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Pharmacokinetics and brain penetration of carbapenems in mice
J Infect Chemother 22 (2016) 346e349
Contents lists available at ScienceDirect
Journal of Infection and Chemotherapy journal homepage: http://www.elsevier.com/locate/jic
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Pharmacokinetics and brain penetration of carbapenems in mice Kazuaki Matsumoto a, *, Yuji Kurihara a, b, Yuko Kuroda a, Seiji Hori c, Junko Kizu a a
Division of Practical Pharmacy, Keio University Faculty of Pharmacy, 1-5-30 Shibakoen, Minato-ku, Tokyo 105-8512, Japan Molecular Medical Bioscience Laboratory, Yokohama City University Graduate School of Medical Life Science, 1-7-29 Suehiro-cho, Turumi-ku, Yokoyama 230-0045, Japan c Department of Infectious Diseases and Infection Control, Jikei University School of Medicine, 3-25-8 Nishi-Shinbashi, Minato-ku, Tokyo 105-8461, Japan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 July 2015 Received in revised form 12 November 2015 Accepted 24 November 2015 Available online 22 January 2016
An adverse effect associated with the administration of carbapenems is central nervous system (CNS) toxicity, with higher brain concentrations of carbapenems being linked to an increased risk of seizures. However, the pharmacokinetics and brain penetration of carbapenems have not yet been examined. Thus, the aim of this in vivo investigation was to determine the pharmacokinetics and brain penetration of carbapenems in mice. Blood samples and brain tissue samples were obtained 10, 20, 30, 60, and 120 min after the subcutaneous administration of carbapenems (91 mg/kg). We obtained the following values for the pharmacokinetic parameters of carbapenems in mice: 1.20e1.71 L/h/kg for CLtotal/F, 1.41e2.03 h1 for Ke, 0.34 e0.51 h for T1/2, 0.66e0.95 L/kg for Vss/F, 0.49e0.73 h for MRT, 83.46e110.58 mg/mL for Cmax, plasma, and 0.28e0.83 mg/g for Cmax, brain tissue. The AUC0∞ of the carbapenems tested in plasma were in the following order: doripenem > meropenem > biapenem > imipenem, and in brain tissue were: imipenem > doripenem > meropenem > biapenem. The degrees of brain tissue penetration, defined as the AUC0∞, brain tissue/fAUC0∞, plasma ratio, were 0.016 for imipenem, 0.004 for meropenem, 0.002 for biapenem, and 0.008 for doripenem. The results of the present study demonstrated that, of the carbapenems examined, imipenem penetrated brain tissue to the greatest extent. © 2015, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Keywords: Carbapenems Pharmacokinetics Brain penetration
Carbapenem antibiotics possess a broad antimicrobial spectrum and are commonly used to treat complicated and serious bacterial infections. Although carbapenems are tolerated well by most patients, an important adverse effect associated with their administration is central nervous system (CNS) toxicity [1]. Early clinical trials reported a relationship between the use of imipenem and development of seizures [1], which remains a concern, particularly at high doses of carbapenems [2]. A previous study investigated the risk of seizures with the use of carbapenems and reported rates as high as 6%, especially when dosing was not carefully adjusted with respect to renal function [3]. Therefore, higher brain concentrations of carbapenems have been linked to an increased risk of seizures. The penetration of drugs into brain tissue may vary according to the kind of carbapenems administered. A meta-analysis previously revealed that the odds ratios for the risk of seizures from imipenem,
* Corresponding author. Tel./fax: þ81 3 5400 2656. E-mail address: [email protected] (K. Matsumoto).
meropenem, and doripenem relative to other antibiotics were 3.50 (95% confidence interval (CI) 2.23, 5.49), 1.04 (95% CI 0.61, 1.77), and 0.44 (95% CI 0.13, 1.53), respectively [4]. Furthermore, among the carbapenems examined, meropenem and biapenem induced weaker convulsive activity than imipenem in animals [5,6]. Nevertheless, the pharmacokinetics and brain penetration of carbapenems have not yet been examined. Therefore, the aim of this in vivo investigation was to determine the pharmacokinetics and brain penetration of carbapenems in mice. This study was reviewed and approved by the Animal Experimentation Committee of Kyoritsu University of Pharmacy (#42), and was performed in compliance with its Animal Experimental Guidelines. Male ddY mice (5-week-old) were supplied by Sankyo Labo Service Co., Ltd. (Tokyo, Japan), kept under a 12 h/12 h lightedark cycle for a week with free access to food and water, and were used in experiments at the age of 6 weeks old. Imipenem and cilastatin were purchased from USP (Rockville MD, USA) and Wako Pure Chemical Industies, Ltd. (Osaka, Japan), respectively. Meropenem, biapenem, and doripenem were a gift
