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I J Hidalgo

Publications and source records attributed to I J Hidalgo.

At least 19 recordsLinked to original sources
PubMed2001

Assessing the absorption of new pharmaceuticals.

The advent of more efficient methods to synthesize and screen new chemical compounds is increasing the number of chemical leads identified in the drug discovery phase. Compounds with good biological activity may fail to become drugs due to insufficient oral absorption. Selection of drug development candidates with adequate absorption characteristics should increase the probability of success in the development phase. To assess the absorption potential of new chemical entities numerous in vitro and in vivo model systems have been used. Many laboratories rely on cell culture models of intestinal permeability such as, Caco-2, HT-29 and MDCK. To attempt to increase the throughput of permeability measurements, several physicochemical methods such as, immobilized artificial membrane (IAM) columns and parallel artificial membrane permeation assay (PAMPA) have been used. More recently, much attention has been given to the development of computational methods to predict drug absorption. However, it is clear that no single method will sufficient for studying drug absorption, but most likely a combination of systems will be needed. Higher throughput, less reliable methods could be used to discover 'loser' compounds, whereas lower throughput, more accurate methods could be used to optimize the absorption properties of lead compounds. Finally, accurate methods are needed to understand absorption mechanisms (efflux-limited absorption, carrier-mediated, intestinal metabolism) that may limit intestinal drug absorption. This information could be extremely valuable to medicinal chemists in the selection of favorable chemo-types. This review describes different techniques used for evaluating drug absorption and indicates their advantages and disadvantages.

Animals↗
PubMed1999

Interplay between CYP3A-mediated metabolism and polarized efflux of terfenadine and its metabolites in intestinal epithelial Caco-2 (TC7) cell monolayers.

PURPOSE: To further characterize cytochrome P450 (CYP) and P-glycoprotein (Pgp) expression in monolayers of the Caco-2 cell clone TC7, a cell culture model of the human intestinal epithelium. To study the interplay between CYP3A and Pgp as barriers to intestinal drug absorption in TC7 cells using terfenadine and its metabolites as substrates. METHODS: mRNA expression of eight CYPs and Pgp was investigated in TC7 and parental Caco-2 (Caco-2p) cell monolayers using RT-PCR. The CYP3A kinetics was determined in microsomes from both cell lines. The transport, metabolism and efflux of terfenadine and its metabolites were investigated in TC7 monolayers. RESULTS: Both TC7 and Caco-2p cells expressed mRNA for Pgp and several important CYPs. However, mRNA for CYP3A4 was detectable anly from TC7 cells. The relative affinity of CYP3A for terfenadine metabolism in the two cell lines was comparable, but the maximum reaction rate in the TC7 cells was 8-fold higher. The rate of transport of terfenadine and its metabolites hydroxy-terfenadine (HO-T) and azacyclonol across TC7 monolayers was 7.1-, 3.5- and 2.1-fold higher, respectively, in the basolateral to apical direction than it was in the apical to basolateral (AP-BL) direction. Inhibition studies indicated that the efflux was mediated by Pgp. Ketoconazole increased the AP-BL transport terfenadine dramatically by inhibiting both terfenadine metabolism and Pgp efflux. CONCLUSIONS: Cell culture models such as TC7 provide qualitative information on drug interactions involving intestinal CYP3A and Pgp.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

The role of an alpha-amino group on H+ -dependent transepithelial transport of cephalosporins in Caco-2 cells.

