Pharmacokinetics of colistin methanesulfonate (CMS) in healthy Chinese subjects after single and multiple intravenous doses

Pharmacokinetics of colistin methanesulfonate (CMS) in healthy Chinese subjects after single and multiple intravenous doses

Accepted Manuscript Title: Pharmacokinetics of colistin methanesulfonate (CMS) in healthy chinese subjects after single and multiple intravenous doses...

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Accepted Manuscript Title: Pharmacokinetics of colistin methanesulfonate (CMS) in healthy chinese subjects after single and multiple intravenous doses Author: Miao Zhao, Xiao-Jie Wu, Ya-Xin Fan, Ying-yuan Zhang, Bei-Ning Guo, Ji-cheng Yu, Guo-ying Cao, Yuan-cheng Chen, Ju-fang Wu, Yao-guo Shi, Jian Li, Jing Zhang PII: DOI: Reference:

S0924-8579(17)30468-5 https://doi.org/10.1016/j.ijantimicag.2017.12.025 ANTAGE 5334

To appear in:

International Journal of Antimicrobial Agents

Received date: Accepted date:

11-10-2017 24-12-2017

Please cite this article as: Miao Zhao, Xiao-Jie Wu, Ya-Xin Fan, Ying-yuan Zhang, Bei-Ning Guo, Ji-cheng Yu, Guo-ying Cao, Yuan-cheng Chen, Ju-fang Wu, Yao-guo Shi, Jian Li, Jing Zhang, Pharmacokinetics of colistin methanesulfonate (CMS) in healthy chinese subjects after single and multiple intravenous doses, International Journal of Antimicrobial Agents (2018), https://doi.org/10.1016/j.ijantimicag.2017.12.025. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Pharmacokinetics of Colistin Methanesulfonate (CMS) in Healthy Chinese Subjects after Single and Multiple Intravenous Doses

Miao Zhao1,&, Xiao-Jie Wu1,&, Ya-Xin Fan1, Ying-yuan Zhang1, Bei-Ning Guo1, Ji-cheng Yu1, Guo-ying Cao1, Yuan-cheng Chen1, Ju-fang Wu1, Yao-guo Shi1, Jian Li2, Jing Zhang1*

1

Institute of Antibiotics, Huashan Hospital, Fudan University & Key Laboratory of

Clinical Pharmacology of Antibiotics, National Health and Family Planning Commission, Shanghai, China; 2Monash Biomedicine Discovery Institute, Department of Microbiology, Monash University, Clayton, Victoria 3800, Australia

Running Title: Colistin PK in Healthy Chinese Subjects

&

Authors contributed equally

*Address correspondence to: Prof. Jing Zhang, Institute of Antibiotics, Huashan Hospital, Fudan University, 12 Middle Wulumuqi Road, Shanghai 200040, China. Fax: +86-21-6248-4347. Email: [email protected]

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Highlights 

The first PK study of CMS and formed colistin in Chinese population.



Our study revealed that steady state was rapidly achieved for colistin in healthy subjects with the dose of 2.5 mg CBA/kg.



No significant accumulation was observed for CMS or colistin.



The first study to characterise urinary PK of CMS and colistin after a 7-day treatment in humans and the very high concentration of formed colistin in the urine.

Abstract High prevalence of extensive-drug resistance (XDR) gram-negative pathogens has forced clinicians to use colistin as the last-line therapy. Our knowledge on the pharmacokinetics (PK) of colistin methanesulfonate (CMS, an inactive prodrug) and colistin has increased substantially; however, the PK in Chinese population is still unknown due to the lack of a CMS product. This study aimed to evaluate the PK of a new CMS product developed in China for optimizing the dosing regimens. Twenty-four healthy subjects (12 female, 12 male) were enrolled in single- and multiple-dose PK studies. Concentrations of CMS and formed colistin in plasma and urine were measured and PK analysis was conducted using a non-compartmental approach. After a single dose of CMS (2.36 mg colistin based activity (CBA) per kg, 1 h infusion), Cmax values of CMS and formed colistin were 18.0 mg/L and 0.69 mg/L, respectively. The estimated half-life (T1/2) of CMS and colistin were 1.38 h and 4.49 h,

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respectively. Approximately 62.5% of the CMS dose was excreted through urine within 24 h after dosing, while only 1.28% was present in the form of colistin. After multiple-doses of CMS, colistin reached steady state within 24 h; there was no accumulation of CMS, but colistin accumulated slightly (RAUC 1.33). Our study provides the first set of PK data in Chinese population and is essential for the design of CMS dosing regimens in Chinese hospitals. The urinary PK data strongly support the use of intravenous CMS for serious urinary tract infections.

Keywords: Colistin methanesulfonate; pharmacokinetics; urinary recovery.

