Effect of norepinephrine on cefpirome tissue concentrations in healthy subjects

Ilka M. Steiner1, Herbert Langenberger1, Claudia Marsik1, Bernhard X. Mayer1, Milena Fischer1, Apostolos Georgopoulos3, Markus Müller1, Gottfried Heinz4 and Christian Joukhadar1,2,*

1 Department of Clinical Pharmacology, Division of Clinical Pharmacokinetics; 2 Institute of Pharmacology; 3 Department of Internal Medicine I, Division of Infectious Diseases and Chemotherapy; 4 Department of Internal Medicine II, Division of Intensive Care Medicine, University of Vienna Medical School, Allgemeines Krankenhaus; Waehringer Guertel 18–20, A-1090 Vienna, Austria

Received 6 October 2003; returned 17 November 2003; revised 28 November 2003; accepted 13 December 2003


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: To test whether norepinephrine (NOR) affects tissue microcirculation and impairs plasma-to-tissue equilibration of antimicrobial agents.

Materials and methods: Eight healthy male volunteers were enrolled to an analyst-blinded, randomized, two-period two-sequence crossover study. A single intravenous dose of 2 g of cefpirome was administered simultaneously with starting a continuous infusion of NOR (0.16 µg/kg per min) or placebo (PL) over 180 min. The microdialysis technique was used for the assessment of unbound cefpirome concentrations in skeletal muscle tissue and subcutaneous adipose tissue. Free plasma concentrations were related to corresponding tissue concentrations. Haemodynamics were determined by the measurement of mean arterial blood pressure (MAP), heart rate and forearm blood flow (FBF).

Results: Area under the concentration–time-curve (AUC) values of cefpirome for interstitium and plasma were not significantly different between the PL and NOR groups (P > 0.47). Tissue penetration of cefpirome as described by the ratios of the AUCs from 0 to 180 min for tissue to the AUC values for plasma were 0.81 ± 0.34 for the PL group and 0.80 ± 0.26 for the NOR group (P > 0.05). Baseline values of MAP, heart rate and FBF were not significantly different between study days. MAP increased significantly following NOR administration from 73.3 ± 3.5 mmHg at baseline to 94.0 ± 5.2 mmHg during infusion (P = 0.017). NOR exerted no significant effects on FBF.

Conclusions: We have shown that intravenous administration of NOR does not exert a significant effect on peripheral blood flow and tissue penetration of cefpirome in healthy men. This might be attributed to systemic regulatory mechanisms, which probably fully compensate for major changes in blood flow in peripheral tissues.

Keywords: microdialysis, forearm blood flow, haemodynamics, pharmacokinetics


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Critically ill patients frequently suffer from severe bacterial infections leading to sepsis or septic shock and inadequate organ perfusion. This necessitates immediate therapeutic intervention, which is originally based on the restoration of fluid volume and the intravenous administration of potent vasopressors. In these cases, vasopressive therapy with catecholamines, mostly with norepinephrine (NOR), is initiated.1 In low doses, NOR causes distinct vasoconstriction and increases mean arterial blood pressure (MAP) without causing deterioration of vital organ function.2 In doses frequently necessary in septic shock patients, NOR causes general constriction of capillary beds in peripheral tissues and significantly impairs blood flow in skeletal muscle and subcutaneous adipose tissue.

Blood flow, however, was recently shown to be a major determinant of drug distribution within body compartments.3 Pharmaceutical agents such as antimicrobials have to pass the capillary endothelial barrier to equilibrate with the interstitium of peripheral tissues. Therefore, recent clinical trials have hypothesized that the administration of NOR is one potential explanation for low concentrations of antibiotics in target tissues in critically ill patients.4,5 This is of particular clinical relevance, because microbial persistence is likely to occur, and the emergence of bacterial resistance is triggered by concentrations of antimicrobial agents below the minimal inhibitory concentration (MIC) value of the causative pathogen.6,7 Critically ill patients may, therefore, be at increased risk for therapeutic failure.

