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.

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.

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.

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.

Biophysical model of the transcuticular excretion of organic acids, cuticle pH and buffer capacity in gastrointestinal nematodes.

A biophysical model was developed, using Ascaris suum as a model gastrointestinal nematode, to provide quantitative perspectives into the microenvironmental pH within the water-filled, porous, negatively charged cuticle matrix of gastrointestinal nematodes. The central features of the model include (a) the constant rate of excretion of organic acid metabolites across the cuticle, (b) the relationship between cuticle pH and pKa of the organic acids that determines the fraction of unionized and ionized species, and (c) the concentration gradient, mean concentration and buffer capacity within the cuticle that maintain the cuticle pH. The model may be used to predict the extent to which transcuticular absorption of weakly basic and acidic anthelmintics will be affected by transcuticular excretion of organic acid metabolites. Coupled with established models for drug absorption by nematodes and the host gastrointestinal tract, the cuticle pH model provides new insights to the design of drugs with physicochemical properties that favor absorption by nematodes.

Use of a biophysical-kinetic model to understand the roles of protein binding and membrane partitioning on passive diffusion of highly lipophilic molecules across cellular barriers.

The novel antioxidants U-78517F and U-74006F, or lazaroids, are highly lipophilic organic molecules with poor brain uptake. To understand this paradoxical behavior better, continuous monolayers of Madin-Darby canine kidney (MDCK) epithelial cells with distinct apical (AP) and basolateral (BL) plasma membrane domains grown on polycarbonate membrane filters and plastic were used to examine the mechanism of transcellular diffusion. Independent kinetic experiments were used to quantify AP to BL flux, efflux from the AP and BL membranes and AP membrane partitioning as functions of bovine serum albumin (BSA) concentration. Fluxes were appropriately reduced to permeability coefficients (Pe) for the membrane, aqueous boundary layer (ABL) and filter, BSA-drug binding constants, and effective (Ke) and intrinsic (Kintr) membrane partition coefficients in the absence of metabolism. Both Pe and Ke decreased exponentially with increased BSA concentration and a concomitant decrease in free drug concentration. Uptake was ABL-controlled under the conditions used and its Pe was 1,000-fold faster than that for efflux due to a large Kintr. Therefore, diffusion across the cellular barrier was limited kinetically by the equilibrium between protein-bound drug and free drug partitioned into the cell membrane and the rate-limiting desorption of drug from the cell membrane into the aqueous receiver. This suggests that brain uptake of these lipophilic antioxidants is limited by interactions with plasma proteins and, possibly, by unfavorable partitioning from the endothelium into the underlying tissue. The present biophysical kinetic model is proposed as generally useful in studying the penetrative ability of other membrane interacting molecules.

Mechanisms of microenvironmental pH regulation in the cuticle of Ascaris suum.

The excretion kinetics of various organic acids by Ascaris suum were quantified to determine if the excretion of these metabolic end-products could generate and maintain a microclimate pH within the aqueous compartment of the cuticle. Ligated and nonligated A. suum were incubated in media buffered with 0.25 or 2.5 mM Hepes (initial pH 7.5) or 0.5 or 5 mM glycine (initial pH 3.25). The concentration of organic acids and the pH of the media were followed for 24 h. Several volatile fatty acids, including acetic, 2-methylbutyric, 2-methylvaleric, n-valeric, and n-butyric, were excreted at relatively high rates. Propionic, n-caproic, 2-methylcaproic, tiglic acid, and the non-volatile organic acids, lactic and succinic, were excreted more slowly. The organic acids were excreted at a constant rate and in apparently fixed molar concentration ratios. The accumulation of organic acids was associated with changes in pH of the medium until a limiting constant pH, in the vicinity of the pKa of the volatile fatty acids, was reached. The rate of organic acid excretion was not affected by initial medium pH, buffer capacity, or parasite ligation. The rate of pH change induced by the excretion of organic acids was also insensitive to whether ligated or nonligated A. suum were used, but was dependent on the initial buffer capacity of the medium. These results suggest that A. suum excrete the end-products of carbohydrate metabolism across the cuticle. The presence of organic acids in the aqueous pores of the cuticle creates and maintains a microclimate pH of about 5.0 +/- 0.3. This pH will influence the transport properties of weak acids and bases and should be considered in the design of delivery systems for anthelmintics.

Mechanistic studies in the transcuticular delivery of antiparasitic drugs. II: Ex vivo/in vitro correlation of solute transport by Ascaris suum.

