Diffusion cell receptor fluid

Values are the mean + S.E. of the number of determinations in parentheses. The solutions refer to the fluid used in the diffusion cell receptor. 

The permeability of human skin to n-hexane has been determined in vitro in flow-through diffusion cells (Loden 1986). Pieces of full-thickness human skin were exposed to 3H -hcxane in human serum, and the appearance of label in the trans compartment measured for 0.5 or 12 hours. The skin was then sectioned with a microtome into 0.25 mm slices and the quantity of label in the skin measured. The rate of resorption (uptake of substance by the receptor fluid beneath the skin [i.e., the amount that passes through the skin]) was calculated. The rate of resorption for n-hexane through human skin was calculated to be 0.83 ( g cm2/hr). The permeability of n-hexane through human skin was much lower (approximately 100-fold) than for other chemicals tested in this study. For example, rates of resorption (in g cm2/hr) were 99 for benzene and 118 for ethylene glycol. 

OECD has adopted an in vitro test for skin absorption potential (OECD TG 428, Skin Absorption In Vitro Method). According to this guideline, excised skin from human or animal sources can be used. The skin is positioned in a diffusion cell consisting of a donor chamber and a receptor chamber, between the two chambers. The test substance, which may be radio-labeled, is applied to the surface of the skin sample. The chemical remains on the skin for a specified time under specified conditions, before removal by an appropriate cleansing procedure. The fluid in the receptor chamber is sampled at time points throughout the experiment and analyzed for the test chemical and/or metabolites. 

The receptor phase of any diffusion cell should provide an accurate simulation of the in vivo permeation conditions. The permeant concentration in the receptor fluid should not exceed 10 percent of saturation solubility (Skelly et al. 1987), as excessive receptor phase concentration can lead to a decrease in absorption rate and result in an underestimate of bioavailability. The most common receptor fluid is pH 7.4 phosphate-buffered saline (PBS), although if a compound has a water solubility below 10 p.g/mL, then a wholly aqueous receptor phase is unsuitable, and addition of solubilizers becomes necessary (Bronaugh 1985). 

The primary approach to assess dermal absorption is the in vitro diffusion cell. In thi,s model, skin sections (full thickness, deimatomed to a specific thickness) are placed in a two-chambered diffusion cell in which receptor fluid is placed in a reservoir (static cells) or perfused through a receiving chamber (flow-through cells) to simulate cutaneous blood flow. Chemical may either be dosed under ambient conditions neat or dissolved in a vehicle (Franz and Bronaugh cells) or in water (sLdc-by-side diffusion... 

Most studies today are conducted in one-chamber diffusion cells that hold receptor fluid beneath the skin. The top surface of the skin is exposed to the environment and is surroimded by a short wall. A tube extends upward from the receptor fluid for manual sample removal. The Franz cell is the most widely known cell of this type (Franz, 1975). A flow-through diffusion cell (Figure 2.1) is a modification of this design that should have a much smaller receptor fluid chamber to permit easy removal of contents with a moderate flow (1 to 2 ml/h) of receptor fluid (Bronaugh and Stewart, 1985). The continual replacement of the receptor fluid pomits maintenance of skin viability when a physiological buffer is used (Collier etal., 1989). This diffusion cell also has the advantage of automatic samphng with the use of a fraction collector. 

In vitro skin absorption smdies often differ in the receptor fluid used. A buffered saline solution may simply be used in a study with nonviable skin however, a more physiological solution such as HEPES-buffered Hanks balanced salt solution is required to maintain the viability of skin in the diffusion cells (Collier et al., 1989). The viabihty of skin can be maintained for at least 24 h based on glucose utilization of skin, histological evaluations, and the maintenance of estadiol and testosterone metabolism (Collier et al., 1989). 

The absorption and metabolism of retinyl palmitate was examined after application in a volatile solvent (acetone) to viable human skin assembled in diffusion cells. Only a small amount (0.2%) of the applied retinyl palmitate that penetrated the skin was fonnd in the receptor fluid at the end of the 24-h study. However, 18% of the applied dose of radioactivity was found in the skin, and 44% of this dose had been metabolized to retinol (Boehnlein et al., 1994). 

Percutaneous absorption may be measured In vivo or It may be determined In vitro using excised skin mounted In glass diffusion cells. The most frequently used approaches employ radlolabelled compounds. The validity of In vitro measurements relies on the assumption that no metabolism occurs In skin, and that absorption Into the receptor fluid of the diffusion cells approximates absorption from dermal tissue Into blood In vivo. These assumptions are generally unproven, and In vitro measurements ultimately require confirmation In vivo. These limitations notwithstanding, In vitro studies still provide substantially useful Information, and a variety of methods have been developed. The first section of this paper will focus on the general Interpretation of In vitro data and Its application to risk assessment. 

Percutaneous absorption studied in vitro is normally characterized either by a permeability constant or by the time course of the penetration process. Direct measurements of absorption require intermittent sampling of fluid contained in the receptor half of a diffusion cell. Permeability constants are frequently calculated by removing and assaying microaliquots of receptor fluid at various intervals during the early time course of absorption ( static" diffusion cell technique), until steady state (ss) flux is obtained the permeability coefficients are then derived according to Pick s First Law of Diffusion ... 

If the receptor fluid Is not replaced, the concentration In the receptor phase of the diffusion cell will eventually equilibrate with the donor phase concentration, at which point further net absorption will cease. Thus, this technique Is unsatisfactory for measuring the complete time course of absorption. This limitation may be overcome by Intermittently emptying the receptor fluid and replacing with "fresh" solution In order to maintain "sink" conditions ("dynamic diffusion cell technique). 

