Mechanistic analysis of PLGA/HPMC-based in-situ forming implants for periodontitis treatment

In-situ forming implant formulations based on poly(lactic-co-glycolic acid) (PLGA), acetyltributyl citrate (ATBC), minocycline HCl, N-methyl pyrrolidone (NMP) and optionally hydroxypropyl methylcellulose (HPMC) were prepared and thoroughly characterized in vitro. This includes electron paramagnetic resonance (EPR), nuclear magnetic resonance (¹H NMR), mass change and drug release measurements under different conditions, optical microscopy, size exclusion chromatography (SEC) as well as antibacterial activity tests using gingival crevicular fluid samples from periodontal pockets of periodontitis patients. Based on these results, deeper insight into the physico-chemical phenomena involved in implant formation and the control of drug release could be gained. For instance, the effects of adding HPMC to the formulations, resulting in improved implant adherence and reduced swelling, could be explained. Importantly, the in-situ formed implants effectively hindered the growth of bacteria present in the patients’ periodontal pockets. Interestingly, the systems were more effectively hindering the growth of pathogenic bacterial strains (e.g., Fusobacterium nucleatum) than that of strains with a lower pathogenic potential (e.g., Streptococcus salivarius). In vivo, such a preferential action against the pathogenic bacteria can be expected to give a chance to the healthy flora to re-colonize the periodontal pockets.     

Development and validation of a reverse phase liquid chromatography method for the quantification of rasagiline mesylate in biodegradable PLGA microspheres

In the present study, a reverse phase high performance liquid chromatographic method was developed and validated for the determination of rasagiline mesylate in biodegradable microspheres. Chromatographic separation was carried out on a RP-18 column using a mobile phase consisting of acetonitrile:water (5:95, v/v) adjusted at pH 3.1. Flow rate was 1.0 ml min⁻¹ and UV detection at 290 nm. Acyclovir was used as the internal standard. The calibration curve was linear over the range 0.5–20.0 μg ml⁻¹. R.S.D. for precision was <1.8%. Accuracy ranged between 99.01% and 102.55% with a R.S.D. lower than 1.3%. LOD and LOQ were 0.07 μg ml⁻¹ and 0.23 μg ml⁻¹, respectively. The method was simple, rapid, and easy to apply, making it very suitable for routine analysis of rasagiline mesylate in biodegradable PLGA microspheres. It could be also used with reliability for the determination of the drug in other pharmaceutical dosage forms.     

Synchronous microencapsulation of multiple components in silymarin into PLGA nanoparticles by an emulsification/solvent evaporation method

The development of polymeric carriers loaded with extracts suffers from the drawback not to be able to incorporate simultaneously various pharmacological compounds into the formulation. The aim of this study was therefore to achieve synchronous microencapsulation of multiple components of silymarin into poly (lactic-co-glycolic acid) nanoparticle, the most commonly used polymeric carrier with biodegradability and safety. The main strategy taken was to improve the overall entrapment efficiency and to reduce the escaping ratio of the components of different physicochemical properties. The optimized nanoparticles were spherical in morphology with a mean particle size of 150?±?5?nm. Under common preparative conditions, silybin and isosilybin were entrapped in high efficiency, whereas taxifolin, silychristin and silydianin, especially taxifolin, showed less entrapment because they were more hydrophilic. By changing the pH of the outer aqueous phase and saturating it with silymarin, the entrapment efficiency of taxifolin, silychristin and silydianin could be significantly improved to over 90%, the level similar to silybin and isosilybin, thereby achieving synchronous encapsulation. It could be concluded that synchronous encapsulation of multiple components of silymarin was achieved by optimizing the preparative variables.     

