Phosphate prodrugs of PD154075.

In the preparation of phosphate prodrugs of PD154075, several strategies of linking a phosphate group to the indole moiety were studied. A novel linker, p-hydroxymethylbenzoyloxymethoxycarbonyl, was discovered to provide the phosphate prodrug of PD154075 (compound 9) with significantly increased aqueous solubility, sufficient stability in aqueous solution and good bio-reconversion in vivo.

Oral delivery of HIV-protease inhibitors.

Strategies for optimizing the oral delivery of HIV-protease inhibitors draw from drug discovery efforts in molecular design, drug development tools in dosage formulation, and dosage regimen considerations in clinical medicine. This review outlines the evolution of these strategies for drugs that have been approved for human use, drug candidates still in development, and molecules that are no longer in development but from which valuable delivery information was obtained. Molecular design for obtaining desirable pharmacokinetics following oral administration primarily involved maximizing aqueous solubility and minimizing first-pass metabolism. Optimization of molecular design for oral drug delivery purposes is tempered by additional considerations for drug potency, toxicity, potential for interactions, and development of viral resistance. Strategies for improving oral bioavailability through dosage formulation use information from the effects of coadministered meals on drug plasma levels. Patient adherence to dosage regimens remains a major issue in assuring effective oral drug treatment and in preventing the development of resistance. Progress has been made in clinical studies where improved oral bioavailability and reductions in drug plasma level variability have been achieved with appropriate dosage regimen adjustment.

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

PURPOSE: The purpose of this study was to elucidate the mechanisms by which an HMG-CoA reductase inhibitor, atorvastatin (an organic acid with a pKa of 4.46), was transported in the secretory and absorptive directions across Caco-2 cell monolayers. METHODS: Caco-2 cells were grown on polycarbonate membrane inserts in 6-well Snapwell plates (Costar). The permeability of radiolabeled compounds across Caco-2 cell monolayers was determined using a side-by-side diffusion apparatus (NaviCyte) and an automated liquid handler (Hamilton Microlab 2200). The apical uptake of 14C-atorvastatin was also determined in Caco-2 cells. Cyclosporin A (20 microM) was present in the uptake media to block potential P-glycoprotein-mediated atorvastatin efflux. RESULTS: Polarized permeation of atorvastatin was observed with the basolateral-to-apical (B-to-A) permeability being 7-fold greater than the A-to-B permeability (35.6 x 10(-6) and 4.9 x 10(-6) cm/s, respectively). The secretion of atorvastatin was a saturable process with an apparent Km of 115 microM. The B-to-A permeability of atorvastatin was significantly reduced by cyclosporin A (10 microM), verapamil (100 microM), and a P-glycoprotein specific monoclonal antibody, UIC2(10 microg/ml) (43%, 25%, and 13%, respectively). Furthermore, both CsA and verapamil significantly increased the A-to-B permeability of atorvastatin by 60%; however, UIC2 did not affect the A-to-B permeability of atorvastatin. CsA uncompetitively inhibited the B-to-A flux of atorvastatin with a Ki of 5 microM. In addition, atorvastatin (100 microM) significantly inhibited the B-to-A permeability of vinblastine by 61%. The apical uptake of atorvastatin increased 10.5-fold when the apical pH decreased from pH 7.4 to pH 5.5 while the pH in the basolateral side was fixed at pH 7.4. A proton ionophore, carbonylcyanide p-trifluoro-methoxyphenylhydrazone (FCCP) significantly decreased atorvastatin uptake. In addition, atorvastatin uptake was significantly inhibited by benzoic acid, nicotinic acid, and acetic acid each at 20 mM (65%, 14%, and 40%, respectively). Benzoic acid competitively inhibited atorvastatin uptake with a Ki of 14 mM. Similarly, benzoic acid, nicotinic acid, and acetic acid significantly, inhibited the A-to-B permeability of atorvastatin by 71%, 21%, and 66%, respectively. CONCLUSION: This study demonstrated that atorvastatin was secreted across the apical surface of Caco-2 cell monolayers via P-glycoprotein-mediated efflux and transported across the apical membrane in the absorptive direction via a H(+)-monocarboxylic acid cotransporter (MCT). In addition, this study provided the first evidence that negatively charged compounds, such as atorvastatin, can be a substrate for P-glycoprotein.

