Biochemical, physiological and clinical effects of l-methylfolate in schizophrenia: a randomized controlled trial.

Folic acid supplementation confers modest benefit in schizophrenia, but its effectiveness is influenced by common genetic variants in the folate pathway that hinder conversion to its active form. We examined physiological and clinical effects of l-methylfolate, the fully reduced and bioactive form of folate, in schizophrenia. In this randomized, double-blind trial, outpatients with schizophrenia (n=55) received l-methylfolate 15 mg or placebo for 12 weeks. Patients were maintained on stable doses of antipsychotic medications. The pre-defined primary outcome was change in plasma methylfolate at 12 weeks. Secondary outcomes included change in symptoms (Positive and Negative Syndrome Scale (PANSS), Scale for Assessment of Negative Symptoms, Calgary Depression Scale for Schizophrenia), cognition (Measurement and Treatment Research to Improve Cognition in Schizophrenia composite) and three complementary magnetic resonance imaging measures (working memory-related activation, resting connectivity, cortical thickness). Primary, mixed model, intent-to-treat analyses covaried for six genetic variants in the folate pathway previously associated with symptom severity and/or response to folate supplementation. Analyses were repeated without covariates to evaluate dependence on genotype. Compared with placebo, l-methylfolate increased plasma methylfolate levels (d=1.00, P=0.0009) and improved PANSS Total (d=0.61, P=0.03) as well as PANSS Negative and General Psychopathology subscales. Although PANSS Total and General Psychopathology changes were influenced by genotype, significant PANSS Negative changes occurred regardless of genotype. No treatment differences were seen in other symptom rating scales or cognitive composite scores. Patients receiving l-methylfolate exhibited convergent changes in ventromedial prefrontal physiology, including increased task-induced deactivation, altered limbic connectivity and increased cortical thickness. In conclusion, l-methylfolate supplementation was associated with salutary physiological changes and selective symptomatic improvement in this study of schizophrenia patients, warranting larger clinical trials. ClinicalTrials.gov, NCT01091506.

Progression from selective to general involvement of hippocampal subfields in schizophrenia.

Volume deficits of the hippocampus in schizophrenia have been consistently reported. However, the hippocampus is anatomically heterogeneous; it remains unclear whether certain portions of the hippocampus are affected more than others in schizophrenia. In this study, we aimed to determine whether volume deficits in schizophrenia are confined to specific subfields of the hippocampus and to measure the subfield volume trajectories over the course of the illness. Magnetic resonance imaging scans were obtained from Data set 1: 155 patients with schizophrenia (mean duration of illness of 7 years) and 79 healthy controls, and Data set 2: an independent cohort of 46 schizophrenia patients (mean duration of illness of 18 years) and 46 healthy controls. In addition, follow-up scans were collected for a subset of Data set 1. A novel, automated method based on an atlas constructed from ultra-high resolution, post-mortem hippocampal tissue was used to label seven hippocampal subfields. Significant cross-sectional volume deficits in the CA1, but not of the other subfields, were found in the schizophrenia patients of Data set 1. However, diffuse cross-sectional volume deficits across all subfields were found in the more chronic and ill schizophrenia patients of Data set 2. Consistent with this pattern, the longitudinal analysis of Data set 1 revealed progressive illness-related volume loss (~2-6% per year) that extended beyond CA1 to all of the other subfields. This decline in volume correlated with symptomatic worsening. Overall, these findings provide converging evidence for early atrophy of CA1 in schizophrenia, with extension to other hippocampal subfields and accompanying clinical sequelae over time.

Impact of duration of untreated psychosis and premorbid intelligence on cognitive functioning in patients with first-episode schizophrenia.

