Pharmacokinetic–pharmacodynamic modelling of the electroencephalogram effect of imipenem in rats with experimental hypovolaemia or endotoxaemia

A. Limosin1,2, A. Dupuis1,3, I. Lamarche1, O. Mimoz1,4, J. Paquereau5 and W. Couet1,*

1 EE Médicaments Anti-infectieux et Barrière Hémato-encéphalique; 5 Equipe Sommeil: Attention et Respiration, PBS, Faculté de Médecine & Pharmacie, 40 avenue du Recteur Pineau, 86022 Poitiers Cedex; 2 Laboratoire de Pharmacocinétique, PBS; 3 Pharmacie Centrale, CHU La Milétrie, 86022 Poitiers; 4 Département d'Anesthésie et Réanimation Chirurgicale, CHU La Milétrie, 86000 Poitiers Cedex, France

Received 20 February 2004; returned 10 March 2004; revised 22 March 2004; accepted 1 April 2004


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Objectives: The epileptogenic activity of imipenem in rats with experimentally induced hypovolaemia or endotoxaemia was investigated by pharmacokinetic–pharmacodynamic modelling of the electroencephalogram effect.

Methods: Hypovolaemia was induced by removal of 30% of the blood volume and endotoxaemia by intravenous lipopolysaccharide injection.

Results: Imipenem clearance and volume of distribution values of 16.4±1.1 mL/min per kg and 357±49 mL/kg (mean±S.E.M.) in healthy rats (n=5), were significantly reduced in hypovolaemic (n=6) and endotoxaemic (n=6) animals. A dose reduction from 250 mg/kg to 120 mg/kg was necessary in endotoxaemic rats. The pharmacokinetic–pharmacodynamic model with an effect compartment previously developed in healthy rats described the data adequately and pharmacodynamic parameters in hypovolaemic and endotoxaemic rats were not significantly different from corresponding values estimated in the control group.

Conclusion: Hypovolaemia and endotoxaemia only had an effect on imipenem pharmacokinetics.

Keywords: carbapenems , EEG , epileptogenic


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Imipenem is the leading compound of the carbapenem antibiotic family. It possesses a broad spectrum of antibacterial activity against most Gram-positive and Gram-negative aerobic and anaerobic bacteria including Pseudomonas aeruginosa, Bacteroides fragilis and Listeria monocytogenes.1 It is therefore of great value for the treatment of severe infections of all body systems including intra-abdominal infections,2 gynaecological infections,3 lower respiratory tract infections,4 as well as infections in children5 and in febrile neutropenic patients.6 However, imipenem possesses a relatively high convulsant activity which needs to be seriously taken into consideration.7

We have previously developed and assessed the robustness of a pharmacokinetic–pharmacodynamic (PK-PD) modelling approach using quantitative electroencephalogram (EEG) recording to investigate the epileptogenic activity of imipenem in rats.8 The complex relationship between EEG effect and imipenem plasma concentration was successfully described using a PK-PD model with a compartment effect.8 Because imipenem induces seizures most frequently in patients with renal dysfunction,9 this PK-PD modelling approach was used to investigate its epileptogenic activity in rats with experimental renal failure.10 Other risk factors for seizures such as hypovolaemia or endotoxaemia are frequently encountered in critical care patients and may have an impact on imipenem PK as well as on its affinity for the PD receptors at the central nervous system (CNS) level. The CNS sensitivity of barbiturates11 and benzodiazepines12 in rats was found to be increased during hypovolaemia, whereas the theophylline-induced neurotoxicity13 and the depressant activity of zoxazolamine14 were unaffected. Using a PK-PD modelling approach comparable to the one we used to investigate the convulsant activity of imipenem,8 De Paepe et al. have demonstrated that hypovolaemia was responsible for an increased hypnotic effect of propofol in rats which was attributed to both PK and PD changes,15 whereas the effect observed with etomidate was mainly attributed to PK changes,16 and no effect was observed with gamma-hydroxybutyrate.17 These authors also studied the effect of endotoxaemia on the propofol PK-PD relationship in rats using the same experimental approach, and observed an increased efficacy of propofol that was mainly attributed to PK changes.18