Atorvastatin coadministration may increase digoxin concentrations by inhibition of intestinal P-glycoprotein-mediated secretion.

The effect of atovarstatin on digoxin pharmacokinetics was assessed in 24 healthy volunteers in two studies. Subjects received 0.25 mg digoxin daily for 20 days, administered alone for the first 10 days and concomitantly with 10 mg or 80 mg atorvastatin for the last 10 days. Mean steady-state plasma digoxin concentrations were unchanged by administration of 10 mg atorvastatin. Mean steady-state plasma digoxin concentrations following administration of digoxin with 80 mg atorvastatin were slightly higher than concentrations following administration of digoxin alone, resulting in 20% and 15% higher Cmax and AUC(0-24) values, respectively. Since tmax and renal clearance were not significantly affected, the results are consistent with an increase in the extent of digoxin absorption in the presence of atorvastatin. Digoxin is known to undergo intestinal secretion mediated by P-glycoprotein. Since atorvastatin is a CYP3A4 substrate and many CYP3A4 substrates are also substrates for P-glycoprotein transport, the influence of atorvastatin and its metabolites on P-glycoprotein-mediated digoxin transport in monolayers of the human colon carcinoma (Caco-2) cell line was investigated. In this model system, atorvastatin exhibited efflux or secretion kinetics with a K(m) of 110 microM. Atorvastatin (100 microM) inhibited digoxin secretion (transport from the basolateral to apical aspect of the monolayer) by 58%, equivalent to the extent of inhibition observed with verapamil, a known inhibitor of P-glycoprotein transport. Thus, the increase in steady-state digoxin concentrations produced by 80 mg atorvastatin coadministration may result from inhibition of digoxin secretion into the intestinal lumen.

Transport of pregabalin in rat intestine and Caco-2 monolayers.

PURPOSE: The purpose of this study was to determine if the intestinal transport of pregabalin (isobutyl gamma-aminobutyric acid, isobutyl GABA), 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 (beta-alanine, gamma-aminobutyric acid, and methyl aminoisobutyric acid) did not significantly change pregabalin transport. In Caco-2 monolayers 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.

Evidence for overlapping substrate specificity between large neutral amino acid (LNAA) and dipeptide (hPEPT1) transporters for PD 158473, an NMDA antagonist.

PURPOSE: The objective of this research was to investigate the substrate specificity of large neutral amino acid carrier (LNAA) and di/tripeptide (hPEPT1) 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 hPEPT1 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 (hPEPT1) 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.

CHO/hPEPT1 cells overexpressing the human peptide transporter (hPEPT1) as an alternative in vitro model for peptidomimetic drugs.

The present study characterized Chinese hamster ovary cells overexpressing a human intestinal peptide transporter, CHO/hPEPT1 cells, as an in vitro model for peptidomimetic drugs. The kinetic parameters of Gly-Sar uptake were determined in three different cell culture systems such as untransfected CHO cells (CHO-K1), transfected CHO cells (CHO/hPEPT1) and Caco-2 cells. Vmax in CHO/hPEPT1 cells was approximately 3-fold higher than those in Caco-2 cells and CHO-K1 cells, while Km values were similar in all cases. The uptake of beta-lactam antibiotics in CHO/hPEPT1 cells was three to twelve fold higher than that in CHO-K1 cells, indicating that CHO/hPEPT1 cells significantly enhanced the peptide transport activity. However, amino acid drugs also exhibited high cellular uptake in both CHO-K1 and CHO/hPEPT1 cells due to the high background level of amino acid transporters. Thus, cellular uptake study in CHO/hPEPT1 cells is not sensitive enough to distinguish the peptidyl drugs from amino acid drugs. The potential of CHO/hPEPT1 cells as an in vitro model for peptidomimetic drugs was also examined through the inhibition study on Gly-Sar uptake. Peptidomimetic drugs such as beta-lactam antibiotics and enalapril significantly inhibited Gly-Sar uptake whereas the nonpeptidyl compounds, L-dopa and alpha-methyldopa, did not compete with Gly-Sar for cellular uptake within the therapeutic concentrations. In conclusion, the present study demonstrates the further characterization of CHO/hPEPT1 cells as an uptake model as well as inhibition study and suggests their utility as an alternative in vitro model for drug candidates targeting the hPEPT1 transporter.