BACKGROUND: The neurotoxic hypothesis suggests that psychosis is toxic to the brain leading to clinical consequences. In this study, we hypothesized that a longer duration of untreated psychosis (DUP) in first episode schizophrenia (FES) patients is associated with poorer cognitive functioning, and that higher premorbid intelligence buffers against DUP-related cognitive impairment. METHOD: Eighty-one FES patients completed a neuropsychological battery, the Brief Assessment of Cognition in Schizophrenia (BACS). Composite scores of the BACS, which were normalized to a matched healthy control of seventy-three subjects, were used as an index of general cognition. A median split using the Wide Range Achievement Test-Reading Test scores was used to divide the patients into low versus high premorbid IQ groups. Hierarchical linear regression was performed to examine predictors of general cognition, including DUP. RESULTS: Longer DUP was found to be a significant predictor of poorer general cognition. In addition, DUP predicted general cognition in the low premorbid IQ group but not in the high premorbid IQ group. CONCLUSIONS: Our findings demonstrate that longer DUP in FES patients is associated with worse cognitive scores, and that this association is more pronounced in a subgroup of patients who have lower premorbid intelligence. Our results suggest the importance of earlier identification and management of patients with low premorbid IQ, given that their cognition may be more vulnerable to the toxicity of psychosis.

In vivo imaging of adult human hippocampal neurogenesis: progress, pitfalls and promise.

New neurons are produced within the hippocampus of the mammalian brain throughout life. Evidence from animal studies has suggested that the function of these adult-born neurons is linked to cognition and emotion. Until we are able to detect and measure levels of adult neurogenesis in living human brains-a formidable challenge for now-we cannot establish its functional importance in human health, disease and new treatment development. Current non-invasive neuroimaging modalities can provide live snapshots of the brain's structure, chemistry, activity and connectivity. This review explores whether existing macroscopic imaging methods can be used to understand the microscopic dynamics of adult hippocampal neurogenesis in living individuals. We discuss recent studies that have found correlations between neuroimaging measures of human hippocampal biology and levels of pro- or anti-neurogenic stimuli, weigh whether these correlations reflect changes in adult neurogenesis, detail the conceptual and technical limitations of these studies and elaborate on what will be needed to validate in vivo neuroimaging measures of adult neurogenesis for future investigations.

Interrelationships among physicochemical properties, absorption and anthelmintic activities of 2-desoxoparaherquamide and selected analogs.

The interrelationships between physicochemical properties, absorption and potency of 2-desoxoparaherquamide and five analogs, representing a new anthelmintic class, were evaluated in in vitro and in vivo assays. At pH 7.5, rates of drug absorption by the gastrointestinal nematode Haemonchus contortus and jird small intestine, parameterized by the permeability coefficient, P(e), ranged from 1.2-2.4 x 10(-4) cm/min (nematode) to 2.5-5.5 x 10(-3) cm/min (jird). In the jird intestine, absorption was pH-dependent, with P(e) at pH 7.5 being twice that at pH 4.5, reflecting the negative influence of protonation on transport of these weakly basic molecules. Each compound rapidly paralyzed H. contortus during in vitro exposure to therapeutically relevant concentrations (1-10 microm). The kinetics of drug action on motility in vivo mirrored their in vitro effects; motility concentrations were reduced in nematodes collected from jird stomach 3 h following oral drug dosing, by which time > or =50% clearance of the parasites had occurred. The nematode/medium partition coefficient K ranged from 10.1 to 16.1, consistent with the lipophilic nature of the compounds. The time required to reduce motility in vitro by 50% (t50*) and P(e) were used to determine C(n)*, the concentration of drug in the nematode at t50*, as an indicator of intrinsic potency. In the jird, the apparent potencies of the compounds were insensitive to route of administration (i.e. oral = i.v. = i.p. = i.m.) for H. contortus and two other gastrointestinal nematodes, Ostertagia ostertagi and Trichostrongylus colubriformis; topical administration, however, required three to 10-fold higher doses for equivalent efficacy.

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.

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.

Increased lipophilicity and subsequent cell partitioning decrease passive transcellular diffusion of novel, highly lipophilic antioxidants.