http://dx.doi.org/10.1016/j.jiac.2015.11.010 1341-321X/© 2015, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
K. Matsumoto et al. / J Infect Chemother 22 (2016) 346e349
from Sumitomo Dainippon Pharma Co., Ltd. (Osaka, Japan), Meiji Seika Pharma Co., Ltd. (Tokyo, Japan), and Shionogi Co., Ltd. (Osaka, Japan). Other reagents used in this study were of analytical grade. Single-dose plasma pharmacokinetic studies were performed after the subcutaneous administration of 9.1 mg/mL of the different carbapenems and cilastatin dissolved in saline, because imipenem/ cilastatin could dissolve in saline up to 9.1 mg/mL. Cilastatin was administered to avoid the degradation of carbapenems. Blood samples and brain tissue samples, after decapitation under ether anesthesia, were obtained 10, 20, 30, 60, and 120 min after the subcutaneous administration of carbapenems (91 mg/kg) (four animals per time point). The blood samples collected at each sampling time point were centrifuged (3000 rpm, 10 min) immediately after collection, and the obtained plasma was stored frozen at 80 C for later analyses. After the brain tissue sample was washed with saline and weighed, the sample was added at 2 times its volume weight to the mobile phase. The mixture was homogenized using a polytron homogenizer to prepare a brain tissue suspension, which was then centrifuged (13,000 rpm, 10 min). The obtained supernatant was stored frozen at 80 C for later analyses. Carbapenem concentrations in plasma and brain tissue were measured using high-performance liquid chromatography (HPLC) [7]. Ninety microliters of the mobile phase was added to a 10-mL aliquot of a plasma sample. The whole volume was then pipetted into a centrifugal filter (10,000 NMWL Filter Unit; Merck Millipore, Darmstadt, Germany) and centrifuged (14,000 rpm, 20 min), after which a 20-mL aliquot of the filtrate was injected into the HPLC equipment. A 140-mL aliquot of the supernatant obtained from brain tissue was pipetted into a centrifugal filter device and centrifuged (14,000 rpm, 30 min), after which a 20-mL aliquot of the filtrate was injected into the HPLC equipment. HPLC employed a Luna 5um C18(2) 100 Å column (250 4.6 mm; Phenomenex Ltd, Torrance, USA) and detected ultraviolet absorbance at a wavelength of 300 nm. The mobile phase consisted of 0.1 M phosphate buffer (pH 7.8):methanol ¼ 92:8 for imipenem and biapenem, and 78:22 for meropenem and doripenem. The quantification limits were 0.02 mg/mL for imipenem and biapenem and 0.04 mg/mL for meropenem and doripenem. The intra- and inter-day accuracy (as absolute values of the relative errors of the means) and precision (as coefficient of variation values) were within 10%. A non-compartmental pharmacokinetic analysis was conducted to estimate the rate and extent of the penetration of carbapenems from the systemic circulation into brain tissue. Cmax was defined as the observed maximum concentration of carbapenems, and Tmax was the time to Cmax. The area under the drug concentrationetime curve from 0 to infinity (AUC0∞) and mean residence time (MRT) were calculated based on the trapezoidal rule (Microsoft Excel 2010). Using plasma concentration data, total clearance (CLtotal/F) was estimated as dose/AUC0∞, and the volume of distribution at a steady state (Vss/F) was calculated as CLtotal/F*MRT. The elimination half-life (T1/2) was estimated by dividing 0.693 by the elimination rate constant Ke (¼ CLtotal/F/Vss). Since protein binding rates of imipenem, meropenem, biapenem and doripenem in mouse serum were previously reported to be 2.5, 18.9, 3.8 and 25.2%, respectively [8,9], the free AUC0∞ for plasma were calculated: fAUC0∞, plasma ¼ (1 protein binding rates of carbapenems)*AUC0∞, plasma. Data were subjected to an analysis of variance using SPSS version 22 for Windows (IBM Japan Co., Ltd., Tokyo, Japan). Data were analyzed by Tukey's HSD tests. Differences of P < 0.05 were considered significant. The concentrations of carbapenems observed in plasma were shown in Fig. 1. The concentration of doripenem was significantly higher than that of imipenem at 10 min (P < 0.05). Furthermore, the concentration of meropenem was significantly higher than those of
347
Fig. 1. Observed plasma concentrations of carbapenems in mice after a single subcutaneous administration (91 mg/kg). Data are the average values of 4 experiments (±S. D.). *: P < 0.05 significantly different from imipenem, **: P < 0.05 significantly different from biapenem.
