Vincent H.L. Lee

Biopharmaceutics classification system: the scientific basis for biowaiver extensions.

The current BSC guidance issued by the FDA allows for biowaivers based on conservative criteria. Possible new criteria and class boundaries are proposed for additional biowaivers based on the underlying physiology of the gastrointestinal tract. The proposed changes in new class boundaries for solubility and permeability are as follows: 1. Narrow the required solubility pH range from 1.0-7.5 to 1.0-6.8. 2. Reduce the high permeability requirement from 90% to 85%. The following new criterion and potential biowaiver extension require more research: 1. Define a new intermediate permeability class boundary. 2. Allow biowaivers for highly soluble and intermediately permeable drugs in IR solid oral dosage forms with no less than 85% dissolved in 15 min in all physiologically relevant dissolution media, provided these IR products contain only known excipients that do not affect the oral drug absorption. The following areas require more extensive research: 1. Increase the dose volume for solubility classification to 500 mL. 2. Include bile salt in the solubility measurement. 3. Use the intrinsic dissolution method for solubility classification. 4. Define an intermediate solubility class for BCS Class II drugs. 5. Include surfactants in in vitro dissolution testing.

Penetration and enzymatic barriers to peptide and protein absorption

Abstract The recent rise to prominence of therapeutic peptides and proteins as potential drugs of the future, coupled with their oral ineffectiveness due to proteolytic destruction, has focused attention on non-oral routes for routine drug administration. Although there was early optimism about these routes, the bioavailability of peptides and proteins from the non-oral mucosal routes is, in reality, poor when compared with the parenteral route, because of the formidable penetration and enzymatic barriers to mucosal peptide and protein absorption. The purpose of this review is twofold: (1) to describe the nature and magnitude of the penetration and enzymatic barriers to peptide and protein drug absorption, and (2) to review critically the effectiveness and limitations of the approaches that have been used to overcome such barriers.

The characteristics and mechanisms of uptake of PLGA nanoparticles in rabbit conjunctival epithelial cell layers

AbstractPurpose. To delineate the characteristics and mechanisms of up- take of biodegradable poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles in primary cultured rabbit conjunctival epithelial cells (RCECs). Methods. Poly(d,l-lactide-co-glycolide) nanoparticles (PLGA 50:50, 100 nm in diameter) containing 6-coumarin (as a fluorescent marker) were used. The effect of size was studied using various particle sizes (100 nm, 800 nm, and 10 μm). The effect of cytochalasin D, nocodazole, and metabolic inhibitors on nanoparticle uptake was investigated. The capability of nanoparticles to enhance the uptake of an encapsulated protein, BSA bound to Texas red (TR-BSA), was evaluated. Results. Maximal uptake of nanoparticles at 37°C occurred at 2 h, and 100-nm particles had the highest uptake in RCECs in comparison with 800-nm and 10-μm particles. Nanoparticle uptake was saturable over the 0.1-4 mg/ml concentration range. Nanoparticle uptake was confirmed by confocal microscopy and was inhibited significantly by coumarin-free nanoparticles (of similar size), by lower incubation temperature, and by the presence of metabolic inhibitors and cytochalasin D. The uptake of encapsulated TR-BSA in RCECs at 4 h was 28% higher than free BSA application. Conclusion. Our findings suggest that PLGA nanoparticle uptake in primary cultured rabbit conjunctival epithelial cells occurs most likely by adsorptive-type endocytosis.

Monolayers of human alveolar epithelial cells in primary culture for pulmonary absorption and transport studies

