenization speed) showed significant differences in their physicochemical properties (such as pore size, total porosity, particle size and surface distribution of peptide on the prepared microspheres). This, in turn, resulted in significant alteration in the release characteristics. Peptide-polymer interaction, in vitro degradation and microsphere morphology studies were conducted to facilitate understanding of the differences in the drug release characteristics. A rabbit model was used to determine the pharmacokinetic profiles of all the prepared formulations. The obtained in vivo release profiles showed the same rank order as the in vitro release profiles but with low burst release and overall faster in vivo release rates. The low in vivo burst release is considered to be due to the masking effect of the absorption phase from the intramuscular site, and this complicated the development of an IVIVC. Despite these challenges, an affirmative Level A IVIVC over the entire release profile was successfully developed in a rabbit model for peptide microspheres for the first time. The developed IVIVC was also predictive of the RLD product, Lupron Depot®. This work highlights the feasibility of developing IVIVCs for complex parenteral drug products such as peptide microspheres. In conclusion, these results indicate the sensitivity of peptide release, and hence, the safety and efficacy of highly potent peptide microspheres, to minor manufacturing changes. Accordingly, development of IVIVCs for such complex drug products is highly desirable.

Understanding drug distribution and release in ophthalmic emulsions through quantitative evaluation of formulation-associated variables.

Establishing bioequivalence (BE) of ophthalmic emulsions in the absence of in vivo data is challenging. In these emulsions, drug release is a complex process due to drug distribution among various phases which are difficult to characterize. The objective of this study is to investigate the process of drug distribution and mechanism of drug release in the context of formulation-associated variables. A previously reported kinetic method for determining drug partitioning was used to quantitatively evaluate the drug distribution within a simplified biphasic (emulsion) system employing cyclosporine and difluprednate as model drugs. The impacts of formulation variables, such as the amount of polysorbate 80, glycerin, and carbomer copolymer as well as the area of oil-water interface were investigated. Polysorbate 80 was found to have the greatest influence on the drug distribution. It enhanced both the rate and extent of the drug distribution from oil to aqueous phase. Glycerin was found to slightly reduce the rate and extent of drug distribution of cyclosporine into the aqueous phase, probably by suppressing the solubilization capability of the micelles. Carbomer slowed down the diffusion of drug into the oil phase and shifted the equilibrium drug distribution towards the aqueous phase. Furthermore, increase in the interfacial area significantly increased the rate of drug diffusion across the oil-aqueous interface but had negligible effect on the extent of drug distribution. It is noteworthy that the experimental setup utilized a planar interface rather than an interface with curvature, which may have slightly underestimated the influence of globule size on equilibrium drug distribution. The findings of this study give insight into the drug distribution and diffusion in complex ophthalmic emulsions and assist with formulation design as well as development of in vitro methods to support BE assessment of ophthalmic emulsions.

In vitro-in vivo correlation of parenteral PLGA microspheres: Effect of variable burst release.

Development of IVIVCs is a very complicated process, especially for complex drug products such as parenteral PLGA microspheres with multiphasic drug release characteristics. Specifically, microspheres that exhibit an initial burst release phase are even more challenging since the in vitro and in vivo burst release phases may not be comparable if drug absorption is rate-limiting at this stage. Therefore, the objectives of the present work were: 1) to investigate the predictability of developed IVIVCs for the in vivo burst release phase based on the in vitro burst release phase of the formulations; and 2) to evaluate the impact of variable burst release on the predictability of the developed IVIVCs for two different types of microsphere-based drug products. Accordingly, Risperdal Consta® (Risperidone) and Lupron Depot® (Leuprolide acetate, LA) were selected as model products. Compositionally equivalent risperidone and LA formulations with variable burst release phases were prepared with manufacturing process changes (such as solvent systems and mixing methods). The prepared microspheres exhibited differences in critical physicochemical properties (such as particle size, porosity, average pore diameter, and drug distribution) and hence differences in their in vitro release characteristics (such as variable burst release and release rate). The in vitro and in vivo (rabbit model (intramuscular injection) burst release were similar for the risperidone microspheres but were significantly different for the LA microspheres. This had an impact on the complexity of the developed IVIVC models. Level A IVIVCs with the ability to predict various types of burst release were developed using time scaling and shifting factors. Moreover, it was observed that IVIVCs developed using formulations with less variation in burst release had better predictability and vice-versa. Thus, the present research has provided a comprehensive understanding of the impact of the burst release phase on the development, complexity, and predictability of IVIVCs for complex parenteral microspheres containing a variety of therapeutic molecules.

