Improving drug delivery to primary and metastatic brain tumors: strategies to overcome the blood-brain barrier.

Brain tumor diagnosis has an extremely poor prognosis, due in part to the blood-brain barrier (BBB) that prevents both early diagnosis and effective drug delivery. The infiltrative nature of primary brain tumors and the presence of micro-metastases lead to tumor cells that reside behind an intact BBB. Recent genomic technologies have identified many genetic mutations present in glioma and other central nervous system (CNS) tumors, and this information has been instrumental in guiding the development of molecularly targeted therapies. However, the majority of these agents are unable to penetrate an intact BBB, leading to one mechanism by which the invasive brain tumor cells effectively escape treatment. The diagnosis and treatment of a brain tumor remains a serious challenge and new therapeutic agents that either penetrate the BBB or disrupt mechanisms that limit brain penetration, such as endothelial efflux transporters or tight junctions, are required in order to improve patient outcomes in this devastating disease.

Mechanism of sensitization of MDR cancer cells by Pluronic block copolymers: Selective energy depletion.

This paper, for the first time, demonstrates that exposure of cells to the poly(ethylene oxide)-poly(propylene oxide) block copolymer, Pluronic P85, results in a substantial decrease in ATP levels selectively in MDR cells. Cells expressing high levels of functional P-glycoprotein (MCF-7/ADR, KBv; LLC-MDR1; Caco-2, bovine brain microvessel endothelial cells [BBMECs]) are highly responsive to Pluronic treatment, while cells with low levels of P-glycoprotein expression (MCF-7, KB, LLC-PK1, human umbilical vein endothelial cells [HUVECs] C2C12 myoblasts) are much less responsive to such treatment. Cytotoxicity studies suggest that Pluronic acts as a chemosensitizer and potentiates cytotoxic effects of doxorubicin in MDR cells. The ability of Pluronic to inhibit P-glycoprotein and sensitize MDR cells appears to be a result of ATP depletion. Because many mechanisms of drug resistance are energy dependent, a successful strategy for treating MDR cancer could be based on selective energy depletion in MDR cells. Therefore, the finding of the energy-depleting effects of Pluronic P85, in combination with its sensitization effects is of considerable theoretical and practical significance.

Transport of fluorescein in MDCKII-MRP1 transfected cells and mrp1-knockout mice.

The multidrug resistant-associated protein 1 (MRP1) is a membrane-bound transport protein that is involved in the efflux of organic anions and has been implicated in multidrug resistance in cancer. MRP1 has also been reported to be ubiquitously expressed in normal tissues, including the brain. The presence of functional organic anion transporters in the blood-brain and blood-CSF barriers that influence the distribution of various compounds to the brain has long been known. The purpose of this study was to examine the role of MRP1 in the brain distribution of a model organic anion, fluorescein. The substrate specificity of MRP1 for fluorescein was initially determined by examining the accumulation of fluorescein in MDCKII MRP1-transfected cells. The distribution of fluorescein in the brain was then examined in wild-type and mrp1 gene knockout mice. The results show that in MDCKII MRP1-transfected cells, the accumulation of fluorescein was significantly lower (about 40% lower) than that in wild-type MDCKII cells. MRP1 inhibitors such as probenecid, MK-571, and LY402913 enhanced fluorescein accumulation in MDCKII MRP1-transfected cells to a greater extent than in wild-type MDCKII cells. In an in vivo study, after intravenous injection of fluorescein, the fluorescein brain-to-plasma concentration ratio in mrp1 knockout mice was not significantly different than that in wild-type mice. However, when probenecid was co-administered with fluorescein in wild-type mice, the fluorescein brain-to-plasma ratio was significantly increased (1.5-fold). These findings suggest that fluorescein is a substrate for MRP1. Furthermore, the in vivo study also suggests that MRP1 has a limited role in the transport and distribution of fluorescein in the brain. Therefore, other organic anion transport proteins, including the various isoforms of the MRP family, may be responsible for the accumulation and transport of organic anions in the brain.

Effect of capillary efflux transport inhibition on the determination of probe recovery during in vivo microdialysis in the brain.