乳化溶剂挥发法制备重组人血管内皮抑制素缓释微球

目的:制备重组人血管内皮抑制素(恩度)缓释微球,并对微球理化性质及体外释放行为进行初步考察。方法:采用乳化溶剂挥发法(W/O/O)制备恩度载药微球;对微球载药量、粒径、突释、体外释放速率及降解行为进行考察,同时利用凝胶电泳初步评价体外释放过程中恩度的完整性。结果:增加聚乳酸-羟基乙酸嵌段共聚物(PLGA)中羟基乙酸的比例、提高PLGA浓度、降低内水相体积、提高理论载药量均增加微球载药能力;降低内水相体积、提高分散速度均减小突释。增加PLGA中羟基乙酸的比例,30 d时累积释放可增加到65%。降解实验说明释放初期微球主要以扩散方式释放恩度,释放后期主要表现为微球的降解。凝胶电泳结果表明微球制备过程对蛋白质聚集性的影响不大。结论:用PLGA作为载体材料制备微球,可以延缓恩度的释放。     

复乳法制备阿柔比星A的聚乳酸聚乙醇酸共聚物纳米粒影响包封率因素考察

以含阿柔比星A(Aclarubicin A，ACRB-A)的酸性溶液为内水相，采用复乳法制备ACRB-A(PLGA)纳米粒。考察了有机溶剂、油酸的量、稳定剂种类、投药量、乳化剂、Na2SO4的量和外水相的pH值几个主要因素对ACRB-APLGA纳米粒包封率的影响。结果表明，以二氯甲烷和丙酮为有机溶剂、油酸(15mg)、右旋糖酐-70、ACRB-A的浓度(Smg／ml)、以F68和Tween-80为乳化剂、2％的Na2SO4和外水相的pH等于8有利于提高ACRB-A的包封率。经实验条件优化后制备的ACRB-A PLGA包封率为85．41％，纳米粒粒径为272nm，粒径分散指数为0．213。     

A Bayesian adaptive design for cancer phase I trials using a flexible range of doses

We present a Bayesian adaptive design for dose finding in cancer phase I clinical trials. The goal is to estimate the maximum tolerated dose (MTD) after possible modification of the dose range during the trial. Parametric models are used to describe the relationship between the dose and the probability of dose-limiting toxicity (DLT). We investigate model reparameterization in terms of the probabilities of DLT at the minimum and maximum available doses at the start of the trial. Trial design proceeds using escalation with overdose control (EWOC), where at each stage of the trial we seek the dose of the agent such that the posterior probability of exceeding the MTD of this agent is bounded by a feasibility bound. At any time during the trial, we test whether the MTD is below or above the minimum and maximum doses, respectively. If during the trial there is evidence that the MTD is outside the range of doses, we extend the range of doses and complete the trial with the planned sample size. At the end of the trial, a Bayes estimate of the MTD is proposed. We evaluate design operating characteristics in terms of safety of the trial design and efficiency of the MTD estimate under various scenarios and model misspecification. The methodology is further compared to the original EWOC design. We showed by comprehensive simulation studies that the proposed method is safe and can estimate the MTD more efficiently than the original EWOC design.     

Mimicking chronic glaucoma over 6 months with a single intracameral injection of dexamethasone/fibronectin-loaded PLGA microspheres

To create a chronic glaucoma animal model by a single intracameral injection of biodegradable poly lactic-co-glycolic acid (PLGA) microspheres (Ms) co-loaded with dexamethasone and fibronectin (MsDexaFibro). MsDexaFibro were prepared by a water-in-oil-in-water emulsion method including dexamethasone in the organic phase and fibronectin in the inner aqueous phase. To create the chronic glaucoma model, an interventionist and longitudinal animal study was performed using forty-five Long Evans rats (4-week-old). Rats received a single intracameral injection of MsDexafibro suspension (10%w/v) in the right eye. Ophthalmological parameters such as clinical signs, intraocular pressure (IOP), neuro-retinal functionality by electroretinography (ERG), retinal structural analysis by optical coherence tomography (OCT), and histology were evaluated up to six months. According to the results obtained, the model proposed was able to induce IOP increasing in both eyes over the study, higher in the injected eyes up to 6 weeks (p < 0.05), while preserving the ocular surface. OCT quantified progressive neuro-retinal degeneration (mainly in the retinal nerve fiber layer) in both eyes but higher in the injected eye. Ganglion cell functionality decreased in injected eyes, thus smaller amplitudes in PhNR were detected by ERG. In conclusion, a new chronic glaucoma animal model was created by a single injection of MsDexaFibro very similar to open-angle glaucoma occurring in humans. This model would impact in different fields such as ophthalmology, allowing long period of study of this pathology; pharmacology, evaluating the neuroprotective activity of active compounds; and pharmaceutical technology, allowing the correct evaluation of the efficacy of long-term sustained ocular drug delivery systems.     