Oxidative stress is considered a cause or propagator of acute and chronic disorders of the central nervous system. Novel 2, 4-diamino-pyrrolo[2,3-d]pyrimidines are potent inhibitors of iron-dependent lipid peroxidation, are cytoprotective in cell culture models of oxidative injury, and are neuroprotective in brain injury and ischemia models. The selection of lead candidates from this series required that they reach target cells deep within brain tissue in efficacious amounts after oral dosing. A homologous series of 26 highly lipophilic pyrrolopyrimidines was examined using cultured cell monolayers to understand the structure-permeability relationship and to use this information to predict brain penetration and residence time. Pyrrolopyrimidines were shown to be a more permeable structural class of membrane-interactive antioxidants where transepithelial permeability was inversely related to lipophilicity or to cell partitioning. Pyrrole substitutions influence cell partitioning where bulky hydrophobic groups increased partitioning and decreased permeability and smaller hydrophobic groups and more hydrophilic groups, especially those capable of weak hydrogen bonding, decreased partitioning, and increased permeability. Transmonolayer diffusion for these membrane-interactive antioxidants was limited mostly by desorption from the receiver-side membrane into the buffer. Thus, in this case, these in vitro cell monolayer models do not adequately mimic the in vivo situation by underestimating in vivo bioavailability of highly lipophilic compounds unless acceptors, such as serum proteins, are added to the receiving buffer.

Biophysical model for organic acid excretion in Ascaris suum.

To develop a model for the mechanisms of organic acid excretion in nematodes, we measured the concentrations of volatile fatty acids (VFAs), pH and electrical potentials across hypodermal and muscle membranes and across the composite body wall (consisting of hypodermis, muscle and cuticle) of Ascaris suum using standard chromatographic and microelectrode recording techniques. In incubates containing one parasite in 20 ml modified Ascaris Ringer's solution, the level of combined VFAs excreted into the medium increased linearly for about 18 h, then plateaued at a concentration of 4.2 mM; the medium acidified rapidly to a plateau at about pH 5.0 within 4-6 h. Following 24 h incubations, the concentrations of VFAs in the hypodermis, muscle, and pseudocoelomic compartments were 62.4 +/- 8.1, 62.3 +/- 7.8 and 74.4 +/- 3.2 mM, respectively. The pseudocoelomic fluid was more acidic (pH 6.52 +/- 0.06) than the hypodermis (pH 6.78 +/- 0.03) or muscle (pH 6.77 +/- 0.03). These data and the electrical potentials across hypodermal (-57.9 +/- 6.3 mV) and muscle (-30.3 +/- 0.8 mV) membranes were used to determine the equilibrium concentrations for protonated (HVFA) and anionic (VFA-) forms of the acids across these membranes and across the cuticle. Under these conditions, little transmembrane or transmural excretion of HVFAs is expected to occur in A. suum. However, a 16-27 mV driving force for VFA- excretion exists across hypodermal and muscle membranes, and a larger driving force is predicted to exist for these anions across the cuticle. This driving force could provide potential energy for VFA- excretion through anion channels which exist in muscle and hypodermal membranes of this parasite, or for facilitated transport systems.

Absorption and intestinal metabolism of purine dideoxynucleosides and an adenosine deaminase-activated prodrug of 2',3'-dideoxyinosine in the mesenteric vein cannulated rat ileum.

This study investigates the mechanisms of absorption and the role of intestinally localized purine salvage pathway enzymes on the ileal availabilities of 2',3'-dideoxyinosine (ddI), a substrate for purine nucleoside phosphorylase (PNP); 2'-fluoro-2',3'-dideoxyinosine (F-ddI), a non-PNP substrate; and 6-chloro-2',3'-dideoxypurine (6-Cl-ddP), an adenosine deaminase (ADA) activated prodrug of ddI. The potential for increasing the intestinal availability of 6-Cl-ddP through the use of ADA inhibitors, namely, 2'-deoxycoformycin (DCF) and erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), is also explored. Drug permeability coefficients across the intestinal epithelium were determined in in situ perfusions in the mesenteric vein cannulated rat ileum based on both drug appearance in blood (Pblood) and disappearance from the lumen (Plumen) and their paracellular and transcellular components were estimated by comparison to the permeabilities of two paracellular markers, mannitol and urea. Values of Pblood for ddI were determined to be (1.1 +/- 0.3) x 10(-6) cm/s, in close agreement with the value of (1.0 +/- 0.3) x 10(-6) cm/s obtained for F-ddI, a PNP resistant analogue of ddI having virtually the same molecular size and lipophilicity as ddI. This indicates that PNP may not play an important role in the low intestinal absorption of ddI. The Pblood for 6-Cl-ddP, (19 +/- 2) x 10(-6) cm/s, was 4.5-fold lower than Plumen, (84 +/- 12) x 10(-6) cm/s, which means that 77 +/- 6% of 6-Cl-ddP was metabolized during its intestinal transport, thus qualitatively accounting for the low oral bioavailability (7%) of 6-Cl-ddP observed in vivo in rats. Extensive intracellular metabolism of 6-Cl-ddP by ADA was confirmed by the high concentrations of ddI found both in the intestinal lumen and blood during 6-Cl-ddP perfusions and by a rate of ddI appearance in blood which was approximately 10-fold higher than ddI controls. Co-perfusion of the potent, hydrophilic ADA inhibitor DCF (Ki = 0. 001-0.05 nM) with 6-Cl-ddP led to only partial inhibition of intestinal ADA, while complete inhibition was obtained using the less potent but more lipophilic inhibitor EHNA (Ki = 1-20 nM). Hence, EHNA may be used to improve intestinal absorption of 6-Cl-ddP in vivo.