In vitro stability and intestinal absorption characteristics of hexapeptide endothelin receptor antagonists.

Endothelins are potent vasoconstrictor peptides which have a wide range of tissue distribution and three receptor subtypes (ET(A), ET(B) and ET(C)). Among the linear hexapeptide ET(A)/ET(B) receptor antagonists, PD 145065 (Ac-D-Bhg-L-Leu-L-Asp-L-Ile-L-Ile-L-Trp, Bhg = (10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5-yl)-Gly) and PD 156252 (Ac-D-Bhg-L-Leu-L-Asp-L-Ile-(N-methyl)-L-Ile-L-Trp) were selected to evaluate the metabolic stability and intestinal absorption in the absence and/or in the presence of protease inhibitors. In vitro stability of both compounds was investigated in fresh plasma, lumenal perfusate, intestinal and liver homogenates. PD 156252 was more stable than PD 145065 in intestinal tissue homogenate (63.4% vs. 20.5% remaining) and liver homogenate (74.4% vs. 35.5% remaining), while both compounds showed relatively good stability in the fresh plasma (94.5% vs. 86.7% remaining) and lumenal perfusate (85.8% vs. 72.3% remaining). The effect of protease inhibitors on the degradation of PD 145065 and PD 156252 was also investigated. Amastatin, thiorphan, chymostatin and the mixture of these three inhibitors were effective in reducing the degradation of both compounds. The pharmacokinetic parameters of PD 156252, calculated by using a non-compartmental model, were 6.95 min (terminal half-life), 191 mL (Vss), and 25.5 mL/min (Cl(tot)) after intravenous administration in rats. The intestinal absorption of PD 156252 in rats was evaluated in the absence and/or in the presence of protease inhibitors. The results indicate that the major elimination pathway of PD 156252 appears to be the biliary excretion and protease inhibitors increase the intestinal absorption of PD 156252 through increasing metabolic stability.

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

PURPOSE: 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 degrees 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 cell experiments: Cells were grown on plastic dishes for 7-10 d, then rinsed and incubated with drug solution at 37 degrees C. Uptake was quenched, cells were lysed, protein precipitated and drug was analyzed by HPLC. IAM chromatography: 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'C-18 and log D) were superior to ClogP in the correlation analysis.

Evaluation of a targeted prodrug strategy of enhance oral absorption of poorly water-soluble compounds.

PURPOSE: The purpose of this research was to examine a targeted prodrug strategy to increase the absorption of a poorly water-soluble lipophilic compound. METHODS: Three water-soluble prodrugs of Cam-4451 were synthesized. The amino acid (Cam-4562, Cam-4580) or phosphate (Cam-5223) ester prodrugs introduced moieties ionized at physiological pH and targeted intestinal brush-border membrane enzymes for reconversion to the parent. Selectivity for reconversion of the three prodrugs was examined in rat intestinal perfusate and brush-border membrane suspensions. Bioavailability of Cam-4451 in rats was evaluated after administering orally as the parent or as prodrugs in a cosolvent vehicle or in methylcellulose. RESULTS: Cam-5223 was highly selective for reconversion at the brush-border, but was rapidly reconverted in intestinal perfusate. Cam-4562 was not as selective but was more stable in the perfusate, whereas Cam-4580 was neither selective nor stable. Oral bioavailability of Cam-4451 was 14% after dosing as the parent in the cosolvent vehicle, 39% and 46%, respectively, as Cam-4562 and Cam-5223. Oral bioavailability was only 3.6% when the parent was dosed in methylcellulose, whereas the bioavailability was 7-fold higher when dosed as the phosphate prodrug. CONCLUSIONS: Water-soluble prodrugs that target brush-border membrane enzymes for reconversion can be useful in improving drug oral bioavailability.