The role of an alpha-amino group on interaction with the intestinal and renal peptide carriers (PEPT 1 and PEPT 2, respectively) has been the subject of much investigation. Studies have differed in their conclusions about the role of an alpha-amino group on carrier-mediated absorption. Most studies have used brush-border membrane vesicles or perfused intestinal segments. These techniques enable the determination of membrane uptake and luminal disappearance, respectively, but not transepithelial transport. Transepithelial transport should be more predictive of absorption because it includes basolateral efflux, which could be the rate-limiting process in drug absorption. The objective of this study was to evaluate the influence of an alpha-amino group on PEPT 1-mediated transepithelial transport in Caco-2 cells. The apical-to-basolateral permeability coefficients of cephalosporins with or without a free alpha-amino group were determined in the presence and absence of a pH gradient. Permeability coefficients obtained under these conditions were used to calculate a permeability ratio (i.e. P(app) (pH 6.0)/P(app) (pH 7.4)), which should indicate whether PEPT 1 is involved in transport. For cephalosporins with an alpha-amino group (cephalexin, cefaclor, cefadroxil, cephradine, cephaloglycin) the permeability ratios ranged between 1.77 and 2.77. In contrast, the permeability ratios for cephalosporins without an alpha-amino group were 1 (approx.; range = 0.74-1.26). These data suggest that the presence of an alpha-amino group on cephalosporins increases their PEPT 1-mediated transepithelial transport in Caco-2 monolayers.

Amines↗
PubMed1998

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.

ATP-Binding Cassette Transporters↗
PubMed1997

In vivo determination of drug kinetic linearity and individual organ elimination by the accelerated infusion technique.

The objectives of this study were to (1) establish a controlled drug input method, accelerated infusion, for pharmacokinetic analysis in animals and (2) use the accelerated infusion method to determine the biological barriers responsible for the low oral bioavailability of a peptidic compound in rats. The method involves the administration of an infusion at flow rates which increase linearly with time, that is, accelerated infusion (AI). The accuracy of the system in delivering AIs was tested by comparing the observed volumes with the expected volumes. For experiments with the peptidic compound, a solution was administered by AI into the systemic circulation of rats via the carotid artery (IA). The pharmacokinetic linearity of the compound was determined from the linear phase of the concentration-time curve with the systemic clearance (CLs) calculated from the slope of the linear phase. Solutions of the peptidic compound were administered into the portal vein (IPV) or the inferior vena cava (IVC) by AI to assess the hepatic and pulmonary contribution to elimination. AIs into the duodenum (ID) assessed the roles of permeability and metabolism on the absorption of the peptidic compound across the intestinal mucosa. The system was reliable in delivering the desired AI in vitro. The pharmacokinetics of the peptidic compound in rats was linear up to 17 micrograms/ml in blood. Systemic clearance (CLs) of total compound in blood was calculated to be 31 ml/min/kg. Above 17 micrograms/ml, drug concentrations showed an upward deviation from linearity, indicating loss of linearity due to saturation of elimination. Plasma concentration-time profiles for IPV, IVC, and IA infusions were not substantially different. Our conclusions can be summarized as follows: (1) Accelerated infusion is a valuable, reliable approach that can be used to assess pharmacokinetics linearity, first-pass metabolism, and bioavailability. (2) The peptidic compound has a broad, linear kinetic range in rats, and nonlinearity at high concentration is due to saturation of elimination. (3) The compound has low first-pass elimination in the intestinal mucosa, liver, and lung. (4) The factor that determines the oral bioavailability in rats of the peptidic compound used in this study is mucosal permeability.

Animals↗
PubMed1997

Comparison of CYP3A activities in a subclone of Caco-2 cells (TC7) and human intestine.

PURPOSE: To compare the activity of the CYP3A enzyme expressed by TC7, a cell culture model of the intestinal epithelial cell, to the activity of human intestinal CYP3A4, using terfenadine as a substrate. METHODS: The metabolism of terfenadine was investigated in intact cells and microsomal preparations from TC7, human intestine, and liver. The effect of two CYP3A inhibitors, ketoconazole and troleandomycin (TAO), on the metabolism of terfenadine was also examined. RESULTS: Only hydroxy-terfenadine was detected in TC7 microsomal incubations. In contrast, azacyclonol and hydroxy-terfenadine were detected in human intestinal and hepatic microsomal incubations. The Km values for hydroxy-terfenadine formation in TC7 cells, intestine and liver microsomes were 1.91, 2.5, and 1.8, microM respectively. The corresponding Vmax values were 2.11, 61.0, and 370 pmol/min/mg protein. Km values for azacyclonol in intestinal and hepatic samples were 1.44 and 0.82 microM and the corresponding Vmax values were 14 and 60 pmol/min/mg protein. The formation of hydroxy-terfenadine was inhibited by ketoconazole and TAO in human intestine and TC7 cell microsomes. The Km and Vmax values for terfenadine metabolism in intact TC7 cells were similar to those from TC7 cell microsomes. CONCLUSIONS: Our results indicate that TC7 cells are a potentially useful alternative model for studies of CYP3A mediated drug metabolism. The CYP3A expressed by TC7 cells is not CYP3A4, but probably CYP3A5, making this cell line suitable for studies of colonic drug transport and metabolism.