Intracerebral microdialysis probe recovery (extraction fraction) may be influenced by several mass transport processes in the brain, including efflux and uptake exchange between brain and blood. Therefore, changes in probe recovery under various experimental conditions can be useful to characterize fundamental drug transport processes. Accordingly, the effect of inhibiting transport on probe recovery was investigated for two capillary efflux transporters with potentially different membrane localization and transport mechanisms, P-glycoprotein and an organic anion transporter. Fluorescein/probenecid and quinidine/LY-335979 were chosen as the substrate/inhibitor combinations for organic anion transport and P-glycoprotein-medicated transport, respectively. Probenecid decreased the probe recovery of fluorescein in frontal cortex, from 0.21 +/- 0.017 to 0.17 +/- 0.020 (p < 0.01). Quantitative microdialysis calculations indicated that probenecid treatment reduced the total brain elimination rate constant by 3-fold from 0.37 to 0.12 (ml/min. ml of extracellular fluid). In contrast, the microdialysis recovery of quinidine, delivered locally to the brain via the probe perfusate, was not sensitive to P-glycoprotein inhibition by systemically administered LY-335979, a potent and specific inhibitor of P-glycoprotein. Recovery of difluorofluorescein, an analog of fluorescein, was also decreased by probenecid in the frontal cortex but not in the ventricle cerebrospinal fluid. These experimental observations are in qualitative agreement with microdialysis theory incorporating mathematical models of transporter kinetics. These studies suggest that only in certain circumstances will efflux inhibition at the blood-brain barrier and blood-cerebrospinal fluid barrier influence the microdialysis probe recovery, and this may depend upon the substrate and inhibitor examined and their routes of administration, the localization and mechanism of the membrane transporter, as well as the microenvironment surrounding the probe.

The use of transgenic mice in pharmacokinetic and pharmacodynamic studies.

Transgenic technology has made it possible to alter the genetic make-up of a laboratory mouse through the removal or insertion of selected genes. The resulting transgenic mouse provides a means for determining the developmental and functional contributions of selected genes and the proteins they encode. The current article reviews examples of the use of transgenic mice in pharmacokinetic and pharmacodynamic studies. In addition to examining current applications of transgenic technology in the areas of pharmacokinetics and pharmacodynamics, the potential for future advancements as well as limitations of the technology are discussed.

Pluronic P85 enhances the delivery of digoxin to the brain: in vitro and in vivo studies.

Drug delivery across the blood-brain barrier is limited by several mechanisms. One important mechanism is drug efflux, mediated by several transport proteins, including P-glycoprotein. The goal of this work was to examine the effect of a novel drug delivery system, Pluronic block copolymer P85, on P-glycoprotein-mediated efflux from the brain using in vitro and in vivo methods. The hypothesis was that specific Pluronic copolymer systems enhance drug delivery to the central nervous system through the inhibition of P-glycoprotein. The effect of P85 on the cellular accumulation and transport of digoxin, a model P-glycoprotein substrate, was examined in porcine kidney epithelial cells (LLC-PK1) transfected with the human MDR1 gene. The effect of P85 on the directional flux across an in vitro BBB was also characterized. In vivo brain distribution studies were accomplished using wild-type and P-glycoprotein knockout mice. Pluronic increased the cellular accumulation of digoxin 3-fold in LLC-PK1 cells and 5-fold in the LLC-PK1-MDR1-transfected cells. Similar effects were observed for a prototypical P-glycoprotein substrate rhodamine-123. P85 treatment decreased the basolateral-to-apical and increased the apical-to-basolateral digoxin flux across LLC-PK1-MDR1 cell monolayers, and analogous results were observed with the in vitro BBB monolayers. The coadministration of 1% P85 with radiolabeled digoxin in wild-type mice increased the brain penetration of digoxin 3-fold and the digoxin level in the P85-treated wild-type mice was similar to that observed in the P-glycoprotein-deficient animals. These data indicate that Pluronic P85 can enhance the delivery of digoxin to the brain through the inhibition of the P-glycoprotein-mediated efflux mechanism.

Effect of probenecid on fluorescein transport in the central nervous system using in vitro and in vivo models.

