Barbra H. Stewart

A saturable transport mechanism in the intestinal absorption of gabapentin is the underlying cause of the lack of proportionality between increasing dose and drug levels in plasma.

Gabapentin (l-(aminomethyl)cyclohexaneacetic acid) is a neuroprotective agent with antiepileptic properties. The structure is small (molecular weight less than 200), is zwitterionic, and resembles an amino acid with the exception that it does not contain a chiral carbon and the amino group is not alpha to the carboxylate functionality. Gabapentin is not metabolized by humans, and thus, the amount of gabapentin excreted by the renal route represents the fraction of dose absorbed. Clinical trials have reported dose-dependent bioavailabilities ranging from 73.8 ± 18.3 to 35.7 ± 18.3% when the dose was increased from 100 to 1600 mg. The permeability of gabapentin in the rat intestinal perfusion system was consistent with carrier-mediated absorption, i.e., a 75 to 80% decrease in permeability when the drug concentration was increased from 0.01 to 50 mM (0.46 ± 0.05 to 0.12 ± 0.04). Excellent agreement was obtained between the actual clinical values and the predicted values from in situ results for the fraction of dose absorbed calculated using the theoretically derived correlation, Fabs = 1 - exp(−2Peff) by Ami-don et al. (Pharm. Res. 5:651–654, 1988). The permeability values obtained for gabapentin correspond to 67.4 and 30.2% of the dose absorbed at the low and high concentrations, respectively. In the everted rat intestinal ring system, gabapentin shared an inhibition profile similar to that of L-phenylalanine. Characteristics of gabapentin uptake included cross-inhibition with L-Phe, sensitivity to inhibition by L-Leu, stereoselectivity as evidenced by incomplete inhibition by D-Phe, and lack of effect by Gly. Our findings support absorption of gabapentin by a saturable pathway, system L, shared by the large hydrophobic amino acids, L-Phe and L-Leu. The saturable absorption pathway makes a major contribution to the lack of proportionality in plasma levels of drug with increasing dose ob-served in the clinic.

Improved oral drug delivery: solubility limitations overcome by the use of prodrugs

Abstract This chapter describes a strategy to improve the oral delivery of poorly water-soluble drugs by chemical derivatization to a water-soluble prodrug. The strategy utilizes esterification of a drug hydroxyl, amine or carboxyl group with a moiety (progroup) designed to introduce an ionizable function or reduce intermolecular interactions responsible for low solubility. The use of spacer groups to introduce derivatizable functions and/or to position ionizable progroups for unhindered hydrolysis is also described. Prodrug strategies coupling drug solubilization with membrane carrier targeting and the use of collapsible and bifunctional prodrugs are outlined. These approaches are illustrated with studies utilizing model compounds to test the strategy as well as examples from various therapeutic classes of drugs in which aqueous solubility limits drug absorption.

Comparison of Intestinal Permeabilities Determined in Multiple in Vitro and inSitu Models: Relationship to Absorption in Humans

In vitro and in situ experimental models that are descriptive of drug absorption in vivo are valuable tools in the discovery of new chemical entities that are bioavailable after oral administration. The specific objective of the study was to compare the intestinal permeabilities obtained in the three absorption models for consistency, and to assess the utility of the models in predicting the fraction of dose absorbed in human studies. The intestinal absorption models that were compared are widely used: the rat in situ single-pass intestinal perfusion system, the rat everted intestinal ring method, and monolayers of human colon adenocarcinoma cell line (CACO-2). The models were compared using small molecular reference compounds, as well as a series of peptidomimetic (PM) analogs. Each model had strong potential for estimating the fraction absorbed. For small organic molecules, excellent correlation was observed when permeabilities from CACO-2 cells and perfusions, or everted rings and perfusions, were compared. Weaker correlation was observed between everted rings and CACO-2 cells. Permeabilities for the set of reference compounds and PMs were positively correlated between any two of the three systems. Variance between correlations for reference compounds and PMs are likely due to structural features and physicochemical properties that are unique to the latter class of compounds. The results support caution in extrapolating correlations based on findings with small organic molecules to the behavior of complex peptidomimetics. Corroboration of permeabilities with two methods of determination is a useful cross-validation of experimental systems, as well as producing a reliable permeability assessment. CACO-2 cell monolayers and rat single-pass intestinal perfusion combine the highest correlation between systems, most defined relationship with fraction absorbed in humans, and experimental logistics in-line with discovery candidates.

Atorvastatin transport in the Caco-2 cell model: contributions of P-glycoprotein and the proton-monocarboxylic acid co-transporter.

