Richard A. Morrison

Current methodologies used for evaluation of intestinal permeability and absorption.

This review article will focus on the various techniques that are currently employed by drug discovery scientists in evaluating permeability/absorption of drug candidates during the drug candidate selection process. Various preclinical methodologies are available; each having advantages and disadvantages, but it is the judicious use of these techniques that can help identify drug candidates that will be well absorbed in humans. It is well recognized that the human intestinal permeability cannot be accurately predicted based on a single methodology (in vitro: tissue/cell culture, in situ, or in vivo).

Prediction of the Oral Absorption of Low-Permeability Drugs Using Small Intestine-Like 2/4/A1 Cell Monolayers

AbstractPurpose. To characterize the paracellular route of 2/4/A1 monolayers and to compare the permeabilities of incompletely absorbed oral drugs in 2/4/A1 with those in Caco-2 monolayers. Methods. The cells were cultivated on permeable supports. The 2/4/A1 expression of genes associated with tight junctions was compared with that in the small intestine using RT-PCR. The aqueous pore radii were determined using paracellular marker molecules. The permeabilities of a series of incompletely absorbed drugs (defined as having a fraction absorbed 0 to 80%) after oral administration to humans were studied. Results. Occludin and claudin 1 and 3 were expressed in 2/4/A1. The pore radius of 2/4/A1 was 9.0 ± 0.2 Å, which is similar to that in the human small intestine, although the pore radius was smaller (3.7 ± 0.1 Å) in Caco-2. The relationship between permeability and fraction absorbed of 13 drugs was stronger in 2/4/A1 than in Caco-2. The relationships were used to predict the intestinal absorption of another seven drugs. The prediction was more accurate in 2/4/A1 (RMSE = 15.6%) than in Caco-2 (RMSE = 21.1%). Further, Spearmans rank coefficient between FA and permeability was higher in 2/4/A1. Conclusion. The improved 2/4/A1 cell culture model has a more in vivo-like permeability and predicted the oral absorption of incompletely absorbed drugs better than Caco-2 cells.

A rapid and sensitive LC/MS/MS assay for quantitative determination of digoxin in rat plasma

Digoxin is a cardiac glycoside that is widely used for the treatment of congestive heart failure. To evaluate pharmacokinetics of digoxin in rats, a sensitive LC/MS/MS assay was developed and validated for the determination of digoxin concentration in rat plasma. For detection, a Sciex API3000 LC/MS/MS with atmospheric pressure ionization (API) mass spectrometry turbo ion spray inlet in the positive ion-multiple reaction monitoring mode was used to monitor precursor-->product ions of m/z 798.6-->651.6 for digoxin and m/z 577.6-->433.3 for oleandrin, the internal standard (IS). The standard curve was linear (r(2)>or=0.999) over the digoxin concentration range of 0.1-100 ng/ml in plasma for digoxin. The mean predicted concentrations of the quality control samples deviated by <5.8% from the corresponding nominal values; the intra-assay and inter-assay precision of the assay were within 8.6% relative standard deviation. At the lower limit of quantitation (LLQ) of 0.1 ng/ml, the mean deviation of predicted concentrations from the nominal value was within 3.7%. The extraction recoveries of digoxin and internal standard were 82.7+/-3.9 and 105.9+/-2.3%, respectively. The present method was successfully applied to characterization of pharmacokinetic profiles of digoxin in rats after oral administration.

A rapid screening system to determine drug affinities for the intestinal dipeptide transporter 2: affinities of ACE inhibitors.

