Poverty and the extent of child obesity in Canada, Norway and the United States.

The goal of this paper is to compare the extent of child obesity in Canada, Norway and the United States. As child poverty is an important correlate of child obesity, we wish to examine the potential role of international differences in child poverty in explaining international differences in the extent of child obesity. We use three representative microdata surveys containing parental reports of child height and weight collected in the mid-1990s in Canada, Norway and the US. We calculate both the prevalence and proportional severity of child obesity for 6-11-year-old children in each country, and represent the 'extent' of obesity diagrammatically. Differences in patterns of child poverty are similarly depicted. Obesity extent is also compared for poor and non-poor children in Canada and the US. Finally, child obesity in the three countries is compared using only non-poor children where we find that the extent of child obesity is much lower in Norway than in Canada or the US. The pattern apparent for obesity is remarkably similar to that found for child poverty. In Canada and especially in the US, we find a much greater extent of obesity for poor than non-poor children. However, when we compare only non-poor children in the three countries, although the magnitude of difference is smaller, it remains clear that Norwegian children are much less likely to be obese. Policy and research directed towards reducing the extent of child obesity in both Canada and the US should pay particular attention to issues of child poverty.

Physicochemical determinants of passive membrane permeability: role of solute hydrogen-bonding potential and volume.

The relationship of solute structure with cellular permeability was probed. Two series of dipeptide mimetics consisting of glycine, alanine, valine, leucine, phenylalanine, and cyclohexylalanine with amino acids in the D-configuration were prepared. Partition coefficients for the peptidemimetics were obtained in the octanol/water (log P(octanol/water)), hydrocarbon/octanol (Delta log P), and heptane/ethylene glycol (log P(heptane/glycol)) systems in order to explore the contributions of solute volume, or surface area, and hydrogen-bond potential to the permeability of the solutes. Permeability coefficients were obtained in Caco-2 cell monolayers as a model of the human intestinal mucosa. The results were interpreted in terms of a partition/diffusion model for solute transport where membrane partitioning into the permeability-limiting membrane microdomain is estimated from the solvent partition coefficients. Neither log P(octanol/water) nor Delta log P alone correlated with cellular permeability for all the solutes. In contrast, log P(heptane/glycol) gave a qualitatively better correlation. With regard to solute properties, log P(octanol/water) is predominantly a measure of solute volume, or surface area, and hydrogen-bond acceptor potential, while Delta log P is principally a measure of hydrogen-bond donor strength. Log P(heptane/glycol) contains contributions from all these solute properties. The results demonstrate that both hydrogen-bond potential and volume of the solutes contribute to permeability and suggests that the nature of the permeability-limiting microenvironment within the cell depends on the properties of a specific solute. Collectively, these findings support the conclusion that a general model of permeability will require consideration of a number of different solute structural properties.

Activation of apoptotic and inflammatory pathways in dysfunctional donor hearts.

BACKGROUND: Myocardial dysfunction is common after brain death, but the mechanisms remain unclear. Apoptosis is tightly regulated by enzymes termed the caspases. We have investigated the caspases involved in the terminal part of the apoptotic pathway in dysfunctional (nontransplanted) donor hearts and their relation to inflammatory markers and compared them to hearts with good ventricular function (transplanted donors). METHODS: Thirty-one donor hearts assessed for transplantation were examined. Western blotting was used to measure pro-caspase-9, caspase-3, DFF45, the activated nuclease CPAN and poly (ADP-ribose) polymerase, a DNA repair enzyme inactivated by caspase-3. Caspase-3 activity was also measured. Histologic and immunocytochemical analysis for HLA Class II and Real Time polymerase chain reaction for tumor necrosis factor-alpha and interleukin 6 were performed to detect inflammatory activation. RESULTS: Cleaved caspase-9 was higher (5.53+/-0.6 vs. 3.64+/-0.4 O.D. units, P<0.01) in nontransplanted compared with transplanted donors and there was a trend for higher pro-caspase-9 (5.20+/-1.0 vs. 4.22+/-0.4 O.D. units, P=NS). Levels of pro-caspase-3 were higher in nontransplanted (9.66+/-0.5 vs. 5.15+/-0.5 O.D. units, P<0.00001) donors and cleavage products of caspase-3 were elevated in 14 of 14 nontransplanted and 2 of 17 transplanted donors. Intact DFF-45 (8.94+/-0.36 vs. 6.14+/-0.30 O.D. units, P<0.000005), its spliced product (2.38+/-0.35 vs. 0.4+/-0.21 O.D. units, P=0.0001) and the nuclease caspase-activated nuclease (2.01+/-0.3 vs. 0.66+/-0.16 OD units, P=0.001) were higher in nontransplanted donors. The caspase-3 substrate poly (ADP-ribose) polymerase was higher in nontransplanted (1.16+/-0.13 vs. 0.61+/-0.22 O.D. units, P=0.57) donors. CONCLUSIONS: The caspases are elevated in dysfunctional donor hearts compared with hearts with good ventricular function with a possible link to inflammatory activation supporting the concept that brain death causes inflammatory activation which can lead to apoptosis with a possible important effect on function.