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INTRODUCTION The rapid spread of antibiotic resistance poses a serious threat to global public health and the era of antibiotics may come to an end if the discovery of novel antibiotics continues falling behind [1, 2]. ‘Old’ antibiotics, like colistin, have been increasingly used for the treatment of extensively drug resistant (XDR) gram-negative bacteria [3]. Colistin is a lipopeptide antibiotic which was approved for clinical use in the late 1950s but fell out of favour in the 1970s due to potential nephrotoxicity and neurotoxicity [4]. Without novel classes of antibiotics in the development pipeline [5], colistin has gained a considerable interest since the early 2000s[6]. The prevalence of XDR gram-negative pathogens have also increased rapidly in China in recent years. During 2005 to 2014 carbapenem resistance rates in A. baumannii and K. pneumoniae increased from 31% to 66.7% and from 2.9% to 13.4%, accordingly [7]. XDR has become one of the most troublesome issues in the management of bacterial infections in China [8]. Therefore, colistin-based combinations have been recommended by Chinese XDR Consensus Working Group as the therapeutic option for XDR infections [8].

Colistin is usually administered intravenously as a pro-drug, CMS, which converts to the antibacterial entity, colistin in vitro and in vivo [9]. Developing rational dosage regimens for colistin will be critical to optimizing their efficacy while minimizing nephrotoxicity. CMS is a very complex mixture of multiple components [10], and different CMS products may have different PK profiles [11-13]. Since a clear

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association between the timely initiation of appropriate antimicrobial therapy and patient survival has been established for conditions such as ventilator-associated pneumonia and septic shock [14, 15], differences in the concentration-time profiles of formed colistin after intravenous CMS may have a substantial impact on bacterial killing, emergence of resistance and clinical outcomes. In the ‘Bad Bugs, No Drugs’ era, CMS is a critically important component of the antibiotic armamentarium against difficult-to-treat XDR Gram-negative ‘superbugs’. CMS is unavailable in China and the pharmacokinetics (PK) in Chinese population is still unknown. Currently, a new brand of CMS product has been developed for treatment of XDR Gram-negative infections. Our study aimed to evaluate the PK of this new CMS product and formed colistin after intravenous administration in Chinese population. The results provide essential pharmacological information for the product information and optimization of its use in patients.

MATERIALS AND METHODS Study design and dosing regimens. The study was approved by the ethics committee of Huashan Hospital, Fudan University, China. It was an open-label study conducted in Huashan Hospital between February and July 2014. Informed consent was obtained from all subjects before they were enrolled. Healthy subjects between the ages of 19 and 45 years old, with body mass indices (BMI) between 19.0 and 24.0 and no pregnancy were eligible to participate in the study. All the volunteers were general

healthy

as

determined

by

their

medical

history,

physical

tests,

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electrocardiogram (ECG) and laboratory tests. No other medications were taken one week before and during the study in any subjects.

A total of 24 Chinese healthy volunteers were enrolled for a single-dose (n = 12, 2.5 mg colistin base activity (CBA) per kg CMS as a 1 h infusion) and multiple-dose (n = 12, 2.5 mg CBA/kg/12h CMS as a 1 h infusion for 7 days) study. CMS (Lot number: 130706122) was supplied by CHIA TAI TIAN-QING Pharmaceutical Group Co., LTD (Jiangsu, China) as a dry powder and reconstituted in 100 mL sterile saline immediately before dosing. A dose of 150 mg CBA was equivalent to approximately 336 mg of CMS. The maximum dose was 150 mg CBA in the single dose study and 300 mg CBA per day in the multiple-dose study. Safety, tolerability, and PK characteristics were assessed in all patients.

Safety evaluation The safety and clinical symptom were monitored and laboratory tests (including serum chemistry, haematology, urinalysis) were conducted during the study. Estimated creatinine clearance was calculated using the Cockcroft-Gault equation to determine the renal function. Biomarkers of early acute kidney injury such as urine β2microglobulin, urinary NAG (N-acetyl-β-D-glucosaminidase), urinary NAGL (neutrophil gelatinase-associated lipocalin), serum β2- macroglobulin and serum Cystatin C (CysC, cysteine dehydrogenase inhibitor C) were tested along with standard laboratory tests. Clinical symptoms were monitored by the clinician after CMS dosing

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and laboratory tests were conducted on the screening day, admission day, 24 h and 48 h after the end of the dose in the single-dose study; and in the multiple-dose study at 2 h before the administration on days 2, 4 and 7, and 48 h after the administration of the last dose on day 7.

Sample collection As CMS is not stable and can convert to colistin in vitro and in vivo [16, 17], all blood and urine samples were placed on ice immediately after the collection. Blood samples (4 mL) were collected from the cubital vein at pre-dose, during the intravenous infusion (30 min after the start of the infusion), immediately at the end of the infusion (1 h), and at 1.25, 1.5, 2, 2.5, 3, 5, 7, 9, 12, 17, 25, 37 and 49 h after the end of the 1 h infusion in the single-dose study. In the multiple-dose study, the time points were: (1) Days 1 and 7, pre-dose, during the intravenous infusion (30 min after the start of the infusion), immediately at the end of the infusion (1 h), and at 1.25, 1.5, 2, 2.5, 3, 5, 7, 9, 12 h; and (2) Days 2 - 6: pre-dose and immediately at the end of the infusion after the first dose. All blood samples were collected and drawn into Vacuette EDTA K2 tubes, then centrifuged at 2,000 g for 10 min at 4℃. Urine samples were collected at pre-dose, 0 - 3, 3 - 5, 5 - 9, 9 - 12, 12 - 17, 17 - 25, 25 - 37, 37 - 49 h in the single dose study using polypropylene pots to minimize the absorption of colistin to container as previously described [18] which were placed in a refrigerator (4℃) during the collection period. Time points for urine sample collection in the multiple-dose study were: Day 1, pre-dose, 0 - 3, 3 - 5, 5 - 9, 9 - 12 h; and Day 7, the same as those in the

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single-dose study above. All plasma and urine samples were divided into three aliquots and stored at - 70℃ within 60 min after the collection until the analysis by UPLC-MS/MS within a month [18].