The purpose of this study was therefore to extend PK-PD modelling investigations of the imipenem EEG effect previously conducted in healthy rats8 and in rats with experimentally induced renal failure,10 to animals with experimental models of hypovolaemia and endotoxaemia, two disease states frequently encountered in critical care patients treated with imipenem.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Chemicals and reagents

Imipenem monohydrate-sodium cilastatin salt (Tienam; Merck, Sharp & Dohme Laboratories, France) was used to prepare imipenem solutions in 0.9% NaCl, for intravenous (iv) administration. All chemicals used were of analytical grade and analytical solvents were of HPLC grade. Lipopolysaccharides (LPS) (Escherichia coli serotype 0127:B8) were supplied by Sigma.

Animals

Male Sprague–Dawley rats (Depres Breeding Laboratories, St Doulchard, France) weighing 303±35 g were used. The animals were placed in wire cages in a 12 h light–dark cycle for 1 week to adjust to the new environment and to overcome stress possibly incurred during transit. They had free access to food (A04; UAR Laboratories, France) and water. Ethical approval was obtained from the Animal Ethics Committee of the Faculty of Pharmacy (BHE/2001/12/AF).

Surgery

Five days before the experiment, each rat was placed on a stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA). While under anaesthesia [33 mg/kg of ketamine (Ketalar); Parke Davis Laboratories, France and 7 mg/kg of xylazine hydrochloride (Rompun); Sanofi Laboratories, France], a vertical incision was made in the skin to expose the skull and to implant four cortical EEG electrodes. The electrodes were screwed into little holes drilled into the skull at the following positions, in relation to bregma: 2 mm anterior, 2 mm lateral (F1 and F2); 4 mm posterior right, 2 mm lateral (O2); and 4 mm anterior left, 2 mm lateral (reference electrode). The stainless steel electrodes were connected to a miniature plug (Electro Din 5 pins, ref. MDF5B; OHM Electronique, Poitiers, France) fixed to the skull with two types of dental cement. The first cement with aqueous solvent (Aquacem, Denstsply, Germany) was used directly in contact with the skull and a second cement with organic solvent (Heliotone, France) was then used to finalize the mounting. The day before the experiment, two permanent polyethylene catheters were implanted while the rat was under anaesthesia (thiopental sodium, 60 mg/kg; Sanofi Laboratories, France): one in the left femoral vein for drug administration and the other one in the left femoral artery for blood sample collection. Animals were housed individually in plastic cages and had free access to water until drug administration.

Imipenem administration

Imipenem monohydrate-sodium cilastatin salt (Tienam; Merck, Sharp & Dohme Laboratories, France) was used to prepare a 15.9 mg/mL solution of imipenem in 0.9% NaCl, infused using a motor-driven syringe pump (Program 2; Vial Inc.).

Hypovolaemic animals. On the day of the experiment, hypovolaemia was induced in rats (n=6) by removing 30% of the initial blood volume (assumed to be 60 mL/kg) in six increments over 30 min through the arterial line. Infusion was started 30 min after the last blood removal. The imipenem dose was equal to 250 mg/kg.

Endotoxaemic animals. On the day of the experiment, endotoxaemia was induced in rats (n=6) 6 h before imipenem administration (H-6), by intravenous administration of LPS dissolved in isotonic saline (2 mg/mL) at a bolus dose of 4 mg/kg. Two arterial blood samples (400 µL) were withdrawn at H-6 and H0 (start of imipenem infusion) for measurement of creatinine and HCO3 plasma levels, and plasma activities of aspartate aminotransferase and alanine aminotransferase. Imipenem was administered at a 120 mg/kg dose.

