Determination of free interstitial concentrations of piperacillin tazobactam combinations by microdialysis

Transcription

1 Journal of Antimicrobial Chemotherapy (1998) 42, Determination of free interstitial concentrations of piperacillin tazobactam combinations by microdialysis JAC Teresa Dalla Costa, Arno Nolting, Andreas Kovar and Hartmut Derendorf* Department of Pharmaceutics, 10494, College of Pharmacy, University of Florida, Gainesville, FL 32610, USA The investigation of tissue penetration and distribution of antibiotics is of great importance, since infections occur mostly in the tissues. The aim of this study was to investigate the pharmacokinetics of piperacillin and tazobactam, alone and in combination, by measuring total plasma and free interstitial concentrations, and to examine the relationship between free levels of both drugs in blood and those in the extracellular space. Piperacillin and tazobactam were administered, alone and in combination, to anaesthetized rats as a single iv bolus dose. Total plasma concentrations and free extracellular concentrations were quantified by HPLC. In-vivo microdialysis sampling was used to study the free tissue distribution patterns of both drugs. The pharmacokinetics of piperacillin and tazobactam in plasma were consistent with a two-compartment body model. Piperacillin pharmacokinetics were not influenced by co-administration of tazobactam. Tazobactam s volumes of distribution and clearance were decreased by the co-administration of piperacillin and the area under the curve was significantly increased. Comparisons between calculated free concentrations in the peripheral compartment for both drugs and measured free extracellular concentrations revealed excellent agreement. For piperacillin and tazobactam, alone and in combination, predictions of the concentration time profiles of free drug in the peripheral compartment can be made on the basis of plasma data. Introduction Bacterial resistance has rendered some of the older -lactam antibiotics obsolete. The antibacterial effect of -lactam antibiotics depends on their capacity to resist or avoid the barriers imposed by the bacteria. 1 The main mechanism for bacterial resistance to -lactam antibiotics is the production of -lactamases, enzymes that mediate antibiotic degradation. 2 The co-administration of a nonantimicrobial drug capable of inhibiting -lactamase activity in conjunction with a -lactam antibiotic is a strategy that has been used to overcome -lactamase-mediated resistance. Piperacillin has been combined with tazobactam to treat infections caused by -lactamaseproducing bacteria. 3 Piperacillin tazobactam is a very active -lactam -lactamase inhibitor combination, as demonstrated by in-vitro studies. 4 6 The effect of co-administration of piperacillin and tazobactam on the pharmacokinetic behaviour of each of these agents was investigated in humans for different combinations. 7 9 The pharmacokinetics of piperacillin remained unaffected after co-administration with tazobactam in ratios of 1:4 and 1:8. 8 The pharmacokinetics of tazobactam, on the other hand, were significantly affected by the presence of piperacillin. Tazobactam s total body clearance decreased and its volume of distribution (V d ) increased when combined with piperacillin. The plasma concentrations and the half-life of tazobactam were increased after co-administration with piperacillin when compared with tazobactam administration alone. The halflife of tazobactam when administered in combination is similar to the half-life of piperacillin and the plasma levels of the two compounds parallel one another. The changes observed in tazobactam pharmacokinetics when in combination result from the fact that both drugs are eliminated by tubular secretion. Renal elimination of tazobactam and piperacillin accounts for 50 60% of the dose after combined administration. 8 There is competitive inhibition for transport at the renal site in favour of piperacillin. The antibiotic concentrations in plasma are only indirectly important for the cure of infections. The free drug concentrations present at the site of infection are *Corresponding author. Tel: ; Fax: ; hartmut@cop.health.ufl.edu 1998 The British Society for Antimicrobial Chemotherapy 769

