MSD Pierre A M Peeters
email: pierre.peeters2@merck.com

Early Stage Development Paul Passier

MSD Astellas Smeets

B V Zwiers

Alex ; Msd De Zwart

Marcel ; Msd Van De Wetering-Krebbers

Saskia ; Msd Van Iersel

Marlou ; Msd Van Marle

Sjoerd ; Pra

Diels; MSD International Van Den Dobbelsteen

Jean Smeets

Alex Zwiers

Marcel De Zwart

Saskia Van De Wetering-Krebbers

Marlou Van Iersel

Sjoerd Van Marle

Diels Van Den Dobbelsteen

Sugammadex is Cleared Rapidly and Primarily Unchanged via Renal Excretion

Keywords: sugammadex, Org 25969, pharmacokinetics, gamma-cyclodextrin Sugammadex, Org 25969, pharmacokinetics, gamma-cyclodextrin

Sugammadex is a modified γ-cyclodextrin which rapidly reverses rocuronium-and vecuronium-induced neuromuscular blockade. Previous studies suggest that sugammadex is mostly excreted unchanged via the kidneys. This single-center, open-label, nonrandomized study used 14 C-labeled sugammadex to further investigate the excretion, metabolic and pharmacokinetic (PK) profiles of sugammadex in six healthy male volunteers. 14 C-labeled sugammadex 4 mg/kg (0.025 MBq/kg of 14 C-radioactivity) was administered as a single intravenous bolus. Blood, urine, feces, and exhaled air samples were collected at pre-defined intervals for assessment of sugammadex by liquid chromatography-mass spectrometry (LC-MS) and for radioactivity measurements. Adverse events were also assessed. Excretion of sugammadex was rapid with ~70% of the dose excreted within 6 h and ~90% within 24 h. Less than 0.02% of radioactivity was excreted in feces or exhaled air. Ninety-five percent of the radioactivity detected in urine could be attributed to sugammadex, as determined by LC-MS, suggesting very limited metabolism of sugammadex. LC-MS analysis of plasma samples found that sugammadex accounted for 100% of total 14 Cradioactivity in the plasma. In general, PK parameters determined from radioactivity and sugammadex plasma concentrations were very similar. Any adverse events were of mild-to-moderate intensity, and judged unrelated to sugammadex. These findings demonstrate that sugammadex is cleared rapidly, almost http://mc.manuscriptcentral.

Introduction

The modified γ-cyclodextrin, sugammadex, was specifically designed to encapsulate the steroidal neuromuscular blocking agent rocuronium, with structural similarities meaning that it will also encapsulate vecuronium [START_REF] Bom | A novel concept of reversing neuromuscular block: chemical encapsulation of rocuronium bromide by a cyclodextrin-based synthetic host[END_REF]2], and has the ability to rapidly reverse both moderate and deep rocuronium-or vecuronium-induced neuromuscular blockade [3][4][5][6].

Sugammadex is approved in various countries for the reversal of rocuronium-or vecuronium-induced neuromuscular blockade in adults at a recommended dose range of 2-16 mg/kg, depending on the level of blockade at the time of administration. It is also approved for the reversal of moderate rocuronium-induced blockade in pediatric patients at a dose of 2 mg/kg [7][8][9].

Previous studies evaluating the pharmacokinetic (PK) profile of sugammadex have suggested that the majority of an intravenously (i.v.) administered sugammadex dose is excreted unchanged by the kidneys [10][11][12]. At doses of 2, 4, and 8 mg/kg, the mean recovery of sugammadex in urine within 24 h of administration ranged from 58.8% to 80.4% of the administered dose [10]. Using higher than recommended doses of 32, 64, or 96 mg/kg, urinary recovery within 48 h of administration was ~90% of the administered dose [START_REF] Peeters | Safety, Tolerability and Pharmacokinetics of Sugammadex Using Single High Doses (Up to 96 mg/kg) in Healthy Adult Subjects: A Randomized, Double-Blind, Crossover, Placebo-Controlled, Single-Centre Study[END_REF]. Despite this relatively high urinary recovery of sugammadex, quantified by tandem liquid chromatography mass spectrometry (LC-MS), a minor role of metabolism in the clearance of sugammadex cannot be excluded. The use of 14 Clabeled sugammadex enables determination of the extent of metabolism and the relative contribution of the various routes of excretion to elimination.

The aim of the current trial was to further elucidate the extent of sugammadex metabolism and the relative contribution of various routes of excretion at a therapeutic dose of 4 mg/kg in healthy volunteers. To achieve this, the pharmacokinetic parameters

Materials and Methods

Study design

This was a single-center, open-label, non-randomized study of six healthy adult male subjects, conducted at PRA International (Zuidlaren, The Netherlands). A total of six subjects were selected because this number is generally considered adequate for studies evaluating the disposition of 14 C-labeled compounds. The study protocol was approved by an independent ethics committee (Stichting Beoordeling Ethiek Bio-Medisch Onderzoek, Assen, The Netherlands) and was conducted in compliance with the Declaration of Helsinki, the International Conference on Harmonization Guidelines, Good Clinical Practice, and current regulatory requirements.

Subjects

Subjects were eligible for inclusion in the study if they were aged between ≥18 years and ≤65 years, had normal 12-lead electrocardiogram (ECG) intervals (PR between ≥120 ms and ≤210 ms, QRS <120 ms and QTc ≤450 ms), no clinically relevant ECG abnormalities and a heart rate >50 beats per minute after 5 min rest, had a body mass index between ≥18 and ≤30 kg/m 2 , and were in otherwise good health for their age.

Major exclusion criteria were: evidence of renal or hepatic disease; a history of drug or alcohol abuse, or a positive drug or alcohol screen; smoking >5 cigarettes/day; hepatitis B, C or HIV infection; febrile illness within 3 days before administration of sugammadex, stool production less than once every 2 days; a history of sensitivity/idiosyncrasy to chemically-related compounds or excipients which may be used in the study or to any other unspecified drug in the past; and drug therapy (excluding paracetamol) within 2 weeks (or 1 month in the case of inducers or inhibitors of drug-metabolizing enzymes) of

Study drug administration

Each subject received a single i.v. bolus dose of 14 C-labeled sugammadex at a therapeutic dose level of 4 mg/kg (with 0.025 MBq/kg bodyweight of 14 C-radioactivity, about 1.85 MBq per subject); the radiolabel was located on each of the side chains (Figure 1). Study drug administration was performed on the morning of day 1, after an overnight fast of at least 10 h. The use of any concomitant prescription or nonprescription drug (with the exception of intermittent paracetamol) was prohibited during the entire treatment period (i.e. from sugammadex administration until follow-up).

Consumption of alcohol and strenuous physical exercise were prohibited from 24 and 48 h before sugammadex administration, respectively, until collection of the last PK blood sample. Subjects received a standardized meal on day 1 and were not allowed to smoke during the entire study period.

Sample collection

Blood samples for PK evaluation and measurement of radioactivity were collected predose (not for radioactivity) and at regular pre-defined intervals at 2, 5, 10, 20, and 35 min and 1, 1.5, 2.5, 4, 6, 8, 12, 18, 24, and 48 h post-dose. Urine samples were collected for PK evaluation and quantification of radioactivity at pre-dose (-12 to 0 h) and at 0-6, 6-12, 12-24, and 24-48 h post-dose; sampling for quantification of radioactivity continued until at least 48-72 h post-dose. All urine samples were diluted 1:1 volume for volume with blank human plasma to stabilize drug-related material prior to storage. Feces samples were collected for measurement of radioactivity pre-dose (from 3 days before sugammadex administration) and at 0- All samples taken for PK evaluation and measurement of radioactivity were stored at -20°C.

Sample analysis

Sugammadex concentrations in plasma and urine were determined using a validated liquid chromatography-mass spectrometry (LC-MS) assay method after solid phase extraction (SPE). The sample volumes used for analysis were 250 µl and 100 µl for plasma and urine, respectively. Isolute ® 96-well SPE plates with 25 mg Isolute ® HAX sorbent material per well (Sopachem, Wageningen, The Netherlands) were used for SPE of plasma and urine samples.

