Dennis H. Robinson

A physiologically based pharmacokinetic (PBPK) model to characterize and predict the disposition of monoclonal antibody CC49 and its single chain Fv constructs

Optimization of the use of monoclonal antibodies (MAbs) as diagnostic tools and therapeutic agents in the treatment of cancer is aided by quantitative characterization of the transport and tissue disposition of these agents in whole animals. This characterization may be effectively achieved by the application of physiologically based pharmacokinetic (PBPK) models. The purpose of this study was to develop a PBPK model to characterize the biodistribution of the pancarcinoma MAb CC49 IgG in normal and neoplastic tissues of nude mice, and to further apply the model to predict the disposition of multivalent single chain Fv (scFv) constructs in mice. Since MAbs are macromolecules, their transport is membrane-limited and a two-pore formalism is employed to describe their extravasation. The influence of binding of IgG to the protective neonatal Fc receptor (FcRn) on its disposition is also accounted for in the model. The model successfully described (131)I-CC49 IgG concentrations in blood, tumor and various organs/tissues in mice. Sensitivity analysis revealed the rate of transcapillary transport to be a critical determinant of antibody penetration and localization in the tumor. The applicability of the model was tested by predicting the disposition of di- and tetravalent scFv constructs of CC49 in mice. The model gave reasonably good predictions of the disposition of the scFv constructs. Since the model employs physiological parameters, it can be used to scale-up mouse biodistribution data to predict antibody distribution in humans. Therefore, the clinical utility of the model was tested with data for (131)I-CC49 obtained in patients, by scaling up murine parameter values according to known empirical relationships. The model gave satisfactory predictions of CC49 disposition and tumor uptake in man.

Enhanced cellular association of paclitaxel delivered in chitosan-PLGA particles

We have previously demonstrated that the cellular association, cytotoxicity, and in vivo anti-tumor efficacy of paclitaxel are significantly greater when delivered in PLGA microparticles compared to nanoparticles. The purpose of this research is to test the hypothesis that mucoadhesive chitosan promotes adhesion of PLGA particles to mucus on the tumor epithelium, resulting in enhanced cellular association and cytotoxicity of paclitaxel. PLGA particles containing paclitaxel or Bodipy(®) were prepared and chitosan was either adsorbed or chemically conjugated to the particle surface. The cellular association and cytotoxicity of paclitaxel in 4T1 cells was determined. A 4-10 fold increase in cellular association of paclitaxel was observed when chitosan was adsorbed or conjugated to the PLGA particles. Chitosan-conjugated PLGA microparticles were most cytotoxic with an IC(50) value of 0.77 μM. Confocal microscopy demonstrated that chitosan-PLGA microparticles adhered to the surface of 4T1 cells. Pretreatment of either 4T1 cells or chitosan-PLGA particles with mucin resulted in significant increase in cellular association of paclitaxel. A linear correlation was established between theoretical amount of chitosan used and experimentally determined amount of chitosan adsorbed or conjugated to PLGA nanoparticles. In conclusion, cellular association and cytotoxicity of paclitaxel was significantly enhanced when delivered in chitosan-PLGA particles.

Preparation and characterization of biodegradable poly(L-lactic acid)gentamicin delivery systems

Abstract In the last two decades, localized antibiotic therapy has emerged as an important approach to treating orthopedic infections. This paper describes the preparation and in vitro evaluation of biodegradable, poly( l -lactic acid), implants for localized delivery of gentamicin sulfate for the treatment of osteomyelitis. Cylindrical, poly( l -lactic acid) implants containing gentamicin sulfate were obtained by compression of microcapsules prepared by a nonsolvent-induced, coacervation process. Mean particle size distributions of the microcapsules, based on volume, ranged from 278 to 444 μm. The gentamicin sulfate loading of the microcapsules, after a methylene chloride-water extraction procedure, exceeded 95% of the theoretical value. In vitro dissolution studies on microcapsules and implants with drug loading varying from 5 to 67% w/w indicated that the rate of gentamicin sulfate released from both microcapsules and implants increased, while the dissolution half-life (T50) decreased, exponentially, with an increase in drug loading. Profiles of amount of drug dissolved at different times followed a square-root-time relationship. All batches of microcapsules and implants released greater than 80% gentamicin sulfate within 3 weeks. In comparison, previous studies in this laboratory have indicated that conventional, nonbiodegradable polymethylmethacrylate implants, containing gentamicin or tobramycin, show incomplete and poorly controlled drug release during the same time period.