In this study, we aimed to test the hypothesis that administration of NOR markedly affects tissue distribution of antimicrobials. Cefpirome, a fourth generation cephalosporin was used as a model compound. The rationale for selecting cefpirome was based on the fact that its plasma and tissue pharmacokinetics are well documented in patients and healthy volunteers.8,9 The well-established microdialysis technique was used for the measurement of antimicrobial concentrations in interstitial space fluid of soft tissues.1012 Simultaneously, haemodynamics were recorded by measurements of blood flow and arterial blood pressure.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The study protocol was approved by the Ethics Committee of the University of Vienna, Medical School. The study was carried out in accordance with the Declaration of Helsinki (1964) in the revised version of 1996 (Somerset West), the Guidelines of the International Conference of Harmonization (ICH), the Good Clinical Practice (GCP) Guidelines, and Austrian Drug Law (Arzneimittelgesetz). Healthy volunteers were enrolled in the study after their written informed consent was obtained. They received a detailed description of the study before any examination or intervention was undertaken.

Volunteers

Eight Caucasian healthy male volunteers with a mean age of 26.6 ± 5.3 years, a mean body mass index of 22.3 ± 1.1 kg/m2 and a mean weight of 71.1 ± 4.9 kg were enrolled in the study.

In an initial screening visit, they were physically checked; blood pressure and ECG parameters were recorded. Normotension was demanded for inclusion, defined as systolic blood pressure (SBP) < 130 mmHg and diastolic blood pressure (DBP) < 85 mmHg after a 5 min rest in the supine position. Venous fasting blood samples were taken for analysis of complete blood count, laboratory chemical parameters and for hepatitis and HIV serology tests. Volunteers were questioned about past medical history and treatment. The subjects were enrolled in the study unless the investigator considered abnormal values in blood chemistry and physical examination relevant. Subjects were non-smokers and they had to be drug free for at least 3 weeks before the first study day.

Sample size calculation

The sample size calculation was carried out based on the equation published by Stolley & Strom.13 A sample size of eight subjects with paired measurements has 80% power to detect differences of approximately 15% in AUC values9 and arterial blood flow14 between groups.

Study design

A randomized, analyst-blinded, placebo-controlled, two-period, two-sequence, crossover study was carried out. Volunteers were randomly assigned to receive NOR on study day one or on study day two. Wash out period was at least 1 week. Microdialysis and forearm blood flow measurements were carried out on both study days.

During study days, subjects were in a supine position. Three plastic cannulas were inserted into cubital and antecubital veins for drawing blood samples and for the administration of cefpirome combined with NOR or cefpirome combined with placebo (PL).

Cefpirome (Cefrom, Albert Rousell Pharma, Vienna, Austria) was administered as an intravenous single dose of 2 g over a period of 10 min on both study days.

Norepinephrine (Arterenol, Hoechst AG, Frankfurt, Germany) was administered by a primed infusion of step-wise increasing doses for periods of 10 min per dose step. Thereafter, NOR was administered continuously over 180 min at a rate of 0.16 µg/kg per min.

Ringer’s solution (Ringer Lösung ‘Mayrhofer’ Infusionslösung, Mayrhofer Pharma Gesellschaft m.b.H., Linz, Austria) served as placebo.

Microdialysis

For the determination of free interstitial concentrations of cefpirome, in vivo microdialysis was carried out.1012 This method is based on sampling of analytes from the interstitial space by means of a semi-permeable membrane at the tip of a microdialysis probe. Once the microdialysis probe is implanted into the tissue, substances present at a certain concentration (Ctissue) in the interstitial fluid diffuse out of the extravascular fluid into the probe, resulting in a concentration (Cdialysate) in the perfusion medium. For most analytes, equilibrium between interstitial space fluid and the perfusion medium is incomplete (Ctissue > Cdialysate). The factor by which the concentrations are interrelated is termed relative recovery.

For calibration of the microdialysis (MD) probes, in vivo recovery was assessed in each experiment according to the retrodialysis method.15

The principle of this method relies on the fact that diffusion through a semi-permeable membrane is a bidirectional process and quantitatively equal. Therefore, cefpirome was added to the perfusate at a concentration of 30 mg/L.11

The in vivo recovery value was calculated as:

Recovery (%) = 100 – (100 x concentrationdialysate/concentrationperfusate)