AbstractPurpose. The purpose of this study was to elucidate the mechanismsby which an HMG-CoA reductase inhibitor, atorvastatin (an organicacid with a pKa of 4.46), was transported in the secretory and absorptivedirections across Caco-2 cell monolayers. Methods. Caco-2 cells were grown on polycarbonate membrane insertsin 6-well Snapwell plates (Costar). The permeability of radiolabeledcompounds across Caco-2 cell monolayers was determined using aside-by-side diffusion apparatus (NaviCyte) and an automated liquidhandler (Hamilton Microlab 2200). The apical uptake of14C-atorvastatin was also determined in Caco-2 cells. Cyclosporin A (20 μM) waspresent in the uptake media to block potential P-glycoprotein-mediatedatorvastatin efflux. Results. Polarized permeation of atorvastatin was observed with thebasolateral-to-apical (B-to-A) permeability being 7-fold greater thanthe A-to-B permeability (35.6 × 10−6 and 4.9 × 10−6 cm/s,respectively). The secretion of atorvastatin was a saturable process with anapparent Km of 115 μM. The B-to-A permeability of atorvastatin wassignificantly reduced by cyclosporin A (10 μM), verapamil (100 μM),and a P-glycoprotein specific monoclonal antibody, UIC2(10 μg/ml)(43%, 25%, and 13%, respectively). Furthermore, both CsA andverapamil significantly increased the A-to-B permeability of atorvastatinby 60% however, UIC2 did not affect the A-to-B permeability ofatorvastatin. CsA uncompetitively inhibited the B-to-A flux ofatorvastatin with a Ki of 5 μM. In addition, atorvastatin (100 μM) significantlyinhibited the B-to-A permeability of vinblastine by 61%. The apicaluptake of atorvastatin increased 10.5-fold when the apical pH decreasedfrom pH 7.4 to pH 5.5 while the pH in the basolateral side wasfixed at pH 7.4. A proton ionophore, carbonylcyanidep-trifluoro-methoxyphenylhydrazone (FCCP) significantly decreased atorvastatinuptake. In addition, atorvastatin uptake was significantly inhibited bybenzoic acid, nicotinic acid, and acetic acid each at 20 mM (65%,14%, and 40%, respectively). Benzoic acid competitively inhibitedatorvastatin uptake with a Ki of 14 mM. Similarly, benzoic acid,nicotinic acid, and acetic acid significantly, inhibited the A-to-Bpermeability of atorvastatin by 71%, 21%, and 66%, respectively. Conclusion. This study demonstrated that atorvastatin was secretedacross the apical surface of Caco-2 cell monolayers viaP-glycoprotein-mediated efflux and transported across the apical membrane in theabsorptive direction via a H+-monocarboxylic acid cotransporter(MCT). In addition, this study provided the first evidence thatnegatively charged compounds, such as atorvastatin, can be a substrate forP-glycoprotein.

Physicochemical and drug-delivery considerations for oral drug bioavailability

The bioavailability of a compound after oral administration is a function of molecular characteristics, dosage form design, and the barrier functions of the organism. The extent of intestinal absorption is dependent on drug stability, solubility and permeability. First-pass elimination can be effected by intestinal mucosal or hepatic processing. Biological availability is dependent on the physicochemical parameters of the drug, including molecular weight and lipophilicity, as well as on specific structural features such as H-bonding capacity. This review illustrates the relationships between physicochemical parameters, intestinal permeability and hepatic processing, using a set of eight peptidomimetic renin inhibitor analogs. Results from these studies provide suggestions for the design of compounds with improved bioavailability.

Discrimination between drug candidates using models for evaluation of intestinal absorption

Abstract Methods employing in vitro and in situ means can be used to gain early information regarding the potential for intestinal absorption of new chemical entities (NCEs). Physicochemical and biological approaches for evaluation of the intestinal absorption of NCEs are described in this review. Physicochemical measurements such as aqueous solubility, lipophilicity and membrane partioning form an important base for subsequent biological experiments. Biological techniques that include subcellular membrane fractions, intestinal cells, isolated tissue and organ perfusion are detailed. Several of these preparations can be utilized for transport, as well as stability indicators. While some methods are generally applicable to NCEs, others require selectivity on the part of the investigator, depending on the information desired.

The Role of 4-phosphonodifluoromethyl- and 4-phosphono-phenylalanine in the selectivity and cellular uptake of SH2 domain ligands

Incorporation of 4-phosphonodifluoromethyl-phenylalanine (F2Pmp) and 4-phosphono-phenylalanine into SH2 targeted peptides and peptidomimetic ligands was found to effect binding affinity and selectivity of these ligands toward the Src and Abl SH2 domains. Furthermore, dipeptide analogs containing these phosphonate amino acids were used to produce prodrugs with excellent cellular delivery and reconversion rates.