imipenem and biapenem at 60 and 120 min (P < 0.05). Plasma pharmacokinetics parameters were summarized in Table 1, and the following values were obtained: 1.20e1.71 L/h/kg for CLtotal/F, 1.41e2.03 h1 for Ke, 0.34e0.51 h for T1/2, 0.66e0.95 L/kg for Vss/F, 0.49e0.73 h for MRT, and 83.46e110.58 mg/mL for Cmax. Tmax was 10 min. The AUC0∞ of the carbapenems tested in plasma were as follows: doripenem > meropenem > biapenem > imipenem (Table 1). The observed concentrations of carbapenems in brain tissue were shown in Fig. 2. The concentration of imipenem was significantly higher than those of meropenem and biapenem at 10 and 20 min (P < 0.05), while the concentration of doripenem was significantly higher than that of biapenem at 20 min (P < 0.05). The concentrations of imipenem and doripenem were significantly higher than those of meropenem and biapenem at 30 min (P < 0.05). The concentration of imipenem was significantly higher than those of the other carbapenems at 60 min (P < 0.05). The concentration of doripenem was significantly higher than that of biapenem at 60 min (P < 0.05). The ranges of Cmax and Tmax were 0.28e0.83 mg/g and 10e20 min, respectively. The AUC0∞ of carbapenems in brain tissue were in the following order: imipenem > doripenem > meropenem > biapenem (Table 1). Furthermore, fAUC0∞, plasma for imipenem, meropenem, biapenem and doripenem were 52.00, 57.19, 58.02 and 56.92 mg*h/mL, respectively. The degrees of brain tissue penetration, defined as the AUC0∞, brain tissue/fAUC0∞, plasma ratio, were 0.016 for imipenem, 0.004 for meropenem, 0.002 for biapenem, and 0.008 for doripenem. And fCmax, plasma for imipenem, meropenem, biapenem and doripenem were 81.37, 69.34, 86.80 and 82.71 mg/mL, respectively. The Cmax, brain tissue/fCmax, plasma ratio, were 0.010 for imipenem, 0.006 for meropenem, 0.003 for biapenem, and 0.007 for doripenem. We herein showed the detailed pharmacokinetic parameters and penetration into brain tissue of each carbapenem, and demonstrated that, among the carbapenems tested, imipenem had the highest AUC0∞, brain tissue/fAUC0∞, plasma ratio. Previous studies on the mouse pharmacokinetics of carbapenems demonstrated that the AUC24 h of imipenem was 60.6 mg*h/ mL at a subcutaneous dose of 128 mg/kg/day [10], the AUC0∞ of meropenem and biapenem were 61.0 and 77.6 mg*h/mL, respectively, at an intraperitoneal dose of 100 mg/kg/day [11], and the AUC0∞ of doripenem was 75.3 mg*h/mL at an intravenous dose of 100 mg/kg/day [8]. As shown in Table 1, our results were consistent with these findings. However, detailed pharmacokinetic parameters of carbapenems were not shown in the previous studies, and
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K. Matsumoto et al. / J Infect Chemother 22 (2016) 346e349
Table 1 Pharmacokinetic parameters of carbapenems in mice after a single subcutaneous administration (91 mg/kg) (n ¼ 4). CLtotal/F (L/h/kg)
IPM MEPM BIPM DRPM
1.71 1.30 1.52 1.20
± ± ± ±
0.12 0.15 0.12 0.09
Ke (h1)
2.03 1.41 2.03 1.83
± ± ± ±
Vss/F (L/kg)
T1/2 (h)
0.44 0.30 0.13 0.21
0.35 0.51 0.34 0.38
± ± ± ±
0.08 0.09 0.02 0.04
0.87 0.95 0.75 0.66
± ± ± ±
MRT (h)
0.17 0.21 0.05 0.08
0.51 0.73 0.49 0.55
Cmax
AUC0∞
Plasma (mg/mL) ± ± ± ±
0.11 0.13 0.03 0.06
83.46 85.50 90.23 110.58
± ± ± ±
Brain tissue (mg/g) 8.86 10.81 7.89 18.85
0.83 0.41 0.28 0.58
± ± ± ±
0.22 0.11 0.01 0.12
Plasma (mg*h/mL) 53.33 70.52 60.31 76.09
± ± ± ±
4.07 7.15 5.09 6.05
Brain tissue (mg*h/g) 0.84 0.24 0.12 0.46
± ± ± ±
0.29 0.02 0.03 0.07
CLtotal, total clearance; F, bioavailability; Ke, elimination rate constant; T1/2, elimination half-life; Vss, volume of distribution at a steady state; MRT, mean residence time; AUC0∞, area under the plasma concentrationetime curve from 0 to infinity. The pharmacokinetics parameters were calculated using total plasma concentrations.
Fig. 2. Observed brain tissue concentrations of carbapenems in mice after a single subcutaneous administration (91 mg/kg). Data are the average values of 4 experiments (±S. D.). **: P < 0.05 significantly different from biapenem, #: P < 0.05 significantly different from meropenem, ##: P < 0.05 significantly different from doripenem.