Probing the mechanism of bupivacaine drug release from multivesicular liposomes.

The mechanism of drug release from complex dosage forms, such as multivesicular liposomes (MVLs), is complex and oftentimes sensitive to the release environment. This challenges the design and development of an appropriate in vitro release test (IVRT) method. In this study, a commercial bupivacaine MVL product was selected as a model product and an IVRT method was developed using a modified USP 2 apparatus in conjunction with reverse-dialysis membranes. This setup allowed the use of in situ UV-Vis probes to continuously monitor the drug concentration during release. In comparison to the traditional sample-and-separate methods, the new method allowed for better control of the release conditions allowing for study of the drug release mechanism. Bupivacaine (BPV) MVLs exhibited distinct tri-phasic release characteristics comprising of an initial burst release, lag phase and a secondary release. Temperature, pH, agitation speed and release media composition were observed to impact the mechanism and rate of BPV release from MVLs. The size and morphology of the MVLs as well as their inner vesicle compartments were analyzed using cryogenic-scanning electron microscopy (cryo-SEM), confocal laser scanning microscopy and laser diffraction, where the mean diameters of the MVLs and their inner "polyhedral" vesicles were found to be 23.6 ±â€¯11.5 µm and 1.52 ±â€¯0.44 µm, respectively. Cryo-SEM results further showed a decrease in particle size and loss of internal "polyhedral" structure of the MVLs over the duration of release, indicating erosion and rearrangement of the lipid layers. Based on these results a potential MVL drug release mechanism was proposed, which may assist with the future development of more biorelevant IVRT method for similar formulations.

Beyond Q1/Q2: The Impact of Manufacturing Conditions and Test Methods on Drug Release From PLGA-Based Microparticle Depot Formulations.

Drug-loaded polymeric microparticles have been used as long-acting injectable (LAI) depot formulations. To obtain U.S. Food and Drug Administration approval, a generic LAI depot product needs to be qualitatively (Q1) and quantitatively (Q2) the same in terms of inactive ingredients as its reference-listed drug. However, Q1/Q2 sameness as the reference-listed drug does not guarantee the same in vitro drug release profile and in vivo performance, especially when the manufacturing methods are different. There is little consensus on how the in vitro testing needs to be done to examine the release profiles of LAI depot formulations. This study examined the manufacturing differences in making risperidone-loaded poly(lactide-co-glycolide) microparticles and their impact on the release kinetics. It also examined the impacts of in vitro testing methods on the drug release profiles. Two in-house manufactured risperidone poly(lactide-co-glycolide) microparticles and Risperdal Consta® were used in the study. Of the in vitro release methods tested, the orbital agitation method provided the most reproducible release profiles. The results indicate that the in vitro release kinetics depend not only on manufacturing procedures but also on the in vitro testing conditions, such as the agitation speed, vessel-dimensions, solid beads, media exchange volume, and other parameters both under real-time and accelerated testing conditions. In the current case, the in vitro experimental condition seemed to affect the drug release kinetics more than the manufacturing differences. The developed orbital agitation release testing method is simple, robust, and reproducible, which allows the comparison of in vitro release profiles of formulations that are prepared with manufacturing differences.

Reverse Engineering the 1-Month Lupron Depot®.