The influence of manufacturing procedure on the degradation of poly(lactide-co-glycolide) 85:15 and 50:50 implants

Abstract Poly(lactides-co-glycolides) (PLGA) are widely investigated biodegradable polymers and are extensively used in several biomaterials applications as well as drug delivery systems. The PLGA polymers degrade by bulk hydrolysis of ester bonds and breakdown into their constituent monomers, lactic and glycolic acids which are excreted from the body. The purpose of this investigation was to study the effect of manufacturing procedure on the in vitro degradation of two PLGA copolymers, 85:15 and 50:50, which were fabricated as implants. Implants were compressed from microcapsules prepared by nonsolvent induced phase separation using two solvent-nonsolvent systems, viz., methylene chloride-hexane (non-polar) and acetone-phosphate buffer (polar). Studies were performed at pH 4.5, 7.4 and 9.4 and polymer degradation was monitored by measuring the decrease in number- and weight-average molecular weights (M n and M w ), mass loss, formation of lactic and glycolic acids and pH of the degradation medium. Representative scanning electron micrographs (SEMs) were also obtained to study the changes in surface morphology of the implants. Results of these studies indicated that both PLGA 85:15 and 50:50 implants prepared by the non-polar procedure degraded faster than the implants prepared by the polar procedure. The decrease in polymer M n and M w followed pseudo first-order kinetics. Changes in M n and M w occurred before the onset of mass loss, after which implant mass loss was described by pseudo first order kinetics. The appearance of lactic and glycolic acids corresponded to the initiation of mass loss and also resulted in decrease in pH of the bulk degradation medium. The SEMs indicated that water uptake was faster in implants prepared by the non-polar method resulting in a more porous matrix which degrades more rapidly. Finally, the average degradation time for PLGA 85:15 was ≈26 weeks and that for PLGA 50:50 was 6–8 weeks.

Particle size and temperature effect on the physical stability of PLGA nanospheres and microspheres containing Bodipy

The purpose of this study was to investigate the effect of particle size, storage temperature, and duration of storage on the physical stability and morphology of polylactic-co-glycolic acid (PLGA) nanospheres and microspheres. PLGA nanospheres and microspheres containing the fluorescent dye, Bodipy, were prepared in varying sizes by controlling the method and degree of agitation during the emulsification phase of preparation. Mean diameters of the particles were measured by dynamic light scattering. To evaluate the effect of storage temperature and duration of storage on the extent of aggregation, nanospheres and microspheres were stored at 4°C, 25°C, 37°C, and 50°C for 6 days and then monitored using both confocal and scanning electron microscopy. The mean ±SD diameters of PLGA particles containing Bodipy were: 266.9±2.8, 351.6±1.8, 988.8±14.1, and 1865.9±67.0 nm. The extent of aggregation of the particulate delivery system decreased as the mean diameter increased, and increased as the storage temperature increased. The maximum extent of aggregation was observed with the smallest (266 nm) nanospheres. Microspheres did not aggregate. The aggregation of nanospheres was significantly reduced by introducing an additional evaporation step during preparation, suggesting that migration of residual dichloromethane from within the nanospheres may have dissolved the PLGA on the surface. The extent of aggregation of nanospheres increased as the temperature was increased from 4°C to 50°C, and decreased as particle size increased. To avoid aggregation, PLGA nanospheres should be stored at 4°C.