PURPOSE To assess the affinities of a series of ACE inhibitors for the di/tri/oligopeptide transport system (DTS) using a rapid in vitro system. METHODS Monolayers of Caco-2 cells were cultured in plastic wells for 7-9 days and the uptake of Gly-[3H]L-Pro was used as an affinity probe. Gly-[3H]L-Pro (50 nM), together with excess L-Pro (10 mM), to suppress uptake of any [3H]L-Pro produced by degradation of the probe, was incubated with the test compound (usually 1 mM) at pH 6 for 3-mins. The uptake of radiolabel was determined by liquid scintillation counting. RESULTS A 2-dimensional six-domain model of the transporter based on the structure of a phosphinate ACE inhibitor (SQ-29852) was constructed to facilitate interpretation of the competitor affinities. The SQ-29852 molecule was divided into six binding domains (A-F) based on functional groups within these regions and the effects of structural variation in four of these domains (A, C-E) were explored. A series of dipeptide-like compounds varying within specific domains were selected from a large number of commercially available ACE inhibitors and SQ-29852 analogues. Domain A had a preference for an uncharged group, with bulky hydrophobic groups reducing affinity. Domain C exhibited a preference for a positive charge over a neutral function, with the space this functional group occupies contributing to affinity. Domain D favoured lipophilic residues and domain E retained activity when the carboxylic acid was esterified. CONCLUSION The test system is able to reveal structure-activity relationships of peptidomimetic agents and may well serve as a design tool to optimise affinity for the DTS.

Suitability of enalapril as a probe of the dipeptide transporter system : In vitro and in vivo studies

AbstractPurpose. Previous in situ and in vitro studies indicated that the intestinal absorption of enalapril is a saturable carrier-mediated process via the dipeptide transporter system (DTS); however, the oral absorption of enalapril has not been reported to be a saturable process in vivo. Our objectives were to: 1) evaluate the suitability of enalapril as a probe of the DTS, and 2) compare various experimental models as they pertain to studying the DTS. Methods. The in vitro uptake of enalapril by rat intestinal rings and permeability across Caco-2 cells were studied as a function of concentration and in the presence of compounds that are known substrates of the DTS. The effect of enalapril on the uptake of [3H]-glycyl-L-proline (gly-L-pro) by Caco-2 cells was also examined. In vivo studies were conducted in rats (1 to 50 mg/kg) and dogs (0.06 to 6 mg/kg) to evaluate the oral absorption of enalapril over a wide dose range. Results. In vitro intestinal uptake/permeability of enalapril was not saturable nor inhibited by p-lactam antibiotics, gly-L-pro, or SQ-29852. Moreover, a 20,000-fold molar excess of enalapril did not inhibit the uptake of [3H]-gly-L-pro by Caco-2 cells. The in vivo studies in rats and dogs did not demonstrate saturable absorption. Conclusions. The present in vitro and in vivo results indicated that enalapril is primarily absorbed by a non-saturable, passive diffusion process and it is not a suitable model compound for studying the DTS.

Orally active prodrugs of quinoline-4-carboxylic acid angiotensin II receptor antagonists

Abstract Prodrug derivatization of a potent quinoline-4-carboxylic acid angiotensin II receptor antagonist was Undertaken as an approach to achieve improved oral activity. A dioxolenone carboxylic ester and an alkylated tetrazole prodrug both showed greater oral antihypertensive acivity in the salt-deplete spontaneously hypertensive rat and increased oral bioavailability relative to the parent compound.

A rapid screening system to determine drug affinities for the intestinal dipeptide transporter 1: system characterisation

PURPOSE To establish an in vitro system for the rapid assessment of the affinities of potential substrates for the di/tri/oligopeptide transport system (DTS). METHODS Monolayers of Caco-2 cells were cultured in plastic wells for 7-9 days and the uptake of Gly-[3H]L-Pro, a specific and relatively stable substrate for the DTS was used as an affinity probe. Gly-[3H]L-Pro (50 nM), together with excess L-Pro (10 mM), to suppress uptake of any [3H]L-Pro produced by degradation of the probe, was incubated with the test compound (usually 1 mM) at pH 6 for 3 min. The uptake of radiolabel was determined by liquid scintillation counting. RESULTS High specific-uptake (> 85%) of Gly-[3H]L-Pro was obtained with cells grown for 7-9 days. Gly-[3H]L-Pro uptake had a substantial active concentration-dependent component (Km of 0.39 +/- 0.02 mM, Vmax of 0.98 +/- 0.04 nmol min(-1) (mg protein)(-1). This process was shown to be specific for the DTS as evidenced by the significant inhibition by compounds reported to be transported by this system and the lack of inhibition by amino acids. The use of low competitor concentrations (1 mM) enabled a range of inhibition values (0-89%) of a series of competitors (amino acids, dipeptides and beta-lactam antibiotics) to be estimated, illustrating that structurally similar compounds can be ranked for affinity to the DTS. CONCLUSION A screening system, using Caco-2 cells and the dipeptide Gly-[3H]L-Pro as a displaceable probe, was developed to assess a variety of compounds for recognition by the di/tri/oligopeptide transport system. This fully describes the first system that allows structurally related compounds to be ranked on the basis of their affinity for the DTS recognition site.