Investigation of the stabilizer elimination during the washing step of charged PLGA microparticles utilizing a novel HPLC-UV-ELSD method

Quantification of stabilizer content in microparticles and other products is of great importance for formulation development, drug product quality control as well as for reproducible manufacturing. A fast and sensitive HPLC method with evaporative light scattering detection (ELSD) capable of detecting docusate sodium (DOSS), poly (lactic-co-glycolic acid) (PLGA; Resomer RG 503 H) and R-1,2-dioleoyloxy-3-trimethylammonium-propane (DOTAP) in a single run was successfully developed. In contrast to previously described methods, hydrolysis of PLGA as pretreatment is not necessary, thereby enabling accurate quantification of stabilizer next to the intact matrix polymer.This method was used to investigate the impact of washing procedures of polymeric microparticles manufactured either with anionic stabilizer DOSS or with cationic stabilizer DOTAP. High amounts of DOSS were detected in the washing water. This finding was consistent with the result that no DOSS could be detected in the washed and dried microparticles (<limit of detection).In contrast, DOTAP was hardly measurable in the washing water during all washing cycles. However, DOTAP could be quantified in dried particles. The ratio of DOTAP to dry particle mass was approximately 1:10.This is most probably due to the different polymer surfactant interactions (e.g. charge) and the different hydrophilicity of the stabilizers used.     

Preparation of bovine serum albumin loaded poly (D,L-lactic-co-glycolic acid) microspheres by a modified phase separation technique

BSA-loaded mcirospheres were prepared by a modified phase separation method, in which petroleum ether (PE) containing a certain amount of Span 80 rather than poly (dimethylsiloxane) (PDMS) was adopted as coacervating agent. Process parameters such as Span 80 concentration, the volume and addition rate of coacervating agent, polymer concentration, agitation rate during the phase separation process and PE type were evaluated to optimize the protein encapsulation. It was found microspheres with high yield (>80.0%) and entrapment efficiency (>90%) could be obtained using PE containing 5.0% Span 80 as the coacervating agent. Microspheres with small particle size (<10?µm) could be produced successfully with appropriate process parameters. In vitro release study suggested that burst release was significantly influenced by Span 80 concentration, polymer concentration and PE type and the burst release could be reduced to <20% with optimized formulation. A biphasic release behavior in vitro test was observed for the microspheres prepared by this method. GC analysis demonstrated that residual solvent of DCM and petroleum ether was decreased dramatically in comparison with PDMS used as a conventional coacervating agent.     

Enhancement of gene transfection into human dendritic cells using cationic PLGA nanospheres with a synthesized nuclear localization signal