Antibodies↗
PubMed1996

Molecular modeling study of structural requirements for the oligopeptide transporter.

The intestinal oligopeptide transporter (OPT) mediates the absorption of di-/tripeptides, beta-lactam antibiotics, angiotensin converting enzyme (ACE) inhibitors and renin inhibitors. This suggests that the targeting of molecules to this transporter could result in orally-absorbed drugs. Results from a recent study with renal brush border membrane vesicles (BBMV) suggested that an alpha-NH2 group is required for interaction with the renal OPT. In general, structural requirements for interaction with the renal and intestinal OPT are similar. However, these recent findings do not agree with earlier studies, which showed that an alpha-NH2 group is not essential for interaction with the intestinal OPT. Thus, it appears that the renal and intestinal OPT may differ in their recognition of compounds containing an alpha-NH2 group. In this study, molecular modeling was used to determine the tridimensional structures of various cephalosporins for which Ki values had been determined using renal BBMV. All cephalosporins which interact with the OPT have two, energetically equivalent, conformations. Most compounds which do not interact with the OPT cannot adopt the two conformations. A key factor which influences the conformation seems to be the substituent group at the alpha position; an electron drawing group at that position alters the common conformations. For the OPT substrates, the distances between the -NH2 and -COOH groups are comparable to those of the tripeptide, GlyGlyGly; and the distances between -NH2 and carbonyl group in the beta-lactam ring are close to the distance between N-terminal and C-terminal in the dipeptide, GlyGly. The corresponding distances in cephamycin C (in which a -NH2 group is located in a different position) and the tetrapeptide, GlyGlyGlyGly, are longer than those in alpha-NH2 cephalosporins and GlyGlyGly. Cephamycin C and the tetrapeptide have low affinity for the renal OPT, suggesting that the distances between functional groups are critical for affinity. The alpha-NH2 group had no effect on the conformations of the molecules. We concluded that the alpha-NH2 group may interact directly with the renal oligopeptide transporter. Whether this is unique to the renal transporter or could be applied to the intestinal transporter will require further investigation.

Biological Transport, Active↗
PubMed1994

Polarized transport of docetaxel and vinblastine mediated by P-glycoprotein in human intestinal epithelial cell monolayers.

The expression of the multidrug transporter P-glycoprotein has been studied in two human intestinal epithelial cell lines. No functional expression of P-glycoprotein was found in the differentiated HT29-18-C1 cell line. The expression of P-glycoprotein in the Caco-2 cell line was very high, as judged by immunoblotting and by active efflux of vinblastine. The polarized transport of vinblastine in the basolateral to apical direction was temperature and energy dependent, and was reduced by P-glycoprotein inhibitors such as verapamil, chlorpromazine and reserpine. This adds further evidence that the polarized transport of vinblastine across Caco-2 monolayers is mediated by P-glycoprotein. The anticancer drug docetaxel (Taxotere) was transported in a polarized manner: basolateral to apical permeability was 20-fold higher than in the reverse direction. This polarized transport was inhibited by verapamil, chlorpromazine and reserpine, thus demonstrating that docetaxel is a substrate of P-glycoprotein. The implications of these results for the pharmacokinetics and toxicity of taxoids are discussed.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Evaluation of the bile acid transporter in enhancing intestinal permeability to renin-inhibitory peptides.

