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JAC Advance Access originally published online on January 5, 2006
Journal of Antimicrobial Chemotherapy 2006 57(2):312-316; doi:10.1093/jac/dki459
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© The Author 2006. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Intraabdominal tissue concentration of ertapenem

M. Wittau1,*, E. Wagner1, V. Kaever2, T. Koal2, D. Henne-Bruns1 and R. Isenmann1

1 University of Ulm, Department of Visceral Surgery, Steinhövelstrasse 9, 89075 Ulm, Germany; 2 Medical School Hannover, Institute of Pharmacology, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany

* Corresponding author. Tel: +49-731-50027232; Fax: +49-731-50021568; E-mail: mathias.wittau{at}web.de

Received 14 July 2005; returned 14 October 2005; revised 6 November 2005; accepted 23 November 2005


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Objectives: Ertapenem, a class I carbapenem, is approved for the treatment of mild to severe intraabdominal infections, but its in vivo concentrations in intraabdominal tissues are unknown. The purpose of this study was to determine the concentration of ertapenem in intraabdominal tissue.

Patients and methods: After informed consent 48 patients, 23 female and 25 male with a median age of 58 years (34–81), requiring surgical intervention at intraabdominal organs were enrolled. Patients received 1 g of ertapenem intravenously for perioperative prophylaxis. Tissue samples were taken after resection of parts of the organs. Plasma samples were taken when tissue samples were taken. Drug concentrations were determined by liquid chromatography/mass spectrometry. An ANCOVA test (analysis of covariance) was performed to assess organ-specific differences in ertapenem concentration and penetration ratios.

Results: Mean ± SD ertapenem tissue concentration (mg/kg) was 16.0 ± 8.8 in the gall bladder, 12.1 ± 5.3 in the colon, 7.0 ± 5.7 in the small bowel, 4.5 ± 2.3 in the liver and 3.4 ± 2.9 in the pancreas. The mean tissue/plasma ratio was 0.19 (colon), 0.17 (small bowel), 0.17 (gall bladder), 0.088 (liver) and 0.095 (pancreas). The ANCOVA test revealed statistically significant organ-specific differences in ertapenem tissue concentration in the gall bladder versus liver/pancreas and in tissue penetration for the colon versus liver/pancreas.

Conclusions: These pharmacokinetic results support the assumption that ertapenem is suitable for the treatment of intraabdominal infections.

Keywords: pharmacokinetics , carbapenems , tissue penetration


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The treatment of serious bacterial infections often requires parenteral antibiotic therapy. One of the most potent classes of antimicrobial agents is the carbapenems. They are usually reserved for the treatment of serious infections or multiresistant pathogens.1 Carbapenems are ß-lactam antibiotics with a broad spectrum of antimicrobial activity against Gram-positive, Gram-negative and anaerobic species.2 They are typically stable against hydrolysis by most chromosomal- and plasmid-mediated ß-lactamases, including the extended-spectrum ß-lactamases (ESBLs).1

Ertapenem is a structurally unique carbapenem. Owing to the 1ß-methyl group, it has an increased stability against renal dehydropeptidase-I (DHP-I) and therefore does not have to be combined with cilastatin for protection against renal DHP-I. Furthermore, the benzoate anionic side chain makes the compound highly protein bound and its half-life is ~3.8 h.3 Pharmacokinetics of ertapenem in healthy young adults support its use as a once-daily antibiotic at a dose of 1 g intravenously (iv).3,4 Ertapenem is active against a wide variety of organisms but has limited activity against Pseudomonas aeruginosa and no activity against Enterococcus faecium, Enterococcus faecalis and methicillin-resistant Staphylococcus aureus. Owing to its antimicrobial spectrum, ertapenem seems to be an effective drug for the treatment of intraabdominal infections where anaerobes or aerobic Gram-negative bacteria are the predominant pathogens.3 However, ~84–96% of ertapenem is bound to plasma proteins and this protein binding decreases as plasma concentrations of ertapenem increase.4 Since only the unbound fraction of ertapenem can penetrate into the tissue, only 4–16% of the mean plasma concentration is responsible for the antimicrobial activity. Thus, the concentration of ertapenem in intraabdominal tissue must exceed the MIC90 for the microorganisms found in intraabdominal infections.