Automated sample preparation for drugs in plasma using a solid-phase extraction workstation.

An automated solid-phase extraction workstation was used to develop, characterize and validate two separate HPLC methods for quantifying drugs in plasma. Method development was facilitated by workstation functions which allowed wash solvents of varying organic composition to be mixed and tested automatically. The precision estimates for the two methods were within 6.0 and 2.0% RSD across their respective calibration ranges. Accuracies for replicate determinations of quality controls were between -1.2 and +4.8 RE over ng ml-1 calibration ranges, respectively. Optimized recoveries were quantitative and were generally greater than 90% for the four analytes tested, and depended to a great extent, as expected, on the composition of the wash solvent. Sample throughput benchmarks for the two methods ranged from 3 to 10 min per sample, depending on the extent of air drying used. Because of parallel sample processing, 60 samples could be extracted in as little as 17 min.

Regulatory role of CD8 in major histocompatibility complex-unrestricted tumoricidal activity of mouse T cells activated with anti-CD3 monoclonal antibody.

Antigen-nonspecific CD8+ cytotoxic T cells induced with anti-CD3 monoclonal antibody (mAb) are able to kill tumor cells in a major histocompatibility complex (MHC)-unrestricted fashion. However, the role of CD8 in the MHC-independent tumoricidal activity of anti-CD3-activated killer T (AK-T) cells has not been investigated. Here we show that anti-CD8 alpha mAb inhibits, in a dose-dependent fashion, lysis of P815 and YAC-1 tumor cells by mouse AK-T cells. The inhibition of MHC-unrestricted cytotoxicity by anti-CD8 alpha mAb cannot be attributed to interference with an adhesion-like function of CD8 towards class I MHC molecules on the target cells because anti-CD8 alpha mAb (i) had equal inhibitory effects on the cytolysis of tumor target cells regardless of their relative level of class I MHC molecule expression and (ii) did not interfere with the formation of conjugates between AK-T cells and class I MHC-bearing P815 tumor cells. However, anti-CD8 alpha mAb abrogated AK-T cell granule exocytosis in the presence of P815 tumor cells, indicating a regulatory role for CD8 in the signal transduction events which result in lysis of the tumor target cells. Immunoblot analysis of the post-nuclear fraction of lysates from AK-T cells exposed to P815 tumor cells in the presence of anti-CD8 alpha mAb revealed reduced phosphorylation of tyrosine residues on a protein with an Mr of approximately 62 kDa. Taken together, these data suggest that CD8 is able to affect the tumoricidal activity of MHC-unrestricted AK-T cells independent of class I MHC molecules on the target cell.

Design of a potent combined pseudopeptide endothelin-A/endothelin-B receptor antagonist, Ac-DBhg16-Leu-Asp-Ile-[NMe]Ile-Trp21 (PD 156252): examination of its pharmacokinetic and spectral properties.

The endothelins (ETs) are a family of bicyclic 21-amino acid peptides that are potent and prolonged vasoconstrictors. It has been shown that highly potent combined ETA/ETB receptor antagonists can be developed from the C-terminal hexapeptide of ET (His16-Leu17-Asp18-Ile19-Ile20-Trp21), such as Ac-(D)Dip16-Leu-Asp-Ile-Ile-Trp21 (PD 142893) and Ac-DBhg16-Leu-Asp-Ile-Ile-Trp21 (PD 145065). However, these compounds are relatively unstable to enzymatic proteolysis as determined in an in vitro rat intestinal perfusate assay. This instability is thought to be due to carboxypeptidase activity. In fact, incubation of PD 145065 with carboxypeptidase inhibitors greatly increased its half-life in rat intestinal perfusate. By performing a reduced amide bond and N-methyl amino acid scan, it was discovered that N-methylation of Ile-20 resulted in a compound (Ac-DBhg16-Leu-Asp-Ile-[NMe]Ile-Trp21, PD 156252) that retained full receptor affinity at both endothelin receptor subtypes along with enhanced proteolytic stability and cellular permeability. Interestingly, N-methylation of this bond allows the cis configuration to be readily accessible which greatly alters the preferred structure of the entire molecule and may be responsible for the observed enhanced metabolic stability.

