Norman F.H. Ho

The Influence of Peptide Structure on Transport Across Caco-2 Cells

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 Influence of Peptide Structure on Transport Across Caco-2 Cells. II. Peptide Bond Modification Which Results in Improved Permeability

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 differences 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 peptide structure and transport across epithelial cell monolayers

Abstract The successful development of orally bioavailable peptides and peptide-like substances as therapeutic agents will require an understanding of how structure influences absorption across the intestinal mucosa. In an attempt to define such relationships, homologous series of peptides were prepared which varied in lipophilicity, chain length and number of polar functionalities, and permeability studies conducted across Caco-2 cell monolayers as a model of the intestinal mucosa. The results suggested that the number of polar groups in the peptide, which presumably require desolvation before transfer of the peptide into the cell membrane, was a principal determinant of transport. Consistent with this hypothesis, two experimental methods of determining desolvation potential were found to correlate well with the observed permeability results for the peptides. The insights gained from these studies were used in an attempt to rationally modify a renin inhibitory peptide, in order to improve its permeability across the intestinal mucosa. Based on the results of this work, it is argued that a peptide must possess a delicate balance of affinity for the aqueous-membrane interface and a reasonably low desolvation energy in order for it to efficiently cross an epithelial cell membrane.

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.

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.

MECHANISTIC STUDIES IN THE SIMULTANEOUS FLOW AND ADSORPTION OF POLYMER-COATED LATEX PARTICLES ON INTESTINAL MUCUS I: METHODS AND PHYSICAL MODEL DEVELOPMENT#

Abstract The adsorption of particles and concurrent steady-state flow of a dilute suspension from an infinite reservoir over the mucous surface of intestinal strips were quantitatively studied. The micronsize particle systems included negatively charged poly (vinyltoluene) and hydroxylated Dynosphere® particles with and without a positively charged polybrene polymer coated layer. The suspensoids were characterized by zeta potential measurements. An in vitro setup and technique likened to a thin falling liquid film system was developed wherein an excised intestinal segment cut lengthwise is spread on a plastic flute and positioned at an incline, and a suspension is allowed to flow down the intestinal strip. The falling liquid film was estimated to be 54 μm in thickness from nonsteady-state flow kinetic experiments. Particle concentrations entering the segment from the dilute suspension reservoir and leaving the intestinal segment were determined with the Coulter counter to quantify the steady-state fraction of particles adsorbed as they relate to intestinal length, flow rate, ionic strength, zeta potential and particle concentration. The flowing liquid film technique was found to be quantitatively sensitive and, consequently, allowed one to focus on the mechanism of the approach of micron-size particles to the mucous surface without the major concern of particle flocculation in the bulk liquid. The logarithm of the fraction of nonadsorbed particles remaining in the liquid film decreased linearly with intestinal length at all flow rates employed. The fraction adsorbed decreased with increasing flow rates on account of the shorter transit times. The steady-state region of the fraction of nonadsorbed particles remaining versus time plots persisted for a relatively long time indicating the existence of a particle concentration gradient along the length of the intestinal strip. The fact that the fraction of particles adsorbed was not affected by particle concentrations ranging from 4.5–14× 10 6 particles per ml suggests that there is sufficiently available unoccupied surface area for incoming particles to be adsorbed onto mucus. It has been estimated that no more than 5% of the area of the intestinal strip is occupied by adsorbed particles. Failure to desorb particles from the mucous surface by perfusing the intestinal segment indicates tight binding between the particles and mucus. A physical model was deduced from the experimental results wherein the steady-state fraction of adsorbed particles is related to length of the intestinal strip, flow rate and mass transfer-adsorption coefficient. The mass transfer resistances of negatively charged particles decreased with the addition of sodium chloride and approached the minimum resistance obtained by the positively charged, polybrenecoated latex particles, whose mass transfer resistance was independent of electrolyte concentration. This supports a mechanism involving the diffusion of the negatively charged particles within an electrostatic field of force as the particles approach the negatively charged mucous surface. Passage of particles over the potential energy barrier is required for the successful collision and tight binding with mucus.

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 ~104-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 approaches to mechanistic transepithelial studies of peptides

Abstract Systematic studies were conducted to understand the physicochemical and biophysical mechanisms governing the membrane transport of peptides. These studies focused primarily on the Peyers patch of the small intestine, particularly the M cells, with respect to the vesicular transport mechanisms, and also the buccal, intestinal and Caco-2 (human-derived colon carcinoma cell) membrane systems with respect to the passive diffusional mechanism of peptides. The development of quantitatively sensitive experimental methodologies was required and, in conjunction with the specific membrane system, was initially assessed with non-peptide compounds of well-known active or passive transport properties. Using a miniature closed perfusion cell system positioned over the large Peyers patch of the rabbit coupled with cannulation of the mesenteric blood and lymph vasculatures, adsorptive endocytosis uptake and concurrent appearance kinetics in the blood and lymph were followed with cationic poly(d-lysine) (PDL, 55 kDa) conjugated with [ 14 C] formaldehyde. The influx of PDL by the apical membrane was 100-fold larger than the efflux into blood; none was detected in lymph. A mixture of metabolic inhibitors, 2-deoxyglucose and Na azide, caused partial inhibition of endocytosis. By using a fluorescein isothiocyanate-PDL conjugate and fluorescence microscopy, PDL was found to be localized predominantly on the apical membrane surface of all intestinal epithelia. It also was found to be accumulated intracellularly by epithelia, most likely M-cells, that occupy the dome region of the Peyers patch and by cells within the lymphoid follicles. The results suggested that PDL was trapped by the cells and lymphoid follicles and that the rate-determining step in the appearance of PDL in the mesenteric lymph is the migration of lymphocytes from the lymph space associated with M-cells. Transport studies were conducted to determine structure-passive absorption relationships of small model peptides using the buccal, intestinal mucosal and/or Caco-2 cell monolayer membranes. In the course of buccal absorption studies, amino acids and their BOC-derivatives, and ancillary non-peptide compounds were included to aid in data interpretation. Based on the amphoteric peptide series, ( d - Phe) n Gly, and the neutral series, Ac( d - Phe ) n NH 2 , where n = 1, 2 or 3, terminal charges on zwitterionic peptides have a negative effect on membrane permeability even though the effective partition coefficient in n-octanol/water is relatively high. Although the partition coefficient is increased by incremental additions of d - Phe , the membrane permeability tends to decrease. This may be related not only to molecular size but also to the number ofsolvated amide bonds. Partition coefficients by the n-octanol/water scale appear to be poor predictors of membrane absorption of peptides.

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.

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 × 10−4 cm/sec at pH 5.5 and 2.1 × 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.