To date, there are only limited data on tissue concentrations of ertapenem.5,6 Data on tissue concentrations in intraabdominal organs are not available. The purpose of this study was to determine the concentration of ertapenem in intraabdominal tissue.


   Patients and methods
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Patients

Hospitalized patients 18 years or older requiring surgical intervention (open or laparoscopic surgery) at the liver, colon, pancreas or gall bladder were eligible for this study.

Exclusion criteria were as follows: pregnancy or lactation in women, emergency surgery, history of serious allergy or intolerance to ß-lactam antibiotics, systemic antimicrobial therapy within a 7 day period before study entry, ongoing intraabdominal infections, terminal illness, chronic immunosuppressive therapy, severe diseases of the liver, e.g. cirrhosis of the liver with ALT or AST >6x upper limit of normal (ULN) and bilirubin >3x ULN, severe renal insufficiency with a creatinine clearance ≤30 mL/min, neutrophil count <1000 cells/mm3, platelets <75 000 cells/mm3 and coagulation studies (INR) >1.5x ULN.

Forty-eight patients, 23 female and 25 male with a median age of 58 years (range 34–81 years), were enrolled in the study. Median weight was 73 kg (range 41–109 kg), median body mass index was 25.6 (range 16.5–32.5) and median creatinine clearance was 84.5 mL/min (range 49.9–153 mL/min).

The underlying diseases of the patients are listed in Table 1.


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  		Table 1.. Underlying diseases of the patients

 
Study design and antimicrobial treatment

This prospective, unicentric, open-label study was conducted at the University of Ulm, Department of Visceral Surgery, from June 2003 to October 2004. The ethics committee of the University of Ulm approved the protocol and written informed consent was obtained from all study participants.

All patients received 1 g of ertapenem iv 30 min before the surgical procedure over a 30 min period for perioperative prophylaxis.

Collection of tissue samples and blood samples

Tissue samples were taken after resection of the gall bladder and/or parts of the liver and/or pancreas and/or colon and small bowel. Tissue samples were only taken from macroscopically normal parts of the resected tissue, i.e. not from malignant tissue. In order to avoid blood contamination, adherent blood was removed by rinsing the samples with sterile saline for a few seconds. After this, samples were immediately shock frozen in liquid nitrogen. Samples were stored after resection at –80°C for 6–8 weeks and then analysed for total drug concentration.

Bile samples were taken after resection of the gall bladder and immediately shock frozen in liquid nitrogen.

Blood samples were taken when tissue samples were taken. Blood samples were immediately chilled at 4°C. Plasma was obtained by centrifugation of blood samples at 4000 rpm for 10 min at 4°C. Plasma samples were stored at –80°C in two aliquots and later analysed for total drug concentration.

Bioanalytical methods

Ertapenem was quantified by a newly developed high-performance liquid chromatography/mass spectrometry (HPLC/MS) method.7 Plasma and bile samples were immediately stored at –80°C and stabilized by addition of 100 mM 2-(4-morpholino)ethylsulphonate (MES) buffer (pH 6.5) 1/1 (v/v) after thawing. Frozen tissue samples were ground in a liquid nitrogen frozen mortar. Pulverized tissue (200 mg) was treated immediately with 600 µL of 100 mM MES buffer (pH 6.5). The tissue/MES sample was homogenized three times for 10 s by using an Ultraturrax machine at 4°C. To 100 µL of plasma/MES, bile/MES or human tissue/MES samples in polypropylene tubes 400 µL of methanol including 12.5 µg/mL of internal standard (IS) ceftazidime was added for protein precipitation. Samples were immediately vortexed (20 s) and centrifuged for 10 min at 20 800 g at 4°C. Supernatant (400 µL) was transferred into 1.5 mL sample vials, and concentrated to dryness using nitrogen gas stream at room temperature. Dried samples were dissolved with 150 µL of H2O to realize appropriate start conditions for HPLC separation. After centrifugation for 5 min at 20 800 g at 4°C, 100 µL of supernatant was transferred into a 1.5 mL sample vial with a 200 µL volume microinsert. During LC/MS analysis samples were kept at 4°C.