The 1-month Lupron Depot® (LD) encapsulating water-soluble leuprolide in poly(lactic-co-glycolic acid) (PLGA) microspheres is a benchmark product upon which modern long-acting release products are often compared. Despite expiration of patent coverage, no generic product for the LD has been approved in the USA, likely due to the complexity of components and manufacturing processes involved in the product. Here, we describe the reverse engineering of the LD composition and important product attributes. Specific attributes analyzed for microspheres were as follows: leuprolide content by three methods; gelatin content, type, and molecular weight distribution; PLGA content, lactic acid/glycolic acid ratio, and molecular weight distribution; mannitol content; in vitro drug release; residual solvent and moisture content; particle size distribution and morphology; and glass transition temperature. For the diluent, composition, viscosity, and specific gravity were analyzed. Analyzed contents of the formulation and the determined PLGA characteristics matched well with the official numbers stated in the package insert and those found in literature, respectively. The gelatin was identified as type B consistent with ~ 300 bloom. The 11-µm volume-median microspheres in the LD slowly released the drug in vitro in a zero-order manner after ~ 23% initial burst release. Very low content of residual moisture (< 0.5%) and methylene chloride (< 1 ppm) in the product indicates in-water drying is capable of removing solvents to extremely low levels during manufacturing. The rigorous approach of reverse engineering described here may be useful for development of generic leuprolide-PLGA microspheres as well as other new and generic PLGA microsphere formulations.

Development of in vitro-in vivo correlation of parenteral naltrexone loaded polymeric microspheres.

Establishment of in vitro-in vivo correlations (IVIVCs) for parenteral polymeric microspheres has been very challenging, due to their complex multiphase release characteristics (which is affected by the nature of the drug) as well as the lack of compendial in vitro release testing methods. Previously, a Level A correlation has been established and validated for polymeric microspheres containing risperidone (a practically water insoluble small molecule drug). The objectives of the present study were: 1) to investigate whether a Level A IVIVC can be established for polymeric microspheres containing another small molecule drug with different solubility profiles compared to risperidone; and 2) to determine whether release characteristic differences (bi-phasic vs tri-phasic) between microspheres can affect the development and predictability of IVIVCs. Naltrexone was chosen as the model drug. Three compositionally equivalent formulations of naltrexone microspheres with different release characteristics were prepared using different manufacturing processes. The critical physicochemical properties (such as drug loading, particle size, porosity, and morphology) as well as the in vitro release characteristics of the prepared naltrexone microspheres and the reference-listed drug (Vivitrol®) were determined. The pharmacokinetics of the naltrexone microspheres were investigated using a rabbit model. The obtained pharmacokinetic profiles were deconvoluted using the Loo-Riegelman method, and compared with the in vitro release profiles of the naltrexone microspheres obtained using USP apparatus 4. Level A IVIVCs were established and validated for predictability. The results demonstrated that the developed USP 4 method was capable of detecting manufacturing process related performance changes, and most importantly, predicting the in vivo performance of naltrexone microspheres in the investigated animal model. A critical difference between naltrexone and risperidone loaded microspheres is their respective bi-phasic and tri-phasic release profiles with varying burst release and lag phase. These variations in release profiles affect the development of IVIVCs. Nevertheless, IVIVCs have been established and validated for polymeric microspheres with different release characteristics.

Accelerated in vitro release testing method for naltrexone loaded PLGA microspheres.

The objective of the present study was to develop a discriminatory and reproducible accelerated release testing method for naltrexone loaded parenteral polymeric microspheres. The commercially available naltrexone microsphere product (Vivitrol®) was used as the testing formulation in the in vitro release method development, and both sample-and-separate and USP apparatus 4 methods were investigated. Following an in vitro drug stability study, frequent media replacement and addition of anti-oxidant in the release medium were used to prevent degradation of naltrexone during release testing at "real-time" (37°C) and "accelerated" (45°C), respectively. The USP apparatus 4 method was more reproducible than the sample-and-separate method. In addition, the accelerated release profile obtained using USP apparatus 4 had a shortened release duration (within seven days), and good correlation with the "real-time" release profile. Lastly, the discriminatory ability of the developed accelerated release method was assessed using compositionally equivalent naltrexone microspheres with different release characteristics. The developed accelerated USP apparatus 4 release method was able to detect differences in the release characteristics of the prepared naltrexone microspheres. Moreover, a linear correlation was observed between the "real-time" and accelerated release profiles of all the formulations investigated, suggesting that the release mechanism(s) may be similar under both conditions. These results indicate that the developed accelerated USP apparatus 4 method has the potential to be an appropriate fast quality control tool for long-acting naltrexone PLGA microspheres.