An NMR study of conformations of substituted dipeptides in dodecylphosphocholine micelles: implications for drug transport.

Efficient transport of intact drug (solute) across the intestinal epithelium is typically a requirement for good oral activity. In general, the membrane permeability of a solute is a complex function of its size, lipophilicity, hydrogen bond potential, charge, and conformation. In conjunction with theoretical/computational and in vitro drug transport studies, seven dipeptide (R(1)-D-Xaa-D-Phe-NHMe) homologues were each dissolved in a micellar d(38)-dodecylphosphocholine solvent system. In this homologous dipeptide series, factors such as size, lipophilicity, hydrogen-bond potential, and charge were either tightly controlled or well-characterized by other methods in order to investigate by nmr how conformational factors relate to transport. Nuclear Overhauser effect spectroscopy experiments and amide-NH-H(2)O chemical exchange rates showed that the five more lipophilic dipeptides were predominately associated with micelle, whereas the two less lipophilic analogues were not. Rotating frame nuclear Overhauser effect spectroscopy derived interproton distance restraints for each analogue, along with (3)J(HH)-derived dihedral restraints, were used in molecular dynamics/simulated annealing computations. Our results suggest that-other factors being equal-flexible dipeptides having a propensity to fold together nonpolar N- and C-terminal moieties allow greater segregation of polar and nonpolar domains and may possess enhanced transport characteristics. Dipeptides that were less flexible or that retained a less amphiphilic conformation did not have comparably enhanced transport characteristics. We suggest that these conformational/transport correlations may hold true for small, highly functionalized solutes (drugs) in general.

Transport of model peptides across Ascaris suum cuticle.

Several FMRFamide-related peptides (FaRPs) found in nematodes exert potent excitatory or inhibitory effects on the somatic musculature of Ascaris suum and other nematode species when injected into the pseudocoelom or applied directly to isolated neuromuscular preparations. These peptides, however, generally fail to induce detectable effects on the neuromusculature when applied externally to intact nematodes. The apparent lack of activity for these peptides when administered externally in whole-organism assays is likely a function of both absorption and metabolism. To delineate the factors that govern transport of peptides across the cuticle/hypodermis complex of nematodes, we measured the rates of absorption of a series of structurally related model peptides using isolated cuticle/hypodermis segments from A. suum and two-chamber diffusion cells. [14C]-Labeled peptides were prepared from D-phenylalanine, with the amide nitrogens sequentially methylated to give AcfNH2, Acf3NH2, Acf(NMef)2NH2, and Ac(NMef)3NHMe. These model peptides were designed to allow systematic analysis of the influence of peptide size, hydrogen bonding and lipophilicity on transport. Results of these studies show that, within this series, permeability across the cuticle increases with addition of each methyl group. The permeability coefficient of Ac(NMef)3NHMe, with four methyl groups, was 10-fold greater than that of the smaller peptide, AcfNH2, even though both peptides contain five hydrogen bonds. When compared with vertebrate membranes, transport of the model peptides across A. suum cuticle was about 10-fold slower. A biophysical model for transcuticular transport of peptides predicted that nematode FaRPs, which are larger, less methylated and less lipophilic than the model peptides tested, would not be absorbed across the cuticle of nematodes. This prediction was confirmed for the excitatory FaRP, AF2 (KHEYLRFamide), which did not diffuse across the cuticle/hypodermis complex, but diffused rapidly across lipid-extracted cuticle preparations.

