An improved assay procedure and a new chemically stable ligand for cytosolic retinoic acid binding protein.

A high through-put method to assay for binding of retinoids to their cytosolic binding protein is described. The protein-bound retinoid is quantified following its complete separation from the free ligand by a single gel filtration chromatography step on Sephadex G-25 columns. The method allows a single person to process up to 100 samples per day. A new, substituted benzo[b]thiophene carboxylic acid derivative (tritiated) is proposed as an alternative ligand for cytosolic retinoic acid binding protein. This new analog (CD270), which contains no olefinic double bonds, is characterized by its chemical stability to light and atmospheric oxidation. This molecule shows binding characteristics similar to those of retinoic acid, both in terms of binding affinity and binding specificity (Kd of about 2 nM), and offers the additional advantage that the unbound molecule is absorbed on Sephadex G-25, while the protein-bound ligand is unaffected by the carbohydrate gel matrix. When the affinities of retinoic acid and several analogs were determined by competition binding experiments, similar results were obtained when either tritiated retinoic acid or tritiated CD270 were used as the labeled ligand. The tritiated, heterocyclic retinoic acid analog, CD270, is thus proposed as an alternative ligand for cytosolic retinoic acid binding protein.

Receptor-linked adenylate cyclase in the membranes of cultured human epidermal keratinocytes.

The beta-adrenergic receptors, previously shown to be present on the membranes of cultured human epidermal keratinocytes, were found to be functionally coupled to membrane-bound adenylate cyclase. Using membrane preparations, the enzyme could be activated by guanosine triphosphate (GTP), the stable GTP analog GPP(HN)p, and NaF, all of which are known to activate the adenylate cyclase without interacting with membrane receptors. Binding of catecholamine agonists (epinephrine, norepinephrine, and isoproterenol) to the beta-adrenergic receptors is followed by an increase in the activity of adenylate cyclase. This activation could be reversed (or prevented) by beta-adrenergic antagonists, but was unaffect by the presence of alpha-adrenergic ligands (either agonists or antagonists). The activation by catecholamines appears to be directly related to receptor occupancy, since the activation constant (Ka) of adenylate cyclase for the three catecholamines was found to be very similar to the equilibrium dissociation constant (Kd) determined from competition binding experiments. The activation of adenylate cyclase under these conditions appears to be restricted to the catecholamine agonists only. The non-catecholamine beta-adrenergic agonists (salbutamol, terbutaline) did not show any measurable activation of adenylate cyclase, even though these agonists were shown previously to bind to the beta-adrenergic receptors on keratinocyte membranes with the expected affinities.

High-yield purification of plasma membranes from transformed human keratinocytes in culture.

The density pertubation technique with cationic silica microbeads was applied to prepare highly purified plasma membranes from cultured human keratinocytes. Trypsinized cells were coated successively with the beads (diameter approximately 50 nm, gravity greater than 2 g/cm3) and polyacrylic acid before they were lysed by osmotic shock and mechanical shear. The plasma membranes remained in the form of large open sheets which could easily be separated from other cell organelles and the cytosol by low-speed centrifugation. The membrane preparation was characterized by scanning and transmission electron microscopy, marker enzyme activities, one-dimensional sodium dodecyl sulfate polyacrylamide electrophoresis, and the specific beta-adrenergic receptor count. A yield of 79 +/- 9% was calculated by comparing the amount of beta-adrenoceptors in the purified membrane preparation with that of a crude cellular particulate fraction. The specific beta-adrenoceptor count of these two preparations was 1.2 +/- 0.02 and 0.2 +/- 0.05 pmol/mg protein, respectively, indicating a 6-fold improved purification with this microbead technique. The purified membranes were essentially free from contamination of other cell organelles.

Characterization of the beta-adrenergic receptors of cultured human epidermal keratinocytes.

The presence of beta-adrenergic receptors has been demonstrated in membrane preparations from passaged human epidermal keratinocytes. The receptors were characterized in terms of density and binding properties. Using the titrated beta-adrenergic antagonists dihydroalprenolol and propranolol, the equilibrium dissociation constant (Kd) was found to be about 1.4 nM for the two antagonists with a receptor density of approximately 280 fmol/mg membrane protein. Stereospecificity of the binding sites was shown by the much lowered affinity to D-isoproterenol as compared to that of L-isoproterenol. By the use of subtype specific antagonists, the receptors were classified as beta 2 adrenoceptors. This finding is supported by the relative order of affinities of the agonists isoproterenol greater than epinephrine greater than norepinephrine. The Kd value for dihydroalprenolol was approximately the same when determined from equilibrium binding studies or from association and dissociation kinetics, suggesting that the ligand binding is a single step bi-molecular reaction.

A rapid method for measuring drug enrichment in epidermis.