Paracellular diffusion in Caco-2 cell monolayers: effect of perturbation on the transport of hydrophilic compounds that vary in charge and size.

We applied the principles of molecular-size-restricted diffusion within a negative electrostatic field of force to follow the changes in the aqueous pore radius of tight junctions (TJs) induced by perturbants and the accompanying influence on the permeation of neutral (urea and mannitol), cationic (methylamine and atenolol), and anionic (formate and lactate) compounds that vary in size. The perturbants included palmitoyl-DL-carnitine (PC), which opens TJs by an unknown Ca++-independent mechanism, and ethyleneglycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), a Ca++ chelator. Mass transfer resistances of the collagen-coated filter support and the aqueous boundary layers were factored out to yield paracellular permeability coefficients (P[P]). As viewed from the P(P) values of urea and mannitol, EGTA exhibited insignificant effects on pore size at low concentrations compared with control, and then caused a dramatic opening of the TJs over a narrow concentration range (1.35-1.4 mM). The P(P) values for urea and mannitol remained constant at >1.4 mM EGTA. However, PC produced dose-dependent responses from O to 0.15 mM that plateaued at >0.15 mM. In general, cations permeated the cellular TJs faster and anions slower than their neutral images. The effects of changes in pore size (4.6 to 14.6 A in effective radius) on the ability of these solutes to permeate the TJs were analyzed by the Renkin molecular sieving function. These studies established an experimental, theoretical, and quantitative template to assess perturbants of the TJ and define the limits, short of detrimental effects, at which the TJs may be sufficiently perturbed for maximal enhancement of permeation of solutes varying in size and charge.

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.

Intestinal absorption of sodium dodecyl sulfate in the rodent: evidence for paracellular absorption.

Experiments were performed to define the mechanism of intestinal absorption of dodecyl sulfate (DS), an amphipathic organic anion whose chemical structure resembles that of dodecanoate, a C12 fatty acid anion. With jejunal segments perfused in single-pass fashion in the anesthetized rat, steady-state absorption of DS was concentration dependent, with the apparent permeability constant (P(app)) ranging from 4 to 22 x 10(-5) cm/s. When DS concentration was held constant and net water absorption was induced by decreasing perfusate osmolality, DS absorption increased in direct proportion to water absorption, suggesting absorption by solvent drag via the paracellular route. However, DS absorption continued even when water secretion was induced by a hypertonic perfusate. Consequently, for all experiments, DS absorption could be empirically described as the sum of two terms: 1) absorption in the absence of water absorption (P(app) = 5.6 x 10(-5) cm/s) and 2) absorption induced by water movement [(delta P(app)/delta water absorption) = 0.2 x 10(-5) cm x s(-1) x microl segment(-1) x min(-1)]. In a polarized epithelial monolayer of renal epithelial cells (Madin-Darby canine kidney cells), DS was absorbed predominantly by a paracellular pathway, as the absorption rate increased threefold when paracellular junction pore size was increased by the addition of cytochalasin D. The calculated apparent radius was 2.9 A, indicating that the cross section of the molecule, not its length, determined the rate of absorption. It is concluded that absorption of DS in the intact animal occurs slowly and mostly via the paracellular route, because the fixed negative charge on the molecule retards rapid passive entry into the enterocyte, as occurs with protonated fatty acids. That absorption of DS persisted despite net water secretion suggests a low level of transcellular absorption across the jejunal enterocyte also occurs.