PURPOSE: The purpose of this study was to characterize the function of multidrug resistance-associated proteins (MRPs) (or MRP-like organic anion transport systems) in the blood-brain harrier (BBB) and blood-cerebrospinal fluid barrier (BCSFB) using both an in vitro BBB model and an in vivo microdialysis model. METHODS: In vitro functional studies were performed using bovine brain microvessel endothelial cells (BBMEC). The accumulation of fluorescein, an anionic fluorescent dye, in BBMEC was determined with and without the presence of inhibitors of various efflux transport proteins. In vivo microdialysis simultaneously monitored fluorescein concentrations in cortical extracellular fluid and cerebrospinal fluid. The effect of probenecid on the in vivo distribution of fluorescein was studied using a balanced crossover design in the rat. RESULTS: In vitro experiments showed that probenecid, indomethacin, LY-329146, and all MRP inhibitors significantly increased (two- to threefold) the accumulation of fluorescein in BBMEC, whereas LY-335979, a P-gp inhibitor, had no effect on the accumulation of fluorescein. Probenecid significantly increased fluorescein plasma concentration and the plasma free fraction in vivo. The distribution of fluorescein across the BBB and BCSFB was enhanced by 2.2- and 1.9-fold, respectively, when probenecid was coadministered, even after correction for increased fluorescein plasma concentrations and free fraction. CONCLUSIONS: These results demonstrate that MRPs or MRP-like transport system(s) may play an important role in fluorescein distribution across both BBB and BCSFB. This study showed that microdialysis proved to be a powerful in vivo technique for the study of transport systems in the central nervous system, and in vitro/in vivo correlations are possible using these model systems.

Microdialysis in the study of drug transporters in the CNS.

Quantitative microdialysis in the central nervous system (CNS) has recently provided evidence for the existence of transporters as they relate to the brain distribution of a variety of drugs. Support for the existence of drug transporters in the blood-brain barrier (or in the blood-CSF barrier) comes from investigations that have found: unbound drug concentrations in brain fluids that are lower than corresponding levels in plasma; saturability of transport clearances across the blood-brain barrier and; the regulation of transport by putative inhibitors. Additional confirmatory evidence for the existence of active transport or carrier-mediated processes has also been derived from models that relate observed drug levels in the CNS with those in plasma or blood. The conclusion that reduced drug levels in brain fluids generally indicate the existence of active efflux transport is questioned. In the case of relatively polar compounds with modest blood-brain barrier permeability, lower unbound concentrations in brain may be a consequence of dilution by turnover of brain fluids. This review summarizes recent reports (grouped by class of compounds) where investigators have used microdialysis to examine the distribution of therapeutic agents to the CNS, and have reached conclusions regarding the functional presence of drug transporters in the brain.

Expression of various multidrug resistance-associated protein (MRP) homologues in brain microvessel endothelial cells.

Multidrug resistance-associated protein (MRP) actively transports a broad range of anionic compounds out of the cell. To date, six different homologues of MRP (i.e. MRP1-MRP6) have been identified. The current study examines the expression of the various MRP homologues in both primary cultured bovine brain microvessel endothelial cells (BBMEC) and the capillary-enriched fraction from bovine brain homogenates. RT-PCR analysis demonstrated the presence of MRP1, MRP4, MRP5 and MRP6 in both BBMEC and the capillary-enriched fractions of brain homogenates. While low levels of MRP3 were detected in the BBMEC, it was not observed in the capillary-enriched fraction. In addition, RT-PCR and Western blot studies indicated an absence of MRP2 expression in both blood-brain barrier preparations. The presence of several different MRP homologues in the brain microvessel endothelial cells may be important in controlling the permeability of the blood-brain barrier to organic anions.

Cyclosporin A has low potency as a calcineurin inhibitor in cells expressing high levels of P-glycoprotein.

Cyclosporin A (CsA) is a widely-used immunosuppressant drug whose therapeutic and toxic actions are mediated through inhibition of calcineurin (CN), a calcium- and calmodulin-dependent phosphatase. Inhibition of CN by CsA requires drug binding to its protein cofactor in the inhibition, cyclophilin. Because cyclophilin is a high affinity target for CsA it is expected that this protein can act as a reservoir for the drug in the cell and may be able to inhibit cellular efflux of CsA. P-glycoprotein (P-gp) is known to increase the rate of CsA efflux from CsA loaded cells but it is not clear if the P-gp drug efflux pump can compete effectively with cyclophilin at therapeutically relevant concentrations of CsA. To test the hypothesis that increased expression of P-gp confers protection against CsA-dependent inhibition of CN phosphatase activity, KB-V cells expressing varying levels of P-gp were analyzed to determine the potency of CsA as a CN inhibitor. When intact cells were treated with CsA, a positive correlation was observed between P-gp expression and resistance to CsA-dependent inhibition of CN: the IC50 is approximately 20-fold higher in the multidrug resistant epidermal carcinoma cell line, KB-V, which expresses P-gp at a high level than in the parental, KB, cell line expressing very low levels of P-gp. The resistance displayed by KB-V cells is abrogated by co-administration of the P-gp inhibitor verapamil, whereas verapamil has no effect on CsA potency in control KB cells. In cell lysates from KB-V cells with different amounts of P-gp CsA exhibits equivalent potency, indicating that the difference in sensitivity to CsA among the cell types requires maintenance of cell integrity. These observations support the view that resistance to CN inhibition by CsA occurs in cells with moderately elevated P-gp activity. Therefore, P-gp activity appears to be an important determinant of CsA cellular specificity for both therapeutic and toxic effects.

