Chao-Pin Lee

Electrospun Nanofibers in Oral Drug Delivery

In order to enhance the delivery of drugs with limited absorption due to poor solubility/dissolution, approaches are being developed to improve the dissolution rates and solubility of drug molecules. These approaches include identification of water-soluble salts of parent drugs, preparation of stable amorphous drug formulations, inclusion of solubility-enhancing agents in the dosage form, and particle size reduction. Technologies to reduce drug particle size to sub-micrometer range are being applied to product development more frequently. Electrospinning is being considered as one of the technologies which can produce nanosized drugs incorporated in polymeric nanofibers. In vitro and in vivo studies have demonstrated that the release rates of drugs from these nanofiber formulations are enhanced compared to those from original drug substance. This technology has the potential to be used for enhancing the oral delivery of poorly soluble drugs.

Synthesis of analogs of l-valacyclovir and determination of their substrate activity for the oligopeptide transporter in Caco-2 cells

L-Valacyclovir, a prodrug of acyclovir, is a substrate for the peptide transporter (PepT1) in the intestinal mucosa, which accounts for its higher than expected oral bioavailability. The substrate activity of L-valacyclovir for PepT1 is surprising, particularly when one considers that the molecule has the structural features of a nucleoside rather than a peptide. In an attempt to better understand the structure-transport relationships (STR) for the interactions of L-valacyclovir with PepT1, analogs of this molecule with structural changes in the guanine moiety were synthesized and their substrate activity for PepT1 in Caco-2 cell monolayers was determined. The analogs synthesized include those that had the guanine moiety of L-valacyclovir substituted with purine, benzimidazole, and 7-azaindole. All three analogs (purine, benzimidazole, and 7-azaindole) exhibited affinity for PepT1 in binding studies, but only the purine analog (as the L-valine ester) showed PepT1-associated transcellular transport across Caco-2 cell monolayers. The benzimidazole and 7-azaindole analogs (as their L-valine esters) were rapidly metabolized by esterase when applied to the apical surface of Caco-2 cells, which probably explains their low penetration as the intact prodrugs via PepT1.

Selection of development candidates based on in vitro permeability measurements

Abstract Based on the desire to develop orally active drug candidates, approaches have been evaluated to identify molecules with characteristics which not only allow them to be potent at their specific receptor but also to have the appropriate membrane properties to allow absorption across the cells lining the gastrointestinal tract. The Ussing chamber approach has been instrumental in advancing our understanding of the mechanisms involved in drug transport and is also being employed to screen large numbers of compounds for selection of clinical candidates. From these types of studies, it has been demonstrated that synthesis of an amino acid prodrug of the nucleoside antiviral, acyclovir, provides an approach for enhancing uptake across the apical cell membrane with subsequent intracellular hydrolysis and release of the parent molecule across the basolateral membrane. It is these types of approaches which should provide a more rapid and rational method for identification of clinical candidates in the future.

Metabolism, uptake, and transepithelial transport of the diastereomers of Val-Val in the human intestinal cell line, Caco-2.

AbstractPurpose. The purpose of this study was to determine whether the binding of the diastereomers of Val-Val to the apical oligopeptide transporter(s) could be correlated with their cellular uptake and transepithelial transport. Methods. The Caco-2 cell culture system was used for all experiments. The binding of the diastereomers of Val-Val was evaluated by determining their ability to inhibit [3H]cephalexin uptake. The stability of the diastereomers was determined in a homogenate of Caco-2 cells and in the apical bathing solution over Caco-2 cell monolayers. The cellular uptake and transepithelial transport properties of the individual diastereomers were studied using Caco-2 cell monolayers. Results. 10 mM concentrations of L-Val-L-Val, L-Val-D-Val, D-Val-L-Val and D-Val-D-Val inhibited cellular uptake of [3H]cephalexin (0.1 mM) by 92%, 37%, 70%, and 18%, respectively. When the cellular uptake of Val-Val diastereomers (1 mM) were evaluated, the intracellular concentrations of L-Val-D-Val and D-Val-L-Val were 15 and 50 times higher, respectively, than that of D-Val-D-Val. The cellular uptake of L-Val-D-Val and D-Val-L-Val was inhibited by Gly-Pro (10 mM) (>95%), whereas Gly-Pro had no effect on the cellular uptake of D-Val-D-Val. L-Val-L-Val was not detected in the Caco-2 cells, probably due to its metabolic lability. When the transepithelial transport of the Val-Val diastereomers (1 mM) was determined, L-Val-D-Val, D-Val-L-Val and D-Val-D-Val transport rates were similar. The transepithelial transport of L-Val-D-Val and D-Val-L-Val was inhibited by Gly-Pro (10 mM) 36% and 30%, respectively, while Gly-Pro inhibited carnosine (1 mM) transepithelial transport by 65%. Gly-Pro had no effect on the transepithelial transport of D-Val-D-Val. Conclusions. These results suggest that the major transepithelial transport route of L-Val-D-Val, D-Val-L-Val and D-Val-D-Val is passive diffusion via the paracellular route. The binding of Val-Val diastereomers to the oligopeptide transporter(s) is a good predictor of their cellular uptake, however, the binding is not a good predictor of their transepithelial transport. It appears that the stereochemical requirements for the transporter that mediates efflux of the peptide across the basolateral membrane may be different from the requirements for the apical transporter that mediates cellular uptake.