Mechanism, structure-activity studies, and potential applications of glutathione S-transferase-catalyzed cleavage of sulfonamides.

The mechanism of sulfonamide cleavage of PNU-109112, a potent HIV-1 protease inhibitor, by glutathione-S-transferase (GST) was investigated in the presence of reduced GSH. GST-catalyzed sulfonamide cleavage takes place via the nucleophilic attack of GSH on the pyridine moiety of the substrate with formation of the GS-para-CN-pyridinyl conjugate, the corresponding amine, and sulfur dioxide. Structure activity studies with a variety of sulfonamides indicate that an electrophilic center alpha to the sulfonyl group is required for cleavage. Substituents that withdraw electron density from the carbon atom alpha- to the sulfonyl group facilitate nucleophilic attack by the GS(-) thiolate bound to GST. The rate of sulfonamide cleavage is markedly affected by the nature of the electrophilic group; replacement of para-CN by para-CF(3) on the pyridine ring of PNU-109112 confers stability against sulfonamide cleavage. On the other hand, stability of sulfonamides is less dependent on the nature of the amine moiety. These principles can be applied to the synthesis of sulfonamides, labile toward cellular GST, that may serve as prodrugs for release of bioactive amines. Tumors are particularly attractive targets for these sulfonamide prodrugs as GST expression is significantly up-regulated in many cancer cells. Another potential application could be in organic synthesis, where protection of amines as the corresponding activated sulfonamides can be reversed by GST/GSH under mild conditions.

Strategies toward predicting peptide cellular permeability from computed molecular descriptors.

The therapeutic efficacy of an orally administered drug is dictated not only by its pharmacological properties such as potency and selectivity, but also its pharmacokinetic properties such as its access to the site of activity. Thorough evaluation of the physicochemical and biological barriers to drug delivery is essential to the selection and successful development of drug candidates. We have demonstrated previously that cellular permeability, as a primary component of drug delivery, is principally dependent upon the desolvation potential of the polar functionalities in the molecule and, secondarily, upon the solute lipophilicity [Conradi, R.A., Hilgers, A.R., Ho, N.F.H., Burton, P.S. (1992). The influence of peptide structure on transport across Caco-2 cells. II. Peptide bond modification which results in improved permeability. Pharm. Res. 9, 473-479]. Increasingly sophisticated computational methods are becoming available for describing molecular structural features proposed to correlate with such molecular physicochemical determinants of permeability. Herein we examine the relationships of various computationally derived molecular geometric descriptors for a set of peptides and peptidomimetics, in the context of experimentally measured hydrogen-bond potentials and lipophilicities, with their cellular permeabilities. These descriptors include molecular volume, polar and non-polar surface areas and projected molecular cross-sectional areas. Particular attention is paid to the roles of solvation treatments and other computational factors in descriptor generation, deconvolution of cellular transport mechanisms and statistical analyses of the resulting data for the development of valid, structure-based and mechanistically meaningful models of cellular permeability. No significant correlation of cellular permeability with computed descriptors was found. This was primarily because of our inability to identify surrogates for hydrogen-bond desolvation potential for the solutes from among these descriptors.

Properties of native and in vitro glycosylated forms of the glucagon-like peptide-1 receptor antagonist exendin(9-39).

The intestinal hormone glucagon-like peptide-1-(7-36)-amide (GLP-1) has recently been implicated as a possible therapeutic agent for the management of type 2 non-insulin-dependent diabetes mellitus (NIDDM). However, a major difficulty with the delivery of peptide-based agents is their short plasma half-life, mainly due to rapid serum clearance and proteolytic degradation. Using a peptide analog of GLP-1, the GLP-1 receptor antagonist exendin(9-39), we investigated whether the conjugation of a carbohydrate structure to exendin(9-39) would generate a peptide with intact biological activity and improved survival in circulation. The C-terminal portion of exendin(9-39) was reengineered to generate an efficient site for enzymatic O-glycosylation. The modified exendin(9-39) peptide (exe-M) was glycosylated by recombinant UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 1 (GalNAc-T1) alone or in conjunction with a recombinant GalNAc alpha2,6-sialyltransferase (Sialyl-T), resulting in exe-M peptides containing either the monosaccharide GalNAc or the disaccharide NeuAc alpha2,6GalNAc. The nonglycosylated and glycosylated forms of exe-M competed with nearly equal potency (> 90% of control) with the binding of [125I]GLP-1 to human GLP-1 receptors expressed on stably transfected COS-7 cells. In addition, each peptide was equally effective for inhibiting GLP-1-induced cyclic adenosine monophosphate (cAMP) production in vitro. Studies with rats demonstrated that the modified and glycosylated forms of exendin(9-39) could antagonize exogenously administered GLP-1 in vivo. Interestingly, glycosylated exendin(9-39) homologs were more than twice as effective as the nonglycosylated peptide for inhibiting GLP-1-stimulated insulin production in vivo, suggesting a longer functional half-life in the circulation for glycosylated peptides. Results from in vivo studies with 3H-labeled peptides suggest that the glycosylated peptides may be less susceptible to modification in the circulation.