Measurements of CMS and formed colistin in plasma and urine samples Concentrations of CMS and formed colistin in plasma and urine were measured using a validated UPLC-MS/MS method [18]. Weak-cation exchange solid-phase extraction (SPE) was employed to separate CMS and colistin in the complex biological matrix. After alkalized by 5% ammonia, samples were loaded on WCX SPE cartridges. For urine samples, 1% bovine serum albumin (BSA, MP Biomedicals, USA) was added before loading to prevent any potential nonspecific adsorption of colistin to the plastic ware. Colistin was then eluted with acetonitrile/water (30/70, containing 6% formic acid) and collected into an SPE 96-Deep Square Well for injection into the UPLC-MS/MS system, which includes a Waters ACQUITY UPLC system (Waters, USA) equipped with an auto-sampler thermos at 4°C and an Applied Biosystems API4000 QTRAP mass spectrometer (Applied Biosystems Sciex, USA). Colistin was detected in the electrospray ionization (ESI) - positive ion mode. Seven-point calibration curves were constructed for plasma and urine samples at colistin concentrations ranging from 0.05 to 5 mg/L. Biological samples were hydrolysed (sulfuric acid 0.5 M for 15 min) for CMS analysis. Intra- and inter-day precision and accuracy for plasma and urine samples were within 10.8% and 12.6%, respectively.

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PK and statistical analyses PK of CMS and formed colistin was carried out by a non-compartmental method using WinNonlin (Pharsight, CA, version 6.03). The peak concentration (Cmax), concentration at 12 h (C12h), and the time at which Cmax is observed (Tmax) were calculated. The terminal rate constant (Ke) was determined by regression analysis of the terminal log-linear part of the concentration-time curve for each subject. The elimination half-life (T1/2) was calculated as 0.693/Ke. The area under the plasma concentration-time curve (AUC0-12) was determined using the linear/log trapezoidal rule. AUC from zero to infinity (AUC0-∞) was calculated as AUC0-last+Clast/ke for both single-dose and multiple-dose studies. The accumulation ratio (R) was calculated as AUC0-12(day 7)/AUC0-12(single dose).

As CMS converts to colistin in vivo, it is highly likely that colistin in the urine was from the hydrolysis of CMS after excretion from the kidneys. However because CMS was virtually cleared from the body by 8 h after the dose, it was possible to obtain an estimate of colistin clearance from urine collected during the 8 - 48 h time interval without incurring the risk of error caused by post-excretion hydrolysis of CMS [16]. Assuming that renal clearance did not vary during the experiment in a given individual, it was possible to calculate the amount of colistin actually excreted in urine within each collected fraction and the amount excreted as CMS, as descried by the equations proposed by Couet et al.[19]. Therefore, the renal clearance (CLr) of CMS and colistin was calculated as Ae0-8/AUC0-8 and Ae8-48/AUC8-48, where Ae is the

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amount of CMS and colistin excreted in the urine samples specified. These time intervals were used to minimize the influence of CMS degradation to colistin[19]. The fraction of CMS excreted in urine (fe) was calculated as the ratio between renal and total clearances of CMS. As CMS is either directly excreted in urine or converted systematically into colistin, the fraction of CMS converted into colistin in blood (fm) was calculated as fm = 1 − fe. The independent samples t-test was employed for the comparison of PK characters in female and male, and P values of <0.05 were considered statistically significant.

RESULTS Demographic characteristics and safety assessments Table 1 summarises the demographic characteristics of the 24 volunteers enrolled in this study. The mean dose was 2.36 ± 0.13 (2.19 - 2.50) mg CBA/kg in the single-dose study and 2.35 ± 0.15 (2.01 - 2.50) mg CBA/kg in the multiple-dose study. No significant clinical changes in vital signs and laboratory tests were observed after a single dose of CMS (data not shown) in both the single- and multiple-dose studies. After administration of CMS for 7 days, 9 cases of tongue numbness, 2 cases of constipation and 1 case of facial itching occurred, which were determined as possible drug adverse reactions by clinicians. In addition, urinary concentrations of β2-microglobulin and NAG increased after multiple-dosing as early as on day 2 in different subjects (Table 2). All of the above symptoms were minor and recovered within 48 h after the last dose without any treatment.

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Pharmacokinetic analysis Plasma concentration-time curves of CMS and formed colistin after a single dose or multiple doses are shown in Figure 1. CMS reached Cmax (18.0 ± 2.89 mg/L) immediately after 1 h infusion in the single-dose study and the concentration decreased rapidly afterwards. At 12 h after the start of infusion (C12h) the concentration of CMS was 0.079 ± 0.029 mg/L, while the concentration of formed colistin decreased slowly from 0.661 ± 0.069 mg/L (Cmax) at 3.96 ± 1.1 h (Tmax) to 0.274 ± 0.070 mg/L (C12h). The Cmax and C12h of CMS from day 1 to day 7 were almost identical with zero accumulation. While for colistin, Cmax increased from 0.839 ± 0.107 mg/L to 1.17 ± 0.22 mg/L, and C12h increased from 0.283 ± 0.067 mg/L to 0.379 ± 0.093 mg/L from day 1 to day 7, respectively.