Commercially available microdialysis probes (CMA 10, Microdialysis AB, Stockholm, Sweden) with a molecular weight cut-off of 20 kDa, an outer diameter of 0.5 mm, and a membrane length of 16 mm were inserted into subcutaneous adipose tissue and skeletal muscle of the thigh. Before probe insertion, the skin was cleaned and disinfected. The surface of the skin was punctured by 20-gauge intravenous plastic cannulas without anaesthesia. The steel trochars were removed and the MD probes were inserted into the tissues via use of guidance cannulas. The plastic cannula was removed, leaving the probe under the surface of the skin. Then the MD probes were connected to microinfusion pumps (CMA 100, Stockholm, Sweden or Precidor; Infors-AG, Basle, Switzerland) which provided a constant flow-rate of 1.5 µL/min throughout the study period. Two baseline samples at an interval of 15 min were collected during MD probe calibration. The whole MD system was manually rinsed after the termination of MD probe calibration. Then the MD probes were re-connected to the microinfusion pumps and were constantly perfused with Ringer’s solution for a period of approximately 30 min. Finally, a sample was collected over 15 min immediately before cefpirome and NOR or PL administration was started. Thereafter, the sampling interval was extended to 20 min. All samples were subjected to chemical analysis. There was no detectable cefpirome in any sample collected before cefpirome administration.

Blood sampling

Following MD probe calibration, samples of dialysates and venous blood were drawn at 20 min intervals over a period of 180 min. Samples were kept on ice for a maximum of 30 min until centrifugation. The venous catheter was rinsed with Ringer’s solution after each sampling. Blood samples were centrifuged at 4°C, 2500g for 5 min; cells were discarded and plasma was obtained. Plasma and dialysate samples were frozen at –80°C until analysis.

Measurements of blood pressure and heart rate

Systolic and diastolic blood pressure and heart rate (HR) were measured in 30 min intervals on both study days.

Forearm blood flow measurements

Forearm blood flow (FBF) was measured by venous occlusion plethysmography. The principle of the technique has been described previously.16,17 All measurements were carried out using a mercury-filled silicone strain-gauge plethysmograph (Hokanson EC6 Plethysmograph, Bellevue, WA, USA). The rate of forearm swelling during venous occlusion is related to arterial inflow. Venous outflow of the forearm was abruptly stopped by inflating an occlusion cuff above the elbow to a pressure of 50 mmHg. This pressure was sufficient to occlude the veins while not affecting arterial inflow. The relationship between raising forearm volume and arterial inflow is only valid if the veins are not distended. Therefore, the forearm was placed at the level of the right atrium. The rate of swelling is a measure of total arterial blood flow. In all subjects, the dominant forearm was used, because blood flow differs between dominant and non-dominant upper limbs.18 Measurements were carried out at 30 min intervals on both study days.

Drug assay

Free cefpirome concentrations in plasma and cefpirome concentrations in microdialysates were analysed after ultrafiltration by micellar electrokinetic chromatography according to a previously published method.19 Quantification limits for plasma and dialysates were 1 and 0.3 mg/L, respectively. Reproducibility measurements yielded day-to-day coefficients of variation below 7%.

Pharmacokinetic (PK) calculations

PK analysis was carried out using computer software (Kinetica 3.0, Innaphase Sarl, Paris, France) and data were calculated using non-compartmental approaches. Area under the concentration–time-curve (AUC) values for plasma and interstitium were calculated from non-fitted data by employing the linear trapezoidal rule. The volume of drug distribution (V) and total drug clearance (CL) were calculated for plasma by use of standard formulae as follows: V = dose/AUC x kel, CL = kel x V; where kel represents the elimination rate constant. The half-life calculated for the terminal slope (t1/2ß) was calculated by the equation t1/2ß = ln(2)/kel.

The following PK parameters were determined: AUC, maximum concentration (Cmax), time to maximum concentration (tmax). The main PK data are summarized in Table 1.


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Table 1. Main pharmacokinetic data
 
Statistical calculations

Statistical analysis was carried out using commercially available software (Statistica, StatSoft, Inc., Tulsa, OK, USA). As parameters were non-normally distributed, Wilcoxon matched pairs tests were used for comparison between study groups. Analyses of variance (Friedmann-ANOVA) for repeated measurements were used. A P value <0.05 was considered the level of significance. All data are presented as mean ± standard deviation (S.D.).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
One subject suffered from strong headache immediately after the start of NOR administration and decided to stop participation in the study. Thus, data sets from only seven volunteers were eligible for PK and FBF analysis.

The plasma protein binding of cefpirome was approximately 10%. All plasma data presented in the manuscript relate to the unbound fraction of cefpirome.