LC/MS was performed by direct coupling from an HPLC column [Synergi 4µ Polar-RP 80A Mercury (10 x 2.0 mm)] used in combination with an HPLC column SecurityGuardTM (Phenomenex, Aschaffenburg, Germany) to the MS system (LC-MSD, Agilent 1100, Waldbronn, Germany) equipped with electrospray ion source. At zero time an aliquot of 20 µL of sample was injected and analysed by following analysis. Pump A supplied 500 µL/min start eluent (100% eluent A = H2O, 2 mM NH4Oac, 0.1% acetic acid, pH 3.8). HPLC separation was proceeded by using a linear HPLC gradient with a constant total flow rate of 500 µL/min, that means gradient formation from 0.0 min (100% eluent A, 0% eluent B = 100% methanol) to 5.0 min (10% eluent A, 90% eluent B). For system re-equilibration for analysis of the next sample the total HPLC eluent was changed back to 100% eluent A at 5.1 min (500 µL/min). A total analysis time of 8 min was obtained. MS detection in negative SIM detection mode (IS voltage, 4000 V; temperature, 350°C; gas supply, 100 psi; nebulizer gas, 35 psi; drying gas, 10 L/min) allows detection of ertapenem (tR = 3.7 min; SIM, 473.9 amu) and IS ceftazidime (tR = 3.1 min; SIM, 465.8 amu). Ertapenem (calibration curve 0.1–50.0 µg/mL) was a gift from MSD (Whitehouse Station, NJ, USA) and ceftazidime used as IS was purchased from Sigma–Aldrich (Steinheim, Germany).

Koal et al.7 showed the method validation results for accuracy and precision in plasma samples. Relative standard deviation (RSD) values for intraday and interday precision determined at three concentrations are shown with excellent RSD <10%. Accuracy was investigated by means of laboratory in-house extra-spiked plasma samples and samples show acceptable accuracies >90%.

Statistical methods

Data are displayed as means ± SD, except the data of the ertapenem concentration in the bile and patient characteristics (median, minimum and maximum). To distinguish between the effect of time on ertapenem concentration (or tissue-to-plasma ratio) and the organ-specific differences in ertapenem concentration (or in tissue-to-plasma ratio) an ANCOVA test (analysis of covariance) was performed (SAS, version 8.2). P values <0.05 were statistically significant (adjusted for multiple testing according to Bonferroni).


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After administration of ertapenem no serious adverse experiences were observed.

The mean ± SD tissue concentrations of ertapenem in the different organs are listed in Table 2. The highest ertapenem concentration was observed in the gall bladder with a mean of 16.0 ± 8.8 mg/kg and the lowest tissue concentration was observed in the pancreas with a mean of 3.41 ± 2.9 mg/kg.


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  		Table 2.. Mean tissue concentration of ertapenem (ETP) following single 1 g dose iv

 
Since tissue samples were taken at different times, tissue concentrations were stratified according to the time after ertapenem administration (Table 3). The mean ertapenem tissue concentration of most organs decreases with the time after administration, indicating a time-dependent effect on ertapenem tissue concentration. In addition, the ertapenem concentration in each organ at the same time was different, indicating organ-specific differences in the ertapenem tissue concentration.


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  		Table 3.. Mean tissue concentration of ertapenem at different times following single 1 g dose iv

 
In order to distinguish between the effect of time on ertapenem concentration and the organ-specific differences in ertapenem concentration an ANCOVA test was performed. As expected, the time after administration had a statistically significant influence on ertapenem tissue concentration (P = 0.0014). Statistically significant organ-specific differences in ertapenem concentration were found in the gall bladder compared with liver (P = 0.006) and gall bladder compared with pancreas (P = 0.014).

The mean ± SD tissue-to-plasma concentration ratios (penetration ratios) are listed in Table 4. The highest penetration ratio was observed for the colon with a mean of 0.191 and the lowest penetration ratio was observed for the liver with a mean of 0.088. Since tissue samples were taken at different times, tissue-to-plasma ratios of the different organs at different times were also analysed with an ANCOVA test. In contrast to the ertapenem tissue concentration, time had no statistically significant influence on ertapenem penetration ratio (P = 0.428). However, statistically significant organ-specific differences in ertapenem penetration ratio were found in the colon compared with liver (P = 0.0236) and colon compared with pancreas (P = 0.0168).