To evaluate the bile acid transporter as a means of enhancing the ability of renin-inhibitory peptides (RIPs) to penetrate the intestinal mucosa, two RIP-cholic acid conjugates and an RIP-taurocholic acid conjugate were synthesized. Conjugation was through the N-terminus of an RIP and the 3-position of the bile acid, via a six-carbon spacer. An RIP derivative containing the spacer without the bile acid moiety was also synthesized. The bile acid-RIP conjugates and the RIP derivative were shown to be potent inhibitors of human renin in vivo and to have in vivo hypotensive activity equivalent to that of the parent RIP (ditekiren) in a human renin-infused rat model. The ability of these RIP derivatives to bind to the bile acid transporter and be transported across an epithelial cell monolayer was evaluated in an in vitro model of the intestinal mucosa consisting of Caco-2 cell monolayers grown on microporous membranes. One of the RIP-cholic acid conjugate (KI = 60 +/- 10 microM) and the RIP-taurocholic acid conjugate (KI = 19 +/- 5 microM), but not the RIP derivative, were shown to be potent inhibitors of the apical (AP) to basolateral (BL) transport of [14C]-taurocholic acid ([14C]-TA). At concentrations up to 250 microM these RIP-bile acid conjugates had no effect on the diffusion of [3H]-PEG (800-1000), which is a marker of the paracellular pathway. The permeability coefficients of the RIP-bile acid conjugates, determined using Caco-2 cell monolayers, were shown to be six times less than that of [3H]-PEG (800-1000). In addition, the transport of one of the RIP-cholic acid conjugates was investigated in perfused rat ileum in which the mesenteric vein was cannulated. The conjugate was not detected in blood samples taken from the mesenteric vein, while its concentration in intestinal perfusate remained almost constant during the perfusion experiment. These results suggest that while the peptide-bile acid conjugates retain binding affinity for the intestinal bile acid transporter, the molecules are not themselves transported.

Animals↗
PubMed1991

Substrate specificity and some properties of phenol sulfotransferase from human intestinal Caco-2 cells.

The phase II metabolic reactions, sulfation and glucuronidation, were studied in a human colon carcinoma cell line (Caco-2), which has been developed as a model of intestinal enterocytes. Phenol sulfotransferase (PST, EC 2.4.2.1) was isolated from Caco-2 cells cultured for 7, 14 and 21 days. The enzyme catalyzed the sulfation of both p-nitrophenol and catecholamines (e.g., dopamine) as well as most catecholamine metabolites. The affinity (Km) of PST for dopamine was much higher than for p-nitrophenol, and the specific activity of PST with both substrates increased with the age of the cells. The thermal stability of Caco-2 PST increased with cell age and was not dependent on the acceptor substrate used. The thermolabile PST from 7-day old cells was more sensitive to NEM than was the thermostable enzyme from 21-day old cells. No UDP-glucuronyltransferase (EC 2.4.1.17) activity was detected in 7-, 14- and 21-day old Caco-2 cells with any of the methods used.

Arylsulfotransferase↗
PubMed1991

Characterization of the unstirred water layer in Caco-2 cell monolayers using a novel diffusion apparatus.

Caco-2 monolayers grown on Transwell polycarbonate membranes have been characterized as a valuable tool in drug transport studies. Despite the clear advantages of this system, the lack of stirring may create an unstirred water layer (UWL) whose resistance may limit the transcellular transport of lipophilic molecules. The objective of this study was to evaluate a novel diffusion cell where the transport buffer is mixed by gas lift and to determine the mixing flow rate needed to reduce the thickness (h) of the UWL adjacent to cell monolayers. The transport of the leakage marker, mannitol, remained at least 15-fold lower than the flux of testosterone, indicating that the stirring flow rates used did not affect the integrity of the monolayers. The permeability (P) of testosterone (log PC 3.13) across monolayers mounted on this diffusion cell was 4.07, 10.90, and 14.18 x 10(-5) cm/sec at flow rates of 0, 15, and 40 ml/min, respectively, and the apparent UWLs were calculated to be 1966, 733, and 564 microns. P and h in the stagnant Transwell were 3.08 x 10(-5) cm/sec and 2597 microns, respectively. On the other hand, h was significantly smaller in the unstirred, cell-free membranes than in their cell-containing counterparts. P was correlated with lipophilicity and, in the case of the more lipophilic compounds, with the mixing flow rate.