Chromatographic separations were carried out using an HP1100 For radioactivity analysis, reference standards of 14 C-labeled sugammadex and [1-methyl- 14 C]-caffeine were used to prepare quality control samples. Human plasma, urine, and feces matrices were used for the analysis of blank samples in analytical runs and to prepare quality control samples. All scintillation counting were performed using a Packard Tri-Carb TM 3100 liquid scintillation analyzer (Downers Grove, IL) equipped with low level counting mode (LLCM) and normal counting mode (NCM). All calculations were performed in Microsoft Excel (Redmond, WA). Results were recorded in ng sugammadex equivalents/ml for plasma, dpm/ml for urine, dpm/g for feces, and in dpm for exhaled air and medication (quality control) Samples were assayed as follows:

Medication (quality control) samples

Samples were assayed using an aliquot of the i.v. medication solution, transferred into a 20 ml counting vial and mixed with 10 ml of the scintillation cocktail (Ultima Gold). After 20 min, the sample was counted for 10 min on NCM of the scintillation counter. The administered radioactive dose was calculated by multiplying the mean (n = 3) measured radioactivity level per g by the exact administered weight of the i.v. solution.

Plasma samples

Samples were assayed using a 1000 µl aliquot of the plasma sample, transferred into a 20 ml counting vial and mixed with 10 ml of the scintillation cocktail (Ultima Gold). After waiting at least 30 min, samples were counted on the LLCM of the scintillation counter until a statistical error (2 s) of 0.5% was obtained with a maximum counting time of 10 min.

Urine samples

Samples were assayed using a 1000 µl aliquot of the urine sample, transferred into a 7 ml counting vial and mixed with 5 ml of the scintillation cocktail (Ultima Gold). After waiting at least 30 min, samples were counted until a statistical error (2s) of 0.5% was obtained with a maximum counting time of 10 min. Samples with a 14 C activity level of < 50000 dpm/ml were counted on the LCCM; samples above 50000 dpm/ml were counted on the NCM.

Feces samples

Samples were transferred into a fitting bucket per subject and per 24 h time period and homogenized with a minimum of water (1-2 weight equivalents). After homogenization, two 15 g aliquots were taken for determination of total 14 C-radioactivity and two 50 g aliquots were taken for possible metabolite profiling. An aliquot of ~0.5 g of the feces homogenate to be analyzed was then dried at 50°C for at least 3 h and combusted for a max of 1 min using a Packard 307 Sample Oxidizer (Perkin Elmer Life Sciences, Groningen, The Netherlands). CarboSorb-E (7 ml) was used to absorb carbon dioxide.

At the end of the combustion cycle, the absorber was mixed with 13 ml of the scintillant Perma Fluor E+, and samples were then placed in the liquid scintillation counter for at least 30 min before counting. Samples were counted until a statistical error (2 s) of 0.5% was obtained with a maximum counting time of 10 min. Samples with a Urine concentrations of sugammadex and radioactivity were used to calculate the cumulative amount excreted in the urine up to the end of the last collection interval (Ae cum,tlast ) as a percentage of the total dose administered. These parameters were then used to calculate renal clearance (CL R =Ae cum,tlast /AUC 0-∞ ) and the fraction of the dose excreted unchanged in the urine (fe=Ae cum,tlast /dose). Cumulative excretion was also calculated for the exhaled air and feces samples based on level of radioactivity.

Statistical Analysis

Descriptive statistics were calculated for the plasma concentrations of sugammadex and radioactivity by time point for the cumulative excretion of radioactivity and sugammadex, and for PK parameters.

The PK analyses were performed using SAS version 8.2 (SAS Institute Inc., Cary, NC).

Safety

The tolerability and safety of sugammadex were evaluated based on adverse event (AE)

Results

Enrollment and subject baseline characteristics

All six subjects who entered the study received the study medication and were evaluable for PK assessment. All six subjects were healthy Caucasian males with a mean (standard deviation [SD]) age of 52.8 (16.3) years, a mean (SD) weight of 75.8 (9.9) kg and a mean (SD) BMI of 23.8 (1.9) kg/m 2 .

Safety and tolerability

Each of the subjects experienced one or more AEs, all of which were of mild or moderate intensity and resolved without sequelae. There were no SAEs or discontinuations because of AEs and none of the AEs were considered by the investigator to be related to sugammadex. The most frequent AEs were abnormally frequent urination (n = 3) and headache (n = 2). No clinically significant abnormalities in ECG findings, vital signs, or clinical laboratory results were reported.

Pharmacokinetic profile

Excretion of sugammadex was rapid, with ~70% (mean [range] of 73 [27-92]%) of the radioactive dose excreted within 6 h, 92 (81-102)% within 24 h, and 95 (83-105)% within 48 h. The mean total cumulative excretion of sugammadex, as analyzed both by total 14 C-radioactivity and LC-MS, is presented in Figure 2. At 48 h the mean (range) total cumulative amount of sugammadex (quantified by LC-MS) recovered in the urine during 48 h was 91 (85-94)% of the dose (Table 1). So, on average, 95% of the radioactivity detected in the urine could be attributed to sugammadex, as analyzed by LC-MS, suggesting there was very limited, if any, metabolism of sugammadex. In plasma, sugammadex measured by LC-MS, accounted for 100% of the total 14 C-radioactivity, illustrated by the overlapping concentration curves (Figure 3). This suggests that there were either no radiolabeled metabolites of sugammadex in the plasma or that they were present at extremely low concentrations. Generally, the PK parameters determined from radioactivity and sugammadex plasma concentrations were very similar (Table 1). The small difference in t ½ between these two methods of detection can be explained by the lower limit of quantification of the radioactivity measurements versus the LC-MS method of detection, resulting in a later time interval for determination of the t ½ . In accordance with the similar plasma concentrations observed (Figure 3), comparison of the AUC 0-∞ of 14 C and sugammadex revealed that on average 100% of the radioactivity in the plasma could be attributed to sugammadex.

Discussion

This study aimed to determine the pharmacokinetics and excretion profile of 14 Csugammadex and compare them with sugammadex concentrations as determined by LC-MS analysis. Radiolabeled sugammadex was excreted rapidly and almost exclusively via the kidneys. Total 14 C detection mirrored the concentrations determined by LC-MS both in the plasma and also in urine (Figures 2 and3). Furthermore, in the urine collected over 48 h following dosing, the mean cumulative amount of sugammadex quantified by LC-MS in the urine was 91% of the dose. Moreover, 95% of the radioactivity detected in the urine was attributed to sugammadex. This suggests that there was minimal to no metabolism of sugammadex, which is consistent with the findings of previous studies evaluating sugammadex in surgical patients and healthy volunteers [10][11][12][START_REF] Peeters | Safety, Tolerability and Pharmacokinetics of Sugammadex Using Single High Doses (Up to 96 mg/kg) in Healthy Adult Subjects: A Randomized, Double-Blind, Crossover, Placebo-Controlled, Single-Centre Study[END_REF]. Less than 0.02% of the dose was excreted in exhaled air and feces, suggesting that in man these routes of excretion play no role in the clearance of sugammadex. Interestingly, in rat and dog, a minor role for excretion in feces is observed with 4.22-6.82% and 0.33-0.80%, respectively, of the dose collected in the feces. For 14 C α-cyclodextrin and 14 C γ-cyclodextrin given i.v. to rat [START_REF] De Bie | Disposition of [14C]gamma-cyclodextrin in germ-free and conventional rats[END_REF][START_REF] Van Ommen | Disposition of [14C]-alpha-cyclodextrin in germ-free and conventional rats[END_REF] excretion into the feces has also been demonstrated to be a minor route of excretion (3-8% of the dose); however, as shown in the current study, this route is not applicable for sugammadex in man.