PK parameters of CMS and colistin are shown in Table 3. AUC0-12h of CMS was 35.5 ± 4.33 mg·h/L in the single-dose study and AUC0-12h of colistin was 5.87 ± 0.76 mg·h/L. Half-life of colistin (4.49 ± 0.56 h) was much longer than CMS (1.38 ± 0.21 h) as proved in previous studies [20]. Multiple doses of CMS did not substantially affect the PK characteristics of both CMS and formed colistin (P>0.05). There was no accumulation of CMS over 7 days (RAUC, 0.98 ± 0.09). Interestingly, colistin exposure accumulated slightly with an RAUC of 1.33 ± 0.15 and the Cmax of colistin was higher on day 7 than day 1 (RCmax1.39 ± 0.18), which would not affect the dosing regimen of CMS. As T1/2 of colistin was relatively short, colistin reached the steady state at

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around 24 h.

Recoveries of CMS and colistin in urine are presented in Figure 2. The urinary recovery of CMS was much higher than colistin in all subjects. After a single dose of CMS, 62.5 ± 4.65% of the dose was excreted though urine within 24 h, most of which was excreted within the first 2 h (Figure 2). While for colistin, only a very small amount of the dose (1.28 ± 0.58%) excreted into urine and the highest excretion rate occurred at 4-8 h after dosing. Similar results were obtained with the multiple-dose study (Figure 2).

PK parameters were analysed by gender stratification. No significant difference in Cmax, C12h, Tmax, AUC, T1/2, CLt, CLr, V or fe was detected between male and female subjects in both single- and multiple-dose studies. Significant difference in the accumulated Ae0-48 of colistin (0.91 ± 0.47% vs 1.65 ± 0.43%) after a single dose (P<0.05) was observed between female and male; however, this difference was not observed after multiple doses of CMS.

DISCUSSION CMS became available for clinical use in the late 1950s and was never subjected to contemporary drug development procedures [4, 21]. Several PK studies were conducted in recent years and our knowledge on the clinical PK of CMS and formed colistin has significantly increased [12, 13, 19, 22-26]. CMS is supplied as mixtures of

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a large number of components produced by fermentation of colistin and subsequent chemical modification, during which sulfomethylation occurs at between one and all five of the primary amine groups on colistin [4]. Potentially, the complex composition of CMS can lead to different PK [11], pharmacodynamics (PD) and toxicodynamics. Major PK differences have recently been observed in rats between four brands of CMS from different countries [11], which were also observed in humans [12, 13, 19]. After CMS administration in critically ill patients, colistin concentrations increase slowly and it takes approximately two days to reach the steady state for a brand of CMS in Greece (Norma) [13]. This slow increase of colistin concentrations was not observed in a French study with a different brand of CMS (Sanofi Aventis) in healthy subjects [19] or critically ill patients [12]. Such discrepancy highlights the necessity to perform PK studies for new CMS products. No CMS products were available in China and CMS administered in this study is the first CMS product developed by a Chinese pharmaceutical company, and this study is also the first PK study of CMS and formed colistin in a Chinese population.

In the dosing regimen used in present study, CMS was well tolerated without any serious adverse effects or significant clinically findings in the routine laboratory tests. Several minor clinical symptoms occurred after repeating doses in healthy subjects, but recovered within 48 h after the last dose without any intervention. Three biomarkers were employed in this study to monitor any potential early acute kidney injury after intravenous CMS. Urinary NAG is predominantly in the proximal tubule

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and its activity increases after acute exposure to other nephrotoxins [27]. NAGL is expressed by renal tubular epithelium and the rise in urinary concentrations indicates acute renal injury [28]. Serum Cystatin C is also closely related to the kidney function and, in acute tubular necrosis it allows better prediction of the need for renal replacement therapy than serum creatinine concentrations [28]. In the present study, only NAG and Urinary β2-microglobulin were detected to increase after multiple doses of CMS (Table 2). Increase of urinary NAG concentrations after CMS dosing was also reported in adult patients with cystic fibrosis [29] and in Japanese healthy male subjects [30], which suggests that CMS might cause early-stage damages to renal proximal tubules that would not have detected by serum creatinine concentrations. However, increase of urinary NAG concentrations was minor and was non-clinically significant. Although there were minor intra-patient variations in the NAG and Urinary β2-microglobulin concentrations, most of them were within the normal range. Since CMS is mainly used in critically ill patients with different renal conditions and receiving multiple drugs, careful monitoring of the renal function with sensitive and reliable laboratory biomarkers is strongly recommended for intravenous CMS.