Effective delivery of DNA encoding antigen into the dendritic cells (DCs), which are non-dividing cells, is very important for the development of DNA vaccines. In a previous study, we developed the PLGA nanospheres that contained a cationic nanomaterial and showed high transfection efficiency in COS7 cells, which divide. In the present study, to produce an effective vector for the DNA vaccines, the gene expression and intracellular trafficking of pDNA complexed with PLGA/PEI nanospheres, in combination with an NF-κB analog as a nuclear localization signal (NLS) and electroporation were evaluated in human monocyte-derived DCs (hMoDCs). Cellular uptake of pDNA both in COS7 cells and hMoDCs was enhanced using the PLGA/PEI nanospheres. On the other hand, the PLGA/PEI nanospheres significantly promoted the transfection in COS7 cells, but had almost no effect on transfection in hMoDCs. The intranuclear transport of pDNA by PLGA/PEI nanospheres in COS7 cells was significantly higher than that in hMoDCs. These results indicate that pDNA complexed with PLGA/PEI nanospheres cannot enter into the nuclei of non-dividing cells. However, PLGA/PEI nanospheres combinated with NLS and electroporation (experimental permeation enhancer) greatly elevated the transfection efficiency by improvement of not only intracellular uptake but also intranuclear transport of pDNA in the hMoDCs. Thus, this delivery system using nanospheres combined with synthesized NLS might be applicable to DC-based gene vaccines when much non-invasive application such as needle-free injector should be required.     

Preparation of polymeric particles in CO2 medium using non-toxic solvents: Formulation and comparisons with a phase separation method

The aim of this work was to elaborate formulation strategies to encapsulate a protein into biodegradable polymeric particles for sustained release purpose. In this paper, two encapsulation methods will be presented, one dealing with a phase separation phenomenon while the other involving an emulsification/extraction process in CO₂ medium. In those methods, only non-volatile injectable solvents such as glycofurol or isosorbide dimethyl ether were used to dissolve the polymer. Moreover, experimental designs were built up to help us to go further in the understanding of the processes and to better predict output responses in design space. Spherical particles were successfully generated with a satisfactory encapsulation yield. Further characterization steps such as in vitro, in vivo releases will be carried out to validate the interest of our encapsulation methods in the development of drug delivery systems.     

Simultaneous enantioseparation of a basic active pharmaceutical ingredient compound and its neutral intermediate using reversed phase and normal phase liquid chromatography with a new type of polysaccharide stationary phase

Simultaneous enantioseparation of a basic API compound, (R)-2-Amino-N-[2-[1,2-dihydro-1-(methylsulfonyl) spiro [3H-indole-3,4′-piperidin]-1′-yl]-2-oxo-1-[(phenylmethyloxy) ethyl]-2-methylpropanamide monomethanesulfonate (compound-A) and its neutral penultimate intermediate, (R)-2-BOC-Amino-N-[2-[1,2-dihydro-1-(methylsulfonyl) spiro [3H-indole-3,4′-piperidin]-1′-yl]-2-oxo-1-[(phenylmethyloxy) ethyl]-2-methylpropanamide monomethanesulfonate (compound-B) was investigated using reversed phase (RPLC) and normal phase liquid chromatography (NPLC). After an initial screening, a Sepapak-4 column, a new type of polysaccharide chiral stationary phase (CSP), was selected for further method development based on hits on separation selectivity for both compounds under RPLC and NPLC. After comparing the pros and cons, a method utilizing the Sepapak-4 chiral column (150 mm × 4.6 mm, 3 μm particle size) in RPLC mode was finally developed. Separations were performed in gradient elution mode starting at 50% A (10 mM, NH₄COOH at pH 6.5)/50% B (50/50 EtOH/MeCN) to 25% A (10 mM, NH₄COOH at pH 6.5)/75% B (50/50 EtOH/MeCN). The flow rate was 1.0 mL/min; the column temperature was 50 °C; the UV wavelength was 220 nm and the mass spectrometric detection was APCI in the positive ionization mode. The reaction mixture sample was directly diluted in ethanol. Baseline enantioseparation were achieved for both compound-A and its intermediate simultaneously with resolution greater than 2.0. The method was validated in terms of injection precision, linearity, limit of detection (LOD), limit of quantitation (LOQ), accuracy, and ruggedness. The specificity of the method was further evaluated by spiking a mixture of enantiomers of compound-A and its intermediate into a reaction matrix containing all of the synthetic reagents. No matrix interference was observed across the elution windows of compound-A and its intermediate. Additionally, the peak purity of each enantiomer was evaluated by mass spectra, indicating the specificity of the method.     