2 T. Dalla Costa et al. responsible for the antimicrobial effect. The antibiotics have to distribute from the blood into the infected tissue in order to be active against microorganisms. Only the free, unbound fraction of the antibiotic is available to interact with bacteria. Hence, the determination of plasma pharmacokinetics must be conducted together with the determination of free levels of the drug in the tissue. Microdialysis is a suitable technique for sampling actual free interstitial concentrations since it does not disturb the physiological conditions. Its potential application for pharmacokinetic studies has increased over the years not only for research in animals 5,7,10 14 but also for investigations in humans In a previous publication 19 we reported the results of the investigation of piperacillin pharmacokinetics in rats based on total plasma levels and free interstitial levels in muscle obtained by microdialysis. The objectives of the present study were to elucidate the pharmacokinetics of tazobactam alone, to determine the pharmacokinetics of tazobactam combined with piperacillin when administered to rats in different dose combinations (1:4 and 1:8), to test the hypothesis that the concentration time profile of piperacillin in plasma and tissue is not affected by the administration of tazobactam, and to test the hypothesis that diffusion is the mechanism that drives the distribution of piperacillin and tazobactam between blood and tissues when administered alone or in combination. In order to reach these goals the pharmacokinetics of both drugs were investigated by two approaches: the traditional one based on plasma data analysis, and a novel approach using microdialysis to investigate the free interstitial levels available at the site of action. Materials and methods Chemicals and reagents Piperacillin and p-aminobenzoic acid were purchased from Sigma Pharmaceuticals (St Louis, MO, USA) and used as received. Tazobactam was donated by Lederle (Münster, Germany). Antipyrine was purchased from Aldrich Chemical Company (Milwaukee, WI, USA). HPLC-grade acetonitrile and tetrabutylammonium hydroxide (TBA) were purchased from Fisher Scientific (Fair Lawn, NJ, USA). All other reagents were of analytical grade and purchased from Fisher Scientific. Drug assay The assay for determination of tazobactam in biological fluids by HPLC was developed from previously published procedures. 20,21 The HPLC assay used for piperacillin was previously described. 19 Both assays were validated by performing three calibration curves on each of three different days and analysing quality control samples. For tazobactam a linear calibration curve was obtained in the range mg/l using peak height ratios. For piperacillin a linear calibration curve was obtained in the range mg/l using peak area ratios. Between-day and within-day variability was determined for four quality control concentrations for each drug and was not greater than 10%. The lower limit of sensitivity for piperacillin and tazobactam in plasma was 2 mg/l and 3 mg/l, respectively. For the microdialysis studies the lower limit of sensitivity was 1 mg/l for both drugs. Experimental design Tazobactam and piperacillin are administered to humans in two dose ratios, 1:4 and 1:8. In a previous study to investigate the pharmacokinetics of piperacillin alone in rats, two different doses were used, namely 60 and 120 mg/kg. 19 The dose ratios studied in this project were based on the same doses of piperacillin in order to allow comparisons. Male Wistar rats weighing g were divided into seven groups of six animals each. The pharmacokinetics of tazobactam alone was studied for three different doses: 15, 30 and 60 mg/kg body weight. To determine the influence of piperacillin on tazobactam pharmacokinetics, three combinations were studied: tazobactam 15 mg/kg with piperacillin 60 mg/kg (1:4) or piperacillin 120 mg/kg (1:8), and tazobactam 30 mg/kg with piperacillin 120 mg/kg (1:4). The influence of tazobactam on piperacillin pharmacokinetics was determined for two combinations, tazobactam 15 mg/kg or 30 mg/kg combined with piperacillin 60 mg/kg body weight (1:4 or 1:2). Although the tazobactam piperacillin dose ratio 1:2 is not used for treatment of infections, it was investigated in this project to determine whether even higher doses of tazobactam would have an effect on piperacillin pharmacokinetics. In all studies the drugs were administered as iv bolus injection. Surgical procedure The animal procedure was approved by the Institutional Animal Care and Use Committee of the University of Florida (IACUC). Rats were anaesthetized with ethylcarbamate (1.25 g/kg ip). After complete anaesthesia the animals were immobilized in a supine position and a catheter was inserted into the carotid artery (polyethylene catheter with an inner diameter of 0.3 mm and an outer diameter of 0.7 mm). The artery was irrigated with heparinized saline. The left hind leg muscle was used for the insertion of the microdialysis probe after skin removal. The microdialysis probe was allowed to equilibrate inside the muscle for 1 h before drug administration. Drugs were injected as iv bolus injection (0.5 ml/100 g) via the femoral vein of the right hind leg. After the completion of the injection (time zero), blood and microdialysis tissue samples were drawn. Microdialysis samples were collected 770

