Biomedical subjects
Publications and source records attributed to N F Ho.
In this article, David Thompson, Norman Ho, Sandra Sims and Timothy Geary look at the problem o f quantitating drug absorption by gastrointestinal nematodes.
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.
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.
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.
In order to study the influence of hydrogen bonding in the amide backbone of a peptide on permeability across a cell membrane, a series of tetrapeptide analogues was prepared from D-phenylalanine. The amide nitrogens in the parent oligomer were sequentially methylated to give a series containing from one to four methyl groups. The transport of these peptides was examined across confluent monolayers of Caco-2 cells as a model of the intestinal mucosa. The results of these studies showed a substantial increase in transport with each methyl group added. Only slight difference in the octanol-water partition coefficient accompanied this alkylation, suggesting that the increase in permeability is not due to lipophilicity considerations. These observations are, however, consistent with a model in which hydrogen bonding in the backbone is a principal determinant of transport. Methylation is seen to reduce the overall hydrogen bond potential of the peptide and increases flux by this mechanism. These results suggest that alkylation of the amides in the peptide chain is an effective way to improve the passive absorption potential for this class of compounds.
The relationship between structure and permeability of peptides across epithelial cells was studied. Using confluent monolayers of Caco-2 cells as a model of the intestinal epithelium, permeability coefficients were obtained from the steady-state flux of a series of neutral and zwitterionic peptides prepared from D-phenylalanine and glycine. Although these peptides ranged in lipophilicity (log octanol/water partition coefficient) from -2.2 to +2.8, no correlation was found between the observed flux and the apparent lipophilicity. However, a strong correlation was found for the flux of the neutral series and the total number of hydrogen bonds the peptide could potentially make with water. These results suggest that a major impediment to peptide passive absorption is the energy required to break water-peptide hydrogen bonds in order for the solute to enter the cell membrane. This energy appears not to be offset by the favorable introduction of lipophilic side chains in the amino acid residues.
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.
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.
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.
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On the basis of the enterohepatocycling phenomenon of bile acids involving the intestines, liver, and gallbladder, it was conceptualized that bile acids could serve as a molecular carrier of drugs by taking advantage of the bile acid active transport mechanism. It was further proposed that derivatization or analogation of bile acids at the C3-OH position was the desired route because of the reactive hydroxyl group and, moreover, because of the active transport requirement of retaining the C17 side chain with a single terminal acidic function. Using 3-tosylcholic, 3-benzoylcholic, and 3-iodocholic acids, in situ liver absorption, biliary excretion, and intestinal absorption studies in the rat were successful in establishing the concept that C3-derivatives and analogs of bile acids are, potentially, novel molecular delivery systems for intestinal and liver-site directed absorption.
A discrepancy has been noted in the relationship between the relative skin permeabilities of phenols and their lipophilicities as expressed in commonly used octanol:water partition coefficients (PCoctanol:water). The lack of correlation between partitioning and permeability is seen with the nitrophenols, particularly 4-nitrophenol. In the present study, the permeability coefficients-of 4-nitrophenol and several other phenols through skin and a model lipophilic membrane made of silicone rubber were found to be independent of concentration, ruling out concentration-dependent molecular aggregation as the cause of the partitioning-permeability incongruity. An unexpectedly low permeation rate was observed for the diffusion of 4-nitrophenol through the synthetic, silicone rubber membrane, confirming the anomalous position of this phenol in permeability relative to the octanol:water partitioning scale. However, when oil:water (o/w) partition coefficients for the phenolic compounds based on either n-hexane, CH2Cl2, CHCl3, or silicone rubber as the water immiscible phase are used, permeability coefficients for the skin and the synthetic membrane followed expected permeation-partitioning dependencies. On this basis, it appears that PCoctanol:water does not properly reflect the lipophilicity of the phenols with respect to partitioning into skin and silicone rubber during mass transfer.
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The transport of cholesterol solubilized in polyoxyethylene(10)-nonylphenol ether with benzyldimethyltetradecylammonium chloride additive (4:1, w/w) was studied in a diffusion cell separated by a silicone rubber membrane. Overall kinetics revealed the presence of an interfacial barrier. Additions of NaCl or Na2SO4 abolished the barrier to the extent that total cholesterol flux was essentially limited by membrane diffusion considerations. The results are consistent with the concept of micelles diffusing in an electrical field followed by a collision-complex transfer of cholesterol in the aqueous-membrane interfacial region. The electrostatic force of repulsion arises from the overlap of diffuse electrical double layers emanating from the charged mixed micelle and the cationic surfactant adsorbed on the membrane. The influence of surfactant concentration on cholesterol transport kinetics was consistent with electrostatic phenomena. The derived physical model focused on interfacial electrical properties in the donor chamber by maintaining a high concentration (greater than or equal to 0.1 M) of strong electrolyte in the receiver. A linear regression of the logarithm of theoretical transport resistance, total resistance less membrane and receiver boundary layer resistances, versus (ionic strength)1/2 in the donor resulted in a reasonable estimate of the total surface potential of the micelle and membrane surfaces as well as the net dispersion attraction constant.