Transport of Pregabalin in Rat Intestine and Caco-2 Monolayers

AbstractPurpose. The purpose of this study was to determine if the intestinal transport of pregabalin (isobutyl -γ-aminobutyric acid, isobutyl GAB A), a new anticonvulsant drug, was mediated by amino acid carriers with affinity for large neutral amino acids (LNAA). Methods. Pregabalin transport was studied in rat intestine and Caco-2 monolayers. An in vitro Ussing/diffusion chamber model and an in situ single-pass perfusion model were used to study rat intestinal transport. An in vitro diffusion chamber model was used to evaluate Caco-2 transport. Results. In rat ileum, pregabalin transport was saturable and inhibited by substrates of intestinal LNAA carriers including neurontin (gabapentin), phenylalanine, and proline. Weak substrates of intestinal LNAA carriers (β-alanine, -γ-aminobutyric acid, and methyl aminoisobutyric acid) did not significantly change pregabalin transport. In Caco-2 mono-layers that showed a high capacity for phenylalanine transport, pregabalin transport was concentration- and direction-independent and equivalent in magnitude to the paracellular marker, mannitol. The in vitro and in situ rat ileal permeabilities of the LNAA carrier-mediated compounds neurontin, pregabalin, and phenylalanine correlated well with the corresponding in vivo human oral absorption. Conclusions. The transport of pregabalin was mediated by LNAA carriers in rat ileum but not in Caco-2 monolayers. Caco-2 was not an appropriate model for evaluating the in vivo human oral absorption of pregabalin and neurontin.

Hydrophobicity of HIV protease inhibitors by immobilized artificial membrane chromatography: application and significance to drug transport.

AbstractPurpose. The feasibility of using hydrophobicity measurements as screens for intracellular availability in T-cells or intestinal permeability in Caco-2 cells was examined. Methods. T-cell experiments: Cells were counted, collected, then incubated with drug solution at 37°C. At selected time intervals, uptake was quenched by transferring a sample into oil, followed by rinsing, lysis of cells, protein precipitation and analysis by HPLC. Caco-2 cellexperiments: Cells were grown on plastic dishes for 7−10 d, then rinsed and incubated with drug solution at 37°C. Uptake was quenched, cells were lysed, protein precipitated and drug was analyzed by HPLC. IAMchromatography: Stock solutions were injected onto an IAM column for HPLC. Mobile phase consisted of varying amounts of acetonitrile in buffer (pH 7.4). The capacity factor, k′IAM, was calculated using citric acid to measure the void volume and was obtained by extrapolation to pure buffer. Results. Nine HIV protease inhibitors were studied for uptake by CEM T-cell suspensions or Caco-2 cell monolayers. Capacity factors (log) between IAM and C-18 columns were positively correlated for this series. Caco-2 uptake rates correlated well with T-cell uptake rates when normalized by protein mass. Single-variable regression using IAM or C-18 columns was acceptable for analysis of T-cell data. Correlation coefficients between T-cell uptake and log k′IAM or log k′C-18 were not improved with multivariable regression. Correlation between Caco-2 uptake and log k′IAM was enhanced when molecular weight and hydrogen-bonding potential were included in multivariable regression analysis (from r2 of 0.39 to 0.91). Correlations obtained using log k′IAM, log k′C-18.or log distribution coefficient (log D) were comparable when regressed against Caco-2 uptake using this approach. Calculated log partition coefficient (ClogP) provided the poorest correlation in the multivariable analysis (r2 = 0.57 for T-cell uptake and r2 = 0.71 for Caco-2 cell uptake). Conclusions. Uptake of HIV protease inhibitors by T-cell suspensions or Caco-2 cell monolayers was positively correlated. Uptake by T-cell suspensions was adequately described by hydrophobicity alone. Description of uptake by Caco-2 cell monolayers required multivariable regression analysis in which molecular weight and hydrogen bonding were included. Experimental measures of hydrophobicity (log k′IAM, log k′C18 and log D) were superior to ClogP in the correlation analysis.

Evidence for Overlapping Substrate Specificity Between Large Neutral Amino Acid (LNAA) and Dipeptide (hPEPTl) Transporters for PD 158473, an NMDA Antagonist

AbstractPurpose. The objective of this research was to investigate the substrate specificity of large neutral amino acid carrier (LNAA) and di/tripeptide (hPEPTl) transporters with respect to PD 158473, an NMDA antagonist. Methods. Cellular uptake studies were carried out using two types of Chinese Hamster Ovary (CHO). CHO-K1 cells represent the wild type with inherent large neutral amino acid (LNAA) activity. CHO-PEPT1 cells were generated by stable transfection of hPEPTl gene into CHO cells. Therefore, these cells possess both LNAA activity and di/tripeptide transporter activities as a result of the transfection. Cellular uptake of PD 158473 was quantified using a HPLC method previously developed in our laboratory. Results. The utility of the CHO-PEPT1 cell model was demonstrated by determining the uptake kinetics of Gly-Sar, a prototypical dipeptide transporter substrate. Uptake kinetics of PD 158473 displayed two carrier-mediated transport components in CHO-PEPT1 cells, while in CHO-K1 cells the relationship was consistent with classic one component Michaelis-Menten kinetics. These results confirmed the affinity of PD 158473 for both LNAA and di/tripeptide transporters. Further, results from inhibition experiments using these two cell types indicate that the high affinity-low capacity system was the LNAA carrier and the low affinity-high capacity carrier was the di/tripeptide transporter. Conclusions. This study demonstrates overlapping substrate specificity between LNAA carrier and di/tripeptide transporter (hPEPTl) for PD 158473, an amino acid analog. Establishing Structure Transport Relationship (STR) for this overlap will aid in a design strategy for increasing oral absorption or targeting specific drugs to selected tissues.