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.

Influence of organic acid excretion on cuticle pH and drug absorption by Haemonchus contortus.

To determine if a cuticle microenvironment pH is maintained by adult Haemonchus contortus, organic acid excretion kinetics and absorption kinetics of selected model weak acids and a weak base were measured in incubation media that varied in buffer capacity (0.25-20 mM HEPES or 5 mM glycine) and initial pH (7.5 or 3.5). To evaluate the importance of the cuticle as a pathway for organic acid excretion and drug absorption the pharynx was paralyzed with 1 nM ivermectin. H. contortus changed the media pH from initial values of 7.5 or 3.25 to an asymptotic value of approximately 5.6. The rate of pH change depended on the buffer capacity, but was not affected by chemical ligation with ivermectin. The intrinsic rate of excretion of organic acids (0.045 +/- 0.016 micromol/cm2 x h) was constant during the first 8-12 h of incubation and was independent of initial pH, buffer capacity or ivermectin ligation. The rates of absorption of the model weak acids, benzoic acid and p-nitrophenol, and the model weak base, aniline, were not affected by initial pH, buffer capacity or ivermectin ligation. These results suggest that H. contortus excretes organic acid endproducts of carbohydrate metabolism across its cuticle, and that these acids maintain a microenvironment pH within the water-filled pores of the cuticle that controls the rate of adsorption of weakly acidic or basic drugs.

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.

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.

Theoretical perspectives on anthelmintic drug discovery: interplay of transport kinetics, physicochemical properties, and in vitro activity of anthelmintic drugs.

This multidisciplinary study demonstrates the utility of the biophysical model approach to assess biological activity of anthelmintics in light of drug-delivery principles. The relationships between drug absorption and efficacy for a set of structurally disparate anthelmintics were determined in cultures of Haemonchus contortus, a nematode that parasitizes the ruminant gastrointestinal tract. Uptake, parameterized by the permeability coefficient, Pe, was shown to occur by absorption across the cuticle. Rates of drug appearance in nematode carcasses paralleled rates of drug disappearance from the medium, and absorption reached an apparent equilibrium within a few hours. The parasite/medium partition coefficient, K, was derived from the ratio of drug concentration in the parasite vs the medium at equilibrium. Pe and K values for each anthelmintic were correlated with lipophilicity (as measured by the partition coefficient (PC) in n-octanol/water) and both parameters plateaued at log PC approximately 2.5, with maximum Pe approximately 8 x 10(-4) cm/min and log K < or = 2.0. Absorption kinetics were related to in vitro potency by monitoring motility of H. contortus. The time required to reduce motility by 50% (t* 50) and Pe were used to calculate Cn*, the drug concentration in the parasite at t* 50, as an indicator of intrinsic potency. The quantitative interplay of apparent biological activity expressed as t* 50, dose, and intrinsic potency highlights the important contribution of drug-uptake kinetics.

Transcellular permeability of chlorpromazine demonstrating the roles of protein binding and membrane partitioning.

Transcellular permeability of the neuroleptic-anesthetic chlorpromazine (CPZ) was examined using a cell type (MDCK) that forms a confluent monolayer of polarized cells resulting in distinct apical (AP) and basolateral (BL) membrane domains separated by tight junctions. Because CPZ is membrane interactive, transmonolayer flux was analyzed as two kinetic events: cell uptake from the AP donor solution and efflux into the BL side receiver. Using the rate of cell uptake in the presence of different concentrations of BSA, an intrinsic cell partition coefficient of 3700 +/- 130 and an operational dissociation binding constant of 0.4 +/- 0.05 mM were calculated. In contrast to uptake, efflux of CPZ from either the AP or the BL side of the cell monolayer was approximately 10(4)-fold slower and was dependent upon the avidity of CPZ for the protein acceptor in the receiver solution. These results emphasized the importance of simultaneously measuring disappearance of a lipophilic molecule from the donor solution and its appearance in the receiver and demonstrated how interactions with proteins on either side of the cellular barrier influence permeability. Appearance kinetics showed that the composition of the receiving environment is critical to model a particular in vivo situation and implied that the intrinsic permeability of membrane-interactive molecules in vitro does not necessarily predict penetration beyond the initial cellular barrier in vivo.