A rapid reversed phase high performance liquid chromatographic method for the determination of docetaxel (Taxotere®) in human plasma using a column switching technique

A rapid, simple and sensitive isocratic high performance liquid chromatography (HPLC) method was developed to measure the concentration of docetaxel in plasma samples with UV detection at 227 nm. The method uses a column switching technique with an Ultrasphere C₁₈ column (75×4.6 mm ID, 3μ, Altex, USA) as clean-up column and a CSC-nucleosil C₈ column (150×4.6 mm ID, 5μ, CSC, Montreal, Canada) as the analytical column. The mobile phase consisted of Phosphate buffer (30 mM, pH=3)-acetonitrile (47:53, v/v) with the flow rates of 1.1 and 1.3 ml min⁻¹ for clean-up and analytical columns, respectively. Paclitaxel was used as an internal standard. The plasma samples were extracted using a solid phase extraction method with Ammonium acetate (30 mM, pH=5)-acetonitrile (50:50, v/v) as final eluent. The extraction method showed a recovery of 92% for docetaxel. In this system, the retention times of docetaxel and Paclitaxel were 7.2 and 8.5 min, respectively. The detection limit of docetaxel in plasma is 2.5 ng ml⁻¹. This analytical method has a very good reproducibility (7.2% between-day variability at a concentration of 10 ng ml⁻¹). It is applicable in clinical and pharmacokinetic studies.     

Reliable on-line sample preparation of basic compounds from plasma using a reversed phase restricted access media in column-switching LC

We investigated on-line sample preparation of basic compounds from plasma using a methylcellulose-immobilized reversed-phase restricted-access media in column-switching liquid chromatography (LC). Dilution of the plasma sample with phosphate buffered saline prevented or delayed the formation of fibrin clots at 4 °C and resulted in reproducible on-line sample preparation over a 30-h period. The use of an ion-pair reagent in the extraction LC enhanced recoveries of hydrophilic basic compounds. The ability of the methods to quantify compounds in plasma were validated and the method was successfully applied to the pharmacokinetic study of a hydrophilic basic compound injected into the bloodstream of rats.     

Microencapsulation of insulin using a W/O/W double emulsion followed by complex coacervation to provide protection in the gastrointestinal tract

Abstract

Microcapsules containing insulin were prepared using a combination of a W/O/W double emulsion and complex coacervation between WPI (used as a hydrophilic emulsifier) and CMC or SA with further spray drying of the microcapsules in order to provide protection in the gastrointestinal tract. The microcapsules prepared exhibited high encapsulation efficiency and showed the typical structure of a double emulsion. After spray drying of these microcapsules, the integrity of the W/O/W double emulsion was maintained and the biological residual activity remained high when using the combination of 180?°C inlet air temperature and 70?°C outlet air temperature. The microcapsules exhibited low solubility at pH 2 and high solubility at pH 7 so they might protect insulin at acid pH values in the stomach and release it at intestinal pH values. The microcapsules developed in this study seem to be a promising oral delivery vehicle for insulin or other therapeutic proteins.     

The unusual gradient elution for reversed phase HPLC of a strong chelator as an active drug substance

The new approach to drug development, especially for cardiovascular and brain diseases, brought to synthesis of new lipophillic derivatives of strong calcium chelator BAPTA — DP-b99 and DP-109. Due to their chelating ability, these compounds require metal-free stationary phases, and their high hydrophobicity resulted in unusually steep gradient elution. Novel HPLC methods for analysis of these two compounds were developed. Purospher® RP-C18, 5 μm, 125×3.0 mm and XTerra™ RP18, 3.5 μm, 100×4.6 mm columns with a steep gradient from: 1% acetic acid to acetonitrile were used for DP-b99, and Hypersil HyPurity™ C4, 5 μm, 100×4.6 mm column with a steep gradient from 1% Acetic acid to 5% THF in methanol — for DP-109. Versatile detection techniques could be used with these LC procedures. The methods appeared to be sensitive, selective, reproducible and stability indicating. They could be easily upgraded to bioanalytical methods with LC-MS technique.     