An ideal pharmacokinetic model of the percutaneous absorption process should be capable of describing not only the time course of penetration through skin and Into blood (or receptor fluid In a diffusion cell), but also the time course of disappearance from the skin surface and accumulation (reservoir effect) of penetrant within the skin membrane. Neither Pick s Plrst Law of Diffusion nor a simple kinetic model considering skin as a rate limiting membrane only Is satisfactory, since neither can account for an accumulation of penetrant within skin. To resolve this dilemma, we have analyzed the In vitro time course of absorption of radiolabeled benzoic acid (a rapid penetrant) and paraquat (a poor penetrant) through hairless mouse skin using a linear three compartment kinetic model (Figure 5). The three compartments correspond to the skin surface (where the Initial dose Is deposited), the skin Itself (considered as a separate compartment), and the receptor fluid In the diffusion cell. The Initial amount deposited on the skin surface Is symbolized by XIO, and K12 and K23 are first order rate constants. 

The output function characterizing the movement of penetrant from the skin compartment Into the receptor fluid of the diffusion cell was determined as follows. Thirty minutes after Initial application, the skin surface was rinsed as described above to remove any surface residual. The diffusion cell was emptied, rinsed and refilled with fresh solution. All radlolabel recovered over the next 12 hours was assumed to have originated from the "filled" skin compartment, rather than the skin surface. "Best fit" parameters were obtained by computational methods as before. 

We have measured the absorption of radiolabeled DDT and p u athion through excised human abdominal skin using an in vitro diffusion cell procedure. The poor water solubility properties of these compounds (Table II) suggested that a nonionic surfactant (oleth 20) would be necessary in the receptor fluid. The compounds were applied to skin in an acetone vehicle at a concentration of 4 ug/cm. The surface of the skin was cleansed with acetone at 24 h to remove unabsorbed material. The absorption of the pesticides was followed for a total of 7 days until the amount of compound entering the receptor fluid was minimal (Figures 3 and 4). 

Permeation studies are accomplished by insertion the fabricated organogels with suitable skin or synthetic manbrane in between receptor and donor compartment in a vertical diffusion cell such as Franz diffusion ceU or keshary-chien diffusion cell. The system is applied to the hydrophilic side of the manbrane and then mounted in the diffusion cell with lipophilic side in contact with receptor fluid. The receiver compartment is maintained at specific temperature (usually 32°C 5°C for skin) and is continuously stirred at a constant rate. The samples are withdrawn at different time intervals, and equal amount of buffer is replaced each time. The samples are diluted appropriately and absorbance is determined spectrophotometrically. Then the amount of drug permeated per centimeter square at each time interval is calculated." ... 

The test substance is applied in an appropriate formulation on the skin sample, which is usually placed in a diffusion cell (Figure 9.1.6) (Balaguer et al, 2006). The diffusion cell consists of an upper donor and a lower receptor chamber, separated by a skin preparation. The cells are made preferably from an inert non-adsorbing material. Temperature control of the receptor fluid is crucial throughout the experiment. The skin surface temperature in the diffusion cell should be kept at the in vivo skin temperature of 32 °C. 

The amounts of penetrated substance found in the receptor fluid are considered to be sys-temicaUy available. The epidermis (except for the stratum comeum) and dermis are considered as a sink, therefore the amounts found in these tissues are equally considered as absorbed and are added to those found in the receptor fluid. The amounts that are retained by the stratum comeum at the time of sampling are not considered to be dermally absorbed and, thus, they do not contribute to the systemic dose. The absorption rate and mass balance should be calculated separately for each diffusion cell. Only then, the mean SD can be calculated. 

Neurotransmitter/Receptor Binding. At this point, the neurotransmitter chemical is free in the synapse (extracellular fluid) and drifts (diffuses) in all directions. Some of the neurotransmitter molecules float across the synapse and bind to receptors on the surface of the adjacent nerve cell. Each neurotransmitter has its own unique three-dimensional shape and binds with certain receptors but not others. The binding between a neurotransmitter and a receptor is similar to fitting a key into a lock. When the neurotransmitter binds the receptor, the signal has been passed to the neighboring nerve cell. This is the process of neurotransmission. 

The intracellular signaltransduction of ofi-adrenoceptors is effectuated by a G-protein-dependent activation of the phospholipase C. This enzyme cleaves phosphatidylinositol, a phospholipid present in cell membranes, into inositol-1,4-5-triphosphate (IP3) and diacylglycerol (DAG). IP3 is a strong inductor of intracellular calcium release which leads to an increase of smooth muscle tone or the liberation of hormones stored in vesicles. Noradrenaline which is released by exocytosis, spreads by diffusion only. Only a small fraction of the total amount of the transmitter released will actually reach the postsynaptic membrane and bind to its specific receptors. Another fraction escapes the synapic cleft by diffusion and is finally enzymatically degraded in the interstitial fluid. Another fraction is taken up postsynaptically and metabolized enzymatically by the target cells (uptake 2). By far most of the transmitter (90%) is actively taken up by the releasing neuron itself (uptake 1 or neuronal re-uptake). In the... 

A special case of signal transduction is represented by a class of small, reactive signaling molecules, such as NO (see chapter 6.10). NO is synthesized in a cell in response to an external signal and is dehvered to the extracellular fluid. Either by diffusion or in a protein-boimd form, the NO reaches neighboring cells and modification of target enzymes ensues, resulting in a change in the activity of these enzymes. NO is characterized as a mediator that lacks a receptor in the classical sense. 

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