their penetration into brain tissue was not evaluated. In the present study, we found that AUC0∞, brain tissue/fAUC0∞, plasma ratios were 0.002e0.016, which were very low (Table 1). The penetration of imipenem into brain tissue was 2.0e7.7-fold greater than that of the other carbapenems. Imipenem was previously shown to be transported through the bloodebrain barrier (BBB) principally via passive diffusion, and not a specific carrier-mediated process [12]. Physicochemical properties such as molecular weight and lipophilicity are the dominant factors in determining unidirectional transport through the BBB [12]. The molecular weights were in the following order: imipenem < biapenem < meropenem < doripenem, while their partition coefficient values were imipenem > biapenem j doripenem j meropenem. Therefore, among the carbapenems examined here, imipenem was considered the most likely to be transported via passive diffusion. On the other hand, Suzuki et al. reported that imipenem had minimal affinity for the organic anion transporter, resulting in its slow elimination from the CNS [12]. Although the affinities of carbapenems for the organic anion transporter may differ, they have not yet been examined; therefore, further studies are needed to investigate differences in the penetration of carbapenems into brain tissue. A previous mouse intracerebroventricular injection study reported that imipenem induced clonic convulsions in a dosedependent manner (2.5e10 mg/mouse), whereas meropenem and doripenem did not induce convulsions at doses of up to 100 mg/ mouse [6]. In dog intracerebroventricular injection studies, although doripenem did not affect electroencephalograms or behavior even at the highest dose tested (1000 mg/dog), 100 mg/dog of imipenem and 300 mg/dog of meropenem caused clonic convulsions [6]. Previous studies have suggested that the convulsive actions of b-lactam antibiotics may be related to their abilities to
inhibit g-aminobutyric acid (GABAA) receptor binding [5,13]. In in vitro binding studies, the 50% inhibitory concentration values for the specific binding of [3H]muscimol, a GABAA receptor agonist, in mouse synaptic membranes were 0.48, 15.63, and 46.44 mM for imipenem, meropenem, and doripenem, respectively [6]. Doripenem had the lowest affinity for GABAA receptors. Day et al. reported that although imipenem (400 mg/kg) significantly lowered the convulsive threshold of pentylenetetrazol in mice, no significant differences were observed between meropenem (400 mg/kg), biapenem (400 mg/kg), and saline (control) [5]. Meropenem and biapenem did not inhibit [3H]muscimol binding to the GABAA receptor complex in rat brain homogenates at concentrations of 1e10 mM, whereas imipenem induced a significant degree of inhibition [5]. These findings indicated that imipenem had the highest affinity for GABAA receptors. Thus, imipenem is the most likely to cause seizures because, among the carbapenems examined here, it penetrated brain tissue to the greatest extent and had the highest affinity for GABAA receptors. In this study, cilastatin, an inhibitor of dehydropeptidase-1 activity, was added to each carbapenem solution in order to avoid the degradation of carbapenems. Each carbapenem (91 mg/kg) was subcutaneously administered to mice concomitantly with cilastatin (91 mg/kg). Previous studies reported that cilastatin itself is not epileptogenic [14], and that it does not interfere with the pharmacokinetics of imipenem in the CNS [15]. An allometrically scaled dose of 91 mg/kg in the mouse would be expected to be equivalent to a human equivalent dose of 7.4 mg/kg (i.e. 91/12.3 ¼ 7.4). If a dose of 7.4 mg/kg were given to a 70 kg human patient the allometrically equivalent dose given to the human would be 517 mg of carbapenem. In conclusion, the results of the present study demonstrated that, among the carbapenems tested, imipenem penetrated brain tissue to the greatest extent. Conflict of interest Seiji Hori has received a speaker's honoraria and grant support from Daiichi Sankyo Co., Ltd. All other authors report no conflicts of interest. References [1] Calandra GB, Brown KR, Grad LC, Ahonkhai VI, Wang C, Aziz MA. Review of adverse experiences and tolerability in the first 2,516 patients treated with imipenem/cilastatin. Am J Med 1985;78:73e8. [2] Winston DJ, Ho WG, Bruckner DA, Champlin RE. Beta-lactam antibiotic therapy in febrile granulocytopenic patients. A randomized trial comparing cefoperazone plus piperacillin, ceftazidime plus piperacillin, and imipenem alone. Ann Intern Med 1991;115:849e59. [3] Zhanel GG, Ketter N, Rubinstein E, Friedland I, Redman R. Overview of seizureinducing potential of doripenem. Drug Saf 2009;32:709e16. [4] Cannon JP, Lee TA, Clark NM, Setlak P, Grim SA. The risk of seizures among the carbapenems: a meta-analysis. J Antimicrob Chemother 2014;69:2043e55.