A main finding of the present study is that peak concentrations of colistin were much lower, compared to the other PK studies in healthy subjects [19, 30]. With a similar dose, Cmax in Japanese subjects was much higher than that in present study, which were 2.55 ± 1.28 mg/L after a single dose of CMS (2.5 mg CBA/kg) and 4.38 ± 1.56 mg/L after repeat doses (2.5 mg CBA/kg, twice daily for 2.5 days) [30]. However, the

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Cmax in our study were only 0.661 ± 0.069 mg/L after a single dose (2.5 mg CBA/kg) and 1.17 ± 0.22 mg/L after multiple doses at day 7 (2.5 mg CBA/kg, twice daily for 7 days). The dose in the present study (2.5 mg CBA/kg, i.e. 150 mg CBA with the weight of 60 kg) was about 5 times as that in the study performed by Couet et al (80 mg CMS, equivalent to 30 mg CBA) [19]; however, the values of Cmax were similar. Also, the very flat colistin concentration-time profiles [23, 31] in critically ill patients were not observed in these healthy subjects. Low concentrations of formed colistin in our present study could be due to at least two reasons. One is higher total clearance in our study (315 - 491 mL/min, i.e. 0.30-0.48 L/h/kg) than that in the French study (48.7 mL/min) [19]. However, the values of CLr in both studies were comparable, 2.35-4.40 mL/min in present study (i.e. 0.0023-0.0042 L/h/kg) vs 1.9 mL/min in the French study [19]. The difference in the CL values means that the non-renal clearance of colistin is much higher in present study, the mechanism of which remains to be elucidated. Interestingly, the concentrations of colistin at steady state (Css,avg) in our study are similar to that in patients from the thus-far largest multicentre PK study [31]. Css,avg values fell into the same range, i.e. around or less than 1 mg/L as that in patients with similar creatinine clearance (> 100 mL/min)[31]. We employed the equations recommended in the largest PK study to estimate the concentration of colistin after multiple doses of CMS with regimen used in present study. A predicted Css at 1.03 ± 0.16 mg/L was obtained, which was very close to the observed maximum concentrations (1.17 ± 0.22 mg/L) on day 7 in the present study. The consistency between the present study and the thus-far largest multicentre PK study [31] implies

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that the latest dosing guidance in critically ill patients can be applicable in Chinese population.

CMS is mainly used for infections caused by multi-resistant Gram-negative bacteria such as A. baumannii, Pseudomonas aeruginosa, and K. pneumoniae [5, 32]. In the present study, the Cmax of colistin is close or lower than the MIC50 value, which is ≤0.5 mg/L for A. baumannii, 1 mg/L for P. aeruginosa, and ≤0.5 mg/L for K. pneumoniae[33]. If plasma protein binding of colistin (~50%) is considered [34, 35], the free Cmax of colistin is only 0.343-0.585 mg/L in the 24 healthy subjects. Although more colistin may convert from CMS in a renal impaired patient and thus higher Cmax, to achieve a free Cmax of 2 mg/L the actual fm needs to increase 3-5 folds in renal impaired patients even if the PK of colistin is linear in these patients. Therefore, in Chinese population we may need to consider a higher dose of CMS for patients with good renal function, if safety permits, and monitor the renal function closely. Furthermore, recent pharmacodynamics studies demonstrated the rapid emergence of resistance to colistin after treatment [36] and combination therapy is strongly recommended for patients with good renal function or when the causative pathogen has an MIC ≥1 mg/L [31]. The concentration of colistin required for bactericidal activity against XDR A. baumannii decreased by 2 times when combined with meropenem, minocycline and rifampicin [37]. Synergistic combination therapy may also decrease the CMS dose and thereby reducing its nephrotoxicity. Well-designed prospective clinical studies on polymyxin combinations are urgently needed. In addition, concentrations of both CMS

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(18.9 - 561 mg/L) and colistin (6.39 - 118 mg/L) in urine were very high within 8 h after the end of infusion, indicating that intravenous CMS with the current dosing regimen is an option for urinary tract infections caused by MDR Gram-negative pathogens.

CONCLUSION In summary, this is the first PK study of CMS and formed colistin in Chinese population. Our study revealed that steady state was rapidly achieved for colistin in Chinese healthy subjects with the dose of 2.5 mg CBA/kg every 12 h. Similar in patients with cystic fibrosis, the half-life of CMS was much shorter than colistin. No significant accumulation in plasma was observed for CMS or colistin within a week. Our study is the first to characterise the urinary PK of CMS and colistin after a 7-day treatment in humans and the very high concentration of formed colistin in the urine strongly support the use of intravenous CMS for serious urinary tract infections.

ACKNOWLEDGEMENTS This study was presented at ASM Microbe/Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC) in San Diego, CA, USA, Sep 17-21, 2015 (Poster ID: 2018), and was also presented at the 2nd International Conference on Polymyxins in San Diego, CA, USA (September 22-24, 2015).