3 Pharmacokinetics of piperacillin tazobactam over a 20 min interval. Blood samples were collected immediately before drug administration and at 2, 5, 10, 15, 20, 30, 45, 60, 90 and 120 min. Blood samples ( L) were harvested into heparinized tubes. After centrifugation, plasma samples for tazobactam analysis were immediately processed as described in the drug assay section. The residues were kept in glass tubes at 5 C until assayed. Plasma samples for piperacillin analysis were frozen and stored at 5 C until assayed. Microdialysis conditions Microdialysis was used to determine free tissue concentrations of the drugs under investigation. A single microdialysis probe was inserted into the left leg muscle of the rat when tazobactam was administered alone. For the investigation of tissue concentrations after administration of the combination of tazobactam and piperacillin, two probes were inserted into the same muscle. Each probe was previously calibrated in vitro for either piperacillin or tazobactam. The microdialysis pump was set at a flow rate of 1.5 L/min. Ringer s solution (148 mm Na, 2.3 mm Ca 2, 4 mm K, and 157 mm Cl ) was used as perfusion fluid. Dialysate samples were collected at 20 min intervals and immediately analysed by HPLC. Since microdialysate concentrations are time averaged over the collection interval these values were translated into the concentration at a single time point by assuming that the concentration obtained is the actual concentration at the middle-point of the time interval. Microdialysis system and probe calibration in vitro The microdialysis system consisted of a Harvard Apparatus Pump 22 connected to a microlitre syringe (1 ml, gas-tight) to provide the perfusate solution. The syringe was connected to the flexible loop probe (tip length 4 mm, molecular weight cut-off 6000 Da) (ESA, Inc., Bedford, MA, USA) by using fused-silica connecting tubes. The lag time due to the dead volume between the sampling site and the point of dialysate collection was calculated to be 27 s. The lag time was considered negligible and no corrections were made in the timepoints. Before each in-vivo experiment, the microdialysis probes were calibrated in vitro. The probes were put into Ringer s solution containing either tazobactam or piperacillin 100 mg/l and allowed to equilibrate for 1 h at 37 C. Ringer s solution was perfused at 1.5 µl/min. After the equilibration period, three samples were collected at 20 min intervals and analysed by HPLC. The recovery was determined as the ratio of dialysate concentration over outside concentration 100. Under these conditions the recoveries of tazobactam and piperacillin were found to be in the range 18 32% and 8 14%, respectively. Tazobactam protein binding determination The protein binding was determined as the ratio between free tissue concentration and total plasma concentration obtained at steady state following a constant intravenous infusion. At steady state the free concentrations of the drug in tissue and plasma are in equilibrium and the ratio between free concentration in tissue and total concentration in plasma can be used to assess the fraction bound to plasma proteins. For the infusion experiments a loading dose followed by a maintenance dose of tazobactam alone were given in order to reach a steady state concentration in plasma of 50 mg/l. Seventy-five minutes after the tazobactam loading dose, piperacillin was also administered to the same animal in order to determine the influence of this drug on the protein binding of tazobactam. Piperacillin was administered as a loading dose followed by a maintenance dose to reach a steady state concentration in plasma of 200 mg/l (1:4 tazobactam:piperacillin ratio). The maintenance doses were given in form of a constant intravenous infusion in the femoral vein. A flow rate of 2 ml/h was maintained with a Flo-Gard 8000 volumetric infusion pump (Travenol Laboratories, Deerfield, IL, USA). Microdialysis was performed as described for the single dose administration. One hour of probe equilibration in the muscle was allowed before drug administration. Microdialysis samples were collected every 20 min. Blood samples were collected before dosing and at 15, 30, 45, 60 and 75 min to analyse tazobactam levels administered alone and at 85, 100, 120, 140, 180, 210 and 240 min for analysis of tazobactam administered in combination. A total of three animals were used for the infusion studies. The loading and maintenance doses were calculated based on pharmacokinetic parameters estimated from single intravenous dose administration of tazobactam or piperacillin with the following equations: LD V c Cp ss (1) where LD is the loading dose, V c is the volume of distribution of the central compartment and Cp ss is the target concentration in plasma. The infusion rate, K 0, was calculated as follows: where Cl is the total clearance. K 0 Cp ss Cl (2) Microdialysis probe calibration in vivo The in-vivo recovery of the microdialysis probe for tazobactam was determined by using the point-of-no-net-flux method. 22 For one group of three animals, a loading dose followed by a maintenance dose of tazobactam alone were administered to obtain a steady state concentration of 50 mg/l. The same infusion conditions described for 771