Mechanistic analysis of triamcinolone acetonide release from PLGA microspheres as a function of varying in vitro release conditions.

In vitro tests for controlled release PLGA microspheres in their current state often do not accurately predict in vivo performance of these products during formulation development. Here, we introduce a new mechanistic and multi-phase approach to more clearly understand in vitro-in vivo relationships, and describe the first "in vitro phase" with the model drug, triamcinolone acetonide (Tr-A). Two microsphere formulations encapsulating Tr-A were prepared from PLGAs of different molecular weights and end-capping (18kDa acid-capped and 54kDa ester-capped). In vitro release kinetics and the evidence for controlling mechanisms (i.e., erosion, diffusion, and water-mediated processes) were studied in four release media: PBST pH 7.4 (standard condition), PBST pH 6.5, PBS+1.0% triethyl citrate (TC), and HBST pH 7.4. The release mechanism in PBST was primarily polymer erosion-controlled as indicated by the similarity of release and mass loss kinetics. Release from the low MW PLGA was accelerated at low pH due to increased rate of hydrolysis and in the presence of the plasticizer TC due to slightly increased hydrolysis and much higher diffusion in the polymer matrix. TC also increased release from the high MW PLGA due to increased hydrolysis, erosion, and diffusion. This work demonstrates how in vitro conditions can be manipulated to change not only rates of drug release from PLGA microspheres but also the mechanism(s) by which release occurs. Follow-on studies in the next phases of this approach will utilize these results to compare the mechanistic data of the Tr-A/PLGA microsphere formulations developed here after recovery of microspheres in vivo. This new approach based on measuring mechanistic indicators of release in vitro and in vivo has the potential to design better, more predictive in vitro release tests for these formulations and potentially lead to mechanism-based in vitro-in vivo correlations.

Mechanisms of in vivo release of triamcinolone acetonide from PLGA microspheres.

Little is known about the underlying effects controlling in vitro-in vivo correlations (IVIVCs) for biodegradable controlled release microspheres. Most reports of IVIVCs that exist are empirical in nature, typically based on a mathematical relationship between in vitro and in vivo drug release, with the latter often estimated by deconvolution of pharmacokinetic data. In order to improve the ability of in vitro release tests to predict microsphere behavior in vivo and develop more meaningful IVIVCs, the in vivo release mechanisms need to be characterized. Here, two poly(lactic-co-glycolic acid) (PLGA) microsphere formulations encapsulating the model steroid triamcinolone acetonide (Tr-A) were implanted subcutaneously in rats by using a validated cage model, allowing for free fluid and cellular exchange and microsphere retrieval during release. Release kinetics, as well as mechanistic indicators of release such as hydrolysis and mass loss, was measured by direct analysis of the recovered microspheres. Release of Tr-A from both formulations was greatly accelerated in vivo compared to in vitro using agitated phosphate buffered saline +0.02% Tween 80 pH7.4, including rate of PLGA hydrolysis, mass loss and water uptake. Both microsphere formulations exhibited erosion-controlled release in vitro, indicated by similar polymer mass loss kinetics, but only one of the formulations (low molecular weight, free acid terminated) exhibited the same mechanism in vivo. The in vivo release of Tr-A from microspheres made of a higher molecular weight, ester end-capped PLGA displayed an osmotically induced/pore diffusion mechanism based on confocal micrographs of percolating pores in the polymer, not previously observed in vitro. This research indicates the need to fully understand the in vivo environment and how it causes drug release from biodegradable microspheres. This understanding can then be applied to develop in vitro release tests which better mimic this environment and cause drug release by the relevant mechanistic processes, ultimately leading to the development of mechanism based IVIVCs.