K. Matsumoto et al. / J Infect Chemother 22 (2016) 346e349 [5] Day IP, Goudie J, Nishiki K, Williams PD. Correlation between in vitro and in vivo models of proconvulsive activity with the carbapenem antibiotics, biapenem, imipenem/cilastatin and meropenem. Toxicol Lett 1995;76: 239e43. [6] Horiuchi M, Kimura M, Tokumura M, Hasebe N, Arai T, Abe K. Absence of convulsive liability of doripenem, a new carbapenem antibiotic, in comparison with beta-lactam antibiotics. Toxicology 2006;222:114e24. [7] Kurihara Y, Kizu J, Hori S. Simple and rapid determination of serum carbapenem concentrations by high-performance liquid chromatography. J Infect Chemother 2008;14:30e4. [8] Hori T, Nakano M, Kimura Y, Murakami K. Pharmacokinetics and tissue penetration of a new carbapenem, doripenem, intravenously administered to laboratory animals. In Vivo 2006;20:91e6. [9] Yamashita N, Kawashita K, Nomura K, Takeuchi H, Hikida M, Naruse T. Pharmacokinetics of biapenem in laboratory animals. Chemother (Tokyo) 1994;42:S243e50. [10] Stearne LE, Buijk SL, Mouton JW, Gyssens IC. Effect of a single percutaneous abscess drainage puncture and imipenem therapy, alone or in combination, in
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treatment of mixed-infection abscesses in mice. Antimicrob Agents Chemother 2002;46:3712e8. Yamada K, Yamamoto Y, Yanagihara K, Araki N, Harada Y, Morinaga Y, et al. In vivo efficacy and pharmacokinetics of biapenem in a murine model of ventilator-associated pneumonia with Pseudomonas aeruginosa. J Infect Chemother 2012;18:472e8. Suzuki H, Sawada Y, Sugiyama Y, Iga T, Hanano M, Spector R. Transport of imipenem, a novel carbapenem antibiotic, in the rat central nervous system. J Pharmacol Exp Ther 1989;250:979e84. Shimada J, Hori S, Kanemitsu K, Shoji Y, Nakashio S, Yanagawa A. A comparative study on the convulsant activity of carbapenems and betalactams. Drugs Exp Clin Res 1992;18:377e81. Williams PD, Bennett DB, Comereski CR. Animal model for evaluating the convulsive liability of beta-lactam antibiotics. Antimicrob Agents Chemother 1988;32:758e60. Washburn DE, Perfect JR, Durack DT. Pharmacokinetics of N-formimidoyl thienamycin and influence of a renal dipeptidase inhibitor in experimental meningitis. J Antimicrob Chemother 1983;12:39e45.
Contents lists available at ScienceDirect
Journal of Infection and Chemotherapy journal homepage: http://www.elsevier.com/locate/jic
Note
Pharmacokinetics and brain penetration of carbapenems in mice Kazuaki Matsumoto a, *, Yuji Kurihara a, b, Yuko Kuroda a, Seiji Hori c, Junko Kizu a a
Division of Practical Pharmacy, Keio University Faculty of Pharmacy, 1-5-30 Shibakoen, Minato-ku, Tokyo 105-8512, Japan Molecular Medical Bioscience Laboratory, Yokohama City University Graduate School of Medical Life Science, 1-7-29 Suehiro-cho, Turumi-ku, Yokoyama 230-0045, Japan c Department of Infectious Diseases and Infection Control, Jikei University School of Medicine, 3-25-8 Nishi-Shinbashi, Minato-ku, Tokyo 105-8461, Japan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 July 2015 Received in revised form 12 November 2015 Accepted 24 November 2015 Available online 22 January 2016
An adverse effect associated with the administration of carbapenems is central nervous system (CNS) toxicity, with higher brain concentrations of carbapenems being linked to an increased risk of seizures. However, the pharmacokinetics and brain penetration of carbapenems have not yet been examined. Thus, the aim of this in vivo investigation was to determine the pharmacokinetics and brain penetration of carbapenems in mice. Blood samples and brain tissue samples were obtained 10, 20, 30, 60, and 120 min after the subcutaneous administration of carbapenems (91 mg/kg). We obtained the following values for the pharmacokinetic parameters of carbapenems in mice: 1.20e1.71 L/h/kg for CLtotal/F, 1.41e2.03 h1 for Ke, 0.34 e0.51 h for T1/2, 0.66e0.95 L/kg for Vss/F, 0.49e0.73 h for MRT, 83.46e110.58 mg/mL for Cmax, plasma, and 0.28e0.83 mg/g for Cmax, brain tissue. The AUC0∞ of the carbapenems tested in plasma were in the following order: doripenem > meropenem > biapenem > imipenem, and in brain tissue were: imipenem > doripenem > meropenem > biapenem. The degrees of brain tissue penetration, defined as the AUC0∞, brain tissue/fAUC0∞, plasma ratio, were 0.016 for imipenem, 0.004 for meropenem, 0.002 for biapenem, and 0.008 for doripenem. The results of the present study demonstrated that, of the carbapenems examined, imipenem penetrated brain tissue to the greatest extent. © 2015, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Keywords: Carbapenems Pharmacokinetics Brain penetration
Carbapenem antibiotics possess a broad antimicrobial spectrum and are commonly used to treat complicated and serious bacterial infections. Although carbapenems are tolerated well by most patients, an important adverse effect associated with their administration is central nervous system (CNS) toxicity [1]. Early clinical trials reported a relationship between the use of imipenem and development of seizures [1], which remains a concern, particularly at high doses of carbapenems [2]. A previous study investigated the risk of seizures with the use of carbapenems and reported rates as high as 6%, especially when dosing was not carefully adjusted with respect to renal function [3]. Therefore, higher brain concentrations of carbapenems have been linked to an increased risk of seizures. The penetration of drugs into brain tissue may vary according to the kind of carbapenems administered. A meta-analysis previously revealed that the odds ratios for the risk of seizures from imipenem,
* Corresponding author. Tel./fax: þ81 3 5400 2656. E-mail address: [email protected] (K. Matsumoto).
