Evidence for lidocaine-induced enzyme inactivation.

The effects of lidocaine on hepatic enzyme activity were studied using the isolated perfused rat liver. The in vivo liver activity was examined by infusing lidocaine via the jugular vein, followed by organ isolation and drug perfusion 24 h later. The liver was studied in vitro by perfusing the organ with lidocaine until steady state was reached, then allowing the drug and metabolites to wash out of the organ, followed by a second infusion of lidocaine to probe enzyme activity. In both types of experiments, pretreatment with lidocaine caused a reduction in deethylation, and led to a more rapid attainment of steady state. The experimental concentration-time profiles and literature data were successfully described by a mathematical model.

Experimental studies of transient mass transfer and reaction in the liver: interpretation with a heterogeneous compartment model.

The uptake and metabolism of lipophilic compounds by the liver were studied by administering a model compound, lidocaine, to the isolated rat liver. Lidocaine was continuously infused into the liver until steady state was reached. Subsequent step changes in the inlet concentration were used to obtain information on rates of cellular uptake and release and to assess the extent of mixing within the organ. A simple heterogeneous model combining mass transfer and enzyme reactions was required to simulate the effluent levels of lidocaine and two primary metabolites, monoethylglycinexylidide and 3-hydroxylidocaine. The rate constants for uptake and release of lidocaine were 1200 and 46 min-1, respectively. The rate-limiting step was intracellular reaction, with a rate constant of 0.49 min-1. Although the rate of lidocaine uptake was fast, it was 50 times slower than the rate of facilitated uptake of galactose, a fact suggesting passive transport of lidocaine between the tissue and the vasculature. The rates of mass transfer of lidocaine and its metabolites differed, but the ratios of the rate of uptake to the rate of release were the same. The results suggested that all three species had an affinity for the cellular region of the liver; concentrations in tissue were approximately five times greater than concentrations in effluent. Because of the large capacity of the organ for uptake of lidocaine and its metabolites, concentrations from washout experiments were controlled by linear mass transfer from the tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Local efficacy of cyclosporine in corneal transplant therapy.

Studies were conducted to evaluate the efficacy of direct injections of cyclosporine (CsA) into the anterior chamber for the prevention of corneal allograft rejection in Dutch Belted rabbits. The mean survival time (MST) of grafts progressively increased from 50 to 89 days as the CsA concentration in the dose was increased from 1 to 10 mg/mL. Injection of 30 microL of 20 mg/mL CsA in olive oil prolonged graft survival to beyond 125 days without any signs of rejection. By comparison, the MST of allografts in control animals which received no therapy was 32 +/- 5 days, and the MST in animals administered a placebo of olive oil only was 31 +/- 4 days. The observed concentration dependence of the MST on CsA concentration is likely related to the time over which the drug delivery rate provides sufficient drug to achieve a therapeutic concentration in the aqueous humor; these studies suggest that the minimum delivery rate to the anterior chamber is between 200 and 325 ng/day. The efficacy of CsA was due to local delivery, and was likely not a systemic effect, because CsA was not detected in the systemic circulation at any time. This indicates that direct delivery of CsA to the eye can be useful in prolonging corneal graft survival, while minimizing systemic side effects. Separate experiments revealed that episodes of advanced rejection could not be reversed by a 30 microL dose of 20 mg/mL CsA to the anterior chamber, indicating the importance of avoiding long periods of subtherapeutic dosing.

Pharmacokinetic differences between ocular inserts and eyedrops.

Controlled release ocular inserts have been found to increase the amount of drug which is absorbed into the aqueous humour when compared to eyedrops. Systemic absorption following delivery using a controlled release insert has been found to be dependent on the release rate of the insert. The objective of this study was to determine if ocular inserts affect drug absorption into other ocular tissues such as the conjunctiva and iris-ciliary body. Ocular absorption studies were performed using albino rabbits and ethylene-vinyl acetate controlled release devices containing timolol maleate. A compartmental model previously developed to simulate ocular absorption following eyedrop administration was modified and used to simulate these experiments. The conjunctival absorption coefficient calculated by the model and the AUC of the conjunctiva per mumol of delivered drug were found to be 2.7 and 42 times higher, respectively, for the ocular insert as compared to eyedrop administration. The increased conjunctiva absorption was likely the result of reduced tear mixing, which caused a high local concentration of timolol between the insert and the conjunctiva. The AUC of the iris-ciliary body per mumol of delivered drug was found to be 24 times higher for the ocular inserts as compared to eyedrop administration. The AUC of the iris-ciliary body was found to be 1.4 times higher than the AUC of the aqueous humour for eyedrop administration, but 9 times greater for delivery via the ocular inserts. Thus, the increased absorption into the iris-ciliary body and aqueous humour observed for ocular inserts is partially the result of an increase in the amount of drug which enters these tissues via penetration across the conjunctiva and sclera.