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DECLARATIONS Funding: This study was supported by the New Drug Creation and Manufacturing Program of the Ministry of Science and Technology of China (2012ZX09303004-001) and the National Natural Science Foundation of China (81373493). We thank CHIA TAI TIAN-QING PHARMACEUTICAL GROUP CO., LTD (Jiangsu, China) for kindly providing CMS. Competing Interests: None Ethical Approval: The study was approved by the ethics committee of Huashan Hospital, Fudan University, China

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REFERENCES [1] Viens AM, Littmann J. Is Antimicrobial Resistance a Slowly Emerging Disaster? Public Health Ethics. 2015;8:255-65. [2] Fowler T, Walker D, Davies SC. The risk/benefit of predicting a post-antibiotic era: is the alarm working? Ann N Y Acad Sci. 2014;1323:1-10. [3] Karaiskos I, Giamarellou H. Multidrug-resistant and extensively drug-resistant Gram-negative pathogens: current and emerging therapeutic approaches. Expert Opin Pharmacother. 2014;15:1351-70. [4] Nation RL, Li J, Cars O, Couet W, Dudley MN, Kaye KS, et al. Framework for optimisation of the clinical use of colistin and polymyxin B: the Prato polymyxin consensus. The Lancet Infectious Diseases. 2015;15:225-34. [5] Tran TB, Velkov T, Nation RL, Forrest A, Tsuji BT, Bergen PJ, et al. Pharmacokinetics/pharmacodynamics of colistin and polymyxin B: are we there yet? Int J Antimicrob Agents 2016; doi: 10.1016/j.ijantimicag.2016.09.010. [6] Willmann M, Bezdan D, Zapata L, Susak H, Vogel W, Schroppel K, et al. Analysis of a long-term outbreak of XDR Pseudomonas aeruginosa: a molecular epidemiological study. J Antimicrob Chemother. 2015;70:1322-30. [7] Hu FP, Guo Y, Zhu DM, Wang F, Jiang XF, Xu YC, et al. Resistance trends among clinical isolates in China reported from CHINET surveillance of bacterial resistance, 2005-2014. Clin Microbiol Infect. 2016;22 Suppl 1:S9-14. [8] Chinese XDRCWG, Guan X, He L, Hu B, Hu J, Huang X, et al. Laboratory diagnosis, clinical management and infection control of the infections caused by extensively drug-resistant Gram-negative bacilli: a Chinese consensus statement. Clin Microbiol Infect. 2016;22 Suppl 1:S15-25. [9] Bergen PJ, Li J, Rayner CR, Nation RL. Colistin methanesulfonate is an inactive prodrug of colistin against Pseudomonas aeruginosa. Antimicrob Agents Chemother. 2006;50:1953-8. [10] Velkov T, Thompson PE, Nation RL, Li J. Structure--activity relationships of polymyxin antibiotics. J Med Chem. 2010;53:1898-916. [11] He H, Li JC, Nation RL, Jacob J, Chen G, Lee HJ, et al. Pharmacokinetics of four different brands of colistimethate and formed colistin in rats. J Antimicrob Chemother. 2013;68:2311-7. [12] Gregoire N, Mimoz O, Megarbane B, Comets E, Chatelier D, Lasocki S, et al. New colistin population pharmacokinetic data in critically ill patients suggesting an alternative loading dose rationale. Antimicrob Agents Chemother. 2014;58:7324-30. [13] Plachouras D, Karvanen M, Friberg LE, Papadomichelakis E, Antoniadou A, Tsangaris I, et al. Population pharmacokinetic analysis of colistin methanesulfonate and colistin after intravenous administration in critically ill patients with infections caused by gram-negative bacteria. Antimicrob Agents Chemother. 2009;53:3430-6. [14] Kumar A, Roberts D, Wood KE, Light B, Parrillo JE, Sharma S, et al. Duration of hypotension before initiation of effective antimicrobial therapy is the critical determinant of survival in human septic shock. Crit Care Med. 2006;34:1589-96. [15] Luna CM, Aruj P, Niederman MS, Garzon J, Violi D, Prignoni A, et al. Appropriateness and delay to initiate therapy in ventilator-associated pneumonia. Eur Respir J. 2006;27:158-64. [16] Wallace SJ, Li J, Nation RL, Prankerd RJ, Velkov T, Boyd BJ. Self-assembly behavior of colistin and its prodrug colistin methanesulfonate: implications for solution stability and solubilization. J Phys Chem B.