Thromboxane receptor antagonist BMS-180291: A new pre-clinical lead

Abstract The synthesis and initial pharmacology of interphenylene 7-oxabicyclo[2.2.1]heptane oxazole thromboxane (TxA2) receptor antagonist BMS-180291 is described. BMS-180291 has been characterized as an orally bioavailable, potent and selective TxA2 antagonist with a long duration of action.

Sites of First-Pass Bioactivation (Hydrolysis) of Orally Administered Zofenopril Calcium in Dogs

The relative contribution of the gut, liver, and lungs as sites of first-pass bioactivation (hydrolysis) of the orally administered ester prodrug, zofenopril calcium (SQ 26,991), to the active angiotensin converting enzyme (ACE) inhibitor, SQ 26,333, was determined. With a five-way study design, two dogs each received a single 1.6-mg/kg dose of zofenopril [as its soluble potassium salt (SQ 26,900)] via the following routes of administration: intraarterial, intravenous, intraportal, and oral. Each dog also received an equimolar oral dose of zofenopril calcium (1.5 mg/kg). Concentrations of zofenopril in plasma were quantitated with a GC/MSD assay. Extraction ratios (E) for zofenopril by the gut, liver, and lungs were calculated based on the ratios of the area under the curve (AUC) values of zofenopril in arterial plasma after administration by the various routes. As individual eliminating organs, the gut and liver each had a high intrinsic capability to hydrolyze zofenopril; E values ranged from 45 to 89%. The lungs were found to have low, but measurable, hydrolytic activity with estimated E values that ranged from 5 to 26%. Overall, about 95% of the orally administered dose of zofenopril calcium was hydrolyzed during the first pass. Because the prodrug is sequentially exposed to the gut, liver, and lungs, the contribution of the gut to the overall first-pass hydrolysis (ca. 87%) was estimated to be significantly greater than that of the liver (<10%) or lungs (<2%). Zofenopril was rapidly eliminated after parenteral administration; mean residence time values were 2 min and the elimination half-life values (intraarterial route only) were 9 min. The total-body clearance (Cltotal) of zofenopril, determined after intraarterial injection, was rapid (ca. 25 ml/min/kg) and was similar to the Cltotal value reported previously for fosinopril sodium, another prodrug ACE inhibitor. Although the Elungs value was relatively low, the lungs receive 100% of cardiac output and were estimated to contribute significantly to systemic bioactivation of zofenopril. The major sites of systemic bioactivation appear to be the lungs (ca. 44 to 65%) and liver (ca. 31%), whereas the hydrolysis of zofenopril by the blood per se does not apparently contribute to Cltotal.

Continuous Blood Withdrawal as a Rapid Screening Method for Determining Clearance and Oral Bioavailability in Rats

AbstractPurpose. To develop a methodology for continuous blood withdrawal in rats suitable for drug discovery screening purposes and perform limited validation studies with a series of test compounds. Methods. A reliable methodology for continuous blood withdrawal in rats was developed. The method is dependent on continuous heparin infusion during withdrawal and the minimization of constrictive, thrombogenic sites. Plasma drug concentrations from either intermittent sampling or continuous withdrawal experiments were determined with HPLC analysis. Results. The continuous withdrawal method was successfully adapted to rats such that blood samples could be reliably collected over a 6-hr experiment. The clearance and oral bioavailability values for theophylline, atenolol, propranolol, warfarin. BMS-182874 and BMS-A were determined from continuous withdrawal or intermittent sampling experiments. The results from the two methods were comparable, with each compound reliably placed in the same low, medium or high category based on clearance or oral bioavailability characteristics. Conclusions. The continuous withdrawal method proved to be a viable alternative to the classic intermittent sampling technique. The method should prove useful in drug discovery screening, where the evaluation of large numbers of compounds for systemic clearance or oral bioavailability is often necessary.