In this trial, the mean total cumulative amount of radioactivity recovered in the urine during 48 h was 95 (range 83-105) %, suggesting that at the most only a small fraction of the radioactivity was unaccounted for. There may be many causes of incomplete recovery of the radiolabel in mass balance trials (such as technical limitations and tissue retention), and it is actually rare for the mass balance in radiolabeled excretion studies to truly achieve 100% recovery [START_REF] Roffey | What is the objective of the mass balance study? A retrospective analysis of data in animal and human excretion studies employing radiolabeled drugs[END_REF]. The minimal role of metabolism in the clearance of sugammadex is fully in line with the excretion profile of other cyclodextrins, which has been noted to be almost entirely renal [START_REF] De Bie | Disposition of [14C]gamma-cyclodextrin in germ-free and conventional rats[END_REF][START_REF] Van Ommen | Disposition of [14C]-alpha-cyclodextrin in germ-free and conventional rats[END_REF][START_REF] Monbaliu | Disposition of hydroxypropyl-β-cyclodextrin in experimental animals[END_REF]18], particularly in man. Subsequent profiling for metabolites in urine and plasma samples demonstrated that the major part of total radioactivity could be attributed to the parent compound, sugammadex (data on file).

Studies in volunteers and surgical patients receiving sugammadex to reverse rocuronium-or vecuronium-induced blockade have also demonstrated that the predominant route of elimination for the complex is via the renal route, and in the presence of sugammadex, the major route of elimination of rocuronium changes from the hepatic to the renal route [10][11][12]. Based on data from surgical patients and healthy volunteers, the cumulative excretion of rocuronium in the urine over 24 h is in the region of 12-26% and increases to up to 74% after administration of sugammadex 4-8 mg/kg [10][11][12][START_REF] Alvarez-Gomez | Pharmacokinetics and pharmacodynamics of rocuronium bromide in adult patients[END_REF][START_REF] Van Den Broek | Clinical pharmacology of rocuronium (Org 9426): study of the time course of action, dose requirement, reversibility, and pharmacokinetics[END_REF][START_REF] Proost | Urinary, biliary and faecal excretion of rocuronium in humans[END_REF]. Given the importance of renal elimination, the efficacy and safety of sugammadex has been assessed in patients with impaired renal clearance, most of whom were receiving dialysis. The available data suggest that sugammadex is well tolerated and effective in patients with severe renal impairment, albeit with reduced plasma clearance of sugammadex and a significantly prolonged terminal elimination half life and MRT [START_REF] Staals | Multicentre, parallel-group, comparative trial evaluating the efficacy and safety of sugammadex in patients with end-stage renal failure or normal renal function[END_REF][START_REF] Staals | Reduced clearance of rocuronium and sugammadex in patients with severe to end-stage renal failure: a pharmacokinetic study[END_REF]. However, despite longer retention of the sugammadex-rocuronium complex, no signs of recurarization were reported among renal patients in the 48 h after administration of rocuronium followed by sugammadex [START_REF] Staals | Multicentre, parallel-group, comparative trial evaluating the efficacy and safety of sugammadex in patients with end-stage renal failure or normal renal function[END_REF]

Introduction

The modified γ-cyclodextrin, sugammadex, was specifically designed to encapsulate the steroidal neuromuscular blocking agent rocuronium, with structural similarities meaning that it will also encapsulate vecuronium [START_REF] Bom | A novel concept of reversing neuromuscular block: chemical encapsulation of rocuronium bromide by a cyclodextrin-based synthetic host[END_REF]2], and has the ability to rapidly reverse both moderate and deep rocuronium-or vecuronium-induced neuromuscular blockade [3][4][5][6].

Sugammadex is approved in various countries for the reversal of rocuronium-or vecuronium-induced neuromuscular blockade in adults at a recommended dose range of 2-16 mg/kg, depending on the level of blockade at the time of administration. It is also approved for the reversal of moderate rocuronium-induced blockade in pediatric patients at a dose of 2 mg/kg [7][8][9].

Previous studies evaluating the pharmacokinetic (PK) profile of sugammadex have suggested that the majority of an intravenously (i.v.) administered sugammadex dose is excreted unchanged by the kidneys [10][11][12]. At doses of 2, 4, and 8 mg/kg, the mean recovery of sugammadex in urine within 24 h of administration ranged from 58.8% to 80.4% of the administered dose [10]. Using higher than recommended doses of 32, 64, or 96 mg/kg, urinary recovery within 48 h of administration was ~90% of the administered dose [START_REF] Peeters | Safety, Tolerability and Pharmacokinetics of Sugammadex Using Single High Doses (Up to 96 mg/kg) in Healthy Adult Subjects: A Randomized, Double-Blind, Crossover, Placebo-Controlled, Single-Centre Study[END_REF]. Despite this relatively high urinary recovery of sugammadex, quantified by tandem liquid chromatography mass spectrometry (LC-MS), a minor role of metabolism in the clearance of sugammadex cannot be excluded. The use of 14 Clabeled sugammadex enables determination of the extent of metabolism and the relative contribution of the various routes of excretion to elimination.

The aim of the current trial was to further elucidate the extent of sugammadex metabolism and the relative contribution of various routes of excretion at a therapeutic dose of 4 mg/kg in healthy volunteers. To achieve this, the pharmacokinetic parameters

Materials and Methods

Study design

This was a single-center, open-label, non-randomized study of six healthy adult male subjects, conducted at PRA International (Zuidlaren, The Netherlands). A total of six subjects were selected because this number is generally considered adequate for studies evaluating the disposition of 14 C-labeled compounds. The study protocol was approved by an independent ethics committee (Stichting Beoordeling Ethiek Bio-Medisch Onderzoek, Assen, The Netherlands) and was conducted in compliance with the Declaration of Helsinki, the International Conference on Harmonization Guidelines, Good Clinical Practice, and current regulatory requirements.

Subjects

Subjects were eligible for inclusion in the study if they were aged between ≥18 years and ≤65 years, had normal 12-lead electrocardiogram (ECG) intervals (PR between ≥120 ms and ≤210 ms, QRS <120 ms and QTc ≤450 ms), no clinically relevant ECG abnormalities and a heart rate >50 beats per minute after 5 min rest, had a body mass index between ≥18 and ≤30 kg/m 2 , and were in otherwise good health for their age.

Major exclusion criteria were: evidence of renal or hepatic disease; a history of drug or alcohol abuse, or a positive drug or alcohol screen; smoking >5 cigarettes/day; hepatitis B, C or HIV infection; febrile illness within 3 days before administration of sugammadex, stool production less than once every 2 days; a history of sensitivity/idiosyncrasy to chemically-related compounds or excipients which may be used in the study or to any other unspecified drug in the past; and drug therapy (excluding paracetamol) within 2 weeks (or 1 month in the case of inducers or inhibitors of drug-metabolizing enzymes) of

Study drug administration

Each subject received a single i.v. bolus dose of 14 C-labeled sugammadex at a therapeutic dose level of 4 mg/kg (with 0.025 MBq/kg bodyweight of 14 C-radioactivity, about 1.85 MBq per subject); the radiolabel was located on each of the side chains (Figure 1). Study drug administration was performed on the morning of day 1, after an overnight fast of at least 10 h. The use of any concomitant prescription or nonprescription drug (with the exception of intermittent paracetamol) was prohibited during the entire treatment period (i.e. from sugammadex administration until follow-up).

Consumption of alcohol and strenuous physical exercise were prohibited from 24 and 48 h before sugammadex administration, respectively, until collection of the last PK blood sample. Subjects received a standardized meal on day 1 and were not allowed to smoke during the entire study period.

Sample collection

Blood samples for PK evaluation and measurement of radioactivity and metabolic profiling were collected pre-dose (not for radioactivity) and at regular pre-defined intervals at 2, 5, 10, 20, and 35 min and 1, 1.5, 2.5, 4, 6, 8, 12, 18, 24, and 48 h postdose. The exceptions to this were that blood samples for radioactivity measurements were not collected pre-dose and samples for metabolite profiling were not collected at 2 and 5 min post-dose. Urine samples were collected for PK evaluation and quantification of radioactivity and metabolite profiling at pre-dose (-12 to 0 h) and at 0-6, 6-12, 12-24, and 24-48 h post-dose; sampling for quantification of radioactivity continued until at least 48-72 h post-dose. All urine samples were diluted 1:1 volume for volume with blank human plasma to stabilize drug-related material prior to storage. Feces samples were collected for measurement of radioactivity and metabolite profiling pre-dose (from 3 days before sugammadex administration) and at 0-12, 12-24, and 24-48 h post-dose.

