Monitoring in situ liver metabolism in rats using microdialysis. Comparison of microdialysis mass-transport model predictions to experimental metabolite generation data.

The generation of metabolites from two model compounds, phenacetin and acetaminophen, included in the perfusion fluid of a microdialysis probe implanted into rat liver was studied. When 60 microM phenacetin was included in the perfusion fluid using a flow rate of 1.0 microL/min, acetaminophen and acetaminophen sulfate were recovered at concentrations that ranged between 0.4 and 1.6 microM. Acetaminophen sulfate ([AS]gain) diffused back into the microdialysis probe on a micromolar percentage basis of 8.9+/-2.4% (n = 3) when acetaminophen was passed through the probe at a concentration between 11 and 12 microM. When 220-240 microM acetaminophen was passed through the probe, the percentage of acetaminophen sulfate recovered was 4.8+/-1.4% (n = 3) (P < 0.1 compared to the 11 microM group). No acetaminophen glucuronide was detected in the dialysate samples. A mathematical model that describes mass transport in microdialysis sampling was used to predict the concentration of metabolite that could be recovered into the dialysate after the loss of a substrate compound that undergoes metabolism. The model predicts a metabolite recovery of 23.6% using estimates for phenacetin metabolism and 21.5% using estimates for acetaminophen metabolism. The results presented here indicate that microdialysis has potential to be used to study local in situ metabolism and with further refinements of the microdialysis mass-transport model may be used to estimate in vivo metabolic formation rates.

A mechanistic study of griseofulvin dissolution into surfactant solutions under laminar flow conditions.

The in vivo dissolution of many poorly soluble drugs is enhanced by the action of surfactants secreted into the upper gastrointestinal (GI) tract. These substances may act by solubilizing individual drug molecules into two separate liquid phases: the free aqueous phase and a micellar phase in which the drug is incorporated into a complex of two or more surfactant molecules. This complex process, micellar solubilization, was the subject of this in vitro study, wherein griseofulvin (gris) dissolution was observed in flowing surfactant solutions. Aqueous solutions of sodium dodecyl sulfate (SDS), an anionic surfactant, were pumped over a gris tablet embedded in a laminar flow device to simulate flow in the human upper GI tract. SDS solutions were well above the critical micellar concentration (cmc approximately 6-7 mM), and flow rates ranged from 4 to 7 mL/min. Gris solubility in premicellar (4 mM), near-micellar (6 mM), and micellar (>6 mM) SDS solutions was also determined. The measured solubility of gris increased linearly with SDS concentrations above the cmc. Drug solubility in SDS concentrations below the cmc was also higher than that in water. Gris diffusion coefficients were measured using pulsed-field gradient NMR spectroscopy. To determine the controlling mechanism for surfactant-enhanced dissolution, a mathematical model was developed. The model solution, an equation for drug dissolution rate, was compared with experimental data to demonstrate that drug transport away from the solid surface is the slow step in the process. Measured gris diffusion coefficients and solubility values were used as constants in the mathematical model solution and were combined to calculate an effective gris diffusion coefficient. Using these experimentally determined properties, model-calculated dissolution rates were within 7% of the measured values. As hypothesized, dissolution rates were found to be directly proportional to the transport properties of the system (effective drug diffusion coefficient and fluid flow rate) as well as to the drug solubility. To further verify transport-limited dissolution, the measured dissolution rates were found to be proportional to the surrounding medium flow rate to the 1/3 power, as predicted by the model dissolution rate equation.

Factors that influence microdialysis recovery. Comparison of experimental and theoretical microdialysis recoveries in rat liver.

Inhibition of metabolic processes was used to assess the possible change in the recovery of material from a microdialysis probe implanted in vivo in rat liver. Phenacetin and antipyrine were perfused through a microdialysis probe implanted in the liver. Inhibition of phenacetin and antipyrine metabolism was achieved through an iv bolus dose of the cytochrome P450 suicide substrate 1-aminobenzotriazole (1-ABT). 1-ABT inhibited phenacetin clearance by 90%, thus also inhibiting metabolism by 90%. There was no statistical difference in the recovery of phenacetin and antipyrine across the microdialysis membrane in the liver between the control and metabolically inhibited animals. Partial differential equations were developed that describe the transport of analyte from the microdialysis probe and solved by an implicit finite-difference method to aid in the understanding of the above-mentioned microdialysis experiments. Predictions of microdialysis recovery obtained from the numerical model are compared with those found experimentally. The model could predict trends in the data, but not the actual experimental values. This suggests that predictions from this microdialysis model are essentially heuristic and as presently formulated can be used only to show mechanisms that affect recovery, but they cannot be used to accurately predict recovery. Prediction of actual recovery requires knowledge of the values of the parameters that describe chemical properties such as the in vivo diffusion coefficient, metabolism rate constant, and capillary exchange rate constant. For microdialysis experiments performed in the liver, capillary exchange and the rate of liver blood flow appear to be the dominant processes that facilitate net transport from a microdialysis probe rather than metabolic processes. These results indicate that microdialysis recoveries measured after inhibition of a concentration-dependent kinetic process via pharmacological challenge will change only when the kinetic process that is being challenged is large compared to the contribution of all concentration-dependent kinetic processes, including other metabolism routes, capillary exchange, or uptake that remove the analyte from the tissue space. It is concluded that the microdialysis recovery of a substance from the liver is not generally affected by liver metabolism.