4 T. Dalla Costa et al. protein binding determination were followed. One hour after the loading dose was administered (steady-state condition established), three microdialysis samples were collected with plain Ringer s solution as perfusate. For the subsequent samples, tazobactam was added in different concentrations (7.5, 15 or 30 mg/l) to the perfusion fluid. After 1 h equilibration with the new perfusate concentration, the net dialysate concentration was determined by HPLC for three samples. The net concentration in the dialysate solution was plotted against the initial perfusate concentration. The intercept of the plot with the x-axis equals the free concentration of tazobactam in the tissue at steady state. The slope of the line is the in-vivo recovery. Estimation of pharmacokinetic parameters The pharmacokinetic parameters of tazobactam alone and in combination as well as the parameters of piperacillin in combination were estimated for each animal with classical non-compartmental equations. 23 The terminal elimination rate constant (k e ) was estimated from the log-linear plot of concentration versus time. The area under the concentration time curve (AUC) and the area under the first moment curve (AUMC) were calculated using the trapezoidal rule. The mean residence time (MRT), halflife (t 1/2 ), volumes of distribution (V c, Vd ss and Vd area ) and total clearance (Cl) were also determined. The compartmental analysis was performed with the computer program SCIENTIST (MicroMath, Salt Lake City, UT, USA). The average results for both drugs alone and in combination were fitted with a two-compartment body model according to the following equation: C p A e t B e t (3) Statistical analysis The values of pharmacokinetic parameters obtained from the non-compartmental approach were compared by analysis of variance (ANOVA). When significant differences were observed, Duncan s multiple range test was applied for individual comparisons. For the compartmental analysis the model selection criterion (MSC) was used to determine the goodness of the curve fit. Results Piperacillin pharmacokinetics The results of the non-compartmental analysis of piperacillin 60 mg/kg in plasma and in tissue, alone 19 and in combination with tazobactam 15 and 30 mg/kg (1:4 and 1:2 ratios) are summarized in Table I. There was no statistically significant difference between the noncompartmental parameters estimated for piperacillin in either combination (1:2 and 1:4), leading to the conclusion that if tazobactam has an effect on piperacillin pharmacokinetics this effect is not dose dependent. When piperacillin alone was compared with piperacillin in combination, no significant difference was observed, showing that the administration of tazobactam does not affect piperacillin pharmacokinetics. The volume of distribution in the central compartment (V c ) was the only parameter that showed a significant difference when piperacillin alone was compared with piperacillin (1:4) or piperacillin combined average. Concentration time profiles of free piperacillin in plasma and tissue after administration of 60 mg/kg in combination with tazobactam are shown in Figure 1. The where C p is the total plasma concentration at time t, and are the hybrid constants for the distribution and elimination phases respectively, and A and B are the corresponding zero-time intercepts. All data points were weighted equally for the compartmental fitting. The concentrations of free piperacillin and tazobactam in the peripheral compartment were predicted based on plasma pharmacokinetic parameters obtained from the plasma data with the following equation: 24 C tissue fu D k 21 (e t e t ) (4) V c ( ) where D k 21 A B V c where fu is the unbound fraction of the drug in plasma, D is the dose administered as iv bolus injection and k 21 is the first-order rate constant from the peripheral to the central compartment. (5) Figure 1. Concentration time profiles for free piperacillin concentrations in plasma ( ) and interstitial fluid ( ) after administration of 60 mg/kg iv bolus combined with tazobactam. Plasma concentrations were fitted to a two-compartment body model. The line for free tissue concentrations represents the prediction based on plasma data. Points represent mean SD of 12 animals. 772