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2010;114:4836-40. [17] Dudhani RV, Nation RL, Li J. Evaluating the stability of colistin and colistin methanesulphonate in human plasma under different conditions of storage. J Antimicrob Chemother. 2010;65:1412-5. [18] Zhao M, Wu XJ, Fan YX, Guo BN, Zhang J. Development and validation of a UHPLC-MS/MS assay for colistin methanesulphonate (CMS) and colistin in human plasma and urine using weak-cation exchange solid-phase extraction. J Pharm Biomed Anal. 2016;124:303-8. [19] Couet W, Gregoire N, Gobin P, Saulnier PJ, Frasca D, Marchand S, et al. Pharmacokinetics of colistin and colistimethate sodium after a single 80-mg intravenous dose of CMS in young healthy volunteers. Clin Pharmacol Ther. 2011;89:875-9. [20] Bergen PJ, Landersdorfer CB, Zhang J, Zhao M, Lee HJ, Nation RL, et al. Pharmacokinetics and pharmacodynamics of 'old' polymyxins: what is new? Diagn Microbiol Infect Dis. 2012;74:213-23. [21] Li J, Nation RL, Turnidge JD, Milne RW, Coulthard K, Rayner CR, et al. Colistin: the re-emerging antibiotic for multidrug-resistant Gram-negative bacterial infections. Lancet Infect Dis. 2006;6:589-601. [22] Imberti R, Cusato M, Accetta G, Marino V, Procaccio F, Del Gaudio A, et al. Pharmacokinetics of colistin in cerebrospinal fluid after intraventricular administration of colistin methanesulfonate. Antimicrob Agents Chemother. 2012;56:4416-21. [23] Garonzik SM, Li J, Thamlikitkul V, Paterson DL, Shoham S, Jacob J, et al. Population pharmacokinetics of colistin methanesulfonate and formed colistin in critically ill patients from a multicenter study provide dosing suggestions for various categories of patients. Antimicrob Agents Chemother. 2011;55:3284-94. [24] Ziaka M, Markantonis SL, Fousteri M, Zygoulis P, Panidis D, Karvouniaris M, et al. Combined intravenous and intraventricular administration of colistin methanesulfonate in critically ill patients with central nervous system infection. Antimicrob Agents Chemother. 2013;57:1938-40. [25] Jitmuang A, Nation RL, Koomanachai P, Chen G, Lee HJ, Wasuwattakul S, et al. Extracorporeal clearance of colistin methanesulphonate and formed colistin in end-stage renal disease patients receiving intermittent haemodialysis: implications for dosing. J Antimicrob Chemother. 2015;70:1804-11. [26] Karaiskos I, Friberg LE, Pontikis K, Ioannidis K, Tsagkari V, Galani L, et al. Colistin Population Pharmacokinetics after Application of a Loading Dose of 9 MU Colistin Methanesulfonate in Critically Ill Patients. Antimicrob Agents Chemother. 2015;59:7240-8. [27] Moriguchi J, Inoue Y, Kamiyama S, Horiguchi M, Murata K, Sakuragi S, et al. N-acetyl-beta-D-glucosaminidase (NAG) as the most sensitive marker of tubular dysfunction for monitoring residents in non-polluted areas. Toxicol Lett. 2009;190:1-8. [28] Waring WS, Moonie A. Earlier recognition of nephrotoxicity using novel biomarkers of acute kidney injury. Clin Toxicol (Phila). 2011;49:720-8. [29] Etherington C, Bosomworth M, Clifton I, Peckham DG, Conway SP. Measurement of urinary N-acetyl-b-D-glucosaminidase in adult patients with cystic fibrosis: before, during and after treatment with intravenous antibiotics. J Cyst Fibros. 2007;6:67-73. [30] Mizuyachi K, Hara K, Wakamatsu A, Nohda S, Hirama T. Safety and pharmacokinetic evaluation of intravenous colistin methanesulfonate sodium in Japanese healthy male subjects. Curr Med Res Opin. 2011;27:2261-70. [31] Nation RL, Garonzik SM, Thamlikitkul V, Giamarellos-Bourboulis EJ, Forrest A, Paterson DL, et al. Dosing guidance for intravenous colistin in critically-ill patients. Clin Infect Dis. 2017;64:565-71.

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[32] Vardakas KZ, Falagas ME. Colistin versus polymyxin B for the treatment of patients with multidrug-resistant Gram-negative infections: a systematic review and meta-analysis. Int J Antimicrob Agents.2017;49:233-38. [33] Gales AC, Jones RN, Sader HS. Contemporary activity of colistin and polymyxin B against a worldwide collection of Gram-negative pathogens: results from the SENTRY Antimicrobial Surveillance Program (2006-09). J Antimicrob Chemother. 2011;66:2070-4. [34] Honore PM, Jacobs R, Waele ED, Gorp VV, Spapen HD. Colistin pharmacokinetics/pharmacodynamics and acute kidney injury: A difficult but reasonable marriage. Indian J Crit Care Med. 2014;18:415-6. [35] Cheah SE, Wang J, Nguyen VT, Turnidge JD, Li J, Nation RL. New pharmacokinetic/pharmacodynamic studies of systemically administered colistin against Pseudomonas aeruginosa and Acinetobacter baumannii in mouse thigh and lung infection models: smaller response in lung infection. J Antimicrob Chemother. 2015;70:3291-7. [36] Cheah SE, Li J, Tsuji BT, Forrest A, Bulitta JB, Nation RL. Colistin and Polymyxin B Dosage Regimens against Acinetobacter baumannii: Differences in Activity and the Emergence of Resistance. Antimicrob Agents Chemother. 2016;60:3921-33. [37] Liang W, Liu XF, Huang J, Zhu DM, Li J, Zhang J. Activities of colistin- and minocycline-based combinations against extensive drug resistant Acinetobacter baumannii isolates from intensive care unit patients. BMC Infect Dis. 2011;11:109.

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Figure 1. Plasma concentration-time profiles of CMS and formed colistin after a single intravenous dose (2.5 mg CBA/kg, a and b) and multiple doses of CMS (2.5 mg CBA/kg, twice daily for 7 days, c and d).

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Figure 2. Urinary recoveries of CMS and formed colistin in each interval after a single (a and b) and multiple (c and d) doses.