PLGA microspheres for the ocular delivery of a peptide drug, vancomycin using emulsification/spray-drying as the preparation method: in vitro/in vivo studies.

The aim of this study was an in vitro/in vivo investigation on poly(lactide-co-glycolide) (PLGA) microspheres as carriers for the topical ocular delivery of a peptide drug vancomycin (VA). The microspheres were prepared by an emulsification/spray-drying technique that can be proposed as an alternative to the double emulsion method for preparation of peptide-loaded microparticles. The drug encapsulation efficiencies were close to the theoretical values (84.2-99.5%); the average particle size, expressed as dvs, was about 11 microm. The microspheres were able to modulate the in vitro drug release of VA with a behavior dependent on their composition: the highest drug content corresponded to the highest release rate. In vivo studies were carried out by assessing the pharmacokinetic profile of VA in the aqueous humor of rabbits after topical administration of aqueous suspensions of microspheres. High and prolonged VA concentrations and increased AUC values (2-fold) with respect to an aqueous solution of the drug were observed. Increasing the viscosity of the microsphere suspension by addition of a suspending-viscosizing agent (hydroxypropylcellulose) did not produce an increase of the ocular bioavailability. PLGA microspheres can be proposed as a system for ocular delivery of peptide drugs.     

Biodegradable submicron carriers for peptide drugs: Preparation of dl-lactide/glycolide copolymer (PLGA) nanospheres with nafarelin acetate by a novel emulsion-phase separation method in an oil system

PLGA nanospheres, biodegradable polymeric carriers for peptide drugs, were prepared by a novel emulsion-phase separation method. The preparation was carried out in an oil phase system in order to improve the entrapment efficiency of water-soluble peptide. An LH-RH analogue (nafarelin acetate (NA)) was employed as a model peptide drug to investigate the encapsulation efficiency. An aqueous solution of the drug was emulsified by addition with stirring to a dichloromethane-acetone mixture containing dissolved PLGA. The gradual addition of Triester oil (caprylate and caprate triglyceride) into the resultant w/o emulsion induced phase separation of PLGA at the interface of aqueous droplets. It was found that the aqueous droplets effectively worked as a coacervation-inducing agent of the polymer. PLGA coacervates precipitated around the aqueous emulsion droplets containing the peptide which were hardened by evaporation of the solvent, producing spherical drug carriers. The presence of surfactant significantly reduced the size of the aqueous droplets, resulting in submicron-sized PLGA spheres (mean diameter, 500–800 nm). The recovery of drug entrapped in the nanospheres was markedly increased compared with our previous preparation technique in a water system. Further, optimum conditions in the present method for preparing nanospheres were established to enhance the recovery of nanospheres and the efficiency of drug entrapment.     

Investigation of alternative compounds to poly(E-MA) as a polymeric surfactant for preparation of microcapsules by phase separation method

Various water-soluble polymers were used to examine an alternative emulsifier for poly(ethylene-alt-maleic anhydride), used in the preparation of crosslinked polyurea microcapsules. Microcapsules were successfully prepared by using the water-soluble polymers with large molecular weight alternating copolymers, namely poly(olefin-maleic anhydride), poly(olefin-maleic acid), and poly(acrylic acid). On the other hand, no microcapsule resulted from olefin-maleic acid with small molecular weight alternating copolymers. From these results, the following guidelines were obtained for the selection of polymeric surfactants suitable for crosslinked polyurea microcapsule. A polymeric surfactant must have maleic acid or a carboxyl group in order to form a crosslinked polyurea microcapsule membrane. Furthermore, to form a stronger capsule membrane it is desirable to have a maleic anhydride group. It is also important for membrane formation that the polymeric surfactant has a suitable molecular weight.