AbstractPurpose. To develop a cell culture model of human alveolar epithelial cells in primary culture for the in vitro study of pulmonary absorption and transport. Methods. Type II pneumocytes isolated from normal human distal lung tissue by enzyme treatment and subsequent purification were plated on fibronectin/collagen coated polyester filter inserts, and cultured using a low-serum growth medium. Characterization of the cell culture was achieved by bioelectric measurements, cell-specific lectin binding, immunohistochemical detection of cell junctions, and by assessment of transepithelial transport of dextrans of varying molecular weights. Results. In culture, the isolated cells spread into confluent monolayers, exhibiting peak transepithelial resistance of 2,180 ± 62 Ω X cm2 and potential difference of 13.5 ± 1.0 mV (n = 30−48), and developing tight junctions as well as desmosomes. As assessed by lectin-binding, the cell monolayers consisted of mainly type I cells with some interspersed type II cells, thus well mimicking the situation in vivo. The permeability of hydrophilic macromolecular FITC-dextrans across the cell monolayer was found to be inversely related to their molecular size, with Papp values ranging from 1.7 to 0.2 X 10−8 cm/sec. Conclusions. A primary cell culture model of human alveolar epithelial cells has been established, which appears to be a valuable in vitro model for pulmonary drug delivery and transport studies.

Influence of preparation conditions on acyclovir-loaded poly-d,l-lactic acid nanospheres and effect of PEG coating on ocular drug bioavailability.

AbstractPurpose. The evaluation of nanosphere colloidal suspensions containing acyclovir as potential ophthalmic drug delivery systems was carried out. The influence of polymer molecular weight and type and concentration of various surfactants on nanosphere properties was studied. The ocular pharmacokinetics of acyclovir-loaded nanoparticles was evaluated in vivo and compared with an aqueous suspension of the free drug. Methods. Nanospheres were made up of poly-d,l-lactic acid (PLA). The colloidal suspension was obtained by a nanoprecipitation process. The surface properties of PLA nanospheres were changed by the incorporation of pegylated 1,2-distearoyl-3-phosphatidylethanol- amine. The mean size and zeta potential of the nanospheres were determined by light scattering analysis. The acyclovir loading capacity and release were also determined. In vivo experiments were carried out on male New Zealand rabbits. The ocular tolerability of PLA nanospheres was evaluated by a modified Draize test. The aqueous humor acyclovir levels were monitored for 6 h to determine the drugs ocular bioavailability for the various formulations. Results. A reduction of the mean size and a decrease of the absolute zeta potential of PLA nanospheres resulted from increasing the surfactant concentration. The higher the polymer molecular weight, the smaller the nanosphere mean size. PEG-coated and uncoated PLA nanospheres showed a sustained acyclovir release and were highly tolerated by the eye. Both types of PLA nanospheres were able to increase the aqueous levels of acyclovir and to improve the pharmacokinetics profile, but the efficacy of the PEG-coated nanospheres was significantly higher than that of the simple PLA ones. Conclusions. PEG-coated PLA nanospheres can be proposed as a potential ophthalmic delivery system for the treatment of ocular viral infections.

Aminopeptidase activity in homogenates of various absorptive mucosae m the albino rabbit: implications in peptide delivery

Abstract A methodology was developed to determine the type and activity of aminopeptidases in the conjunetival, nasal, buccal, rectal and vaginal homogenates, relative to duodenal and ileal homogenates, so as to define the aminopeptidase barrier to peptide absorption from non-oral routes. 4-Methoxy-2-naphthylamides of leucine, alanine, arginine and glutamic acid were used as substrates. Based on the pattern of substrate hydrolysis and the effect of activators and inhibitors on the rate of substrate hydrolysis, four to five aminopeptidases were estimated to be present in the mucosal homogenates studied. Aminopeptidase N was present in all these mucosae to the extent of 50–100% of ileal activity, whereas aminopeptidase A was present to the extent of 4–20% of ileal activity. Overall, the differences in aminopeptidase activity among the various non-oral routes were not large. This suggests that, before guidelines can be established to select a given route for optimal peptide delivery, further work is necessary to determine if the non-oral routes differ in non-aminopeptidase activity and in membrane permeability.