Expression of multidrug resistance-associated protein (MRP) in brain microvessel endothelial cells.

Multidrug resistance-associated protein (MRP) is a recently identified drug efflux transport system that actively transports organic acids and selected glucuronide or glutathione conjugates out of the cell. The current study presents, for the first time, both functional and biochemical data demonstrating the presence of MRP in the brain microvessel endothelial cells that form the blood-brain barrier (BBB). Using known MRP inhibitors, such as indomethacin and probenecid, fluorescein accumulation in primary cultured bovine brain microvessel endothelial cell (BBMEC) monolayers was significantly enhanced compared to control. The specificity of the MRP inhibitors on cellular fluorescein accumulation was confirmed using both MRP positive (Panc-1) and MRP negative (KBv) cell lines. Furthermore, western blot analysis using a specific antibody for MRP (MRPm6) and RT-PCR studies using a complementary sequence probe for human MRP demonstrate the expression of MRP in BBMEC. Previous studies have demonstrated the significance of the P-glycoprotein drug efflux transporter in the BBB. Given its function as a drug efflux transport system, it is anticipated that MRP in the BBB will also have an important role in limiting the exposure of the brain to many endogenous and exogenous compounds, including both toxic and therapeutic agents.

The design and validation of a novel intravenous microdialysis probe: application to fluconazole pharmacokinetics in the freely-moving rat model.

PURPOSE: The purpose of this study was to design and validate a concentric, flexible intravenous microdialysis probe to determine drug concentrations in blood from the inferior vena cava of a freely-moving animal model. METHODS: An intravenous microdialysis probe was constructed using fused-silica tubing and an acrylonitrile/sodium methallyl sulfonate copolymer hollow fiber. The probe was tested in vitro for the recovery of fluconazole and UK-54,373, a fluconazole analog used for probe calibration by retrodialysis. Subsequent in vivo validation was done in rats (n = 7) that had a microdialysis probe inserted into the inferior vena cava via the femoral vein, and the femoral artery was cannulated for simultaneous blood sampling. Comparisons of fluconazole pharmacokinetic parameters resulting from the two sampling methods were performed at 2 and 10 days after probe implantation. RESULTS: There were no statistical differences between the microdialysis sampling and conventional blood sampling methods for the T1/2, Cl, Vdss, and dose-normalized AUC by paired t-test (p > 0.05) for repeated dosing at day 2 and day 10 after probe placement. The probe recovery, as determined by retrodialysis, significantly decreased over the ten day period. This finding indicates the necessity for frequent recovery determinations during a long-term blood microdialysis experiment. CONCLUSIONS: These results show that microdialysis sampling in the inferior vena cava using this unique and robust probe design provides an accurate method of determining blood pharmacokinetics in the freely-moving rat for extended experimental periods. The probe design allows for a simple surgical placement into the inferior vena cava which results in a more stable animal preparation for long-term sampling and repeated-measures experimental designs.

Application of microdialysis in pharmacokinetic studies.

The objective of this review is to survey the recent literature regarding the various applications of microdialysis in pharmacokinetics. Microdialysis is a relatively new technique for sampling tissue extracellular fluid that is gaining popularity in pharmacokinetic and pharmacodynamic studies, both in experimental animals and humans. The first part of this review discusses various aspects of the technique with regard to its use in pharmacokinetic studies, such as: quantitation of the microdialysis probe relative recovery, interfacing the sampling technique with analytical instrumentation, and consideration of repeated procedures using the microdialysis probe. The remainder of the review is devoted to a survey of the recent literature concerning pharmacokinetic studies that apply the microdialysis sampling technique. While the majority of the pharmacokinetic studies that have utilized microdialysis have been done in the central nervous system, a growing number of applications are being found in a variety of peripheral tissue types, e.g. skin, muscle, adipose, eye, lung, liver, and blood, and these are considered as well. Given the rising interest in this technique, and the ongoing attempts to adapt it to pharmacokinetic studies, it is clear that microdialysis sampling will have an important place in studying drug disposition and metabolism.