Piperazinyl oxazolidinone antibacterial agents containing a pyridine, diazene, or triazene heteroaromatic ring.

Oxazolidinones are a novel class of synthetic antibacterial agents active against gram-positive organisms including methicillin-resistant Staphylococcus aureus as well as selected anaerobic organisms. Important representatives of this class include the morpholine derivative linezolid 2, which is currently in phase III clinical trials, and the piperazine derivative eperezolid 3. As part of an investigation of the structure-activity relationships of structurally related oxazolidinones, we have prepared and evaluated the antibacterial properties of a series of piperazinyl oxazolidinones in which the distal nitrogen of the piperazinyl ring is substituted with a six-membered heteroaromatic ring. Compounds having MIC values

Generalized essential telangiectasia in the presence of gastrointestinal bleeding.

Generalized essential telangiectasia was well defined more than 30 years ago. There have been no reported cases of associated gastrointestinal (GI) bleeding. Recurrent hemorrhage in the setting of telangiectases, including GI bleeding, is more typically associated with hereditary hemorrhagic telangiectasia. We report a unique case of a woman with generalized essential telangiectasia and GI bleeding from a watermelon stomach. We include a brief review of the literature of watermelon stomach, generalized essential telangiectasia, and hereditary hemorrhagic telangiectasia.

Mechanistic roles of neutral surfactants on concurrent polarized and passive membrane transport of a model peptide in Caco-2 cells.

The transport of the model peptide Acf(NMef)2NH2 across Caco-2 cell monolayers was studied in the apical (AP) to basolateral (BL) and the BL to AP direction in the presence of Polysorbate 80 or Cremophore EL in the AP compartment. Increasing surfactant concentrations resulted in increasing AP-->BL peptide permeability and decreasing BL-->AP permeability. In either direction, limiting permeabilities were achieved at concentrations less than the critical micellar concentrations (cmc's) of the surfactants, and remained constant at much higher concentrations. These plateau permeabilities were not equivalent in the two directions. This residual assymetry was abolished by increasing the peptide concentration. Altogether, the observations support the presence of at least two pumps in Caco-2 cells for this peptide, polarized in the BL-->AP direction. These experimental results were analyzed within the context of a quantitative biophysical model incorporating concurrent passive diffusion across the AP and BL membranes accompanied by surfactant-inhibitable active polarized efflux across the AP membrane. The model was also used to locate the additional transport activity at the BL membrane as an uptake pump. Under conditions of complete inhibition, the intrinsic passive diffusional permeability of Acf(NMef)2NH2 was found to be 13 x 10(-6) cm/s, essentially identical with results reported earlier with this peptide utilizing verapamil as an inhibitor. With respect to the mechanism of surfactant inhibition of the apical efflux transport, the monomeric species was found to be responsible with no contribution from micelles. Modeling the mode of inhibition as a noncompetitive Michaelis-Menten process gave identical Kis of 0.5 microM for the two surfactants. Finally, increase of either surfactant beyond 750 microM resulted in a decrease of peptide permeability in the AP-->BL direction. This was attributed to weak association of the peptide with micelles in the AP compartment, which effectively decreased the thermodynamic activity of the peptide at surfactant concentrations greater than 20 times their cmc. Both the experimental approach and accompanying theoretical model demonstrated in this work will allow for further characterization of the inhibitory potencies of surfactants for the nonpassive efflux pathway in vitro and in vivo.