Exhaled air samples were collected pre-dose and at pre-defined intervals post-dose (1, 2, 6 and 12 h) for radioactivity measurement after administration of 14 C-labeled sugammadex.

All samples taken for PK evaluation and measurement of radioactivity and metabolite profiling were stored at -20°C.

Sample analysis

Sugammadex concentrations in plasma and urine were determined using a validated liquid chromatography-mass spectrometry (LC-MS) assay method after solid phase extraction (SPE). The sample volumes used for analysis were 250 µl and 100 µl for plasma and urine, respectively. Isolute ® 96-well SPE plates with 25 mg Isolute ® HAX sorbent material per well (Sopachem, Wageningen, The Netherlands) were used for SPE of plasma and urine samples. For radioactivity analysis, reference standards of 14 C-labeled sugammadex and [1-methyl-14 C]-caffeine were used to prepare quality control samples. Human plasma, urine, and feces matrices were used for the analysis of blank samples in analytical runs and to prepare quality control samples. All scintillation counting were performed using a Packard Tri-Carb TM 3100 liquid scintillation analyzer (Downers Grove, IL) equipped with low level counting mode (LLCM) and normal counting mode (NCM). All calculations were performed in Microsoft Excel (Redmond, WA). Results were recorded in ng sugammadex equivalents/ml for plasma, dpm/ml for urine, dpm/g for feces, and in dpm for exhaled air and medication (quality control) Samples were assayed as follows:

Medication (quality control) samples

Samples were assayed using an aliquot of the i.v. medication solution, transferred into a 20 ml counting vial and mixed with 10 ml of the scintillation cocktail (Ultima Gold). After 20 min, the sample was counted for 10 min on NCM of the scintillation counter. The administered radioactive dose was calculated by multiplying the mean (n = 3) measured radioactivity level per g by the exact administered weight of the i.v. solution.

Plasma samples

Samples were assayed using a 1000 µl aliquot of the plasma sample, transferred into a 20 ml counting vial and mixed with 10 ml of the scintillation cocktail (Ultima Gold). After waiting at least 30 min, samples were counted on the LLCM of the scintillation counter until a statistical error (2 s) of 0.5% was obtained with a maximum counting time of 10 min.

Urine samples

Samples were assayed using a 1000 µl aliquot of the urine sample, transferred into a 7 ml counting vial and mixed with 5 ml of the scintillation cocktail (Ultima Gold). After waiting at least 30 min, samples were counted until a statistical error (2s) of 0.5% was obtained with a maximum counting time of 10 min. Samples with a 14 C activity level of < 50000 dpm/ml were counted on the LCCM; samples above 50000 dpm/ml were counted on the NCM.

Feces samples

Samples were transferred into a fitting bucket per subject and per 24 h time period and homogenized with a minimum of water (1-2 weight equivalents). After homogenization, two 15 g aliquots were taken for determination of total 14 C-radioactivity and two 50 g aliquots were taken for possible metabolite profiling. An aliquot of ~0.5 g of the feces homogenate to be analyzed was then dried at 50°C for at least 3 h and combusted for a max of 1 min using a Packard 307 Sample Oxidizer (Perkin Elmer Life Sciences, Groningen, The Netherlands). CarboSorb-E (7 ml) was used to absorb carbon dioxide.

At the end of the combustion cycle, the absorber was mixed with 13 ml of the scintillant Perma Fluor E+, and samples were then placed in the liquid scintillation counter for at was obtained with a maximum counting time of 10 min. Samples with a 14 C activity level of <160000 dpm/ml were counted on the LCCM; samples >160000 dpm/ml were counted on the NCM.

Exhaled air samples

Ten ml of the scintillation cocktail (Emulsifier Safe) was mixed with the CO 2 -trapped exhaled air sample to be analyzed. After at least 90 min, samples were counted on the LLCM until a statistical error (2 s) of 0.5% was obtained with a maximum counting time of 20 min.

On day 3, subjects were discharged from the study center if <1% of the radioactive dose (sum of the urine and feces) was measured in the days excreta and if the total cumulative excretion was >90% of the radioactive dose. Subjects remained in the study center if the above criteria were not met, for a maximum of 7 further days. A follow-up assessment was performed on day 10. Furthermore, if radioactivity exceeded 50 dpm/ml in the urine and/or 75 dpm per 100 mg feces at the time of discharge, subjects were requested to bring 24-h urine samples to the clinic every 2-3 days until the radioactivity decreased to below these limits. The plasma samples taken 20 min after sugammadex administration and urine samples collected at 0-6 and 6-12 h after sugammadex administration were analyzed as individual samples. A representative plasma sample of 1-6 h post-sugammadex administration was prepared for each subject by pooling equal volume aliquots from each of the 1, 1.5, 2.5, 4, and 6 h samples. Furthermore, a representative urine sample of 6-48 h after sugammadex administration was prepared for one subject with protracted renal excretion by pooling equal percentages by volume of the 6-12, 12-24, and 24-48 h samples.

Samples were then used for metabolite profiling after partial purification by solid phase extraction by a validated HPLC method. In samples in which radioactivity was sufficiently high, radioactive components were quantified using the on-line radioactivity detector software (Laura V1.4a, Lablogic, Bioscan, Washington, DC). Where sample radioactivity levels were too low for quantification by this method, fractions of HPLC eluate/scintillator were collected at 0.5 min intervals for the full duration of the analysis using an automatic fraction collector and the radioactive concentration determined. The total radioactivity associated with each radioactive component detected was expressed as a proportion of the total radioactivity eluted. Qualitative LC-MS analysis of a 0-6 h urine sample, as well as reference standards of 14 C-labeled sugammadex, unlabeled

Pharmacokinetic analysis

From the plasma concentration of sugammadex and radioactivity (expressed in mass equivalents of sugammadex) the following non-compartmental PK parameters were calculated: maximum plasma concentration, time to maximum plasma concentration, terminal elimination half life (t ½ ), effective t ½ (t ½, effective= log e 2 • MRT[mean residence time]), area under the concentration versus time curve from time zero to infinity (AUC 0- ∞ ), total plasma clearance, and volume of distribution at steady state.

Urine concentrations of sugammadex and radioactivity were used to calculate the cumulative amount excreted in the urine up to the end of the last collection interval (Ae cum,tlast ) as a percentage of the total dose administered. These parameters were then used to calculate renal clearance (CL R =Ae cum,tlast /AUC 0-∞ ) and the fraction of the dose excreted unchanged in the urine (fe=Ae cum,tlast /dose). Cumulative excretion was also calculated for the exhaled air and feces samples based on level of radioactivity.

Statistical Analysis

Descriptive statistics were calculated for the plasma concentrations of sugammadex and radioactivity by time point for the cumulative excretion of radioactivity and sugammadex, and for PK parameters.

The PK analyses were performed using SAS version 8.2 (SAS Institute Inc., Cary, NC).

Results

Enrollment and subject baseline characteristics

All six subjects who entered the study received the study medication and were evaluable for PK assessment. All six subjects were healthy Caucasian males with a mean (standard deviation [SD]) age of 52.8 (16.3) years, a mean (SD) weight of 75.8 (9.9) kg and a mean (SD) BMI of 23.8 (1.9) kg/m 2 .

Safety and tolerability

Each of the subjects experienced one or more AEs, all of which were of mild or moderate intensity and resolved without sequelae. There were no SAEs or discontinuations because of AEs and none of the AEs were considered by the investigator to be related to sugammadex. The most frequent AEs were abnormally frequent urination (n = 3) and headache (n = 2). No clinically significant abnormalities in ECG findings, vital signs, or clinical laboratory results were reported.