Control of poorly soluble drug dissolution in conditions simulating the gastrointestinal tract flow. 2. Cocompression of drugs with buffers.

The objective of this study was to determine the primary formulation properties that affect the dissolution rate of poorly soluble nondisintegrating drugs. This work focused on compression of orally administered acidic drugs with ionizable buffers. Naproxen was used as the poorly soluble model drug, and calcium salts of carbonic, citric, and phosphoric acids were used as formulation buffers. Gastrointestinal tract (GI) dissolution was simulated in a laminar flow apparatus by exposing a drug/buffer tablet to aqueous solution of a given pH at a constant flow rate. Although formulation with a buffer resulted in reduced available drug surface area, the absolute drug dissolution rate and flux increased with increased buffer content to a maximum from tablets having equal weights of drug and buffer. This buffer-induced enhancement was seen not only in GI tract simulation (pH 7), but also at pH 2 (stomach conditions), where acidic drugs remain in their poorly soluble form upon dissolution. The flux increase was much greater than that achieved by using the same amount of an inert excipient in the solid formulation. Dissolution rates were also increased by decreased drug and buffer particle sizes and increased fluid flow rate. Drug dissolution rates were inversely proportional to intrinsic buffer solubilities: The model drug actually dissolved fastest when the buffer solubility was lower than that of the drug. Dissolution rates were apparently insensitive to the relative proximity of the drug and buffer ionization constants.

Control of poorly soluble drug dissolution in conditions simulating the gastrointestinal tract flow. 1. Effect of tablet geometry in buffered medium.

The dissolution rate of a solid drug from the gastrointestinal (GI) tract is affected by the properties and flow dynamics of the liquid medium surrounding the tablet, as well as by the chemical nature of the drug. In this study, naproxen was used as a poorly soluble model drug. The dissolution medium was buffered with acetate, citrate, or phosphate buffer of varied concentrations and pH. GI flow conditions around a stationary tablet were simulated in a laminar flow device by anchoring the tablet on the floor of its channel having a rectangular cross section. Fresh, buffered solution was passed across the tablet and the effluent was collected for analysis and calculation of the dissolution rate. The dissolution rate was found to vary nonlinearly with the exposed tablet height, reaching a maximum at a tablet height approximately half the channel height. This maximum rate was attributed to an optimal combination of (1) eddy mixing and local turbulence generated by the flow impingement on the bluff object (tablet) and (2) the exposed tablet surface area available for dissolution. This effect was further confirmed by using dye-enhanced visual analysis of flow patterns at varied flow rates and exposed tablet heights. Elevation of the tablet to approximately the channel half-height significantly magnified the dissolution rate increase observed on exposure to buffered medium. Thus, tablet height and exposed surface area are major factors in determining dissolution rate, especially in conditions where the dissolving species reacts with the solvent. These results suggest that standard in vitro dissolution rate methods do not qualitatively indicate incremental changes in rate with altered tablet geometry or dissolution medium.

Dependence of dissolution rate on surface area: is a simple linear relationship valid for co-compressed drug mixtures?

A quantitative analysis of the dependence of dissolution rate on the relative surface area occupied by two non-interacting drug mixtures from co-compressed slabs is described. The results from the experimental dissolution rates of each component from naproxen/phenytoin co-compressed slabs under laminar flow conditions, when corrected for the area occupied by that component in the slab, contradict the stagnant layer model predictions, where dissolution rates are assumed to be directly proportional to the occupied surface area. Simulations from non-mixed co-compressates of naproxen and phenytoin indicated that dissolution rates are proportional to bL2/3, as reported for pure compounds in the laminar dissolution apparatus by Shah and Nelson. However, for a well mixed co-compressate, which differs with the non-mixed case only in the distribution of particles, this proportionality did not hold. The deviation was explained by 'carryover' of material from one section of the component to the next due to fluid flow, resulting in an increase in apparent effective length of the component in the slab (Leff).

A convective-diffusion model for dissolution of two non-interacting drug mixtures from co-compressed slabs under laminar hydrodynamic conditions.

A numerical convective-diffusion dissolution model has been extended to describe dissolution of two neutral non-interacting drugs co-compressed in a slab geometry. The model predicted the experimental dissolution rates of naproxen/phenytoin mixtures and hydrocortisone/nitrofurantoin mixtures quite accurately, except for phenytoin in the naproxen/phenytoin mixture at low weight proportions. A non-linear dependence of dissolution rate on weight proportion with a positive deviation from linearity was observed. An increase in flow rate increased the dissolution rate and the cube-root dependency of dissolution rate on the flow rate for a given weight proportion of the component in the slab, as proposed earlier by Shah and Nelson for pure compounds, was also observed here, suggesting that the changes in dissolution profile were caused by changes in surface area only. As expected from the model an increase in particle size of the powders used to make the slab decreased the dissolution rate. This was explained by an increase in the average length of the component resulting in a bigger 'carryover' of material from one section of the component in the slab to the next section of the same component, due to convection, and hence lower flux.