Control animals. Arterial blood samples were collected for biochemical determinations as described for endotoxaemic rats, before receiving imipenem at a dose of 250 mg/kg.

EEG measurements

Each rat was maintained in a plastic bowl. Simultaneously, a miniature plug was connected to a moving connector to record the EEG signal. Bipolar EEG leads were continuously recorded using the Brain Atlas (Biologic Systems Corp., Chicago, IL, USA). The signal was band-pass filtered from 0.3 to 70 Hz. The EEG signal from the right hemisphere cortical lead (F2-O2) was simultaneously sampled at 256 Hz and analysed online by fast Fourier transform (FFT) to determine the EEG total power in the frequency band from 0.5 to 30 Hz (Data Wave System Co., Thornton, CO, USA). The FFTs were calculated every 2 s, giving a first EEG power trend which could be visualized before being stored on the hard disk. Subsequently, after artifact removal from this power trend, a data reduction was calculated by averaging this first FFT trend every 1 min, resulting in a secondary trend. Consequently, each data point for the second trend was the mean of 30 consecutive points for the first trend.

Blood sampling

Arterial blood samples (200–250 µL) were collected in heparinized vials immediately before and at 15, 30 (end of infusion), 45, 60, 90 and 120 min after starting infusion. An extra sample was collected at 180 min in the endotoxaemia group. Blood was replaced by an equal volume of 0.9% NaCl solution. After collection, blood samples were centrifuged at 4000 rpm for 10 min at 4°C. Before storage, samples were diluted (1:1, v/v) with a stabilizer [0.5 M HEPES buffer, pH 6.8/ethylene glycol/HPLC-grade water (1:0.5:0.5, v/v/v)] and kept frozen at –80°C until analysis to avoid imipenem degradation.

Imipenem assay

A previously described HPLC assay was used with minor modifications for imipenem determination in plasma samples.8,10 Proteins were precipitated by the addition of methanol (1:2, v/v), the mixture was centrifuged, and the supernatant was injected. Separation was carried out with a Nucleosil C8 [5 µm, 250 by 0.4 mm (inner diameter)] column. The mobile phase consisted of 0.2 M aqueous borate buffer, pH 7.2, containing 15% (v/v) methanol, and the flow rate was 1 mL/min. The retention time of imipenem was equal to 5.5 min. The chromatographic system consisted of a model L 6000 Merck–Hitachi pump and a Waters 484 UV absorbance detector ({lambda}=313 nm). Chromatographic data were analysed using Normasoft Software (ICS, France). The limit of quantification of imipenem in plasma was 5 µg/mL. Intraday coefficients of variation calculated at two concentrations were equal to or less than 10%. Corresponding interday coefficients were equal to or less than 13%.

PK and PK-PD modelling procedures

A one- or two-compartment open model with zero-order input (R0) was used to characterize the plasma concentrations-versus-time profiles of imipenem and to estimate PK parameters including elimination half-life (t1/2), total clearance (CL) and volume of distribution (V) or volume of distribution at steady-state (Vss) for the one- or two-compartment model, respectively. Equations have previously been described for the one-8 and two-compartment19 models. Goodness of fit and model selection were assessed by visual inspection and analysis of Akaike and Schwartz criteria and of the coefficient of variation (%) associated with parameter estimates. PK parameters were then fixed and the PD model was regressed to the EEG data for each individual rat. An effect compartment model20 was applied for analysis of the PK-PD relationship, leading to an estimate of the rate constant ke0 for elimination of the drug from the effect compartment.8 The profile of the EEG effect was described with a spline function derived from the Hill equation, under the assumption that the maximal effect (Emax) and drug concentration corresponding to half of this maximal effect (EC50) are both high and cannot be determined experimentally because of animal death:8,21

In this equation, P is the total power (EEG effect) corresponding to Ce, the drug concentration in the effect compartment, P0 is the baseline effect value, Bn is the combined parameter Emax/EC50n, and n is a factor determining the steepness of the curve. Goodness of fit was assessed by visual inspection, analysis of the residuals, and from the coefficient of variation (%) associated with parameter estimates.8,19

All PK and PK-PD calculations were conducted using the software WinNonLin (version 4.0.1; Pharsight Corporation, Mountain View, CA, USA).