Pharmacological characterization of LY335979: a potent cyclopropyldibenzosuberane modulator of P-glycoprotein.

The above data indicate that LY335979 displays the following characteristics of an 'ideal modulator' of Pgp-mediated multidrug resistance: high affinity binding to Pgp, high potency for in vitro reversal of drug resistance, high therapeutic index (activity was demonstrated at doses ranging from 1-30 mg/kg) observed in in vivo antitumor efficacy experiments, and a lack of pharmacokinetic interactions that alter the plasma concentration of coadministered oncolytic agents. These desirable features strongly suggest that LY335979 is an exciting new clinical agent to test the hypothesis that inhibition of P-glycoprotein activity will result in reversal of multidrug resistance in human tumors.

Fluconazole distribution to the brain: a crossover study in freely-moving rats using in vivo microdialysis.

PURPOSE: The purpose of this study was to determine if the microdialysis sampling technique is feasible to study the central nervous system distributional kinetics of a novel triazole antifungal agent, fluconazole, in an awake, freely-moving rat model, and to determine fluconazole distribution to the extracellular fluid (ECF) of the brain. METHODS: The relative recovery of the microdialysis probes (CMA-12) was determined in vitro and in vivo by retrodialysis using UK-54,373, a fluorinated analog of fluconazole. Sprague-Dawley rats received 10 mg/kg and 20 mg/kg fluconazole IV bolus doses in a crossover design, and brain extracellular fluid fluconazole concentrations were monitored using microdialysis and on-line HPLC analysis. The plasma fluconazole concentration vs. time data were determined using sequential blood sampling and HPLC analysis. RESULTS: There was no statistical difference between relative probe recoveries for both fluconazole and UK-54,373, either in vitro or in vivo, and probe recoveries did not change during the course of the in vivo crossover experiment. Fluconazole rapidly distributes into in the brain ECF and the average brain distribution coefficient (brain/plasma AUC ratio) was 0.60 +/- 0.18 and was independent of dose. Plasma pharmacokinetic parameters were linear in the dose range studied. CONCLUSIONS: Fluconazole rapidly reaches a distributional equilibrium between brain extracellular fluid and plasma, and the distribution to the brain is substantial and not dependent on dose over a two-fold range. Furthermore, the results indicate that microdialysis utilizing UK-54,373 as the in vivo retrodialysis probe calibrator is a feasible method to study the transport of fluconazole into the central nervous system.

The binding of cyclosporin A to human plasma: an in vitro microdialysis study.

PURPOSE: The human plasma binding of cyclosporin A was studied in vitro using the technique of microdialysis. The effect of temperature on the overall binding interaction between cyclosporin A and human plasma was also investigated. METHODS: Flow-through loop-type microdialysis probes were constructed from fused silica tubing and regenerated cellulose tubing with a MWCO of 13000 daltons. Probes were perfused with phosphate buffer (0.5 microliters/min) and the concentration of 3H-cyclosporin A in the well-mixed medium (plasma or buffer) was 1200 ng/ml. Relative recoveries of cyclosporin A from plasma or buffer were determined for each probe by separate experiments to measure the solute gain or loss with reference to the perfusate. RESULTS: Recoveries determined by loss were significantly greater than those determined by gain and in each case temperature dependent, with higher recoveries at higher temperatures. The plasma free fraction of cyclosporin A calculated from the recovery data and the perfusate to plasma concentration ratios was dependent on temperature in a log-linear fashion. Mean +/- s.d. plasma free fractions expressed in percent were 33.5 +/- 4.6, 17.9 +/- 3.6, 6.2 +/- 0.8, 3.0 +/- 0.6, and 1.5 +/- 0.2 at temperatures of 4, 10, 20, 30, and 37 degrees C, respectively. Assuming that the enthalpy of binding is constant over the temperature range studied and pseudo-first order conditions exist, the binding reaction at these temperatures was spontaneous, endothermic (delta H = 74.0 kJ/mole), and entropically driven (delta S = 0.274 kJ/mole/deg). CONCLUSIONS: These results show that the free fraction of cyclosporin A in human plasma is dependent on temperature with the fraction unbound decreasing with temperature in the range of 4 to 37 degrees C. The thermodynamic parameters for the binding of cyclosporin A to plasma components indicate that the reaction is a spontaneous endothermic reaction that is mainly entropy driven, similar to the partitioning of lipophilic molecules from an aqueous to a hydrophobic phase. Moreover, these results show that microdialysis is a feasible method to determine the binding interactions between plasma and cyclosporin A, which indicates the method may be suitable for other difficult binding studies where the solutes have nonspecific binding to separation devices.