PK results

Main PK parameters are presented in Table 1. The concentration versus time profiles of cefpirome in plasma, muscle tissue and subcutaneous adipose are shown in Figure 1(a–c), respectively. No significant difference (P > 0.05) in PK parameters of cefpirome was found between groups for AUC0-180min, Cmax, tmax and t1/2ß in plasma and interstitium. In addition, no significant differences (P > 0.05) in V and CL values were detected between groups. The concentration versus time profiles of cefpirome were identical for subcutaneous adipose and muscle tissue in both groups.



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Figure 1. (a) Concentration versus time profiles of cefpirome in plasma for the placebo group (open squares) and the NOR group (filled squares). (b) Concentration versus time profiles of cefpirome in muscle tissue for the placebo group (open triangles) and the NOR group (filled triangles). (c) Concentration versus time profiles of cefpirome in subcutaneous adipose tissue for the placebo group (open diamonds) and the NOR group (filled diamonds). All data are presented as mean ± S.D.

 
Tissue penetration parameters are presented in Table 2. Tissue penetration as described by the ratios of AUC0-180 for muscle to AUC0-180 for plasma and AUC0-180 for subcutis to AUC0-180 for plasma were not significantly different for the NOR and the placebo groups (P > 0.05).


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Table 2. Tissue penetration as described by the mean ratio of AUC for tissue to AUC for plasma (AUCtissue/AUCplasma)
 
Results of MD probe calibration

In vivo recovery values for skeletal muscle tissue were 17.2 ± 7.9% in the NOR group and 17.7 ± 5.6% for the PL group. For subcutaneous adipose tissue, recovery values were 19.5 ± 11.7% in the NOR group and 19.2 ± 7.6% in the PL group.

Results of haemodynamic measurements

FBF measurements, MAP and HR were not significantly different between study days at baseline (P > 0.05). No significant changes versus baseline were detected for FBF measurements following NOR administration (P > 0.05; Figure 2). Systolic, diastolic and MAP increased significantly (P = 0.017) compared with baseline in the NOR group, only. Mean MAP increased from 73.3 ± 3.5 mmHg at baseline to 94.0 ± 5.2 mmHg during continuous NOR infusion and remained constant over 180 min (P > 0.05), whereas MAP did not change versus baseline in the PL group (Figure 3).



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Figure 2. Relative changes versus baseline of FBF for the placebo group (open squares) and for the NOR group (filled squares) over time. Data are presented as mean ± S.D.

 


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Figure 3. Time course of mean arterial blood pressure (MAP) for the placebo group (open circles) and for the NOR group (filled circles) over a period of 180 min. Data are presented as mean ± S.D.

 
The increase in blood pressure in the NOR group was paralleled by a descriptive decrease in mean heart rate to 58.3 ± 8.5 beats per min during continuous infusion of NOR and 66.5 ± 8.5 beats per min during PL administration (P = 0.063, not significant; Figure 4).



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Figure 4. Time course of heart rate for the placebo group (open diamonds) and for the NOR group (filled diamonds) over 180 min. Data are presented as mean ± S.D.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Several preconditions must be fulfilled for an antibiotic to become therapeutically effective. Most importantly, the causative pathogen has to be susceptible to the antimicrobial agent and the drug concentration at the target site must exceed its MIC.20 However, blood flow is the most important determinant that governs the distribution of antimicrobial agents within compartments. In particular, the capillary surface area to volume ratio is considered essential for plasma to tissue equilibration. The administration of NOR induces vasoconstriction of capillary beds and reduces the number of capillaries available for drug exchange. Therefore, it was speculated that NOR therapy could markedly impair tissue penetration of antimicrobial agents21 and account for ineffective concentrations of antibiotics at the target site in critically ill patients.5

Against this background, this study was undertaken to improve our understanding of the reasons for low tissue levels of antimicrobial agents observed in critically ill patients with NOR therapy.5,21,22 Therefore, we employed a well-established microdialysis technique and measured concentrations of cefpirome in skeletal muscle and subcutaneous adipose tissue during continuous administration of NOR to healthy volunteers. Healthy subjects were recruited in order to optimally assess the exclusive effect of NOR on tissue penetration of cefpirome and to avoid confounding variables such as inflammation, concomitant medication and different states of disease.