5 Pharmacokinetics of piperacillin tazobactam Table I. Pharmacokinetic parameters of piperacillin 60 mg/kg alone and in combination with tazobactam 30 mg/kg (1:2), tazobactam 15 mg/kg (1:4) and average of both combinations pooled (mean SD) Piperacillin tazobactam Pharmacokinetic parameter Piperacillin alone a 1:2 1:4 Mean Plasma AUC (mg.min/l) Plasma MRT (min) Plasma half-life (min) V c (L/kg) b b Vd ss (L/kg) Vd area (L/kg) Cl (ml/min/kg) Tissue AUC ( g.min/ml) Tissue MRT (min) Tissue half-life (min) a From Nolting et al. 19 b P < values shown represent the average of all 12 animals from the two groups (1:2 and 1:4). The free plasma concentration time profile of piperacillin could be fitted to a twocompartment body model as was shown earlier for piperacillin alone. 19 Free concentrations in tissue were predicted by using equation 4 and are also shown in Figure 1 together with the free interstitial levels measured by microdialysis. The hybrid constants used for these predictions were obtained from the plasma fits, A mg/l, B mg/l, min 1, and min 1. The value used for the fraction of piperacillin unbound (fu 0.55) was determined previously for piperacillin alone. 13 It can be seen that the predicted line is in good agreement with the measured free interstitial concentrations, showing that diffusion is the process that governs the transfer of piperacillin between blood and tissue. After a short distribution phase, the concentrations in tissue and plasma are in equilibrium. The concentration in the peripheral compartment is higher than the concentration in the central compartment due to elimination from the central compartment only. As expected from a diffusion-driven process, the slopes of the terminal phases are similar, showing that the disappearance of the drug from the two compartments occurs at a similar rate. microdialysis data obtained for tazobactam administered alone or in combination with piperacillin. Protein binding determination Tazobactam protein binding was calculated as the ratio between the free interstitial concentration and the total plasma concentration obtained after constant intravenous infusion. The average concentrations in plasma and tissue obtained after three experiments are shown in Figure 2. Under these conditions the fraction unbound was determined to be for tazobactam administered alone and for tazobactam in combination with piperacillin. Because no significant statistical difference was observed, the overall fraction unbound was calculated as Tazobactam probe calibration in-vivo The result of the in-vivo calibration for tazobactam according to the point of no net flux, based on the slope of the regression line obtained, resulted in recoveries in the order of 23 27% for different probes. A correction factor of 1.16 was obtained for the correlation between in-vitro and in-vivo recoveries and was used to normalize the Figure 2. Tazobactam total plasma concentrations ( ) and free interstitial concentrations ( ) at steady state following a constant iv infusion. Tazobactam was administered alone followed by a leading dose of piperacillin and maintenance doses of tazobactam and piperacillin (1:4) at 85 min. Points represent mean SD of three animals. 773

6 T. Dalla Costa et al. The average total plasma concentration of tazobactam alone under the infusion conditions described above was 35 2 mg/l. Based on this total plasma concentration and the unbound fraction determined as above, the expected free interstitial concentration of tazobactam is in the range mg/l. The free concentration determined during the in-vivo calibration in different animals (9 mg/l) is very close to this range. This result further validates the protein binding value determined in this study. Tazobactam pharmacokinetics Tazobactam alone. Tazobactam alone was administered in three different doses, namely 15, 30 and 60 mg/kg. Total plasma concentration time profiles for these three doses are shown in Figure 3a. As can be seen from the plot, a two-compartment body model sufficiently describes the data in all three cases. Results of the compartmental analysis performed with the data are shown in Table II. The MSC ( 3) and the correlation coefficients (0.99) obtained for these fittings confirm the choice of the model. Figure 3. Total plasma concentration time profile of tazobactam alone: 15 mg/kg ( ), 30 mg/kg ( ) and 60 mg/kg ( ) (a); and total plasma concentration time profile of tazobactam 15 mg/kg alone ( ) and combined with piperacillin 60 mg/kg (1:4) ( ) and 120 mg/kg (1:8) ( ) (b). Points represent