Use of rhodamine 123 to examine the functional activity of P-glycoprotein in primary cultured brain microvessel endothelial cell monolayers.

The fluorescent dye, rhodamine 123, was used to evaluate the functional activity of the P-glycoprotein (P-gp) efflux transport system in primary cultured bovine brain microvessel endothelial cell (BBMEC) monolayers. Rhodamine 123 accumulation was increased significantly in BBMEC monolayers treated with the P-gp modifying agent, cyclosporin A (CSA). Rhodamine 123 accumulation was also increased by other P-gp modifying agents. The rank effectiveness of these agents in increasing rhodamine 123 accumulation in BBMEC monolayers was CSA = dipyridamole > verapamil = quinidine. The maximal increase in rhodamine 123 accumulation in CSA treated. BBMEC monolayers was approximately 3 fold greater than in control monolayers and was qualitatively similar to that observed with 3H-vincristine. Comparison of functional activity with the biochemical expression of P-gp in BBMEC monolayers and in an established tumor cell line that over-expresses P-gp indicate that functional activity may be a more descriptive measure of the importance of this drug efflux system than protein expression. Furthermore, these studies suggest that accumulation of rhodamine 123 in BBMEC monolayers can be used to quantitatively examine P-gp activity in the blood-brain barrier.

Effect of the p-glycoprotein inhibitor, cyclosporin A, on the distribution of rhodamine-123 to the brain: an in vivo microdialysis study in freely moving rats.

The p-glycoprotein is a transmembrane efflux transporter found on the luminal side of the capillary endothelial cells that comprise the blood-brain barrier. This study examined the effect of a p-glycoprotein inhibitor, cyclosporin A, on the distribution to the brain of a p-glycoprotein substrate, rhodamine-123, in freely moving rats using intracerebral microdialysis coupled with on-line HPLC analysis. Results from crossover experiments show that the coadministration of cyclosporin A significantly increased the distribution of rhodamine-123 to the brain. The plasma disposition of rhodamine-123 was unchanged by cyclosporin A, indicating that the change in brain exposure was mediated by a process at the level of the blood-brain barrier, possibly by inhibition of the p-glycoprotein efflux transporter. This finding suggests a functional activity of the p-glycoprotein in the blood-brain barrier and validates an in vivo model to examine the role of this transporter in the brain distribution of drugs.

Transsynovial drug distribution: synovial mean transit time of diclofenac and other nonsteroidal antiinflammatory drugs.

The synovial mean transit time of diclofenac was determined by two methods from existing plasma and synovial fluid concentration-time data. These data were obtained from single- and multiple-dosing regimens of diclofenac in patients with osteoarthritis and rheumatoid arthritis. Plasma and synovial fluid concentration-time data taken from the literature for four other nonsteroidal antiinflammatory drugs (etodolac, ibuprofen, indomethacin, and tenoxicam) were also analyzed. The two methods of data analysis rely on the determination of the ratio of the area under the synovial fluid concentration-time curve to the area under the plasma concentration-time curve. Both methods can be considered noncompartmental because in determining the first-order exit rate constant for the synovial fluid (the inverse of the synovial mean transit time), an analysis of the overall distribution and elimination characteristics of the drug is unnecessary. Method 1 makes use of the information contained in the postdistributional synovial fluid to plasma concentration ratio whereas method 2 is a linear pharmacokinetic model using a partial-areas analysis. The single dose mean +/- S.D. synovial fluid exit rate constant for diclofenac was 0.39 +/- 0.33 hr-1 (n = 6), which was not significantly different from that determined by method 2; which was 0.49 +/- 0.52 hr-1. The steady state mean +/- S.D. diclofenac synovial fluid exit rate constants for methods 1 and 2 were 0.43 +/- 0.18 and 0.54 +/- 0.71 hr-1 (n = 8), respectively, which were not significantly different.(ABSTRACT TRUNCATED AT 250 WORDS)