meropenem, and doripenem relative to other antibiotics were 3.50 (95% confidence interval (CI) 2.23, 5.49), 1.04 (95% CI 0.61, 1.77), and 0.44 (95% CI 0.13, 1.53), respectively [4]. Furthermore, among the carbapenems examined, meropenem and biapenem induced weaker convulsive activity than imipenem in animals [5,6]. Nevertheless, the pharmacokinetics and brain penetration of carbapenems have not yet been examined. Therefore, the aim of this in vivo investigation was to determine the pharmacokinetics and brain penetration of carbapenems in mice. This study was reviewed and approved by the Animal Experimentation Committee of Kyoritsu University of Pharmacy (#42), and was performed in compliance with its Animal Experimental Guidelines. Male ddY mice (5-week-old) were supplied by Sankyo Labo Service Co., Ltd. (Tokyo, Japan), kept under a 12 h/12 h lightedark cycle for a week with free access to food and water, and were used in experiments at the age of 6 weeks old. Imipenem and cilastatin were purchased from USP (Rockville MD, USA) and Wako Pure Chemical Industies, Ltd. (Osaka, Japan), respectively. Meropenem, biapenem, and doripenem were a gift
http://dx.doi.org/10.1016/j.jiac.2015.11.010 1341-321X/© 2015, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
K. Matsumoto et al. / J Infect Chemother 22 (2016) 346e349
from Sumitomo Dainippon Pharma Co., Ltd. (Osaka, Japan), Meiji Seika Pharma Co., Ltd. (Tokyo, Japan), and Shionogi Co., Ltd. (Osaka, Japan). Other reagents used in this study were of analytical grade. Single-dose plasma pharmacokinetic studies were performed after the subcutaneous administration of 9.1 mg/mL of the different carbapenems and cilastatin dissolved in saline, because imipenem/ cilastatin could dissolve in saline up to 9.1 mg/mL. Cilastatin was administered to avoid the degradation of carbapenems. Blood samples and brain tissue samples, after decapitation under ether anesthesia, were obtained 10, 20, 30, 60, and 120 min after the subcutaneous administration of carbapenems (91 mg/kg) (four animals per time point). The blood samples collected at each sampling time point were centrifuged (3000 rpm, 10 min) immediately after collection, and the obtained plasma was stored frozen at 80 C for later analyses. After the brain tissue sample was washed with saline and weighed, the sample was added at 2 times its volume weight to the mobile phase. The mixture was homogenized using a polytron homogenizer to prepare a brain tissue suspension, which was then centrifuged (13,000 rpm, 10 min). The obtained supernatant was stored frozen at 80 C for later analyses. Carbapenem concentrations in plasma and brain tissue were measured using high-performance liquid chromatography (HPLC) [7]. Ninety microliters of the mobile phase was added to a 10-mL aliquot of a plasma sample. The whole volume was then pipetted into a centrifugal filter (10,000 NMWL Filter Unit; Merck Millipore, Darmstadt, Germany) and centrifuged (14,000 rpm, 20 min), after which a 20-mL aliquot of the filtrate was injected into the HPLC equipment. A 140-mL aliquot of the supernatant obtained from brain tissue was pipetted into a centrifugal filter device and centrifuged (14,000 rpm, 30 min), after which a 20-mL aliquot of the filtrate was injected into the HPLC equipment. HPLC employed a Luna 5um C18(2) 100 Å column (250 4.6 mm; Phenomenex Ltd, Torrance, USA) and detected ultraviolet absorbance at a wavelength of 300 nm. The mobile phase consisted of 0.1 M phosphate buffer (pH 7.8):methanol ¼ 92:8 for imipenem and biapenem, and 78:22 for meropenem and doripenem. The quantification limits were 0.02 mg/mL for imipenem and biapenem and 0.04 mg/mL for meropenem and doripenem. The intra- and inter-day accuracy (as absolute values of the relative errors of the means) and precision (as coefficient of variation values) were within 10%. A non-compartmental pharmacokinetic analysis was conducted to estimate the rate and extent of the penetration of carbapenems from the systemic circulation into brain tissue. Cmax was defined as the observed maximum concentration of carbapenems, and Tmax was the time to Cmax. The area under the drug concentrationetime curve from 0 to infinity (AUC0∞) and mean residence time (MRT) were calculated based on the trapezoidal rule (Microsoft Excel 2010). Using plasma concentration data, total clearance (CLtotal/F) was estimated as dose/AUC0∞, and the volume of distribution at a steady state (Vss/F) was calculated as CLtotal/F*MRT. The elimination half-life (T1/2) was estimated by dividing 0.693 by the elimination rate constant Ke (¼ CLtotal/F/Vss). Since protein binding rates of imipenem, meropenem, biapenem and doripenem in mouse serum were previously reported to be 2.5, 18.9, 3.8 and 25.2%, respectively [8,9], the free AUC0∞ for plasma were calculated: fAUC0∞, plasma ¼ (1 protein binding rates of carbapenems)*AUC0∞, plasma. Data were subjected to an analysis of variance using SPSS version 22 for Windows (IBM Japan Co., Ltd., Tokyo, Japan). Data were analyzed by Tukey's HSD tests. Differences of P < 0.05 were considered significant. The concentrations of carbapenems observed in plasma were shown in Fig. 1. The concentration of doripenem was significantly higher than that of imipenem at 10 min (P < 0.05). Furthermore, the concentration of meropenem was significantly higher than those of
347
Fig. 1. Observed plasma concentrations of carbapenems in mice after a single subcutaneous administration (91 mg/kg). Data are the average values of 4 experiments (±S. D.). *: P < 0.05 significantly different from imipenem, **: P < 0.05 significantly different from biapenem.