Pharmacokinetic profile

Excretion of sugammadex was rapid, with ~70% (mean [range] of 73 [27-92]%) of the radioactive dose excreted within 6 h, 92 (81-102)% within 24 h, and 95 (83-105)% within 48 h. The median mean total cumulative excretion of sugammadex, as analyzed both by total 14 C-radioactivity and LC-MS, is presented in Figure 2. At 48 h the mean (range) total cumulative amount of sugammadex (quantified by LC-MS) recovered in the urine during 48 h was 91 (85-94)% of the dose (Table 1). So, on average, 95% of the radioactivity detected in the urine could be attributed to sugammadex, as analyzed by LC-MS, suggesting there was very limited, if any, metabolism of sugammadex. In plasma, sugammadex measured by LC-MS, accounted for 100% of the total 14 C-radioactivity, illustrated by the overlapping concentration curves (Figure 3). This suggests that there were either no radiolabeled metabolites of sugammadex in the plasma or that they were present at extremely low concentrations. Generally, the PK parameters determined from radioactivity and sugammadex plasma concentrations were very similar (Table 1). The small difference in t ½ between these two methods of detection can be explained by the lower limit of quantification of the radioactivity measurements versus the LC-MS method of detection, resulting in a later time interval for determination of the t ½ . In accordance with the similar plasma concentrations observed (Figure 3), comparison of the AUC 0-∞ of 14 C and sugammadex revealed that on average 100% of the radioactivity in the plasma could be attributed to sugammadex.

Metabolite profiling

Metabolite profiling of plasma and urine samples revealed the presence of up to nine radioactive peaks (designated M1-M9) (Figure 4A-D). The amount of radioactivity in the feces samples was too low (<0.02% cumulative radioactivity) to allow metabolite profiling. Most radioactivity in all plasma and urine samples was associated with M8 (coeluting with sugammadex and identified by mass spectrometry) and most of the remaining radioactivity was associated with M6 and M7 (collectively associated with 14 to 28% of radioactivity in plasma samples, 8 to 12% of radioactivity in 0-6 h urine samples and 23 to 30% in ≥6 h urine samples [Tables 2A andB <4% of radioactivity and M9 accounted for <3% of radioactivity. M1 and M2 were mostly associated with <1% and occasionally 4% of radioactivity, and were present in only some of the samples. In the sugammadex dose formulation, four peaks were observed (Figure 4E): a major peak representing 94% of the radioactivity and consistent with 14 Csugammadex, and three minor peaks, two of which were consistent with reference standards (and peaks M6 and M7 in the plasma and urine samples), and accounting for 1.1 and 1.6% of total radioactivity, respectively. The fourth peak accounted for 2% of radioactivity, did not co-elute with any reference standards, but was consistent with peak M9 observed in the patient samples and was also present in a similar proportion to that observed in patient samples. Therefore M9 can be excluded as a metabolite, and most likely this peak relates to a minor impurity in the 14 C-sugammadex dose formulation. Org 48302 (substituted with one hydroxyl group on one of the eight glucosyl units instead of the thiopropionate group) did not co-elute with any of the radioactive peaks (data not shown), indicating that this related γ-cyclodextrin is not a metabolite of sugammadex.

Discussion

This study aimed to determine the pharmacokinetics and excretion profile of 14 Csugammadex and compare them with sugammadex concentrations as determined by LC-MS analysis. Radiolabeled sugammadex was excreted rapidly and almost exclusively via the kidneys. Total 14 C detection mirrored the concentrations determined by LC-MS both in the plasma and also in urine (Figures 2 and3). Furthermore, in the urine collected over 48 h following dosing, the mean cumulative amount of sugammadex quantified by LC-MS in the urine was 91% of the dose. Moreover, 95% of the radioactivity detected in the urine was attributed to sugammadex. This suggests that there was minimal to no metabolism of sugammadex, which is consistent with the findings of previous studies evaluating sugammadex in surgical patients and healthy volunteers [10][11][12][START_REF] Peeters | Safety, Tolerability and Pharmacokinetics of Sugammadex Using Single High Doses (Up to 96 mg/kg) in Healthy Adult Subjects: A Randomized, Double-Blind, Crossover, Placebo-Controlled, Single-Centre Study[END_REF]. Less than 0.02% of the dose was excreted by in exhaled air and feces, suggesting that in man these routes of excretion play no role in the clearance of sugammadex. Interestingly, in rat and dog, a minor role for excretion in feces is observed with 4.22-6.82% and 0.33-0.80%, respectively, of the dose collected in the feces. For 14 C α-cyclodextrin and 14 C γ-cyclodextrin given i.v. to rat [START_REF] De Bie | Disposition of [14C]gamma-cyclodextrin in germ-free and conventional rats[END_REF][START_REF] Van Ommen | Disposition of [14C]-alpha-cyclodextrin in germ-free and conventional rats[END_REF] excretion into the feces has also been demonstrated to be a minor route of excretion (3-8% of the dose); however, as shown in the current study, this route is not applicable for sugammadex in man.

In this trial, the mean total cumulative amount of radioactivity recovered in the urine during 48 h was 95 (range 83-105) %, suggesting that at the most only a small fraction of the radioactivity was unaccounted for. There may be many causes of incomplete recovery of the radiolabel in mass balance trials (such as technical limitations and tissue retention), and it is actually rare for the mass balance in radiolabeled excretion studies to truly achieve 100% recovery [START_REF] Roffey | What is the objective of the mass balance study? A retrospective analysis of data in animal and human excretion studies employing radiolabeled drugs[END_REF]. Subsequent metabolite profiling, performed on plasma and urine samples (stored at -20ºC) approximately 8 months after the LC-MS analysis, visualized up to nine radioactive peaks in high performance liquid radiochromatograms of urine and plasma samples from the subjects involved in this trial. Three of these peaks also eluted when a sample of dose formulation underwent high performance liquid radiochromatography, but the radioactivity associated with two of these peaks, consistent with monosulphoxides of sugammadex, was found at considerably lower concentrations than in the patient samples. Although formation of these monosulphoxides by metabolism in vivo up to a few percent cannot be formally excluded, the presence of additional peaks in these samples is most likely the result of selective oxidation of the radiolabeled sugammadex in the plasma and urine samples during storage or sample work-up.