How structural features influence the biomembrane permeability of peptides.

Successful drug development requires not only optimization of specific and potent pharmacological activity at the target site, but also efficient delivery to that site. Many promising new peptides with novel therapeutic potential for the treatment of AIDS, cardiovascular diseases, and CNS disorders have been identified, yet their clinical utility has been limited by delivery problems. Along with metabolism, a major factor contributing to the poor bioavailability of peptides is thought to be inefficient transport across cell membranes. At the present time, the reasons for this poor transport are poorly understood. To explore this problem, we have designed experiments focused on determining the relationship between peptide structure and peptide transport across various biological membranes both in vitro and in vivo. Briefly, peptides that varied systematically in chain length, lipophilicity, and amide bond number were prepared. Permeability results with these solutes support a model in which the principal determinant of peptide transport is the energy required to desolvate the polar amides in the peptide for the peptide to enter and diffuse across the cell membrane. Further impacting on peptide permeability is the presence of active, secretory transport systems present in the apical membrane of intestinal epithelial and brain endothelial cells. In Caco-2 cell monolayers, a model of the human intestinal mucosa, this pathway displayed substrate specificity, saturation, and inhibition. Similar results have been shown in vivo in both rat intestinal and blood-brain barrier absorption models. The presence of such systems serves as an additional transport barrier by returning a fraction of absorbed peptide back to the lumen.

Spontaneous atrophic patches in extremely premature infants. Anetoderma of prematurity.

BACKGROUND: Anetoderma, characterized clinically by macular depressions or outpouchings of skin, is associated with loss of dermal elastic tissue as noted on histopathologic findings. We report on 9 extremely premature infants who developed patches of anetoderma during their course in the neonatal intensive care unit. OBSERVATIONS: All 9 patients were born between the ages of 24 and 29 weeks of gestation and had numerous complications associated with prematurity. Eight of the 9 infants were noted to have developed anetoderma on the trunk and proximal extremities while in the neonatal intensive care unit. The locations of the lesions on the skin were not explained by previous trauma, although many areas corresponded with placement of monitoring leads or with adhesive for a monitoring device. Reduction or absence of elastic tissue supported the diagnosis of anetoderma in 4 of 5 biopsy specimens. CONCLUSION: We report a previously unrecognized type of anetoderma associated with extreme prematurity. The exact cause is uncertain, although reactions to cutaneous monitoring leads or adhesives is suspected.

The use of surfactants to enhance the permeability of peptides through Caco-2 cells by inhibition of an apically polarized efflux system.

PURPOSE: It has recently been reported that the permeability of peptides across Caco-2 cells, an in vitro model of the intestinal mucosa, was limited by an apically polarized efflux mechanism. Since surfactants (e.g. Cremophor EL, Polysorbate 80) have been reported to inhibit similar efflux systems in tumor cells, we determined whether they could enhance the permeability of peptides across monolayers of Caco-2 cells. METHODS: The transport studies of [3H]-mannitol and [14C]-model peptides were carried out across the Caco-2 cell monolayers. TEER values were determined using Voltohmmeter with STX-2 electrode and the equilibrium dialysis studies were conducted using side-by-side dialysis apparatus with cellulose ester membranes. RESULTS: Initially, [3H]-mannitol flux studies were conducted to find concentrations of the surfactants that did not cause damage to the cell monolayer. Based on these studies, Polysorbate 80 and Cremophor EL were selected for further study. The fluxes of [14C]-AcfNH2 (a nonsubstrate for this efflux system) and [14C]-Acf(N-Mef)2NH2 (a substrate for this efflux system) were then measured in the absence and presence of the two surfactants. The permeability of [14C]-AcfNH2 was not affected by the surfactants, while that of [14C]-Acf(N-Mef)2NH2 increased with increasing concentrations of surfactants and then decreased. For example, the Pe values for [14C]-Acf(N-Mef)2NH2 were 3.75 x 10(-6), 8.58 x 10(-6), 10.29 x 10(-6), 7.48 x 10(-6), and 1.46 x 10(-6) cm/sec with Cremophor EL concentrations of 0, 0.01, 0.1, 1 and 10% w/v, respectively. This bimodal effect of surfactants on the Caco-2 cell permeability of this peptide was shown to be due to the interactions between the peptide and micelles at higher concentrations of surfactants, which were demonstrated by the equilibrium dialysis experiments. CONCLUSIONS: These results suggest that surfactants, which are commonly added to pharmaceutical formulations, may enhance the intestinal absorption of some drugs by inhibiting this apically polarized efflux system.