Chemical Approaches to Improve the Oral Bioavailability of Peptidergic Molecules

This review discusses both tools and strategies that may be employed as approaches towards the pursuit of orally active compounds from peptidergic molecules. Besides providing a review of these subjects, this paper provides an example of how these were utilized in a research programme at SmithKline Beecham involving the development of orally active GPIIb/IIIa antagonists. The tools for studying oral drug absorption in‐vitro include variants of the Ussing chamber which utilize either intestinal tissues or cultured epithelial cells that permit the measurement of intestinal permeability. Example absorption studies that are described are mannitol, cephalexin, the growth hormone‐releasing peptide SK&F 110679 and two GPIIb/ IIIa antagonist peptides SK&F 106760 and SK&F 107260. With the exception of cephalexin, these compounds cross the intestine by passive paracellular diffusion. Cephalexin, on the other hand, crosses the intestine via the oligopeptide transporter. Structure‐transport studies are reviewed for this transporter. The tools for studying oral drug absorption in‐vivo involve animals bearing in‐dwelling intestinal or portal vein catheters. A study of the segmental absorption of SK&F 106760 is provided.

Exploitation of the Intestinal Oligopeptide Transporter to Enhance Drug Absorption

AbstractStudies of the mechanisms involved in the transport of di- and tri-peptides by the intestinal oligopeptide transporter suggest a process which involves proton-dependent uptake at the apical cell membrane of the enterocytes with subsequent exit of intact di- or tri-peptides across the basolateral membrane or, alternatively, intracellular hydrolysis and exit of component amino acids across the basolateral membrane. In the development of techniques for investigating interaction of molecules with this transporter, it was demonstrated that peptidomimetics such as β-lactam antibiotics, cephalosporins, angiotensin converting enzyme inhibitors, and renin inhibitors are taken up at the apical cell membrane of entrocytes by the oliopeptide transporter. Molecules which interact with and are taken up by the oligopeptide transporter demonstrate good oral absorption. In addition, prodrugs of α-methyldopa and phosphonoformic acid which interact with the intestinal oligopeptide transporter and which have enhanced...

Structure-Affinity Relationships of Val-Val and Val-Val- Val Stereoisomers with the Apical Oligopeptide Transporter in Human Intestinal Caco-2 Cells

The objective of this study was to elucidate the structural features of the stereoisomers of Val-Val and Val-Val-Val that afford optimal binding affinity for the apical oligopeptide transporter in human intestinal Caco-2 cells. Three-dimensional conformations of cephalexin and Val stereoisomers were optimized using Chem-X molecular modeling software. Molecular features associated with the optimized conformations of the Val stereoisomers were analyzed to identify potential relationships with their binding affinities for the apical oligopeptide transporter. For Val-Val stereoisomers, the distance between the N-terminal amino group and the C-terminal carboxyl group, d(N1-C7), was found to have a linear relationship with their binding affinities at the 95% confidence level. For Val-Val-Val stereoisomers, three molecular features were found to have linear relationships with their binding affinities at the 95% confidence level. These features included: a) the distance between the N-terminal amino group and the C-terminal carboxyl group, d(N1-C11); b) the distance between the N-terminal amino group and the second peptide bond, d(N1-N9); and c) the molecular dipole moment. Principal component analysis on all molecular features of Val-Val-Val stereoisomers identified three components that accounted for 90% of the variance. A linear model built with these three components by multiple linear regression adequately described the binding affinities (r2 = 0.90). Results from the current study suggest that the distance between the N-terminal amino group and the C-terminal carboxyl group is important for interaction with the apical oligopeptide transporter in Caco-2 cells. In addition, the binding affinities of the Val-Val-Val stereoisomers appear to be influenced by additional factors, including the position of the second peptide bond and the molecular dipole moment.

In vitro permeability screening for identification of orally bioavailable endothelin receptor antagonists

Abstract In vitro intestinal permeability screening identified a compound, SB 217242, in the indane carboxylic acid series of endothelin receptor antagonists, with greatly improved permeability over the initial lead compound in this series. Based on mucosal-to-serosal and serosal-to-mucosal permeability studies, and on the fact that SB 217242 transport does not correlate with changes in mannitol permeability, it was concluded that SB 217242 follows a passive transcellular pathway. Permeability studies at different pH values demonstrate that the permeating species is the unionized form of SB 217242. This compound is well absorbed upon intraduodenal dosing in the rat (Ohlstein et al. (1996) J. Pharm. Exp. Ther. 276, 609–615).

Novel inhibitors of the osteoclast specific cysteine protease, cathepsin K

Daniel F. Veber, Dennis S. Yamashita, Hye-Ja Oh, Brian R. Smith, Kevin Salyers, Mark Levy, Chao-Pin Lee, Antonia Marzulli, Phil Smith, Ted Tomaszek, David Tew, Michael McQueney, George B. Stroup, Michael W. Lark, Ian E. James, and Maxine Gowen 6 Department of Medicinal Chemistry, Preclinical Pharmacokinetics, Bioformulations and Drug Delivery, Molecular Recognition, Protein Biochemistry, and Bone & Cartilage Biology, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406, U.S.A.

The use of rabbit intestinal permeability as an in vitro assay in the search for orally active GPIIb/IIIa antagonists

Abstract A series of potent, high affinity GPIIb/IIIa antagonists were evaluated for intestinal permeability in vitro using rabbit intestinal strips mounted in the Ussing Chamber. In this series of compounds, structural modifications were found to impact intestinal permeability. In general, modifications that would contribute to lipophilicity (e.g. olefinic replacement for amide, phenethyl or isopropyl for methyl) did not enhance permeability. When these intestinal permeabilities were compared to mannitol, which displays ~ 20% oral bioavailability in humans (Laker et al. (1982) Eur. J. Clin. Invest. 12, 485–491), it was possible then to select compounds with permeabilities comparable to mannitol for oral (intraduodenal) evaluation in conscious dogs. The influence of intestinal permeability on oral efficacy can be observed within a series of compounds with closely related structures, but is less discernable in across structural series. Intestinal permeability data can provide meaningful, reliable data that can help in analog design and in the selection of compounds for oral evaluation.