Lipophilicity influence on conjunctival drug penetration in the pigmented rabbit : a comparison with corneal penetration

The influence of lipophilicity on the conjunctival penetration of beta blockers in the pigmented rabbit was investigated and compared with that on corneal penetration. The beta blockers were hydrophilic sotalol, atenolol, nadolol, pindolol, and acebutolol; lipophilic metoprolol, timolol, oxprenolol, levobunolol, labetalol, and alprenolol; and the very lipophilic propranolol and betaxolol. Drug penetration was evaluated by using the isolated pigmented rabbit conjunctiva and cornea in the modified Ussing chamber and was monitored by reversed phase HPLC. The conjunctiva was more permeable to all the beta blockers than was the cornea. A sigmoidal relationship, rather than the familiar parabolic relationship, best described the influence of lipophilicity on both conjunctival and corneal drug penetration. The ratio of corneal to conjunctival permeability coefficients was most sensitive to changes in log PC within the region of 1.5 and 2.5. Outside of this region, the ratio was relatively independent of changes in lipophilicity. For several beta blockers, their intrinsic sympathomimetic activity may play a minor role in influencing their conjunctival and corneal penetration.

Delivery systems for penetration enhancement of peptide and protein drugs : design considerations

This paper discusses the challenges to be met in designing delivery systems that maximize the absorption of peptide and protein drugs from the gastrointestinal and respiratory tracts. The ideal delivery system for either route of administration is one that will release its contents only at a favorable region of absorption, where the delivery system attaches by virtue of specific interaction with surface determinants unique to that region and where the delivery system travels at a rate independent of the transitory constraints inherent of the route of administration. Such a delivery system, which is as yet unavailable, will benefit not only peptide and protein drugs, but other poorly absorbed drugs.

Age-dependent Expression of P-Glycoprotein gp17O in Caco-2 Cell Monolayers

AbstractPurpose. To determine whether the expression and activity of the P-glycoprotein (P-GP) drug efflux pump vary with the culture age of Caco-2 cell monolayers. Methods. Caco-2 cell monolayers were grown for 3–27 days on tissue culture-treated Transwells. P-GP efflux function was determined by measuring transmonolayer fluxes of cyclosporin A (CsA) and verapamil, while P-GP expression level was evaluated by Western blot analysis using monoclonal antibody C219. Results. The apparent permeability coefficient (Papp) of CsA (0.5 µM) in the basolateral-to-apical (B → A) direction increased with culture age and was higher than the apical-to-basolateral (A → B) direction at all times. Net secretory Papp significantly increased from day 17 onward compared to that observed during day 3 through 13. Verapamil (100 µM) significantly inhibited CsA transport in the B → A direction from day 17 to 27, while elevating CsA transport in the A → B direction from day 6 to 27. Interestingly, the Papp of verapamil (0.5 µM) in the B → A direction was significantly higher than in the A → B direction from day 6 to 27, rendering increases in net secretory Papp of verapamil with culture age. Western analysis revealed that P-GP expression level was in the order of 4 weeks ≈ 1 week > 3 weeks > 2 weeks at equal loading of cell proteins. Conclusions. P-GP is continuously expressed throughout the culture period, but it may not be fully functional at an early age. Caco-2 cell monolayers of day 17 to 27 appear to be a good model to evaluate the functional role of P-GP in drug efflux.

Enkephalin hydrolysis in homogenates of various absorptive mucosae of the albino rabbit: Similarities in rates and involvement of aminopeptidases

The systemic delivery of peptides and proteins from the nasal, rectal, vaginal, and buccal mucosae has been the subject of active investigation. The objective of this study was to determine the pathway and rate of hydrolysis of methionine enkephalin (TGGPM), leucine enkephalin (TGGPL), and [D-Ala2] met-enkephalinamide (TAGPM) in homogenates of these non-oral mucosae relative to the ileal mucosa. Aminopeptidases appeared to contribute over 85% to the hydrolysis of TGGPM and TGGPL, while dipeptidyl peptidase and dipeptidyl carboxypeptidase contributed much less. Overall, TGGPM was somewhat more susceptible to hydrolysis than TGGPL but was 10 times more so than TAGPM. These enkephalins were most rapidly hydrolyzed in the rectal and buccal homogenates, followed by the nasal and then the vaginal homogenates, but the differences in hydrolytic rates were small. Indeed, these rates did not differ substantially from the ileal mucosa, suggesting that the same enzymatic barrier to enkephalin absorption possibly exists in both the oral and the non-oral mucosae.