Inhibition of protein tyrosine kinases or protein kinase C prevents nonspecific killer T lymphocyte-mediated tumoricidal activity.

The signal transduction events which govern major histocompatibility complex-unrestricted tumour cell destruction by nonspecific killer T lymphocytes induced with anti-CD3 antibody have not yet been determined. In this study we used pharmacologic inhibitors to investigate the role of protein tyrosine kinases (PTK) and protein kinase C (PKC) in this process. The PTK-inhibitors herbimycin A, genistein, and methyl 2,5-dihydroxycinnamate blocked anti-CD3-activated killer T (AK-T) lymphocyte-mediated killing of tumour target cells. The PKC-inhibitors staurosporine, calphostin C, and myristoylated PKC pseudosubstrate peptide, as well as PKC desensitization by phorbol 12-myristate 13-acetate pretreatment, also suppressed the cytolytic effector function of AK-T lymphocytes. Lack of tumoricidal activity was not due to reduced AK-T lymphocyte binding to tumour target cells but was associated with the abrogation of granule exocytosis, indicating that PTK and PKC are involved in the postbinding process which results in delivery of the 'lethal hit' by AK-T lymphocytes.

Development and validation of an ion-pair reversed-phase high-performance liquid chromatographic method for the separation of phosphonodipeptides.

The objective of this research was to develop a rapid, sensitive and reliable method for the separation of phosphonodipeptide prodrugs and parent compounds to facilitate the evaluation of cell permeation using in vitro cell culture models. Separation was accomplished isocratically within 10.0 min using a C18 (150x4.6 mm I.D., 3 microm) reversed-phase column. The mobile phase consisted of 5 mM tetrahexyl ammonium (ion-pair reagent) in 0.02 M phosphate buffer pH 6.5-acetonitrile (48.5:51.5, v/v). The flow-rate was 1.1 ml/min with detection at 221 nm. The standard curves were linear (r2>0.999) over the concentration range 1-100 microM. The method was reliable and reproducible, with the limit of quantitation being 1 microM (25 ng on column).

Nonpeptidic HIV protease inhibitors: 4-hydroxy-pyran-2-one inhibitors with functional tethers to P1 phenyl ring to reach S3 pocket of the enzyme.

A systematic study of tethering various groups on 6-phenyl ring of 4-hydroxy-6-phenyl-3-[(2-isopropylphenyl)thio]pyran-2-one was performed to increase the binding affinity with HIV protease. This tethering approach was aimed to fill S3 pocket of the enzyme. Thus, tethering hydrophilic groups resulted in more potent inhibitors. Similarly, various aromatic hydrophobic rings as well as heterocyclic rings were explored as tethering substituents to alter the physical properties as well as to enhance the binding affinity with HIV protease. Inhibitor 24, 4-hydroxy-3-[(2-isopropylphenyl)thio]-6-[4-(3-pyridinylmethoxy+ ++ ) phenyl]-2H-pyran-2-one, was evaluated as a prototypic lead structure to study various physical as well as pharmacological properties of this class of HIV protease inhibitors.

Absorption of Cam-2445, and NK1 neurokinin receptor antagonist: in vivo, in situ, and in vitro evaluations.