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  		Table 4.. Mean tissue-to-plasma ratio of ertapenem (ETP) following single 1 g dose iv

 
Bile samples were obtained from 16 patients. Three of the sixteen samples were considered as outliers in the Stem-and-Leaf plot with ertapenem concentrations of 304.4 mg/L, 265.2 mg/L and 95.2 mg/L, respectively. The ertapenem concentration in the bile varied widely with a median of 11.2 mg/L, a minimum of 1.2 mg/L and a maximum of 49.8 mg/L. Nine of the 13 samples had an ertapenem concentration between 1 and 20 mg/L. No dependence on time was observed (Figure 1).


Figure 1
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  		Figure 1.. Concentration of ertapenem in the bile following single 1 g dose iv.

 
The penetration ratio of ertapenem in the bile also showed a broad variation with a median of 0.126, a minimum of 0.006 and a maximum of 0.417 (Figure 2).


Figure 2
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  		Figure 2.. Ratio bile-to-plasma concentration of ertapenem following single 1 g dose iv.

 

   Discussion
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Ertapenem exhibits concentration-dependent plasma protein binding, ranging from 96% at total plasma concentrations of 10 mg/L to 84% at total plasma concentrations of 300 mg/L.8 Thus, only 4–16% of the total plasma concentration can penetrate into extravascular space and is responsible for antimicrobial activity. To inhibit bacterial growth or even cause the killing of bacteria, the tissue concentration of ertapenem must exceed the MIC90 for most microorganisms found at the site of bacterial infection.

To date, only two studies focused on tissue penetration or concentration of ertapenem in humans. The penetration of ertapenem into skin tissue was studied with suction-induced skin blisters following multiple iv doses in 12 adult subjects. After a 1 g dose of ertapenem a mean maximum concentration of 24.4 mg/L was achieved and the mean concentration after 24 h was 7.8 mg/L,5 which exceeds the MIC90 for susceptible bacteria.8 Burkhardt et al.6 investigated the penetration of ertapenem into different pulmonary compartments of patients undergoing lung surgery. The mean lung tissue concentration of 12 patients was 7.60 ± 4.85 mg/kg. The penetration ratio was 23.6 ± 12.3%.

We present the first data concerning ertapenem tissue concentration in different intraabdominal organs of patients undergoing elective abdominal surgery. The mean tissue concentrations in the gall bladder, colon, small bowel, liver and pancreas were above the MIC90 for susceptible bacteria found in intraabdominal infections, especially Escherichia coli, viridans streptococci, Enterobacteriaceae, Klebsiella spp. and Bacteroides fragilis. The lowest mean tissue concentrations up to 6 h after administration (25% of dosing interval) were 3.3 mg/kg in the pancreas and 3.1 mg/kg in the liver.

The time period of drug concentration above the MIC is the pharmacodynamic parameter best correlated with clinical efficacy for carbapenem antibiotics.9–11 In a murine model of soft-tissue infection, the time period of drug concentration above MIC required for a bacteriostatic effect with ertapenem ranged from 24 to 48% for total drug concentration and from 6 to 25% for unbound drug concentration.8 In our study, the ertapenem concentrations in intraabdominal tissue at 25% of dosing interval were at least 1.5 times above the MIC90 for susceptible bacteria found in intraabdominal infections, 3 times for B. fragilis and 10 times above the MIC90 for E. coli, usually the most common isolate in these infections.12 One central finding of our study was the significantly higher concentration of ertapenem in the gall bladder compared with the liver and pancreas despite the different time of tissue sampling, as shown by the ANCOVA test, indicating organ-specific differences in the ertapenem tissue concentration with the highest concentration of ertapenem in the gall bladder, followed by the colon and small bowel. The penetration ratios of intraabdominal organs with 8.8–19.1% were lower than the penetration ratio of lung tissue reported by Burkhardt et al.6 The incomplete tissue penetration is most likely related to the high plasma protein binding. Statistically higher penetration ratios were found in the colon compared with the liver and pancreas, indicating organ-specific differences in the penetration of ertapenem into tissues. This finding is supported by the higher penetration ratio of the lung in Burkhardt's study.6 Penetration ratios similar to the colon were found in the gall bladder and small bowel, showing that ertapenem penetrates best into these intraabdominal organs.