mean SD of six animals. Results of the non-compartmental analysis of the plasma and tissue data are summarized in Table III. Linear pharmacokinetics can be observed for the two lowest doses, 15 and 30 mg/kg. As expected, the AUC doubled when the dose was increased from 15 to 30 mg/kg. A trend of decreasing clearance with increasing concentration was observed, but the differences did not prove to be significant, probably due to the high variability observed in these experiments. All the other parameters estimated were not statistically different. Non-linear pharmacokinetic behaviour was observed between the doses 30 and 60 mg/kg. The AUC increased more than three times between these two doses and the difference was statistically significant. The average halflife and MRT also increased, although the differences were not statistically significant. The increase in half-life for the highest dose can also be observed in Figure 3a. The differences shown for the plasma pharmacokinetics were also observed for the tissue data. No data are reported for tissue with a tazobactam 15 mg/kg dose because the concentrations were very close to the lower limit of sensitivity of the analytical method. The results of the non-compartmental analysis in tissue for the two highest doses are shown in Table III. The tissue AUC increased more than three times when the dose was increased from 30 to 60 mg/kg, following the results observed in plasma. The MRT and the half-life in tissue showed a significant increase, confirming the trend observed for the plasma data. As expected, the MRT and the half-life did not differ significantly between plasma and tissue for each dose analysed individually. Since the AUC increased proportionally in plasma and tissue, the fraction unbound determined by the ratio between AUC tissue and AUC plasma was similar for both doses. These values are in good agreement with the tazobactam protein binding determined previously in the infusion experiments. According to the results presented one can infer that tazobactam non-linearity observed between these two doses (30 and 60 mg/kg) is not related to saturation of protein binding sites. The predicted free tissue concentrations of tazobactam following doses of 30 mg/kg and 60 mg/kg administered alone are based on total plasma data together with the free plasma concentrations and are shown in Figure 4a and b. The predictions were calculated by using equation 4 and the hybrid constants (Table II) obtained from plasma data. The fraction unbound used in each case is shown in Table III. As observed for piperacillin, after equilibrium is reached between free concentrations in tissue and in plasma, the concentrations in tissue are slightly higher than in plasma due to elimination from the central compartment. The slopes of the terminal phases are similar in both cases showing that diffusion is the process that drives the distribution of tazobactam between blood and tissue. It can be seen that the predictions are in good agreement with the measured free tissue concentrations, proving that 774

7 Pharmacokinetics of piperacillin tazobactam Table II. Pharmacokinetic parameters obtained by compartmental analysis of the plasma concentration time profiles of tazobactam alone or in combination with piperacillin (mean SD) Tazobactam alone Tazobactam 15 mg/kg combined with 15 mg/kg 30 mg/kg 60 mg/kg piperacillin piperacillin piperacillin Pharmacokinetic 60 mg/kg 120 mg/kg 120 mg/kg parameter (1:4) (1:8) (1:4) A (mg/l) B (mg/l) (min 1 ) (min 1 ) AUC (mg.min/l) Half-life (min) V c (L/kg) Vd ss (L/kg) Vd area (L/kg) Cl (ml/min/kg) Table III. Pharmacokinetic parameters of tazobactam alone and in combination with piperacillin determined by non-compartmental analysis. (Mean SD) Tazobactam alone Tazobactam 15 mg/kg combined with 15 mg/kg 30 mg/kg 60 mg/kg piperacillin piperacillin piperacillin Pharmacokinetic 60 mg/kg 120 mg/kg 120 mg/kg parameter (1:4) (1:8) (1:4) Plasma AUC (mg.min/l) a a a Plasma MRT (min) Plasma half-life (min) V c (L/kg) Vd ss (L/kg) Vd area (L/kg) Cl (ml/min/kg) Tissue AUC (mg.min/l) ND a a ND ND Tissue MRT (min) ND a a ND ND Tissue half-life (min) ND a a ND ND Fraction unbound ND ND ND ND; not determined. a P <0.05. it is possible to use pharmacokinetic parameters estimated from total plasma data to predict free interstitial concentrations of tazobactam administered alone. Tazobactam combined with piperacillin. Tazobactam 15 mg/kg was administered combined with piperacillin in two different ratios, 1:4 and 1:8. The concentration time profiles of plasma data for these two combinations as well as for the same dose administered alone are shown in Figure 3b. The results of the curve fitting are presented in Table II. Tazobactam administered in combination with piperacillin showed higher plasma concentrations than 775