Passive diffusion of weak organic electrolytes across Caco-2 cell monolayers: uncoupling the contributions of hydrodynamic, transcellular, and paracellular barriers.

A systematic approach was used to demonstrate the quantitative interplay of pH, pKa, lipophilicity, charged and uncharged molecular species, molecular size, aqueous diffusivity, and stirring in passive transport across the aqueous boundary layer, microporous filter support, and transcellular and paracellular barriers in Caco-2 cell monolayers. The relationship of permeability of the aqueous boundary layer and hydrodynamic stirring was elucidated from transmonolayer fluxes of testosterone. Adrenergic receptor antagonists including propranolol (PPL), alprenolol (APL), pindolol (PDL), and atenolol (ATL) represented the model series of structurally similar weak bases with pKa values between 8.8 and 9.65. Although intrinsically lipophilic, their apparent log PC (n-octanol/water) at pH 7.4 and 6.5 ranged from -2.6 to 1.3. Effective permeability coefficients (Pe) correlated with log PC at both pH 7.4 and 6.5 showing a single sigmoidal-like curve: PPL > APL > PDL > or = ATL. The Pe approached a minimum plateau value established by the protonated ATL for the paracellular route (pore radius of 12 A) by molecular size-restricted diffusion within a negative electrostatic field of force. The Pe of the weak bases was delineated into component permeability coefficients of the aqueous boundary layer and porous filter support, the intrinsic permeabilities of charged and uncharged species for the transcellular and paracellular routes, and the extent to which the routes were utilized at each pH. This study emphasized a generally applicable approach to quantitatively analyze passive transport data on weak organic electrolytes and neutral molecules generated using cell culture monolayers.

The effect of verapamil on the transport of peptides across the blood-brain barrier in rats: kinetic evidence for an apically polarized efflux mechanism.

When the blood-brain barrier (BBB) transport of a series of model peptides that varied in their physicochemical properties (lipophilicity, size and hydrogen-bonding potential) was determined using an in situ rat brain perfusion technique, an unexpected increase in flux with increasing peptide concentration was observed with one of the peptides. Further, inclusion of verapamil in the perfusion medium also increased permeability of the peptides. These observations were consistent with the presence of a polarized efflux system in the BBB that was saturable, could be competitively inhibited and showed substrate specificity. Such properties are similar to those of P-glycoprotein (P-gp), an apically localized efflux pump that has recently been reported to be present in the endothelial cells that constitute the BBB, and suggest that P-gp may be responsible for this activity. By measuring the BBB transport of the model peptides in the presence of verapamil (a P-gp inhibitor), the intrinsic BBB permeabilities (due to passive diffusion only) were obtained. The presence of verapamil caused a significant increase in the BBB permeabilities of six of the seven model peptides. When the intrinsic permeability coefficients were correlated with several physicochemical parameters, it was shown that hydrogen bonding potential rather than lipophilicity had the greatest influence on the passive diffusion of these model peptides across the BBB. From these studies it can be concluded that inhibition of P-gp, as well as reduction of the hydrogen bonding potential, can be used as strategies to increase peptide transport across the BBB.

A biophysical model of passive and polarized active transport processes in Caco-2 cells: approaches to uncoupling apical and basolateral membrane events in the intact cell.