Statistical analysis

The unpaired Student's t-test was used to compare fitted values of the derived pharmacokinetic and pharmacodynamic parameters for each treated group to the control group. The paired t-test was used to compare biochemical parameters determined at H-6 and H0 in the control and endotoxaemia groups. Homogeneity of variances was assessed by the Bartlett test. In case of non-homogeneity of variances, non-parametric tests, the Mann–Whitney test and the Wilcoxon matched pairs test, were carried out. A significance level of 5% was selected. Values are reported as means±standard errors (S.E.M.).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Control animals

No statistically significant differences were observed between biochemical parameter values measured at H-6 and H0 (Table 1). The earliest individual spikes usually appeared in the middle of the 30 min infusion period. Their frequency and amplitude then increased dramatically, leading to a relatively sudden increase in the total power, accompanied by behavioural troubles, including tremors and partial seizures at about 30 min after infusion was stopped. The EEG signal eventually came back progressively to the baseline. Imipenem plasma concentration at the end of infusion (Cmax) was equal to 374±26 mg/L. Decay of plasma concentrations with time was monoexponential (Figure 1). Compartmental PK parameter values are presented in Table 2. The temporal delay between imipenem plasma concentration and EEG effect was adequately described by an effect compartment model (Figure 1). Corresponding pharmacodynamic parameters are also presented in Table 2.


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Table 1. Effect of endotoxin administration on biochemical parameters measured 6 h before (H-6) and at the beginning of imipenem infusion (H0)

 


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Figure 1. Imipenem plasma concentrations and EEG effect versus time in a typical control rat. The broken line represents the best PK fit to the measured concentrations (open circles), with the following values for PK parameters: V=360 mL/kg and CL = 17.2 mL/min per kg. The solid line represents the best fit to the measured total power of the EEG signal (filled circles) according to the effect compartment model, with the following values for PD parameters: P0=0.53 mV2, B=0.0170 mV2/mg per L, n=4.0 and ke0=0.0073/min.

 

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Table 2. PK and PD parameters characteristic of imipenem infused iv over 30 min to control and hypovolaemic rats at a dose of 250 mg/kg, and to endotoxaemic rats at a dose of 120 mg/kg

 
Hypovolaemic animals

The EEG signal changes and behaviour alterations were comparable to those observed in the control group. The mean imipenem plasma concentration at the end of infusion (Cmax) was equal to 603±17 mg/L. Decay of imipenem plasma concentrations with time was monoexponential (Figure 2) and characterized by PK parameters presented in Table 2. Hypovolaemia caused a significant reduction in volume of distribution and clearance values of imipenem compared to control animals (Table 2). The temporal delay between drug plasma concentration and EEG effect was adequately described by an effect compartment model (Figure 2). No major differences were observed between PD parameters estimated in this hypovolaemic group and the control group (Table 2).



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Figure 2. Imipenem plasma concentrations and EEG effect versus time in a typical hypovolaemic rat. The broken line represents the best PK fit to the measured concentrations (open circles), with the following values for PK parameters: V=179 mL/kg and CL = 10.88 mL/min. The solid line represents the best fit to the measured total power of the EEG signal (filled circles) according to the effect compartment model, with the following values for PD parameters: P0=0.35 mV2, B=0.0086 mV2/mg per L, n=2.3 and ke0=0.0133/min.