The main finding of this study is that concentration–time profiles of cefpirome for subcutaneous adipose and skeletal muscle tissue are not significantly different between the NOR and placebo study days. The mean ratios of AUCtissue to AUCplasma were approximately 0.8 for both PL and NOR groups. Thus, the tissue PKs observed for cefpirome in this study in healthy young volunteers is mimicking the PK profile determined in an elderly healthy control group to septic patients.21 As expected, haemodynamics were significantly affected by NOR administration. Mean MAP increased from 73.3 ± 3.5 to 94.0 ± 5.2 mmHg (P = 0.017) during continuous administration of NOR at a rate of 0.16 µg/kg per min. This infusion rate is relatively low for septic patients, but was highly effective to increase systemic haemodynamic parameters in our healthy study population. This discrepancy is explained by the fact that vascular responsiveness to exogenous catecholamines is substantially reduced in septic conditions in comparison to healthy controls.2325

In healthy subjects, NOR exerts significant cardiac effects because an increase in MAP physiologically reduces the heart rate by activation of the carotid aortic baroreceptor reflex as a mechanism of haemodynamic compensation. As a result, sympathetic tone is diminished and vagal tone is enhanced. Each of these responses leads to a decrease in heart rate, which is of special importance for drugs that have little capacity to directly activate ß-adrenergic receptors. A descriptive decrease in heart rate was also observed in our study population, though the low sample size of seven subjects was not able to statistically confirm this trend (P = 0.063). Arterial blood inflow in peripheral tissues may remain unchanged in healthy volunteers by NOR administration unless regulatory mechanisms decompensate. These data, therefore, suggest that microcirculatory blood flow in peripheral tissues may remain unaffected by an increase in the local perfusion pressure, as blood flow increases with MAP and decreases with vascular resistance.23

A non-pharmacological method to increase blood flow in tissues is local warming. Local warming of tissue effectively leads to dilatation of capillaries by reducing vascular resistance and thereby increases local blood flow26 without activating any systemic compensatory mechanisms. Our study group used this approach in a separate experiment to study the effect of blood flow on drug tissue penetration in humans (unpublished data). In this experiment, we have demonstrated that local warming of a lower extremity results in a significant increase in blood flow and is paralleled by a significant increase in antimicrobial tissue concentration in comparison to the untreated extremity.

One important limitation has to be considered in the interpretation of our results. This study has a power to detect changes of blood flow of ~15% and therefore we cannot exclude that smaller changes might have occurred but remained undetected. However, we consider such small changes clinically irrelevant. In addition, we cannot completely rule out that NOR caused a simultaneous activation of {alpha}- and ß-adrenoceptors exerting opposite effects neutralizing each other on the tissue level. However, this appears unlikely because cutaneous blood vessels almost exclusively express {alpha}-receptors. Smooth muscle cells of blood vessels that supply skeletal muscles have both, {alpha}- and ß2-receptors, but {alpha}1-mediated vasoconstriction clearly predominates ß2-mediated vasodilatation, which is indicated by the increase in MAP in the NOR group.

It is of relevance to note that for systemically administered agents, it is difficult to discriminate between local and systemic compensatory haemodynamic mechanisms, which possibly alter changes in peripheral blood flow measured by plethysmography. This limitation might be overcome by use of methods that allow for local administration of vasoactive compounds27 and the simultaneous measurement of tissue concentration profiles of systemically administered antimicrobial agents at the site of drug action.3 The intra-arterial administration of NOR27 and microdialysis used as a drug delivery device3,26 permits evaluation of the effects of local changes in blood flow on drug distribution into tissues without systemic involvement.

In conclusion, we were able to demonstrate that penetration of cefpirome into soft tissues remained unaffected in healthy men, although significant changes in systemic haemodynamic parameters were observed during NOR administration. Most probably, this is because of compensatory mechanisms such as the decrease in heart rate and increase in vascular perfusion pressure, which maintain blood flow sufficiently high at peripheral sites in healthy volunteers.


    Acknowledgements
 
This study was supported by the ‘Jubiläumsfonds der österreichen Nationalbank’ Project number: 9355.


    Footnotes
 
* Corresponding author. Tel: +43-1-40400-2981; Fax: +43-1-40400-2998; E-mail: christian.joukhadar{at}univie.ac.at Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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