Examination of microdialysis sampling in a well-characterized hydrodynamic system.

The use of microdialysis sampling was examined in a well-characterized hydrodynamic system. A cross-flow microdialysis probe was designed in which the flow of both the dialysis perfusion solution and the sample solution could be carefully controlled. Dialysis membranes of cellulose (Cuprophan), cellulose acetate, and polyacrylonitrile (PAN) were examined in this system using hydroquinone as the test analyte. The permeability of the membranes to hydroquinone ranged from 1.72 x 10(-6) cm2/s for PAN to 2.97 x 10(-7) cm2/s for cellulose acetate. Determination of the dialysis fibers' recovery as a function of the sample flow velocity resulted in a rapid increase in recovery with increase in flow velocity. The recovery plateaued at high sample velocity. These results show that at low sample velocity diffusion through the sample solution is the rate-limiting step in recovery while at higher velocity transport through the membrane becomes rate limiting. Recovery for all three membrane types plateaued above sample velocities of 0.211 cm/s. This is well below the velocity of most biological fluids in which microdialysis sampling has been applied. This result supports previous reports that an in vitro calibration of microdialysis probes is appropriate for use in hydrodynamic environments in vivo such as the blood and bile.

Dissolution of ionizable drugs into unbuffered solution: a comprehensive model for mass transport and reaction in the rotating disk geometry.

A model has been developed to describe the mass transport and reaction of ionizable compounds where mass transfer is caused by convection and diffusion from a rotating disk. Dissolution rates of benzoic acid, 2-naphthoic acid, and indomethacin in aqueous solutions of high ionic strength (I = 0.5 with potassium chloride) at 25 degrees C were investigated. The model includes the effects of diffusion, convection, and simultaneous acid/base reaction at all points in the region adjacent to the dissolving solid. The solution of the transport equations is obtained numerically with an iterative algorithm which uses (a) closure of all material balances and (b) equilibria at the solid/liquid surface as constraints. The model solution yields both the flux of the dissolving acid and the concentration profile of each component. Reduced values of all reaction rate constants are used in the region adjacent to the dissolving surface to allow convergence of the computation. Although nonequilibrium concentration values are calculated, it is shown that the theoretical dissolution rate determined as the solution of the model is insensitive to the magnitude of the rate constants as their maximum useable values are approached. Comparisons of the model results with experimentally determined fluxes show close agreement and confirm that the transport mechanisms in the model formulation are consistent with the measured values. Further, the inclusion of convection allows accurate calculations without utilization of an arbitrary boundary layer thickness. Accurate dissolution rates can be determined using this technique under a wide range of conditions, except at low pH.

Experimental determinations of diffusion coefficients in dilute aqueous solution using the method of hydrodynamic stability.

Diffusion coefficients were experimentally determined in dilute aqueous solution at 25 +/- 0.1 degrees C, ionic strength 0.5 M, using Taylor's method of hydrodynamic stability. The methodology described is accurate enough to show significant differences in diffusion coefficients between the various ionic forms of the same species as a function of degree of ionization. In Taylor's method, diffusion coefficients were measured by allowing two solutions of differing solute concentration to contact in a capillary tube, forming a stable, measurable concentration gradient. The solute diffusion coefficient is a function of the gradient, the solution viscosity, the solution density, and some capillary dimensions. Viscosity was maintained constant across experiments and values of sufficient accuracy were available in the literature. Solution densities were measured with a tuning fork densimeter. Compounds studied were o-aminobenzoic acid, benzoate anion, the four forms of phosphate and citrate, and the zwitterionic forms of glycine, diglycine, and triglycine. Based on the results for the four forms of phosphate and citrate, experimental diffusivity values vary with the ionic state of the diffusant, presumably because of the altered state of hydration as charge varies. For the glycine series, the diffusivity showed an unexpected dependency on molecular weight (size).

Dissolution of carboxylic acids. III: The effect of polyionizable buffers.

The dissolution behavior of three carboxylic acids of variable aqueous solubility but with approximately equal pKa values into aqueous buffered solutions has been studied as a function of pH and of buffer properties. The dissolution from constant-surface-area compressed disks of benzoic acid, 2-naphthoic acid, and indomethacin into solutions of constant ionic strength (mu = 0.5 with potassium chloride) and constant pH (maintained by pH stat) at 25 degrees C using a rotating disk apparatus was evaluated. Models for dissolution of these weak acids into diprotic and triprotic buffering media are developed to predict the flux of the acid as a function of bulk solution pH and the physical and chemical properties of the buffer and acid. The models assume that mass transfer can be represented by a single second order diffusive term and that instantaneous equilibrium between all reactive species exists. Values of flux and pH at the solid-liquid interface are calculated and the fluxes compared to experimentally determined values. Reasonable correlation was found between values predicted by the models and experimental flux values. Major influences on model accuracy are the Ka and physical properties of the buffer.