8 T. Dalla Costa et al. Figure 4. Concentration time profiles of free tazobactam concentrations in plasma (filled symbols) and interstitial fluid (open symbols) after administration of 30 mg/kg (a, circles), 60 mg/kg (b, triangles) iv bolus and 30 mg/kg of tazobactam combined with 120 mg/kg of piperacillin (1:4) (c, squares). Plasma concentrations fitted to a two-compartment body model. The line for free tissue concentrations represents the prediction based on plasma data. Points represent mean SD of six animals. when administered alone. A two-compartment body model can be used to describe the data in all cases. The goodness of fit obtained confirms the appropriateness of the model. The results of the non-compartmental analysis performed for these two combinations are shown in Table III together with the results for tazobactam 15 mg/kg alone. There was a trend towards increased AUC with an increased proportion of piperacillin in the combination. The difference was significant for the combination tazobactam piperacillin 1:8, where the AUC was twice that with tazobactam alone. A statistically significant decrease in tazobactam volumes of distribution (V c, Vd ss and Vd area ) was observed when piperacillin was administered in combination compared with tazobactam alone. As observed in humans, the co-administration of piperacillin decreased the clearance of tazobactam because piperacillin interferes with tazobactam tubular secretion. For tazobactam in combination, the clearance was significantly lower than that for tazobactam alone. The proportion of piperacillin in combination did not seem to affect tazobactam volume of distribution and clearance in a different fashion since between the two combinations studied no statistically significant difference was observed for these parameters. Once tazobactam volume of distribution as well as the clearance are decreased in the same order of magnitude by the administration of piperacillin, the halflife is expected to remain constant. There was no significant difference in half-life for tazobactam alone compared with tazobactam in combination. Similar MRTs were also observed for these cases. A higher dose of tazobactam (30 mg/kg) was also administered in combination with piperacillin 120 mg/kg (1:4). The results of the non-compartmental analysis of this combination are shown in Table III. The comparison between the AUC plasma for tazobactam 30 mg/kg in combination and 30 mg/kg alone confirm the trend observed for the lower dose. The addition of piperacillin in combination increased the plasma AUC for tazobactam, although the difference was not statistically significant. The same can be said for the volumes of distribution (V c, Vd ss and Vd area ) and clearance. These parameters were decreased by the administration of piperacillin but the differences were not statistically significant. As observed for 15 mg/kg, the administration of piperacillin did not affect the MRT or half-life in plasma. The analysis of the results in tissue led to similar conclusions. Piperacillin caused a statistically significant increase in the tazobactam tissue AUC, confirming the results observed in plasma. No statistically significant differences were observed for MRT and half-life in tissue when tazobactam 30 mg/kg alone and in a 1:4 combination with piperacillin were compared. The same parameters are in good agreement when plasma and tissue data are compared for tazobactam combined with piperacillin. Since both plasma and tissue AUC for tazobactam increased by a mean of 44% on concomitant 776

9 Pharmacokinetics of piperacillin tazobactam administration of piperacillin, the resulting fraction unbound estimated from these parameters is similar to the one calculated for tazobactam alone. Free interstitial concentrations of tazobactam for a dose of 30 mg/kg combined with piperacillin 120 mg/kg (1:4) were estimated based on parameters obtained from fitting plasma data to a two-compartment model (Table II) by using equation 4. The measured free concentrations in the interstitial fluid obtained by microdialysis and the predictions based on plasma data are shown in Figure 4c. The fraction unbound used for this prediction is presented in Table III. As observed for tazobactam 30 mg/kg alone, tazobactam in combination also follows a two-compartment body model. The measured free tissue concentrations were in good agreement with the values predicted from plasma data. The rates of drug disappearance from the central and peripheral compartments were similar and elimination occured only from the central compartment. Diffusion is the process that drives distribution of the drug between blood and interstitial