This report is aimed at the biophysical modeling of transmembrane events involving a passive diffusion and directional pumplike mechanism at the apical (AP) and basolateral (BL) membranes of cultured cell monolayers. The essence of the model is based on experimental evidences for the existence of a saturable, apically polarized transport system in Caco-2 cells for peptides which hindered apical to basolateral flux, enhanced basolateral to apical flux, and showed substrate specificity. This system was further inhibited by verapamil, suggesting some homology with P-glycoprotein, the principal mediator of drug resistance in multidrug resistant cancer cells. Preliminary evidence was also obtained suggesting an additional polarized uptake system for the same peptides in the basolateral membrane. Upon saturation and/or inhibition of the active transport mechanisms with verapamil, the peptide fluxes in apical-to-basolateral direction and the basolateral-to-apical direction converged and became controlled by the passive mechanism. Since the intent of the modeling was to provide useful templates for the design of probing experiments and to delineate and quantify mass transfer mechanisms at the AP and BL membranes and their interrelationships, theoretical equations were developed for a host of kinetic boundary conditions: (a) AP-->BL and BL-->AP transfluxes, (b) bidirectional effluxes from substrate-preloaded cells, (c) undirectional efflux across the AP or BL membrane from preloaded cells, and (d) uptake kinetics via the AP or BL membrane leading to equilibrium. Furthermore, flux expressions were reduced to membrane permeability coefficients to accommodate passive diffusion, saturation, inhibition, and directionality. The diffusional mass transport resistances of the aqueous boundary layers and microporous filter support of the cell monolayer were necessarily included.

Quantitative approaches to delineate paracellular diffusion in cultured epithelial cell monolayers.

When using cultured cell monolayers to determine the mechanism of transcellular diffusion of molecules, it may be important to identify the fraction that moves through the paracellular route or passively diffuses through tight junctions. We characterized the apparent diameter of the junctional pore in a variety of epithelial cell monolayers (Caco-2, MDCK, alveolar). Using hydrophilic extracellular permeants varying in molecular radii and charge (neutral, anionic, cationic, zwitterionic), rate-determining steps and factors of the paracellular route were quantitatively delineated by the model for molecular size-restricted diffusion within a negative electrostatic field of force. Protonated amines permeated the pores faster than their neutral images while organic anions were slower. With increasing molecular size the influence of charge diminished. This approach was used to quantify the relationship between permeant radius and transepithelial electrical resistance and to analyze changes in junctional pore size as a function of pharmacological perturbation, such as in the use of absorption promoters or adjuvants.

Hydrogen bonding potential as a determinant of the in vitro and in situ blood-brain barrier permeability of peptides.

With the exception of various central nervous system (CNS)-required nutrients for which specific, saturable transport systems exist, the passage of most water-soluble solutes through the blood-brain barrier (BBB) is believed to depend largely on the lipid solubility of the solutes. Most peptides, therefore, do not enter the CNS because of their hydrophilic character. Recently, utilizing homologous series of model peptides and Caco-2 cell monolayers as a model of the intestinal mucosa, it was concluded that the principal determinant of peptide transport across the intestinal cellular membrane is the energy required to desolvate the polar amide bonds in the peptide (P. S. Burton et al., adv. Drug Deliv. Rev. 7:365, 1991). To determine whether this correlation can be extended to the BBB, the permeabilities of the same peptides were determined using an in vitro as well as an in situ BBB model. The peptides, blocked on the N- and C-terminal ends, consisted of D-phenylalanine (F) residues: AcFNH2, AcF2NH2, AcF3NH2, AcF2(NMeF)NH2, AcF(NMeF)2NH2, Ac(NMeF)3NH2, and Ac(NMeF)3NHMe. A good correlation among the permeabilities of these model peptides across the bovine brain microvessel endothelial cell (BBMEC) monolayers, an in vitro model of the BBB, and their permeabilities across the BBB in situ was observed (r = 0.928, P < 0.05). The permeabilities of these peptides did not correlate with the octanol-buffer partition coefficients of the peptides (r = 0.389 in vitro and r = 0.155 in situ; P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Epithelial cell permeability of a series of peptidic HIV protease inhibitors: aminoterminal substituent effects.

The influence of the aminoterminal substituent in a homologous series of tetrapeptide analogs on transport across Caco-2 cell monolayers was studied. In a series of pyridylcarboxamide regioisomers, the 2-pyridyl isomer was significantly more permeable than either the 3- or 4-congeners. The uniqueness of this peptide was further suggested by examining the partitioning behavior between heptane and ethylene glycol, a system which has been developed as a simple estimate of the desolvation energy or hydrogen bonding potential of a peptide. In this model, the 2-isomer has a much larger partition coefficient than either the 3- or 4-analogs, consistent with its being less solvated than expected based on simple structural considerations. Factors possibly contributing to this decreased effective polarity could be steric interactions or intramolecular hydrogen bonding.