Cells, Cultured↗
PubMed1990

Transport of a large neutral amino acid (phenylalanine) in a human intestinal epithelial cell line: Caco-2.

We have characterized the transcellular transport of a large neutral amino acid (LNAA) in Caco-2 cell monolayers. The apical (AP) to basolateral (BL) transport of phenylalanine (Phe) was approximately 10-times faster than BL-to-AP transport. The evidence for the carrier-mediated AP-to-BL transport of Phe include: (a) temperature dependence and saturability, (b) Phe transport was not affected by a reverse gradient, (c) the activation energy for transport was 12.0 kcal/mol, and (d) an excess amount of unlabeled Phe caused a 75% reduction in transport rate and a delay (lag time) in the appearance of Phe in the BL side. The Vm and Km for Phe transport were 572.4 pmol.mg protein-1.min-1 and 0.56 mM, respectively. Phe transport was decreased in the absence of glucose and in the presence of sodium azide or ouabain. The carrier interacted with LNAAs and with cationic amino acids but not with small neutral or anionic amino acids.

Biological Transport↗
PubMed1990

Transport of bile acids in a human intestinal epithelial cell line, Caco-2.

The transport of taurocholic acid (TA) across Caco-2 cell monolayers was dependent on time in culture and reached a plateau after 28 days, at which time the apical (AP)-to-basolateral (BL) transport was 10-times greater than BL-to-AP transport. The amounts of TA inside the cells following application of 10 nM [14C]TA to the AP or BL side of the monolayers (30 min) were approximately equal (54.4 +/- 2.7 and 64.6 +/- 2.8 fmol/mg protein, respectively). AP-to-BL transport of TA was saturable and temperature-dependent. Vmax and Km for transport were 13.7 pmol/mg protein per min and 49.7 microM, respectively. The transport of TA had an activation energy of 13.2 kcal.mol-1, required Na+ and glucose. AP-to-BL transport of [14C]TA was inhibited by the co-administration (on the AP side) of either unlabeled TA or deoxycholate, but it was not reduced by the presence of unlabeled TA on the BL side.

Bile Acids and Salts↗
PubMed1990

The use of cultured epithelial and endothelial cells for drug transport and metabolism studies.

In an effort to develop novel strategies for delivery of drug candidates arising from rational drug design and recombinant DNA technology, pharmaceutical scientists have begun to employ the techniques of cell culture to study drug transport and metabolism at specific biological barriers. This review describes some of the general factors that should be considered in developing a cell culture model for transport studies and metabolism studies. In addition, we review in detail the recent progress that has been made in establishing, validating, and using cell cultures of epithelial barriers (e.g., cells that constitute the intestinal, rectal, buccal, sublingual, nasal, and ophthalmic mucosa as well as the epidermis of the skin) and the endothelial barriers (e.g., brain microvessel endothelial cells).

Animals↗
PubMed1989

Binding of epidermal growth factor by human colon carcinoma cell (Caco-2) monolayers.

The objective of this study was to investigate whether Caco-2 cells bind and internalize epidermal growth factor (EGF). [125I]EGF was presented to the apical (AP) or basolateral (BL) side of Caco-2 monolayers, grown on microporous membranes, at different times in culture. At day 10, [125I]EGF binding (at 37 degrees C) to the BL membrane was 2-3 times greater than binding to the AP membrane. Of that [125I]EGF bound to the AP membrane 76% was internalized within 3 h while internalization from the BL membrane was 90%. At lower temperatures membrane-bound [125I]EGF increased while internalization decreased. At day 16, AP and BL binding decreased and then remained constant through day 25. [125I]EGF was bound to the BL membrane of 10 days old monolayers with a Kd of 0.67 nM. There was a single binding site whose numbers in the BL membrane was about 5500/cell.

Cell Membrane↗