The predictive value of a recently developed physical model was tested in the topical treatment of cutaneous infections caused by herpes simplex virus type 1 in hairless mice with two ester prodrugs of 9-beta-D-arabinofuranosyladenine (ara-A) (1). The tests were conducted with 2',3'-di-O-acetyl-ara-A (4) and 5'-O-valeryl-ara-A (3) topically applied with and without 15% 1-dodecylazacycloheptan-2-one (2) (Azone), a percutaneous penetration enhancer. In addition to the in vivo studies, in vitro diffusion cell experiments with excised, full-thickness skin from hairless mice were conducted to determine the penetration enhancement effects of 2. As previously observed, 2 was able to induce remarkably large (100- to 1000-fold) flux enhancements in these in vitro experiments. Consistent with predictions based on the physical model studies, formulations of 3 and 4 without 2 had little or no influence on the pathogenesis of the herpes simplex virus type 1 infections; when 2 was present in the formulations, both 3 and 4 had dramatic therapeutic effects consistent with the predictions made with the physical model. Prodrug 4 with 2 was especially efficacious in the prevention of virus-induced lesions and in the survival of all animals. Similar results were obtained with acyclovir plus 2 in this model system.
The influences of electrical and nonelectrical factors in the interfacially controlled dissolution of cholesterol monohydrate by mixed micelles of alpha-(nonylphenyl)-omega-hydroxydeca(oxyethylene) (polyoxyethylene [10]-nonylphenol ether; POEPE; 1) in combination with long-chain n-alkylamines or fatty acids were investigated and quantified under pH conditions where the amines and fatty acids exist in their charged and uncharged forms. The experimental findings were generally consistent with a mechanism involving micelle collision with the cholesterol surface. A significant interfacial barrier was found with neutral micelles; however, the magnitude of the barrier was smaller with uncharged mixed micelles than with the simple micelles of 1. With charged micelles, the interfacial mass transfer resistance decreased with the addition of sodium chloride to the extent that the interfacial barrier was essentially abolished and the dissolution kinetics became convective/diffusion controlled. The results are consistent with the phenomena of diffusion of charged micelles toward a charged surface, following the classical collision kinetic theory of colloids. The ease in the transfer of cholesterol monohydrate molecules from the crystal surface and into the micelles during the collision step was examined by considering factors affecting the crystal surface as well as those associated with the hydrophobic-hydrophilic structure of the micelle of 1 and the distribution of charged and uncharged amphipathic additives within the basic micelle structure of 1.
The dissolution rate of cholesterol monohydrate in various concentrations of sodium chenodeoxycholate (1) was significantly influenced by the addition of strong electrolytes. The mass transfer resistances decreased with increasing electrolyte concentrations and attained an asymptotic minimum value predicted and experimentally established for the convective/diffusion-controlled situation. Reduction of the interfacial barrier to dissolution was many times more sensitive to Mg2+ than to Na+ at equimolar concentrations. Cholesterol monohydrate solubilities increased nonlinearly with increasing 1 in 0.01 M phosphate buffer at pH 8.0 and was not influenced by the presence of strong electrolytes. Measured diffusion coefficients gave supporting evidence that the effective micellar size remained the same within the various experimental systems up to 116 mM chenodeoxycholate. The experimental findings indicated that the interfacial barrier is electrostatic in character. They are consistent with the phenomenon of diffusion of negatively charged micelles toward a negatively charged cholesterol monohydrate surface and the subsequent collision complex transfer of cholesterol molecules at the crystal surface. The results and mechanistic interpretations are also in accord with the previous model studies on cholesterol monohydrate dissolution in the presence of mixed micelles composed of nonionic and ionic surfactants.
This paper explores the physical model approach in studying the taste of drugs and assessing physical formulation factors for improving the undesirable taste of drugs. Various aspects of physiology relevant to the taste phenomenon have been reviewed to provide the biophysical basis for mathematical modeling. The model involves non-steady-state mass transport across the aqueous boundary layer and buildup of solute concentration at the essentially impermeable tongue surface. The theoretical predictions are consistent with experimental studies on the lag time to taste perception by the electrophysiological method and also with "instantaneous" psychophysical taste perception when solute concentrations much greater than the taste threshold are applied on the tongue. Within the framework of the non-steady-state model, novel experimental studies involving the use of a porous half-diffusion cell placed on the surface of an extended human tongue and the recording of the times for psychophysical taste response are proposed to quantify and provide mechanistic understanding of the taste of drugs and also physical formulation factors in overcoming undesirable taste properties.