Development and characterization of tetracycline-poly(lactide/glycolide) films for the treatment of periodontitis

Abstract Local delivery of antibiotics has been shown to be effective in reducing periodontopathic micro-organisms. The objective of this research was to develop a biodegradable, poly( d , l -lactide/glycolide), 85:15, (PLGA) film that was capable of delivering therapeutic concentrations of tetracycline HCl for a duration of two weeks into the intra-crevicular fluid within the inflamed periodontal pocket. Films (10 × 2 × 0.5 mm) containing varying amounts (10 to 25% w/w) of tetracycline HCl were prepared by film casting a dispersion of the drug in a solution of PLGA dissolved in methylene chloride. Differential dissolution studies were performed in buffer, pH 7.3, at 37°C. Both the rate and percent of drug released increased as drug loading and dissolution media pH increased. However, complete release of drug from the films was not obtained as 83.1 ± 7.0 μg of tetracycline HCl per mg of PLGA was retained for all drug loadings. Linear relationships obtained for graphs of the percent released versus both the square root of time ( r 2 ≥ 0.96) and drug loading ( r 2 ≥ 0.99) indicated a matrix-controlled release from a porous, granular, monolithic system. Preliminary results from a clinical study with 8 periodontal, maintenance patients indicate that films containing 25% w/w tetracycline HCl were effective in decreasing the bacterial count in the intra-crevicular fluid and demonstrated a significant ( p ⩽ 0.01) microbial inhibition for two weeks over the control placebo film. The decrease in tetracycline HCl concentration in the gingival fluid was approximated by a first order relationship with the tetracycline HCl disappearance rate constant of 0.19 days −1 .

Synthesis and Solution Properties of Deferoxamine Amides

The poor membrane permeability and oral bioavailability of the iron chelating agent deferoxamine (DFO) mesylate result from the low octanol/water partition coefficient and high aqueous solubility. With the ultimate aim to improve biomembrane permeability while retaining the iron-binding ability of DFO, a series of more lipophilic amides were prepared by reacting the terminal primary amino group with fatty and aromatic acid chlorides or anhydrides. Octanol/water partition coefficients and equilibrium solubilities of these analogs in solvents, chosen to delineate physicochemical interactions, were determined as a function of temperature. Solid-state properties were evaluated by calorimetry. All DFO amide derivatives had higher melting points, indicating that derivatives formed strong intermolecular interactions in the solid phase. Formamidation of the primary amine of deferoxamine resulted in a 200-fold increase in the octanol/water partition coefficient and reduced aqueous solubility at least 2000-fold compared with the parent molecule. The partition coefficient increased and aqueous solubility decreased 2-fold with the addition of each methylene group in the homologous series of aliphatic amides. Solubilities of the derivatives in water-saturated octanol and hexane showed irregular profiles as a function of increasing aliphatic chain length that were attributed to intermolecular packing in the solid state. The temperature dependence of the partition coefficients was interpreted to indicate that interfacial transfer of the deferoxamine amides was, in part, affected by an apparent diminished ability to form energetically favorable interactions in the water-saturated organic phase.

Comparison of anti-tumor efficacy of paclitaxel delivered in nano- and microparticles.

This research compares the anti-tumor efficacy of paclitaxel delivered intratumorally in PLGA nanoparticles, microparticles, or the commercial Paclitaxel Injection((R)). The hypothesis of the research is that larger PLGA microparticles adhere to mucus on the cell surface, release paclitaxel locally, and enhance cellular association of paclitaxel. PLGA-paclitaxel particles of mean diameters 315 nm, 1 microm, and 10 microm were prepared and their drug content, in vitro release, and cellular association of paclitaxel into 4T1 cells quantified. These particles were injected intratumorally into tumor xenografts, and the tumor volumes monitored over 13 days. Mean tumor volumes of the groups that received placebo and the 315 nm nanoparticles increased 2 and 1.5 times, respectively. Tumor growth was arrested in groups that received 1 microm and 10 microm microparticles. Additional cell culture studies were performed to test the hypothesis. The size-dependent increase in cellular concentration of paclitaxel was independent of duration of incubation of PLGA particles with 4T1 cells, and was enhanced 1.5 times by coating the particles or 4T1 cells with mucin. These particles were not internalized by clathrin-mediated endocytosis or macropinocytosis. In conclusion, PLGA microparticles sustained drug release, increased cellular concentration, and enhanced anti-tumor efficacy of paclitaxel compared to nanoparticles and Paclitaxel Injection.