A rapid and simple method is described for measuring the enrichment of small molecules in epidermal tissue. To measure such an enrichment, a small tissue sample (2-10 mg) is allowed to equilibrate with a buffered solution of a labelled substance for periods of 12-36 h. The concentration of the radioactive molecule in the tissue is measured as a decrease of radioactivity in the solution. Concentration measurements in the tissue itself can be performed, but are not required to detect enrichment in the tissue or to assess its magnitude. The specific density of appendage free human epidermis has been determined and was found to be 1.20 g/cm3. Using this value, tissue weight can be translated into volume and concentraton changes in the solution can be recalculated to yield the concentration of the substance in the tissue itself. Close agreement was found between the calculated tissue concentration and the values actually measured, following digestion of the epidermis with NaOH and measuring the activity in the tissue digest. The enrichment of five substances in human epidermis was measured: alpha-estradiol, thiopyronincce, 8-methoxypsoralen (8-MOP), 5-methoxypsoralen (5-MOP), and theophylline. Of these substances, the first four are concentrated by human epidermis and the concentrations reached within the tissue are 10-500 times higher than the concentration of the same substance in the surrounding buffer. The enrichment data has been analysed in an attempt to distinguish between reversible affinity binding to specific tissue sites and partitioning of the substances between buffer and tissue components (lipids, membranes, etc.). In the case of thiopyronin and 8-MOP, reversible binding is indicated with dissociation constants of 10(-7) M and 10(-5) M, respectively, while partitioning distribution could account for the behavior of 5-MOP and alpha-estradiol. The method can be used either as a rapid screening method or as a quantitative analysis for the characterization of tissue enrichment with specific drugs.

Reversible binding of 5- and 8-methoxypsoralen to human serum proteins (albumin) and to epidermis in vitro.

Binding of the two photosensitizers, 8-methoxypsoralen (8-MOP) and 5-methoxypsoralen (5-MOP), to serum proteins and to epidermis was measured. 8-MOP binds to serum proteins with an apparent dissociation constant (Kd) of 4 x 10(-5) M. Under conditions of oral therapy, serum concentrations of the photosensitizer 2 h after administration are usually in the range of 100-1000 ng per ml serum. In this concentration range, 75-80% of the drug was found to be reversibly bound to serum proteins. 5-MOP shows a higher binding affinity to serum proteins and 98-99% of the drug is protein bound. The binding of both psoralen derivatives appears to take place mainly to serum albumin. 5-MOP and 8-MOP bind to different and non-interacting sites on serum proteins and the binding of the one has no effect on the binding of the other methoxypsoralen. Both photosensitizers bind reversibly to human epidermis. 8-MOP concentration in the epidermis is increased by ten to twenty fold compared with the equilibrium buffer. 5-MOP shows a higher binding affinity, resulting in a higher tissue concentration of the photosensitizer. As in serum, the two drugs appear to be bound in the epidermis to independent and non-interacting sites. No binding competition was found between the two methoxypsoralens and hydrocortisone, fluocinonide and acetyl salicylic acid, either in serum or in epidermis, using up to 1000 fold higher concentrations as compared with those of 5-MOP and 8-MOP.

8-Methoxypsoralen (8-MOP) in human skin: penetration kinetics.

Penetration kinetics of 8-methoxypsoralen (8-MOP) into human skin have been investigated under in vitro conditions. The dependence of the resulting tissue concentration on time of penetration, drug concentration in the applied preparation and vehicle composition has been studied. Tissue concentration of the drug was found to increase with time of penetration; however, in both horny layer and epidermis, this increase was followed by a significant decrease in concentration, (after 100 to 300 min), although a large amount of unpenetrated drug was present on the horny layer surface at all times. In contrast to this, 8-MOP accumulated in the lower corium and subcutis and reached concentrations higher then those found in the upper corium and epidermis. This accumulation of the drug is a consequence of the in vitro conditions and its amount is proportional to that amount that under in vivo conditions would have been taken up by the capillary system. Penetration was dependent on vehicle characteristics and was found to increase with increasing polarity of the vehicle. The penetration was found to be directly proportional to the applied concentration; however, the percentage of the drug that actually penetrated was inversely proportional to the applied concentration.

Quantitative determination of percutaneous absorption of radiolabeled drugs in vitro and in vivo by human skin.

We have measured concentrations of about 30 drugs in the living layers of the skin under conditions which provide data which are applicable in therapeutic treatment. Since the skin is a thin organ and small amounts of drug represent high target concentrations, it is necessary to select a sensitive quantitative method; observation of the kinetics of absorption using radiolabeled drugs is the method of choice. Because of possible hazards--and legal and ethical problems--absorption studies in human skin are commonly performed in vitro. Related in vivo investigations demonstrate the relevance and the limitations of the in vitro experiments. The main hindrance against penetration of drugs is by the horny layer. The barrier-function of this layer--if it is undisturbed--may be described by a multilayer model. The reciprocal function, the reservoir function, is important for the efficiency of topical treatment; it also plays a role in determining the unique pharmacokinetics of drug absorption in the skin and percutaneous resorption. If the horny layer is injured, i.e. in diseased skin, both the barrier and the reservoir functions are disturbed. In consequence, drug concentrations in the skin--and percutaneous resorption--may be greatly enhanced, and topically applied drugs may enter preferentially into diseased areas. The form of application, such as ointment, solution, etc. influences the penetration kinetics in such a specific manner that a specific vehicle for a specific drug should always be postulated. The frequently discussed hazards of side effects due to percutaneous resorption of drugs like corticosteroids are a function of the treated area rather than of its penetration capacity. Thus the indication for local or oral treatment of severe dermatoses should be considered in terms of the affected area. The relatively frequent side effects in the skin itself which originate from unnecessarily high drug concentrations and long term treatment must also be taken into account.