Further evidence for this comes from the lower amount of 14 C radioactivity eluted in the M8 sugammadex peak corresponding with unchanged sugammadex during the high performance liquid radiochromatography compared with LC-MS analysis. The selective oxidation of 14 C sugammadex has also been noted when rat plasma was spiked with the radiolabel; the radiochemical purity of [ 14 C]-sugammadex appeared to decrease from ≥99 % to ≥84 % during a 3 h incubation at 37ºC, although unlabeled sugammadex is fully stable under these conditions. It is well known that in particular sulphur in 14 Clabeled compounds is very sensitive to radio oxidation. The minimal role of metabolism in the clearance of sugammadex is fully in line with the excretion profile of other cyclodextrins, which has been noted to be almost entirely renal [START_REF] De Bie | Disposition of [14C]gamma-cyclodextrin in germ-free and conventional rats[END_REF][START_REF] Van Ommen | Disposition of [14C]-alpha-cyclodextrin in germ-free and conventional rats[END_REF][START_REF] Monbaliu | Disposition of hydroxypropyl-β-cyclodextrin in experimental animals[END_REF]18], particularly in man. Subsequent profiling for metabolites in urine and plasma samples demonstrated that the major part of total radioactivity could be attributed to the parent compound, sugammadex (data on file). Studies in volunteers and surgical patients receiving sugammadex to reverse rocuronium-or vecuronium-induced blockade have also demonstrated that the predominant route of elimination for the complex is via the renal route, and in the presence of sugammadex, the major route of elimination of rocuronium changes from the hepatic to the renal route [10][11][12]. Based on data from surgical patients and healthy volunteers, the cumulative excretion of rocuronium in the urine over 24 h is in the region of 12-26% and increases to up to 74% after administration of sugammadex 4-8 mg/kg [10][11][12][START_REF] Alvarez-Gomez | Pharmacokinetics and pharmacodynamics of rocuronium bromide in adult patients[END_REF][START_REF] Van Den Broek | Clinical pharmacology of rocuronium (Org 9426): study of the time course of action, dose requirement, reversibility, and pharmacokinetics[END_REF][START_REF] Proost | Urinary, biliary and faecal excretion of rocuronium in humans[END_REF]. Given the importance of renal elimination, the efficacy and safety of sugammadex has been assessed in patients with impaired renal clearance, most of whom were receiving dialysis. The available data suggest that sugammadex is well tolerated and effective in patients with severe renal impairment, albeit with reduced plasma clearance of sugammadex and a significantly prolonged terminal elimination half life and MRT [START_REF] Staals | Multicentre, parallel-group, comparative trial evaluating the efficacy and safety of sugammadex in patients with end-stage renal failure or normal renal function[END_REF][START_REF] Staals | Reduced clearance of rocuronium and sugammadex in patients with severe to end-stage renal failure: a pharmacokinetic study[END_REF]. However, despite longer retention of the sugammadex-rocuronium complex, no signs of recurarization were reported among renal patients in the 48 h after administration of rocuronium followed by sugammadex [START_REF] Staals | Multicentre, parallel-group, comparative trial evaluating the efficacy and safety of sugammadex in patients with end-stage renal failure or normal renal function[END_REF]. Currently, for patients with mild-to-moderate renal impairment (creatinine clearance ≥30 and <80 ml/min), the dose recommendations for sugammadex are the same as for other adult patients i.e. a single i.v. bolus dose of 2-16 mg/kg [8]. For patients with severe renal impairment (creatinine clearance <30 ml/min), including patients requiring dialysis, sugammadex is not recommended at present. However, further safety and PK studies are ongoing in this patient population. Sugammadex 4 mg/kg (containing 0.025 MBq/kg of 14 C-radioactivity) was well tolerated in all six healthy male subjects in this study. There were no drug-related AEs and no clinically significant abnormalities, changes, or trends were found with respect to

Conclusion

In conclusion, this study in healthy volunteers shows an i.v. dose of sugammadex to be cleared rapidly and almost exclusively via the kidneys, with minimal fecal excretion and minimal to no metabolism, as determined by LC-MS analysis. The mean total recovery of radiolabel from all routes in 48 h at the dose of 4 mg/kg was 95%. Pharmacokinetic parameters were similar whether calculated from total 14

  Sugammadex is Cleared Rapidly and Primarily Unchanged via Renal Excretion

  of sample radioactivity were compared with those derived from a LC-MS assay.

  . All subjects were required to give written informed consent before enrollment in the study.

  cumulative amount of radioactivity excreted in either feces or exhaled air was <0.02% in all patients.

Figure 1 .Figure 2 .Figure 3 .

 123 Figure 1. Structure of 14 C-labeled sugammadex *denotes position of radiolabel 71x70mm (300 x 300 DPI)

  of sample radioactivity were compared with those derived from a LC-MS assay.

  sugammadex. All subjects were required to give written informed consent before enrollment in the study.

  Chromatographic separations were carried out using an HP1100 series Liquid Chromatograph (Agilent Technologies, Palo Alto, CA). Samples were injected by a HTS PAL autosampler (CTC Analytics, Switzerland) and pumped at a flow-rate of 1.0 ml/min through a Polaris C18-A PEEK (polyaryletheretherketone) high performance liquid chromatography analytical column (50 mm length x 4.6 mm internal diameter, 5 µm particle size; Varian, Inc., Palo Alto, CA) with a linear mobile phase gradient of 0.1% v/v formic acid in water and methanol at ratios of 70:30 to 20:80. The liquid chromatography eluate was split 1:10 before entering a Perkin-Elmer Sciex API 3000 triple-quadrupole mass spectrometer (Sciex Thornhill, ON, Canada) equipped with a turbo ion-spray Biosystems, Foster City, CA). The quadrupoles were operated with unit resolution in the negative ion mode. The resulting multiple reaction monitoring chromatograms were integrated using Analyst version 1.2 software (Sciex). The Watson Drug Metabolism Laboratory Information Management System (version 6.3.0.03) software (Innaphase Corporation, Wayne, PA) was used for all calculations. Calibration curves for sugammadex in both human heparin plasma and human urine were constructed using linear weighted regression (weighting factor 1/Y 2 ) and used to calculate sugammadex levels in the calibration, quality control and study samples.

  before counting. Samples were counted until a statistical error (2 s) of 0.5%

  PK analysis was performed by the Clinical Pharmacology and KineticsDepartment of MSD (Oss, The Netherlands). Quantification of radioactivity in all matrices was performed by the Bioanalytical Laboratory of PRA International (Zuidlaren, The Netherlands). Radioactive high performance liquid chromatography (HPLC) for metabolite profiling and identification by mass spectrometry was performed by Huntingdon Life Sciences Ltd (Huntingdon, UK).

  6 and 6-12 h post-sugammadex samples from all subjects, and the 12-24 and 24-48 h post-sugammadex samples from one subject with protracted urinary excretion of sugammadex) and one sample of the original dose formulation were used for metabolite profiling.

  related cyclodextrin, and two monosulphoxides of sugammadex, was performed to aid metabolite identification.

14 Safety

 14 The tolerability and safety of sugammadex were evaluated based on adverse event (AE) reporting, vital signs (including systolic and diastolic blood pressure and heart rate), ECG monitoring (12-lead ECG at screening, baseline, and 2, 35, and 60 min post dose, and follow-up), physical examination, and clinical laboratory values (hematology, blood chemistry, and urinalysis). Information on AEs and serious AEs (SAEs) was obtained from regular questioning of the subjects or on clinical examination. AEs were categorized according to their severity and relation to the study drug and were coded using MedDRA (version 8.1; MedDRA Maintenance and Support Services Organization, Chantilly). Safety data were evaluated for the all-subjects-treated (AST) group.

  cumulative amount of radioactivity excreted in either feces or exhaled air was <0.02% in all patients.

  ]). Peaks M6 and M7 were consistent with two of the reference standards, both monosulphoxides of sugammadex, as determined by LC-MS. Peaks M3, M4, and M5 each accounted for

  data, vital signs, or ECGs, further adding to the safety profile of sugammadex that has been generated to date.

  C radioactivity assessments or by LC-MS estimation of sugammadex concentrations. The formation of the monosulphoxide related γ-cyclodextrins is due to selective post-sampling oxidation of the 14 C-sugammadex radiolabel and metabolism plays no significant role in the clearance of sugammadex.

Table 1 . Pharmacokinetic parameters based on 14 C-radioactivity detection and sugammadex concentrations determined by LC-MS (n = 6 subjects)

 1

	. Currently, for patients with

14 De Bie AT, Van Ommen B, Bär A. Disposition of [14C]gamma-cyclodextrin in germ-free and conventional rats. Regul Toxicol Pharmacol 1998; 27: 150-158. DOI: 10.1006/rtph.1998.1219 15 Van Ommen B, De Bie AT, Bär A. Disposition of [14C]-alpha-cyclodextrin in germ-free and conventional rats. Regul Toxicol Pharmacol 2004; 39: S57-S66. 16 Roffey SJ, Obach RS, Gedge JI, Smith DA. What is the objective of the mass balance study? A retrospective analysis of data in animal and human excretion studies employing radiolabeled drugs. Drug Metab Rev 2007; 39: 17-43. DOI: PJ. Clinical pharmacology of rocuronium (Org 9426): study of the time course of action, dose requirement, reversibility, and pharmacokinetics. J Clin Anesth 1994; 6: 288-296. DOI: 10.1016/0952-8180(94)90075-2 21 Proost JH, Eriksson LI, Mirakhur RK, Roest G, Wierda JM. Urinary, biliary and faecal excretion of rocuronium in humans. Br J Anaesth 2000; 85: 717-723. 22 Staals LM, Snoeck MM, Driessen JJ, Flockton EA, Heeringa M, Hunter JM. Multicentre, parallel-group, comparative trial evaluating the efficacy and safety of sugammadex in patients with end-stage renal failure or normal renal function. Br J Anaesth 2008; 101: 492-497. DOI: 10.effective , effective half-life; t max , time to C max ; V ss , volume of distribution at steady state.