Controlled release captopril microcapsules: Effect of ethyl cellulose viscosity grade on the in vitro dissolution from microcapsules and tableted microcapsules

Captopril microcapsules were prepared using four different viscosity grades of ethyl cellulose (core: wall ratios 1:1, 1:2 and 1:3) by temperature induced coacervation from cyclohexane. In vitro dissolution studies in 0.1 M hydrochloric acid showed that the drug release was dependent on the core to wall ratio, the viscosity grade of the ethyl cellulose and thus the total viscosity of the coacervation system. Viscosity grade of greater than 100 c.p. was unsuitable for microencapsulation by coacervation method at the concentration used. The surface characteristics of a 1:2 core to wall ratio were studied by scanning electron microscopy. The surface of the microcapsules prepared with 10 c.p. viscosity grade was comparatively more porous with larger size pores than 50 c.p. viscosity grade of ethyl cellulose. However, 300 c.p. viscosity grade showed incomplete wall formation. The microcapsules did not fragment during dissolution, alter in shape or size, or show evidence of enlargement of the surface pores. The tensile strength of tablets prepared at constant pressure from each batch of microcapsules (mean diameter 675 microns) increased as both the core to wall ratios and the viscosity of ethyl cellulose increased. The dissolution rate of the drug from tableted microcapsules was significantly delayed. The in vitro release gave best correlation with first order release kinetics when compared to zero-order and square-root-of-time equations.

A bioresorbable, polylactide reservoir for diffusional and osmotically controlled drug delivery

The purpose of this study was to design and characterize a zero-order bioresorbable reservoir delivery system (BRDS) for diffusional or osmotically controlled delivery of model drugs including macromolecules. The BRDS was manufactured by casting hollow cylindrical poly (lactic acid) (PLA): polyethylene glycol (PEG) membranes (10×1.6 mm) on a stainless steel mold. Physical properties of the PLA:PEG membranes were characterized by solid-state thermal analysis. After filling with drug (5 fluorouracil [5FU] or fluorescein isothiocyanate [FITC]-dextranmannitol, 5:95 wt/wt mixture) and sealing with viscous PLA solution, cumulative in vitro dissolution studies were performed and drug release monitored by ultraviolet (UV) or florescence spectroscopy. Statistical analysis was performed using Minitab® (Version 12). Differential scanning calorimetry thermograms of PLA:PEG membranes dried at 25°C lacked the crystallization exotherms, dual endothermal melting peaks. and endothermal glass transition observed in PLA membranes dried at −25°C. In vitro release studies demonstrated zero-order release of 5FU for up to 6 weeks from BRDS manufactured with 50% wt/wt PEG (drying temperature, 25°C). The release of FITC dextrans of molecular weights 4400, 42 000, 148 000, and 464 000 followed zero-order kinetics that were independent of the dextran molecular weight. When monitored under different concentrations of urea in the dissolution medium, the release rate of FITC dextran 42 000 showed a linear correlation with the calculated osmotic gradient (Δπ). PEG inclusion at 25°C enables manufacture of uniform, cylindrical PLA membranes of controlled permeability. The absence of molecular weight effects and a linear dependence of FITC-dextran release rate on Δπ confirm that the BRDS can be modified to release model macromolecules by an osmotically controlled mechanism.