space showing that it is possible to predict free interstitial fluid levels of tazobactam from plasma pharmacokinetics, when the drug is combined with piperacillin. Discussion The main purpose of this study was to investigate the pharmacokinetics of piperacillin and tazobactam in plasma and tissue when administered alone and in combination. Microdialysis was used to measure the free concentrations of both drugs in rat muscle interstitial fluid. The determination of free concentrations in tissue is an important issue when dealing with anti-infective agents because only the drug unbound to proteins is available to interact with the microorganisms at the infection site. The results presented showed that microdialysis is a suitable technique for investigating levels of free tazobactam and piperacillin alone and in combination. The determination of protein binding is another important aspect when studying anti-infective agents. Tazobactam, a -lactamase inhibitor, is very unstable in rat plasma, making it difficult to use standard methods for protein binding determination. This study shows that a pharmacokinetic approach can be used to estimate the protein binding. At steady state, following a constant intravenous infusion, the ratio between free concentration in tissue and total concentration in plasma is similar to the unbound fraction of the drug. Free tissue concentrations predicted from the protein binding of tazobactam determined in this way were comparable to the free concentration in the interstitial fluid observed in the in-vivo calibration of the microdialysis probes from the point of no net flux. Further validation of the tazobactam protein binding estimate was obtained from the ratio between AUC tissue and AUC total plasma. Very good agreement in the results was observed for all three approaches. The pharmacokinetics of piperacillin in plasma and tissue were not influenced by the co-administration of tazobactam. Even in a dose ratio higher than normally administered to patients (1:2), piperacillin pharmacokinetic parameters were not significantly different from the parameters estimated for piperacillin alone. This behaviour agrees with the results of studies performed in humans. Concentration time profiles of piperacillin in combination with tazobactam could be described by a twocompartment body model. The measured levels of free piperacillin in tissue could be predicted from plasma pharmacokinetics, showing that the distribution of this drug between blood and tissue is mainly governed by diffusion. The pharmacokinetics of tazobactam alone were investigated for three different doses. A two-compartment body model could be used to describe the data in all cases. A linear relationship between concentration and the two lower doses (15 and 30 mg/kg) was observed. There was no significant difference in the elimination half-lives from the central compartment for both doses. Non-linear behaviour was observed between doses of 30 and 60 mg/kg. The protein binding for all three doses was constant, indicating that the non-linearity observed is not related to saturation of protein binding sites. The free interstitial concentrations measured confirmed that diffusion is the force that drives tazobactam distribution between blood and tissue. The co-administration of piperacillin affected the pharmacokinetics of tazobactam in rats, as expected. Plasma and tissue concentrations were higher than those achieved with tazobactam administered alone. Piperacillin decreased the volume of distribution and clearance of tazobactam. One explanation for the effect observed in the clearance could be the interference of piperacillin with the tubular secretion of tazobactam as it was shown previously for humans. No changes in protein binding were observed for the combination. Tazobactam combined with piperacillin could also be fitted to a twocompartment pharmacokinetic model and diffusion was again shown to be the driving force for drug distribution. The ultimate goal of the pharmacokinetic studies was to prove that it is possible to predict tissue levels of free piperacillin and tazobactam, alone and in combination, based on parameters derived from plasma data. The good agreement observed between the measured free interstitial concentrations for both drugs alone and in combination and the predicted levels obtained on the basis of plasma pharmacokinetics confirms this hypothesis. In this way, concentration time profiles of free piperacillin and tazobactam combinations in the interstitial fluid (the site of action) could be predicted and simulated against microorganisms in an in-vitro model of infection in order to evaluate their pharmacodynamic effect. 777

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