Table 1 . Pharmacokinetic parameters based on 14 C-radioactivity detection and sugammadex concentrations determined by LC-MS (n = 6 subjects)

 1 Data presented as median and range AUC 0-∞ , area-under-the-plasma concentration-time curve to time infinity; C max , maximum plasma concentration; CL, plasma clearance; CL R , renal clearance; CV, coefficient of variation; LC-MS, liquid chromatography-mass spectrometry; t 1/2 , terminal half-life; t 1/2 effective , effective half-life; t max , time to C max ; V ss , volume of distribution at steady state.

			14 C		Sugammadex (LC-MS)
			(expressed as sugammadex mass
			equivalents)
			Geometric	Geometric CV	Geometric	Geometric CV
			mean	(%)	mean	(%)
	Urine
	CL R (ml/min)	92.7	29	88.3	25
	fraction excreted	96.1	85-106	91.1	85-94
	unchanged (% dose; last collection interval) a
	Plasma
	C max (µg/ml)	33.0	22	32.2	19
	t max (min)	b	4	2-5	5	2-10
	t 1/2 (min)		190	20	152	31
	t 1/2 effective (min)	110	16	100	19
	AUC 0-∞ (µg•min/ml)	2632	25	2629	23
	CL (ml/min)	96.8	25	96.9	24
	V SS (ml)		15360	19	14027	19

a Data presented as arithmetic mean and range; samples were taken until 48 h after dosing for measurement of sugammadex (LC-MS) in urine, and until 332 h after dosing for measurement of

14

C in urine b

http://mc.manuscriptcentral.com/bdd Biopharmaceutics & Drug Disposition

Acknowledgements

Dr Peter Wiegerinck and Eric Sperling (Radiolabel synthesis group, Dept. of Process Chemistry, MSD, Oss, The Netherlands) are acknowledged for the synthesis and analysis of the sugammadex radiolabel. Pharmacokinetic calculations for this manuscript were performed by Dennis Doorstam, BSc (MSD, Oss, The Netherlands).

Medical writing assistance was provided by Neil Venn, PhD (Prime Medica Ltd, Knutsford, UK) during the preparation of this manuscript. The design and conduct of the study, as well as analysis of the study data and opinions, conclusions, and interpretation of the data, were the responsibility of the authors.

Acknowledgements

Dr Peter Wiegerinck and Eric Sperling (Radiolabel synthesis group, Dept. of Process Chemistry, MSD, Oss, The Netherlands) are acknowledged for the synthesis and analysis of the sugammadex radiolabel. Brian John, PhD, Karen Fitzpatrick, MSc, and Andrew Hackett, PhD (Huntingdon Life Sciences, Huntingdon, UK) are acknowledged for the metabolite profiling. Pharmacokinetic calculations for this manuscript were performed by Dennis Doorstam, BSc (MSD, Oss, The Netherlands).

Medical writing assistance was provided by Neil Venn, PhD (Prime Medica Ltd, Knutsford, UK) during the preparation of this manuscript. The design and conduct of the study, as well as analysis of the study data and opinions, conclusions, and interpretation of the data, were the responsibility of the authors.

by MSD, Oss, The Netherlands.

Conflict of interest

ABSTRACT

Sugammadex is a modified γ-cyclodextrin which rapidly reverses rocuronium-and vecuronium-induced neuromuscular blockade. Previous studies suggest that sugammadex is mostly excreted unchanged via the kidneys. This single-center, openlabel, non-randomized study used 14 C-labeled sugammadex to further investigate the excretion, metabolic and pharmacokinetic (PK) profiles of sugammadex in six healthy male volunteers. 14 C-labeled sugammadex 4 mg/kg (0.025 MBq/kg of 14 C-radioactivity) was administered as a single intravenous bolus. Blood, urine, feces, and exhaled air samples were collected at pre-defined intervals for assessment of sugammadex by liquid chromatography-mass spectrometry (LC-MS) PK, and for radioactivity measurements, and metabolite profiling. Adverse events were also assessed.

Excretion of sugammadex was rapid with ~70% of the dose excreted within 6 h and ~90% within 24 h. Less than 0.02% of radioactivity was excreted in feces or exhaled air.

Ninety-five percent of the radioactivity detected in urine could be attributed to sugammadex, as determined by liquid chromatography-mass spectrometry (LC-MS), suggesting very limited metabolism of sugammadex. LC-MS analysis of plasma samples found that sugammadex accounted for 100% of total 14 C-radioactivity in the plasma.

However, as a result of post-sampling oxidation or a minor impurity in the dose formulation, high performance liquid radiochromatography analysis of urine and plasma samples revealed up to nine radioactive peaks. In general, PK parameters determined from radioactivity and sugammadex plasma concentrations were very similar. Any adverse events were of mild-to-moderate intensity, and judged unrelated to sugammadex. These findings demonstrate that sugammadex is cleared rapidly, almost exclusively via the kidney, with minimal or no metabolism.

Conflict of interest


MD Alessandro Antonelli

Regina Tardanico

MD Luca Giovanessi

MD Nicola Pesenti

MD Lorenzo Gatti

MD Tiziano Zambolin

MD Danilo Zani

MD Sergio Cosciani Cunico

MD Claudio Simeone

PREDICTING PROSTATE CANCER AT RE-BIOPSIES IN PATIENTS WITH HIGH GRADE PROSTATIC INTRAEPITHELIAL NEOPLASIA: A STUDY ON 546 PATIENTS

Keywords: high grade prostatic intraepithelial neoplasia, prostate carcinoma, prostate biopsy, rebiopsy

Objectives -To analyse which factors predict the diagnosis of prostate cancer (PCa) after highgrade prostatic intraepithelial neoplasia (HGPIN).

Methods -data of 546 patients with HGPIN submitted up to a 6-monthly series of 3 re-biopsies, according to an institutional protocol, were reviewed.

Results -PCa has been found in 174 cases (31.8%), in 116 at the first, in 58 at a further re-biopsy.

The risk of finding PCa at the first re-biopsy was correlated with the PSA value and with an anomalous digital rectal examination (DRE), at the time of the initial biopsy; the risk at a subsequent re-biopsy was correlated to the number of cores with HGPIN, with a cut-off of 4, and to the ratio with the total number of cores ("PIN density"), with a cut-off of 50%, at time of initial biopsy.

Conclusions -a tailored protocol of controls can be suggested: a) higher PSA value and/or anomalous DRE: early extended or saturation re-biopsy; b) number of cores with HGPIN >=4 and/or PIN density >=50%: delayed re-biopsy; c) no risk factors: PSA and DRE controls.

Introduction

High grade prostatic intraepithelial neoplasia (HGPIN) -defined as the presence of architecturally benign ducts and acini lined by abnormal secretory cells with morphological changes similar to those observed in prostatic cancer (PCa) [START_REF] Bostwick | Prostatic intraepithelial neoplasia and the origins of prostatic carcinoma[END_REF] -is generally considered a precursor lesion of PCa.

However, if compared to the first reports, the cancer detection rate at a re-biopsy has been definitely lowered by the current adoption of a more extended bioptic sampling scheme, which have surely allowed to spare some of the diagnoses previously missed by the first biopsy. Although HGPIN is a common finding with an incidence up to 24% of all prostatic biopsies [START_REF] Epstein | The pathological interpretation and significance of prostate needle biopsy findings: implications and current controversies[END_REF], its surveillance protocol is still debated with inconsistent opinions among experts. This paper reviews our experience in patients with HGPIN submitted to a bioptic follow-up to find out which of the data available at the initial biopsy could be predictive for the diagnosis of cancer at re-biopsy to outline a tailored protocol of controls.

