Craig L. Barsuhn

Properties of a selective kappa agonist, U-50,488H

U-50,488H has been shown to be a naloxone antagonizable analgesic in rodents. However, the dose of naloxone needed for antagonism is higher than it is for morphine. U-50,488H does not produce physical dependence; however it does produce tolerance upon chronic administration. U-50,488H is cross tolerant with bremazocine but not with morphine. Monkeys trained to discriminate ethylketocyclazocine (EKC) from saline show a complete generalization to U-50,488H but not to morphine. The evaluation of U-50,488H in 3H-EKC site-selective binding indicated that U-50,488H has a high affinity for the kappa receptor (Ki = 114 nM) and a low affinity for the mu receptor (Ki = 6100 nM). The ratio of Ku/Kk was 0.08 for morphine, 0.4 for dynorphin, and 53.5 for U-50,488H. The data suggest that U-50,488H is a selective agonist at the opioid kappa receptor.

U-50488H, a pure kappa receptor agonist with spinal analgesic loci in the mouse

U-50,488H is a chemically novel analgesic that is a potent opioid-like agent on the mouse tail flick and electrically stimulated guinea pig ileum tests. U-50,488H is a very weak competitor for naloxone binding sites in brain and ileum. However, the drug has high affinity for kappa receptor binding sites revealed by competition for EKC sites in the presence of dihydromorphine. Morphine has both supraspinal and spinal sites of action since it was a potent analgesic after both intracranial and intraspinal injections. However, U-50,488H works predominantly at the spinal level. Dynorphin may be an endogenous ligand at this site. Studies on cat dorsal horn neurons suggest that U-50,488H analgesia may be due to an increase in threshold for neuron excitation.

The effect of minor tranquilizers on stress-induced increases in rat plasma corticosteroids

In this study a variety of psychoactive drugs were evaluated for their ability to block a stress-induced elevation in rat plasma corticosteroids. Stress was applied by placing the rats in different cages and moving them to a novel environment which resulted in a rapid increase in plasma corticosteroids, near maximal with 30 min, followed by a decrease to normal by 2 h. Meprobamate, phenobarbital, diazepam and several other benzodiazepines, all of which exhibit anxiolytic properties, were able to block the stress-induced increase. U-33,030 and U-31,889, two triazolobenzodiazepine derivatives, were the most potent compounds tested. No other type of psychoactive compound demonstrated this activity.

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.

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.

(D) Routes of delivery: Case studies: (3) Mechanistic insights to buccal delivery of proteinaceous substances

Abstract There are sufficient in vivo demonstrations that the buccal delivery of peptides is a potential alternative route of administration. Passage across the squamous epithelium occurs by simultaneous passive diffusion and metabolism. The inefficient permeability properties of peptides contribute to their low systemic bioavailability, thus requiring that peptides must be highly potent to exhibit biological activity. This review emphasizes: [1] the nature of the physical, anatomical and enzymatic barriers; [2] the interplay of biophysical factors and physicochemical properties of peptides governing transport mechanisms wherein non-peptide compounds are used as standards; [3] the roles of absorption promoters; and [4] gaps in our current approaches and understanding of peptide transport.