Tyrosinase inactivation in organic solvents.

The inactivation of the catecholase activity of mushroom tyrosinase was investigated under nonaqueous conditions. The enzyme was immobilized on glass beads, and assays were conducted in chloroform, toluene, amyl acetate, isopropyl ether, and butanol. The reaction components were pre-equilibrated for 2 weeks with a saturated salt solution at a water activity of 0.90. The initial reaction velocity varied between 1.3 x 10(3) mol product/((mol enzyme)(min)) in toluene and 8.7 x 10(3) mol product/((mol enzyme)(min)) in amyl acetate. The turnover number varied between 8.1 x 10(3) mol product/mol enzyme in toluene and 7.2 x 10(4) mol product/mol enzyme in amyl acetate. In each solvent, the tyrosinase reaction inactivation parameters were represented by a probabilistic model. Changes in the probability of inactivation were followed throughout the course of the reaction using a second model which relates the reaction velocity to the amount of product formed. These models reveal that the inactivation rate of tyrosinase decreases as the reaction progresses, and that the inactivation kinetics are independent of the quinone concentration in toluene, chloroform, butanol, and amyl acetate. Significant effects of quinone concentration were, however, observed in isopropyl ether. The likelihood of inactivation of the enzyme was found to be greatest toward the beginning of the reaction. In the latter phase of the reaction, inactivation probability was less and tended to remain constant until the completion of the reaction.

Theoretical corneal permeation model for ionizable drugs.

The primary route into the eye for many drugs is transcorneal permeation. A better understanding of the mechanisms involved in transcorneal permeation could lead to improvements in drug dosage forms or the development of drug delivery devices which enhance the ocular bioavailability of drugs. A corneal permeation model has been developed which can be used to study the mechanisms involved in corneal permeation. The model uses five compartments in series to simulate the tear film, epithelium, stroma, endothelium and aqueous humour. These tissues were assumed to be adequately represented by plane sheet barriers of physiological thickness. The tear film was assumed to be perfectly mixed and the stroma completely stagnant. Due to inadequate knowledge of the hydrodynamics of the aqueous humour, both stagnant and perfectly mixed extremes were studied. The four routes of drug loss which were considered the most significant and therefore included in the model were lacrimal drainage, conjunctival absorption, aqueous drainage and iris-ciliary body absorption. The equilibrium that can exist between the ionic and non-ionic forms of a drug was found to be an important step in the mechanism of transcorneal permeation. Including the equilibrium condition in the model resulted in aqueous humour drug levels that were over 50 times higher than the levels predicted by a model which did not use the equilibrium mechanism. A relationship between the lipophilicity of each of the two drug forms and its permeability in each layer of the cornea was used in the model. The model was used to predict aqueous humour drug concentrations resulting from a constant release of timolol into the tear film or from the application of timolol, levobunolol and pilocarpine eyedrops. The model produced transient aqueous humour drug levels that closely followed experimental in vivo data from literature. Using the model, it was also possible to predict the amount of instilled drug that is lost through each of the four elimination routes of the eye.

Models of hepatic drug elimination.

The liver is, by nature, heterogeneous. It contains a complex vascular network for blood flow and a stationary phase consisting of enzymes within parenchymal cells. Several physiological processes, therefore, may combine to give observed ranges in drug elimination. Net changes in concentration are a consequence of a series of steps: uptake of substrate into liver cells, enzymatic reactions within the cells, release of metabolites and unconverted substrate from the cells into the sinusoids, and the net flow of the perfusing medium in the vasculature. In addition, substrate binding to proteins in the blood and in the liver can influence hepatic elimination. An understanding of each of these processes is necessary to fully comprehend the overall process of drug elimination, and these processes must be accounted for, either individually or by grouping and approximation, if a model for drug elimination is to be developed. Existing models of hepatic elimination may be classified according to their treatment of mixing within the vasculature and whether or not the model explicitly accounts for mass transfer between the heterogeneous phases of the liver. Four major classes may be defined: 1. Nonparametric homogeneous models, which assume that either complete mixing or no mixing occurs within the vasculature of the organ. 2. Homogeneous mixing models, which allow for a range of mixing phenomena. 3. Heterogeneous micromixing models, which allow for mass transport between the cells and vasculature and describe mixing within the vasculature on a microscopic level. 4. Heterogeneous compartmental models, which also describe interphase mass transfer but assume complete mixing on a microscopic level, and therefore use a time and spatially averaged approach to model mixing. The utility of these models of hepatic elimination will be critically assessed based upon (1) their ability to account for the influence of the aforementioned physiological processes upon elimination; (2) the data requirements of the model, in addition to its mathematical complexity and ease of use; and (3) the range of compounds and metabolites which may be described using the model.

