Daniel A. Norris

The effect of physical barriers and properties on the oral absorption of particulates

This paper provides an in-depth review of the biological, immunological, and physicochemical barriers to the oral absorption of microparticles. The roles of the mucus and mucosal cell layers are discussed along with technologies and strategies that may be used to improve uptake, including attempts to determine the mechanisms of microparticulate uptake into enterocytes and membranous epithelial (M) cells. Considerations unique to the absorption of microparticles, such as particle size, surface characteristics, cell surface interactions, and the driving force for absorption of microparticles, are also addressed. Although the extent of absorption of microparticles is severely limited, this review provides insight into potential methods and technologies that have been and could be used to improve the oral absorption of microparticles.

Characterization of the Regional Intestinal Kinetics of Drug Efflux in Rat and Human Intestine and in Caco-2 Cells

AbstractPurpose. The aim of the present study was to investigate the transport kinetics of intestinal secretory processes in the jejunum, ileum and colon of rats and humans and in Caco-2 cells, in vitro. Methods. Etoposide, vinblastine sulphate and verapamil hydrochloride were chosen as model substrates since they have been reported to undergo efflux in various other tissues. The concentration dependence, inhibition, directionality, temperature dependence, proton/sodium dependence, and ATP dependence of efflux were studied using side-by-side diffusion chambers and brush border membrane vesicles (BBMVs). Intestinal tissue from rats and humans and Caco-2 cells (passage no. 26) were used. Directional steady state effective permeabilities were calculated from drug appearance in the apical (AP) or basolateral (BL) chambers. Kinetic studies were carried out by investigating substrate efflux at concentrations ranging from 0.2 μM to 1000 μM. Since substrate efflux may be a result of more than one transporter, the hybrid efflux Km (Michaelis-constant), Pc (carrier-mediated permeability), and Pm (passive permeability) were determined as a function of intestinal region. Inhibitor studies were performed using quinidine (0.2 mM), a mixed inhibitor of P-glycoprotein (Pgp) and Multidrug Resistance-Associated Protein (MRP), and Leukotriene C4(100 nM), an inhibitor of MRP and the canalicular multispecific organic anion transporter (cMOAT). Temperature dependent efflux was determined by investigating the BL to AP transport at temperatures ranging from 3°C to 37°C. Energies of activation (Ea) were determined from an Arrhenius analysis. Sodium, proton, and ATP dependence were determined using BBMVs. Immunoquantitation of Pgp, MRP and Lung Resistance Protein (LRP) in Caco-2 cells were carried out using Western blot analysis. Results. Active efflux of all substrates was observed in all regions of rat and human intestine and in Caco-2 cells. Directionality was observed with BL to AP transport exceeding AP to BL transport. The BL to AP/AP to BL permeability ratio, the efflux ratio, ranged from 1.4 to 19.8. Heal efflux was significantly higher (p < 0.001) than in other regions. Kinetic studies revealed that hybrid efflux Km values ranged from 4 to 350 μM. In some cases, efflux was not saturable due to the solubility limits of the compounds utilized in this study. In presence of inhibitors, efflux ratios approached 1. BL to AP transport was temperature dependent in rat ileum for all substrates. Ea of intestinal efflux was found to be 11.6, 8.3, and 15.8 kcal/mole for etoposide, vinblastine and verapamil, respectively, suggesting an active, energy-dependent efflux mechanism. Substrate efflux was not sodium or proton dependent but was dependent on ATP. Using Western blot analysis the presence of Pgp, MRP, and LRP was demonstrated in Caco-2 cells and the amount of each transport protein varied as a function of passage number. Conclusions. Using multiple putative efflux substrates, the current results demonstrate that intestinal efflux was regionally dependent, mediated by multiple efflux transporters, the Km’s were in the micro-molar range, and involved an energy dependent mechanism(s).

Effect of size, surface charge, and hydrophobicity on the translocation of polystyrene microspheres through gastrointestinal mucin

Microspheres (MS) have been proposed for use as oral vaccine delivery vehicles (VDV); however, due to poor and variable absorption their clinical utility is limited. The effects of size, ζ-potential, and surface hydrophobicity on the translocation (PT) permeabilities of polystyrene (PS) MS with varying surface functional groups (amidine, carboxyl, carboxylate-modified [CML], and sulfate) were determined through gastrointestinal (GI) mucin. PT were determined, under steady-state conditions, using a modified Ussing-type diffusion chamber and a mucin packet developed for use with the Transwell-Snapwell system. PT followed the Stokes-Einstein relationship, demonstrating the limited ability of larger MS (>0.5 μm) to diffuse through the mucin layer. PT also varied according to the surface characteristics. Even though the ζ-potential did not correlate with the transport of MS through mucin, surface ionization appears to be important in MS translocation. The PS-amidine MS were significantly less hydrophobic and had a higher PT than that of the other MS, suggesting that hydrophobicity is also a significant factor in MS transport through mucin. While these results suggest that mucin may be a significant barrier to the oral absorption of vaccines and VDVs in vivo, the rate-limiting barrier for the absorption of MS will be the intestinal mucosa.

Development of predictive pharmacokinetic simulation models for drug discovery.

As discovery chemistry produces increased numbers of potential drug compounds, the use of ADME (absorption, distribution, metabolism, and excretion) properties is becoming increasingly important in the drug selection and promotion process. A computer simulation model has been developed and validated to predict ADME outcomes, such as rate of absorption, extent of absorption, etc. using a limited number of in vitro data inputs. The oral bioavailability of ganciclovir in dogs and humans was simulated using a physiologically based model that utilized many biopharmaceutically relevant parameters, such as the concentration of ganciclovir in the duodenum, jejunum, ileum, and colon at various dose levels and solubility values. The simulations were run and compared to dog and human in vivo data. The simulation results demonstrated that the low bioavailability of ganciclovir is limited by compound solubility rather than permeability due to partitioning as previously speculated. This technology provides a breakthrough in in silico prediction of absorption and with its continued development and improvement, will aid drug discovery and development scientists to produce better pharmaceutical products.

Determining the absolute surface hydrophobicity of microparticulates using thin layer wicking.

Surface hydrophobicity is an important factor in the transport of microparticulates (MPs) across biological barriers. We have previously shown in our laboratory that the surface properties of polystyrene MPs influence the diffusion and transport through gastrointestinal (GI) mucus and mucin. Unfortunately, most currently used methods for evaluating the surface hydrophobicity of MPs involve a relative measurement resulting in a rank order rather than an absolute hydrophobicity value. Obtaining an absolute assessment of hydrophobicity is necessary in order to obtain meaningful comparisons and correlations across laboratories, polymers, methods of fabrication, and so on. A modified thin layer wicking (TLW) technique was developed and validated to allow for the determination of absolute surface hydrophobicity of intact MPs. The TLW method was validated by constructing a standard curve and comparing the rate of solvent rise through MPs dried on microscope slides to the known contact angle of PLGA polymers. MPs with surface contact angles ranging between 67.04 degrees and 90.18 degrees were evaluated using the TLW technique. The modified TLW technique was also successfully validated using surface-modified polystyrene and OVA MPs. Based on the results of the current study, the modified TLW technique appears to be a reliable and quantitative method for assessing the surface hydrophobicity of intact MPs.