imipenem and biapenem at 60 and 120 min (P < 0.05). Plasma pharmacokinetics parameters were summarized in Table 1, and the following values were obtained: 1.20e1.71 L/h/kg for CLtotal/F, 1.41e2.03 h1 for Ke, 0.34e0.51 h for T1/2, 0.66e0.95 L/kg for Vss/F, 0.49e0.73 h for MRT, and 83.46e110.58 mg/mL for Cmax. Tmax was 10 min. The AUC0∞ of the carbapenems tested in plasma were as follows: doripenem > meropenem > biapenem > imipenem (Table 1). The observed concentrations of carbapenems in brain tissue were shown in Fig. 2. The concentration of imipenem was significantly higher than those of meropenem and biapenem at 10 and 20 min (P < 0.05), while the concentration of doripenem was significantly higher than that of biapenem at 20 min (P < 0.05). The concentrations of imipenem and doripenem were significantly higher than those of meropenem and biapenem at 30 min (P < 0.05). The concentration of imipenem was significantly higher than those of the other carbapenems at 60 min (P < 0.05). The concentration of doripenem was significantly higher than that of biapenem at 60 min (P < 0.05). The ranges of Cmax and Tmax were 0.28e0.83 mg/g and 10e20 min, respectively. The AUC0∞ of carbapenems in brain tissue were in the following order: imipenem > doripenem > meropenem > biapenem (Table 1). Furthermore, fAUC0∞, plasma for imipenem, meropenem, biapenem and doripenem were 52.00, 57.19, 58.02 and 56.92 mg*h/mL, respectively. The degrees of brain tissue penetration, defined as the AUC0∞, brain tissue/fAUC0∞, plasma ratio, were 0.016 for imipenem, 0.004 for meropenem, 0.002 for biapenem, and 0.008 for doripenem. And fCmax, plasma for imipenem, meropenem, biapenem and doripenem were 81.37, 69.34, 86.80 and 82.71 mg/mL, respectively. The Cmax, brain tissue/fCmax, plasma ratio, were 0.010 for imipenem, 0.006 for meropenem, 0.003 for biapenem, and 0.007 for doripenem. We herein showed the detailed pharmacokinetic parameters and penetration into brain tissue of each carbapenem, and demonstrated that, among the carbapenems tested, imipenem had the highest AUC0∞, brain tissue/fAUC0∞, plasma ratio. Previous studies on the mouse pharmacokinetics of carbapenems demonstrated that the AUC24 h of imipenem was 60.6 mg*h/ mL at a subcutaneous dose of 128 mg/kg/day [10], the AUC0∞ of meropenem and biapenem were 61.0 and 77.6 mg*h/mL, respectively, at an intraperitoneal dose of 100 mg/kg/day [11], and the AUC0∞ of doripenem was 75.3 mg*h/mL at an intravenous dose of 100 mg/kg/day [8]. As shown in Table 1, our results were consistent with these findings. However, detailed pharmacokinetic parameters of carbapenems were not shown in the previous studies, and
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K. Matsumoto et al. / J Infect Chemother 22 (2016) 346e349
Table 1 Pharmacokinetic parameters of carbapenems in mice after a single subcutaneous administration (91 mg/kg) (n ¼ 4). CLtotal/F (L/h/kg)
IPM MEPM BIPM DRPM
1.71 1.30 1.52 1.20
± ± ± ±
0.12 0.15 0.12 0.09
Ke (h1)
2.03 1.41 2.03 1.83
± ± ± ±
Vss/F (L/kg)
T1/2 (h)
0.44 0.30 0.13 0.21
0.35 0.51 0.34 0.38
± ± ± ±
0.08 0.09 0.02 0.04
0.87 0.95 0.75 0.66
± ± ± ±
MRT (h)
0.17 0.21 0.05 0.08
0.51 0.73 0.49 0.55
Cmax
AUC0∞
Plasma (mg/mL) ± ± ± ±
0.11 0.13 0.03 0.06
83.46 85.50 90.23 110.58
± ± ± ±
Brain tissue (mg/g) 8.86 10.81 7.89 18.85
0.83 0.41 0.28 0.58
± ± ± ±
0.22 0.11 0.01 0.12
Plasma (mg*h/mL) 53.33 70.52 60.31 76.09
± ± ± ±
4.07 7.15 5.09 6.05
Brain tissue (mg*h/g) 0.84 0.24 0.12 0.46
± ± ± ±
0.29 0.02 0.03 0.07
CLtotal, total clearance; F, bioavailability; Ke, elimination rate constant; T1/2, elimination half-life; Vss, volume of distribution at a steady state; MRT, mean residence time; AUC0∞, area under the plasma concentrationetime curve from 0 to infinity. The pharmacokinetics parameters were calculated using total plasma concentrations.