Mechanisms of lidocaine kinetics in the isolated perfused rat liver. III. Evaluation of liver models for time-dependent behavior.

Three models for the elimination of lidocaine in the isolated perfused rat liver were used to simulate the time course of both lidocaine and its metabolites in a single-pass perfusion system: well stirred, parallel tube, and a two-compartment model to test the effects of enzyme heterogeneity. All models included multiple enzymes and multiple metabolic pathways, as well as varying degrees of tissue binding. Although the well stirred and parallel tube models gave qualitatively different results, neither model predicted that the concentration of monoethylglycinexylidide would pass through a maximum and then decline to a lower steady state value, as observed in continuous perfusion experiments. Although each of the three models tested would give reasonable agreement with steady state observations, the test of the time-dependent behavior of both lidocaine and monoethylglycinexylidide was more discriminating. Each model gave characteristic predictions for the time course of the metabolites.

Mechanisms of lidocaine kinetics in the isolated perfused rat liver. II. Kinetics of steady state elimination.

The steady state kinetics of lidocaine and its metabolites were modeled using nonlinear elimination pathways for multiple enzymes. The main metabolites, monoethylglycinexylidide and 3-hydroxy-lidocaine, were infused in the absence of lidocaine to measure the kinetic parameters for secondary elimination. Data from continuous perfusion of lidocaine in the isolated perfused rat liver at concentrations ranging from 9.6 to 278 microM (N = 16) were used to calculate the kinetic parameters for formation of the main metabolites. The elimination of lidocaine in the liver was approximated by the well stirred model. The whole liver study gave higher elimination rates than were predicted from microsomal studies. The major pathways for elimination of lidocaine in the rat were deethylation and hydroxylation, and subsequent elimination along these pathways accounted for the poor material balance at low dosage levels. The observed competitive inhibition of hydroxylation was in agreement with the predictions of the model.

L-DOPA production from tyrosinase immobilized on nylon 6,6.

The production of L-DOPA immobilized on chemically modified nylon 6,6 membranes was studied in a batch reactor. Tyrosinase was immobilized on nylon using glutaraldehyde as a crosslinking agent. The effects of membrane pore size and glutaraldehyde concentration upon enzyme uptake and L-DOPA production were investigated. Enzyme uptake was unaffected by glutaraldehyde concentration; approximately 70% uptake was observed when 25% w/v (group 1), 5% (group 2), and 3% (group 3) glutaraldehyde were used, indicating that glutaraldehyde was in excess. Similarly, uptake was the same for membranes with 0.20 and 10 microm pore sizes.Membranes produced using different levels of glutaraldehyde exhibited dramatically different capacities for L-DOPA production, despite the fact that enzyme uptake was equivalent. Membranes from groups 2 and 3 (5% and 3% glutaraldehyde) produced L-DOPA at a rate of 1.70 mg L(-1) h(-1) over 170 h in a 500-mL batch reactor. However, no free L-DOPA was detected when group 1 membranes were used. Experimental evidence suggests that L-DOPA was produced, but remained bound to these membranes via excess glutaraldehyde left over from the immobilization process. Membrane pore size also effected L-DOPA production; less production was observed when 10-microm membranes were used, despite equivalent enzyme uptake. The observed difference in production may be due to differences in the pore density on the two types of membranes which could affect the access of the substrate to the immobilized enzyme.The results of these studies indicate that tyrosinase can be effectively immobilized on nylon 6,6. L-DOPA production was optimal when 0.20-microm-pore-size membranes were activated with 3-5% glutaraldehyde. Stability studies indicated a 20% reduction in activity over 14 days when the immobilized enzyme was used under turnover conditions.

A compartmental model for the ocular pharmacokinetics of cyclosporine in rabbits.

Studies were conducted in rabbits to determine the ocular distribution and elimination of cyclosporine, with the objective of developing a comprehensive pharmacokinetic model. Following a bolus dose into the anterior chamber, drug levels were measured in the aqueous humor, cornea, iris/ciliary body, lens, sclera, and conjunctiva. Cyclosporine was rapidly eliminated from the aqueous, but drug levels in ocular tissues persisted for in excess of 48 hours, particularly in the cornea and iris/ciliary body. The terminal elimination half life from these tissues was 45 hr and 30 hr, respectively, providing evidence that these tissues could act as a reservoir for the drug. It was found that a compartmental model accurately described the experimental data. A single compartment was used for each of the tissues and fluids sampled, except for the cornea, which was subdivided into two compartments, representing its tissue and aqueous regions.