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1

Table 1. Demographic information of the 12 healthy subjects (mean ± SD, range). Multiple-dose group Single-dose group

(Q12 h, 7 days)

Male

6

6

Female

6

6

Age (years)

25.6 ± 3.23 (22 - 33)

25.4 ± 3.18 (22-34)

Height (cm)

170 ± 8.29 (154-184)

166 ± 5.37 (155-177)

Body Weight (kg)

61.4 ± 6.80 (47.3-68.5)

63.1 ± 5.80 (51.2-74.5)

BMI* (kg/m2)

21.3 ± 1.25 (19.8-23.9)

22.8 ± 1.00 (21.3-23.9)

CLcr (mL/min)

129 ± 9.80 (111-137)

133 ± 13.8 (113-156)

Dosage (mg CBA per kg)

2.36 ± 0.13 (2.19-2.50)

2.35 ± 0.15 (2.01-2.50)

Gender

2

*BMI: weight in kilograms divided by the square of height in meters; CBA: colistin

3

based activity. CLcr: Cockcroft and Gault equation was used to estimate CLcr in this

4

study.

5 6 7

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8

Table 2. Biomarkers of acute kidney injury assessments in healthy subjects. Biomarker Pre-dose Day 2 Day 4 Day 7 Day 9 Urinary β2-microglobulin (mg/L) M01 subject

<0.21

<0.21

0.22

0.41

<0.21

M02 subject

<0.21

<0.21

<0.21

<0.21

<0.21

M03 subject

<0.21

<0.21

<0.21

0.34

<0.21

M04 subject

<0.21

<0.21

<0.21

<0.21

<0.21

M05 subject

<0.21

<0.21

<0.21

0.60

<0.21

M06 subject

<0.21

<0.21

0.25

0.31

<0.21

M07 subject

<0.21

<0.21

<0.21

2.62

<0.21

M08 subject

<0.21

<0.21

<0.21

<0.21

<0.21

M09 subject

<0.21

<0.21

<0.21

<0.21

<0.21

M10 subject

<0.21

<0.21

<0.21

<0.21

<0.21

M11 subject

<0.21

<0.21

<0.21

<0.21

<0.21

M12 subject

<0.21

<0.21

<0.21

<0.21

<0.21

M01 subject

2.2

1.7

4.1

8.0

0.6

M02 subject

3.1

11.7

3.5

5.4

0.4

M03 subject

2.5

9.3

3.6

5.3

0.8

M04 subject

2.3

1.9

2.6

4.7

9.1

M05 subject

1.7

5.2

6.3

4.4

0.4

M06 subject

2.6

1.6

5.4

2.4

3.2

M07 subject

3.2

4.3

9.0

18.6

6.2

M08 subject

2.5

4.8

4.5

5.8

2.7

M09 subject

2.8

2.2

2.7

2.8

2.0

M10 subject

2.4

9.7

1.8

3.2

17.3

M11 subject

<0.7

2.7

4.9

4.0

4.4

M12 subject

2.0

1.6

4.4

1.4

24.1

Urinary NAG (U/L)

9 10

*Normal range for NAG is 0.7-11.2 U/L as recommended in healthy adult. Normal range for urinary β2-microglobulin is <0.25 mg/L as recommended in healthy adults.

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Table 3. Pharmacokinetic parameters after a single dose of CMS (1-h infusion, mean ± SD). Cmax

Tmax

AUC0-12h

AUC0-∞

C12h

CLr

CL

fm

T1/2

Vd

(mg/L)

(h)

(mg·h/L)

(mg·h/L)

(mg/L)

(L/h/kg)

(L/h/kg)

-

(h)

(L/kg)

18.0±2.89

1.00

35.5±4.33

35.8±4.42

0.079±0.029

0.094±0.014

0.150±0.023

0.371±0.045

1.38±0.21

0.296±0.033

0.687±0.074

3.96±1.10

5.87±0.76

7.68±1.30

0.274±0.070

0.0023±0.0009

0.48±0.10

4.49±0.56

3.03±0.47

CMS Single dose Colistin Single dose

Cmax: peak concentration; Tmax: Time to reach peak concentration; AUC: the area under the plasma concentration-time curve; C12h: concentration at 11h after the end of infusion; CLr: renal clearance; CL: total clearance; fm: the fraction of CMS converted to colistin; T1/2: elimination half-life; Vd: volume of distribution.

Page 26 of 27

Table 4. Comparison of pharmacokinetic parameters in three studies after a single dose CMS (mean ± SD). Present Study

Couet W, et al.2011[19]

Mizuyachi K, et al.2011[30]

Healthy volunteers

12 female, 12 male

12 male

15 male

Dosing regimen

2.4±0.1 mg/kg CBA, i.v. infusion 1h 336 mg CMS=150 mg CBA

80 mg (i.e. 33.4 CBA) CMS, i.v. infusion 1h

2.5 mg/kg CBA i.v. infusion >0.5 h 160 mg CMS=66.7 mg CBA

Cmax (mg/L) Tmax (h) CLr (mL/min) CL (mL/min) fm T1/2 (h) Vd (L)

18.0 1.00 95.8 152 0.371 1.38 18

CMS 4.8 1 103 148 0.3 2 14

17.97 0.49 84 323 0.73 17.6

Colistin

0.83 2.55 2 2 1.9 10.5 48.7 280 3 4 12.4 94.9 Cmax: peak concentration; Tmax: Time to reach peak concentration; CLr: renal clearance; CL: total clearance; fm: the fraction of CMS converted to colistin; T1/2: elimination half-life; Vd: volume of distribution. Cmax (mg/L) Tmax (h) CLr (mL/min) CL (mL/min) T1/2 (h) Vd (L)

0.69 3.96 2.33 178 4.49 68.2

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