Fig. 2. Observed brain tissue concentrations of carbapenems in mice after a single subcutaneous administration (91 mg/kg). Data are the average values of 4 experiments (±S. D.). **: P < 0.05 significantly different from biapenem, #: P < 0.05 significantly different from meropenem, ##: P < 0.05 significantly different from doripenem.
their penetration into brain tissue was not evaluated. In the present study, we found that AUC0∞, brain tissue/fAUC0∞, plasma ratios were 0.002e0.016, which were very low (Table 1). The penetration of imipenem into brain tissue was 2.0e7.7-fold greater than that of the other carbapenems. Imipenem was previously shown to be transported through the bloodebrain barrier (BBB) principally via passive diffusion, and not a specific carrier-mediated process [12]. Physicochemical properties such as molecular weight and lipophilicity are the dominant factors in determining unidirectional transport through the BBB [12]. The molecular weights were in the following order: imipenem < biapenem < meropenem < doripenem, while their partition coefficient values were imipenem > biapenem j doripenem j meropenem. Therefore, among the carbapenems examined here, imipenem was considered the most likely to be transported via passive diffusion. On the other hand, Suzuki et al. reported that imipenem had minimal affinity for the organic anion transporter, resulting in its slow elimination from the CNS [12]. Although the affinities of carbapenems for the organic anion transporter may differ, they have not yet been examined; therefore, further studies are needed to investigate differences in the penetration of carbapenems into brain tissue. A previous mouse intracerebroventricular injection study reported that imipenem induced clonic convulsions in a dosedependent manner (2.5e10 mg/mouse), whereas meropenem and doripenem did not induce convulsions at doses of up to 100 mg/ mouse [6]. In dog intracerebroventricular injection studies, although doripenem did not affect electroencephalograms or behavior even at the highest dose tested (1000 mg/dog), 100 mg/dog of imipenem and 300 mg/dog of meropenem caused clonic convulsions [6]. Previous studies have suggested that the convulsive actions of b-lactam antibiotics may be related to their abilities to
inhibit g-aminobutyric acid (GABAA) receptor binding [5,13]. In in vitro binding studies, the 50% inhibitory concentration values for the specific binding of [3H]muscimol, a GABAA receptor agonist, in mouse synaptic membranes were 0.48, 15.63, and 46.44 mM for imipenem, meropenem, and doripenem, respectively [6]. Doripenem had the lowest affinity for GABAA receptors. Day et al. reported that although imipenem (400 mg/kg) significantly lowered the convulsive threshold of pentylenetetrazol in mice, no significant differences were observed between meropenem (400 mg/kg), biapenem (400 mg/kg), and saline (control) [5]. Meropenem and biapenem did not inhibit [3H]muscimol binding to the GABAA receptor complex in rat brain homogenates at concentrations of 1e10 mM, whereas imipenem induced a significant degree of inhibition [5]. These findings indicated that imipenem had the highest affinity for GABAA receptors. Thus, imipenem is the most likely to cause seizures because, among the carbapenems examined here, it penetrated brain tissue to the greatest extent and had the highest affinity for GABAA receptors. In this study, cilastatin, an inhibitor of dehydropeptidase-1 activity, was added to each carbapenem solution in order to avoid the degradation of carbapenems. Each carbapenem (91 mg/kg) was subcutaneously administered to mice concomitantly with cilastatin (91 mg/kg). Previous studies reported that cilastatin itself is not epileptogenic [14], and that it does not interfere with the pharmacokinetics of imipenem in the CNS [15]. An allometrically scaled dose of 91 mg/kg in the mouse would be expected to be equivalent to a human equivalent dose of 7.4 mg/kg (i.e. 91/12.3 ¼ 7.4). If a dose of 7.4 mg/kg were given to a 70 kg human patient the allometrically equivalent dose given to the human would be 517 mg of carbapenem. In conclusion, the results of the present study demonstrated that, among the carbapenems tested, imipenem penetrated brain tissue to the greatest extent. Conflict of interest Seiji Hori has received a speaker's honoraria and grant support from Daiichi Sankyo Co., Ltd. All other authors report no conflicts of interest. References [1] Calandra GB, Brown KR, Grad LC, Ahonkhai VI, Wang C, Aziz MA. Review of adverse experiences and tolerability in the first 2,516 patients treated with imipenem/cilastatin. Am J Med 1985;78:73e8. [2] Winston DJ, Ho WG, Bruckner DA, Champlin RE. Beta-lactam antibiotic therapy in febrile granulocytopenic patients. A randomized trial comparing cefoperazone plus piperacillin, ceftazidime plus piperacillin, and imipenem alone. Ann Intern Med 1991;115:849e59. [3] Zhanel GG, Ketter N, Rubinstein E, Friedland I, Redman R. Overview of seizureinducing potential of doripenem. Drug Saf 2009;32:709e16. [4] Cannon JP, Lee TA, Clark NM, Setlak P, Grim SA. The risk of seizures among the carbapenems: a meta-analysis. J Antimicrob Chemother 2014;69:2043e55.
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