The measured ertapenem concentrations in the bile and the bile-to-plasma concentration ratio showed a broad variation. These results are difficult to interpret owing to a concentration range of 1.2–49.8 mg/L and a penetration ratio that ranged from 0.6 to 41%. The reason for these findings is not clear. One could explain these findings with the mixed population of patients with regard to age, gender and body mass index. Thus, further studies are required concerning ertapenem concentrations in the bile. Nevertheless, only two of the thirteen samples had an ertapenem concentration below the MIC90 and the median was ~5 times above the MIC90 for susceptible bacteria found in intraabdominal infections.

In conclusion, our data indicate adequate penetration of ertapenem into uninfected intraabdominal organs with tissue concentrations above the MIC90 for susceptible bacteria found in intraabdominal infections. In accordance with clinical studies,12,13 these pharmacokinetic results support the assumption that ertapenem is suitable for the treatment of intraabdominal infections.


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No declarations were made by the authors of this paper.


   Acknowledgements
 
This study was supported by a grant from MSD Sharpe & Dohme, Haar, Germany.


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1. Barker KF. Antibiotic resistance: a current perspective. Br J Clin Pharmacol 1999; 48: 109–24.[CrossRef][Medline]

2. Wiseman LR, Wagstaff AJ, Brogden RN et al. Meronem. A review of its antibacterial activity, pharmacokinetic properties and clinical efficacy. Drugs 1995; 50: 73–101.[Web of Science][Medline]

3. Cunha BA. Ertapenem. A review of its microbiologic, pharmacokinetic and clinical aspects. Drugs Today 2002; 38: 195–213.

4. Majumdar A, Musson DG, Birk KL et al. Pharmacokinetics of ertapenem in healthy young volunteers. Antimicrob Agents Chemother 2002; 46: 3506–11.[Abstract/Free Full Text]

5. Laethem T, De Lepeleire I, McCrea J et al. Tissue penetration by ertapenem, a parenteral carbapenem administered once daily, in suction-induced skin blister fluid in healthy young volunteers. Antimicrob Agents Chemother 2003; 47: 1439–42.[Abstract/Free Full Text]

6. Burkhardt O, Majcher-Peszynska J, Borner K et al. Penetration of ertapenem into different pulmonary compartments of patients undergoing lung surgery. J Clin Pharmacol 2005; 45: 659–65.[Abstract/Free Full Text]

7. Koal T, Deters M, Resch K et al. Quantification of the carbapenem antibiotic ertapenem in human plasma by a validated liquid chromatography–mass spectrometry method. Clin Chim Acta 2005; doi:10.1016/j.cccn.2005.07.004.

8. Nix DE, Majumdar AK, DiNubile MJ. Pharmacokinetics and pharmacodynamics of ertapenem: an overview for clinicians. J Antimicrob Chemother 2004; 53 Suppl 2: ii23–8.[Abstract]

9. Turnidge JD. The pharmacodynamics of ß-lactams. Clin Infect Dis 1998; 27: 10–22.[Web of Science][Medline]

10. Friedland I, Mixson LA, Majumdar A et al. In vitro activity of ertapenem against common clinical isolates in relation to human pharmacokinetics. J Chemother 2002; 14: 483–91.[Web of Science][Medline]

11. Craig WA. Pharmacokinetic/pharmacodynamic parameters: rationale for antibacterial dosing of mice and men. Clin Infect Dis 1998; 26: 1–10.[Web of Science][Medline]

12. Solomkin JS, Yellin AE, Rotstein OD et al. Ertapenem versus piperacillin/tazobactam in the treatment of complicated intraabdominal infections: results of a double blind, randomized comparative phase III trial. Ann Surg 2003; 237: 235–45.[CrossRef][Web of Science][Medline]

13. Yellin AE, Hassett JM, Fernandez A et al. Ertapenem monotherapy versus combined therapy with ceftriaxone plus metronidazole for treatment of complicated intraabdominal infections in adults. Int J Antimicrob Agents 2002; 20: 165–7.[CrossRef][Web of Science][Medline]


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