Using live, intact Ascaris suum and a closed perfusion system, the absorption kinetics and tissue distribution of selected radiolabeled permeants were measured to determine the importance of the transcuticular pathway for drug absorption. The data support the conclusions established by previous in vitro transport studies which utilized excised cuticle-hypocuticle tissue preparations. The external surface of A. suum can be breached by drugs and the rate-determining barrier is the lipoidal hypocuticle tissue, provided the permeant is sufficiently small to traverse the aqueous-filled, negatively charged collagen matrix of the cuticle. The ex vivo permeability coefficients of the model permeants for the cuticle-hypocuticle barrier were in good quantitative agreement with the in vitro permeability coefficients. The lipophilic permeants hydrocortisone and p-nitrophenol were preferentially distributed in the gut tissue, whereas the hydrophilic permeant urea was distributed evenly throughout the organism and was extensively metabolized. Ligated and nonligated A. suum showed no significant differences in either uptake kinetics or tissue distribution of the permeants. This indicates that the transcuticular pathway is the major route of drug absorption as compared to oral ingestion.

Biophysical transport properties of the cuticle of Ascaris suum.

The transport properties of isolated cuticle from Ascaris suum were studied using standard two-chamber diffusion cells and a number of radiolabeled permeants which varied in molecular size, lipophilicity and electrical charge. The permeability coefficient of the collagen matrix (lipid-extracted cuticle) vs. molecular radius relationship showed the interdependence of molecular size and electrical charge of the permeants with respect to the aqueous pores of the negatively charged matrix. The permeability of neutral solutes decreased monotonically with size. Protonated amines permeated the aqueous pores faster than neutral solutes of comparable size, while the permeation of anions was slower. The average pore size was estimated to be 1.5 nm in radius. A biophysical model which accounted for diffusion of molecules within a fixed electrostatic field of force and for molecular sieving by the pore channels was used in the mechanistic interpretation of the data. The effective permeability coefficient of the non-lipid-extracted cuticle was delineated into the permeability coefficients of the water-filled collagen matrix and the lipoidal component of the cuticle to determine which layer was the rate-controlling barrier. While each solute was capable of penetrating the water-filled collagen matrix, the rate-determining step for the majority of compounds was passive diffusion across the lipid component, which controlled 75-99% of transport. The exception was water, for which transport kinetics was 75% matrix-controlled. In general, permeation across the lipid-filled tissue was more favorable for small lipophilic compounds because of molecular restriction not only in the aqueous pores, but also in the lipid-filled pores.

Local topical delivery of drugs: a model incorporating simultaneous diffusion and metabolic interconversion between drug and a single metabolite in the skin.

A comprehensive biophysical model for the topical delivery of a drug and its single, locally active metabolite is proposed. This elaboration of the simpler case, in which the drug converts irreversibly to a pharmacologically active metabolite in the tissue, allows for enzymatic interconversion between drug and metabolite. Exact mathematical expressions give concentration-distance relationships of drug and metabolite as well as fluxes of the two molecules in terms of concentration of drug applied to the stratum corneum, permeability coefficient of drug in the stratum corneum, diffusion coefficients of drug and metabolite in the viable tissues (epidermis and dermis), rate constants for the two enzyme systems, and the thickness of the viable tissue. Constants included in the mathematical expressions can be evaluated independently by appropriate in vitro experiments with freshly excised animal skin. The model can then predict what physiochemical drug constants will lead to maximal levels of active metabolite at the site of activity within the skin.

Human buccal absorption of flurbiprofen.

The buccal absorption of flurbiprofen was studied in normal men to quantify the transport from the oral cavity in humans and to evaluate the closed-perfusion cell apparatus as a means to study drug transport across externally accessible biologic membranes. Flurbiprofen was buccally absorbed by a passive diffusional mechanism and the rate of absorption was pH dependent. Membrane permeability coefficients for flurbiprofen were 4.3 x 10(-4) cm/sec at pH 5.5 and 2.1 x 10(-5) cm/sec at pH 7.0. These findings are in agreement with the pH relationship for buccal transport observed in dog experiments. Delineation of the effective permeability coefficients into components for the aqueous boundary layer and the lipoidal buccal membrane allowed for the prediction of the extent of absorption of the drug over a period of time. It was concluded that the buccal membranes of the human and dog were essentially lipoidal membranes with equivalent permeabilities and no evident aqueous pore pathways.