Cam-2445 is a selective, high-affinity NK1 receptor antagonist that is a potentially useful treatment for arthritis, asthma, migraine, anxiety, psychosis, and emesis. Cam-2445 exhibits low aqueous solubility and high lipophilicity and has a molecular weight of 470. Cam-2445 has poor oral bioavailability and the purpose of this research was to examine the potential barriers to the oral bioavailability of Cam-2445. Cam-2445 was relatively stable at 37 degrees C in 0.1 N HCl, 5 microM alpha-chymotrypsin, rat intestinal perfusate, and in rat jejunal brush border membrane suspension. High permeability was observed from CACO-2 cells and from rat single-pass intestinal perfusions. Cam-2445 was administered as a solution to rats by intravenous (i.v.), oral (p.o.), intraduodenal (i.d.), and intraportal (i.p.v.) routes. The total oral bioavailability was poor at 1.4%. Absorption appeared to be rapid after i.d. dosing; bioavailability was 26%, and 54% of the dose was absorbed intact into the portal system. After i.p.v. dosing, 48% of the dose was available to the systemic circulation. The elimination t1/2 after i.d. dosing (2.91 h) was comparable to that i.v. dosing (2.93 h), whereas it was significantly longer after p.o. dosing (12.4 h). The p.o. dose apparently precipitated in the gastrointestinal (GI) tract, resulting in low oral bioavailability. These results indicated that neither stability in the GI tract nor membrane transport were major obstacles to the absorption of Cam-2445. While hepatic extraction of 52% was significant, the low aqueous solubility of Cam-2445, as well as the differences noted between p.o. and i.d. studies, strongly support GI dissolution and/or precipitation as the limiting factor for the oral bioavailability of the compound.

Transport properties are not altered across Caco-2 cells with heightened TEER despite underlying physiological and ultrastructural changes.

Selected properties of Caco-2 cells were examined after disparate transepithelial electrical resistance (TEER) measurements were observed in two populations of Caco-2 cells. Comparisons were made between the early passages of Caco-2 cells (Caco-2E, passages 35-47) and the later passages of cells (Caco-2L, passages 87-112). Transmission electron microscopy revealed that regions of Caco-2L cells were composed of multiple cell layers rather than the monolayers observed in Caco-2E cells. Epithelial cell height (or barrier thickness) was not significantly different between the two cell populations. Intercellular and intracellular lumina were observed in the Caco-2L cells, but not in the Caco-2E cells. Results of [3H]thymidine incorporation assays showed significantly higher cell proliferation rates in Caco-2L cells relative to Caco-2E cells. Despite morphological and physiological changes, there were no significant differences in the apparent permeabilities for D-mannitol (paracellular diffusion marker), hydrocortisone (transcellular diffusion marker), or dipeptide, Gly-Sar (carrier-mediated transcellular transport marker) between the two populations of cells. The higher TEER values in Caco-2L cells may be the results of a slight perturbation of tight junctions associated with both the multiple cell layers and the presence of intercellular lumina.

In vitro assessment of oral delivery for hexapeptide endothelin antagonists.

Endothelin (ET-1) is a 21-amino acid, vasoconstrictive peptide originally isolated from endothelial cells. It is one member of a class of potent, purportedly paracrine substances that act at receptors in multiple target organs. Antagonists to the receptor subtypes, ETA and ETB, have been designed around the hydrophobic carboxy-terminus of ET-1. The resulting hexapeptides possess low nanomolar receptor affinity, but face formidable challenges to oral delivery, given their peptidic nature. Hence, it was important to discriminate between analogs, as well as to optimize structural features combining binding potency with stability in intestinal fluids and permeability across biological membranes. PD 142893 (Ac-DDip16-Leu-Asp-Ile-Trp21) and PD 145065 (Ac-DBhg16-Leu-Asp-Ile-Ile-Trp21), as well as the N-methyl-isoleucine20 analogs were studies, where DDip = 3,3diphenylalanine and DBhg = 10,11-dihydro-5H-dibenzo[a,d]cycloheptene glycine. Analyses were conducted with specific HPLC methods. Permeabilities across CACO-2 cell monolayers ranged from 2.0x10(-4) to 6.3x10(-4)cm/min. The results suggested that these compounds can be absorbed in vivo, based on comparison of permeabilities with those obtained with reference compounds. Much greater differences were observed between the analogs when stability half-lives were compared after incubation in rat intestinal perfusate. The parent peptides, PD 142893 and PD 145065, were unstable, with half-lives less than 20 min. N-Methylation of Ile20 resulted in large increases in stability half-lives to greater 500 min. Enzyme inhibition studies demonstrated the involvement of carboxypeptidase A in production of the primary metabolite, the des-Trp derivative. Identification of the primary metabolite of the parent peptide was made by differential UV scanning at 214/280 nm and mass spectral analyses.