 
Endotoxaemic animals

Endotoxin administration induced a significant increase in creatinine plasma levels compared to the control group. HCO3 plasma concentrations were significantly reduced and aspartate aminotransferase significantly increased in endotoxin-treated rats (Table 1). The EEG signal changes and behaviour alterations were comparable to those observed in the control group, except that tremors and partial seizures were maintained for a longer period (up to 80–100 min after stopping infusion). The mean imipenem plasma concentration at the end of infusion (Cmax) was equal to 477±37 mg/L. Decay of plasma concentration with time was bi-exponential and best fitted by a two-compartment open model. Imipenem clearance was significantly reduced (Table 2). The temporal delay between imipenem plasma concentration and EEG effect was again adequately described by an effect compartment model (Figure 3), with corresponding PD parameters presented in Table 2.



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Figure 3. Imipenem plasma concentrations and EEG effect versus time in a typical endotoxaemic rat. The broken line represents the best PK fit to the measured concentrations (open circles), with the following values for PK parameters: Vss=218 mL/kg and CL = 2.50 mL/min. The solid line represents the best fit to the measured total power of the EEG signal (filled circles), according to the effect compartment model, with the following values for PD parameters: P0=0.13 mV2, B=0.0093 mV2/mg per L, n=3.0 and ke0=0.0115/min.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Pharmacokinetic parameter values estimated in the control group of this study are in good agreement with those previously published in healthy rats.8,22 Imipenem volume of distribution was estimated to be 303±37 mL/kg, which corresponds almost exactly to the mean value (297 mL/kg) of the extracellular fluid volume in rats.23 Because cilastatin was co-administered with imipenem to prevent hydrolysis,24 total clearance represents essentially renal clearance,22 and was estimated to be 16.6±1.1 mL/min per kg, that is approximately three times the mean value (5.24 mL/min per kg) of the glomerular filtration rate,23 suggesting substantial renal tubular secretion, as already demonstrated in humans.25 The elimination half-life was short (t1/2=12.6±1.1 h) in agreement with previous estimates.8,22 Pharmacodynamic parameters were also consistent with previously reported values in healthy rats.8

The range of ‘convulsant but non-lethal’ imipenem doses being very narrow, and not only sensitive to the dose itself but also to infusion duration,8 selection of an appropriate dosing regimen to investigate the EEG effect in experimental models of diseases is difficult.10 Preliminary experiments were conducted at sub-convulsant doses (data not shown) in order to estimate the extent of PK changes in each particular model of disease, and a dosing regimen was then selected from simulations using the expected PK parameter values and assuming unchanged PD parameters, as previously done with an experimental model of renal dysfunction.10 This led to a dose reduction by approximately two in the endotoxaemia group, whereas no adjustment was found necessary in the hypovolaemia group. For homogeneity purposes, the duration of infusion was kept the same (30 min) in the three groups. Alteration of the EEG signal together with behavioural troubles such as tremors followed by partial seizures were observed in the three groups of rats, indicating that this initial dose selection process was satisfactory.

The experimental model of hypovolaemia in rats, with removal of 18 mL/kg corresponding to 30% of a theoretical initial blood volume estimated to be 60 mL/kg, had previously been used by several authors to investigate the influence of this pathophysiological state on drugs PK-PD,11,12,15,16 although it may be considered as a model of moderate hypovolaemia.16 Yet hypovolaemia induced by this experimental model resulted in a mean reduction in imipenem volume of distribution by 39% (Table 1), which in absolute terms corresponds to 119 mL/kg on average. This is much more than the plasma volume actually removed, which considering a mean haematocrit in rats of 46%,23 was close to 10 mL/kg. Changes in protein binding cannot contribute to this reduced volume of distribution since imipenem is almost exclusively unbound.26 A more pronounced effect of blood removal could be expected on the volume of distribution of drugs more concentrated within the systemic circulation, i.e. with small volumes of distribution, rather than drugs with extensive tissue distribution. However, the volume of distribution at steady-state of etomidate, estimated to be 3.89±0.21 L/kg in healthy rats, which is more than 10-fold that of imipenem in relation to the much greater lipophilicity of the molecule, was reduced by 28% in hypovolaemic rats, which is almost to the same extent as that of imipenem (–39%), using the same experimental model of hypovolaemia.16 Physiological fluid redistribution induced by hypovolaemia should explain these altered volumes of distribution, and since imipenem is virtually totally unbound with a distribution presumably restricted to the extracellular fluid, its volume of distribution in hypovolaemic rats (184±6 mL/kg) should correspond to the extracellular fluid volume in that situation. A reduction in cardiac output and organ perfusion has sometimes been proposed to explain this volume change,16 however, changing blood flow is more likely to have an effect on the rate rather than on the extent of drug distribution. In contrast, reduced blood flows in hypovolaemic rats may explain or at least contribute to the reduced (33% on average) imipenem clearance. Imipenem clearance and volume of distribution were reduced to about the same extent in hypovolaemic rats and therefore the elimination half-life was virtually unchanged (Table 1). In this study, infusion duration (30 min) corresponds to about 2.5 half-lives and although steady-state is not completely reached at that time, the observed increase (+52%) in plasma concentrations at the end of infusion, should essentially be due to clearance reduction. Yet in the clinical setting, a reduction in the volume of distribution should lead to a proportional increase in peak concentration after a bolus or short-term infusion of imipenem, with potential beneficial consequences on its antimicrobial efficacy and less obvious but potentially problematic effects on its epileptogenic activity. Finally, the lack of effect on imipenem PD is consistent with previous findings by Yasuhara & Levy,13 who demonstrated that the PD contribution to the convulsant effect of theophylline was not affected by hypovolaemia.

The experimental model of endotoxaemia, with systemic injection of LPS from E. coli, was initially proposed by Fink & Heard in 1990 to induce endotoxaemia in rats which could mimic sepsis in humans.27 It constitutes a reasonable paradigm for sepsis in humans and has been used recently by De Paepe et al.18 Endotoxaemia was indirectly assessed by measurements of biochemical markers, with results in agreement with those observed by these authors.18 In particular, HCO3 plasma concentration was decreased probably as a result of metabolic acidosis, usually observed during endotoxaemia and compensated for by hyperventilation.18,28 The severity of insult was also assessed by creatinine enhancement, attesting for renal failure after sepsis induction.18,29 However, we observed that a 4 mg/kg dose of LPS, slightly higher than that used by de Paepe et al.18 (3 mg/kg) was necessary to induce these changes. Imipenem plasma concentration at the end of infusion in endotoxaemic rats (Cmax=477±37 mg/L) was noticeably higher on average than in control animals receiving almost twice the dose (Cmax=374±26 mg/L), mainly due to a reduction in its volume of distribution (Table 2). The unexpected early distribution phase observed in endotoxaemic rats could not be precisely characterized due to the lack of data points and should be confirmed. Imipenem clearance was reduced by almost five-fold (Table 2), which can probably be explained by renal impairment in LPS-treated animals. As for hypovolaemia, endotoxaemia had no apparent effect on the PD contribution to imipenem epileptogenic activity. Consequently, the lower dose needed to reach the same degree of neurotoxicity can be mainly attributed to PK changes, especially to the decrease in imipenem clearance and volume of distribution.

In conclusion, the robustness of the effect compartment model initially developed to characterize the PK-PD relationship of the EEG effect of imipenem in healthy rats, has now been confirmed in animals with experimental models of hypovolaemia and endotoxaemia. Major alterations in imipenem PK were observed but no effect of these disease states could be demonstrated on PD parameters.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We thank Pharsight Corporation for free supply of WinNonLin through the PAL program.


    Footnotes
 
* Corresponding author. Tel: +33-5-49-45-43-79; Fax: +33-5-49-45-43-78; Email: william.couet{at}univ-poitiers.fr


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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