Patients and Methods

Actually there are no screening programs for PCa based on PSA in Italy, even its dosage is commonly suggested by general practitioners and urologists in asymptomatic males older than 40-50 years. At our institute prostatic biopsy is indicated for PSA value higher than 4 ng/ml or suspicious prostatic digital rectal examination (DRE) or transrectal prostatic ultrasound (TRUS).

The procedure is performed via a transperineal approach with local anaesthesia. From 2001 to 2005 the sampling scheme at the first biopsy generally provided for 8/10 cores of the peripheral zone, but afterwards it was increased up to a minimum of 12 cores; suspicious areas at DRE or TRUS have been separately sampled; the transition zone has been usually sampled only at the re-biopsies.

After the diagnosis of HGPIN in all the patients eligible to radical therapy (life expectancy longer than 10/15 years) it has been recommended to be submitted to 3 re-biopsies at a 6 months interval and afterwards to periodical clinical controls of DRE and PSA value; in patients unfit for radical therapy the series of re-biopsies was recommended but stopped if a benign histology was found. All the specimens have been evaluated only by a single experienced uro-pathologist (RT).

For the present study, all the cases with a diagnosis of HGPIN observed from 2001 to 2009 and submitted to at least one re-biopsy have been reviewed. The cases with an associated diagnosis of atypical small acinar proliferation (ASAP) or small foci of PCa have been excluded. The occurrence of PCa at the first re-biopsy or at a further re-biopsy has been separately analysed considering clinical (age, PSA, digital rectal examination -DRE -and prostatic ultrasound -TRUS -at each biopsy) bioptic and pathological data. DRE and TRUS findings other than normal have been defined as abnormal (i.e. including into this definition the cases regarded as ambiguous by the examiners). Among bioptic data it was defined as "PIN density" the ratio between the number of cores with HGPIN and the total number of cores sampled.

Statistical analysis -Cut-off values were defined as the lowest value of a continuous variable which could discriminate two groups making statistically significant the difference of the considered variable.

Nominal variables have been compared by Persons's chi-square test and, if applicable, Fisher's exact test; continuous variables by the Mann-Withney U test or, if applicable, Student's T test.

All the analyses have been carried out by using the software SPSS (SPSS inc, version 17, Chicago, IL, USA)

Results

Were analysed 546 patients with a mean age of 64.9 (range 45-87 years) submitted to a median number of 3 biopsies/patient (2-6 biopsies/patient), with a mean number of 10.8 cores/biopsy (6-23 cores/biopsy) at the initial biopsy and 12.9 cores/biopsy (6-37 cores/biopsy) at re-biopsies, a mean interval between consecutive biopsies of 5.1 months (1-39.5 months) and a mean "bioptic" followup time of 14.8 months (2-102 months).

PCa was found in 174 patients (31.8%), in 116 (21.2%) at the first re-biopsy, at a mean interval of 7.8 months, and in 58 (10.6%) after a median of 2 re-biopsies at a mean interval of 21.6 months. At statistical analysis the detection of PCa at the first re-biopsy was significantly related only to the PSA value and abnormal DRE referred to the initial biopsy; for the PSA value, it was identified a cut-off of 7 ng/ml (table no.1). Conversely, the diagnosis of PCa at a further re-biopsy was related to the number of cores with HGPIN and to the PIN density at the initial biopsy and it was possible to identify the cut-off values of 4 cores with HGPIN and of 50% for PIN density (table no.2).

Grouping the patients on the basis of these cut-off values could discriminate with high specificity the risk of finding PCa at the first or at a further biopsy (table no.3). These results were confirmed at a sub-analysis among the 251 patients sampled with at least 12cores: PCa was found in 48 patients at the first re-biopsy (19.1%, mean latency 7.8 months) with a significant correlation with PSA value (8.4 ng/ml vs 7.2 ng/ml, p=0.034) and abnormal DRE (28.8% vs 15.0%, p=0.015) at the initial biopsy, whereas PCa at a further re-biopsy was found in 21 patients (8.3%, mean latency 15.0 months) with a correlation with the number of cores with HGPIN (4.75 vs 2.89, p= 0.025) and the PIN density (0.3238 vs 0.2051, p=0.031) at the initial biopsy. Also the cut-offs of PSA, number of PIN cores and PIN density could be applied to this subset of patients.

Among the cases submitted at least to 2 re-biopsies, the histological findings of the first re-biopsy weren't related to the risk of a subsequent diagnosis of PCa: indeed the detection rate of PCa was 15.7% in the cases with a benign histology and 21.7% in those with a persistence of HGPIN (p=0.444).

Cancers found at the first re-biopsy showed a higher aggressiveness when compared to the ones found at a further re-biopsy. In particular, they had a significantly higher prevalence of bioptic Gleason score >=7 (16.0% vs 5.3%, p=0.049) and among the 88 patients undergone radical prostatectomy a higher prevalence of pathologic Gleason score >=7 (27.9% vs 16.7%, p=0.404) and a higher rate of pT3a (10.9% vs 0.0%, p=0.183); in all the cases no limphnodal metastasis were detected.

Discussion

HGPIN was described in 1969 by McNeal [START_REF] Mcneal | Origin and development of carcinoma in the prostate[END_REF], but its current definition was formulated in 1987 by Bostwick and Brawer [START_REF] Bostwick | Prostatic intraepithelial neoplasia and early invasion in prostate cancer[END_REF] and formalised during a consensus conference on prostatic pre-neoplastic lesions in 1989. There are some evidences supporting the hypothesis that HGPIN is a pre-neoplastic lesion [START_REF] Joniau | Prostatic intrapithelial neoplasia (PIN): importance and clinical management[END_REF]: is multifocal, like PCa; is more frequent in patients with PCa; has an age distribution equal to PCa; may degenerate into PCa with a progressive transition [START_REF] Sanchez-Chapado | Prevalence of prostate cancer and prostatic intraepithelial neoplasia in Caucasian Mediterranean males: an autopsy study[END_REF][START_REF] Epstein | Relationship of severe dysplasia to stage A (incidental) adenocarcinoma of the prostate[END_REF][START_REF] Quinn | Relationship of severe dysplasia to stage B adenocarcinoma of the prostate[END_REF][START_REF] Shin | Extent and zonal distribution of prostatic intraepithelial neoplasia in patients with prostatic carcinoma in Japan: analysis of whole-mounted prostatectomy specimens[END_REF]. However, some clinical studies showed that patients with HGPIN have a risk of further cancer diagnosis low or not higher than that observed in cases with a benign diagnosis [START_REF] Gallo | Prognostic significance of high-grade prostatic intraepithelial neoplasia (HGPIN): risk of prostatic cancer on repeat biopsies[END_REF][START_REF] Epstein | Prostate needle biopsies containing prostatic intraepithelial neoplasia or atypical foci suspicious for carcinoma: implications for patient care[END_REF][START_REF] Chin | Is repeat biopsy for isolated high-grade prostatic intraepithelial neoplasia necessary?[END_REF][START_REF] Moore | Prognostic significance of high grade prostatic intraepithelial neoplasia and atypical small acinar proliferation in the contemporary era[END_REF][START_REF] Bastien | Prostate cancer detection rate in patients with repeated extended 21-sample needle biopsy[END_REF]. In any case, after the introduction of extended bioptic schemes with 10/12 samples the rate of cancer diagnosis after HGPIN decreased from the values up to 80% of the sextant biopsy era to the current 20-30%: probably, thanks to a wider sampling the diagnoses at re-biopsy that were previously not detected at the initial biopsy were avoided [START_REF] Herawi | Risk of prostate cancer on first re-biopsy within 1 year following a diagnosis of high grade prostatic intraepithelial neoplasia is related to the number of cores sampled[END_REF]. Therefore, it is still debated if it is really necessary to repeat a biopsy for all the patients with HGPIN and EAU guidelines [START_REF] Heidenreich | EAU guidelines on prostate cancer[END_REF] generically suggest an early re-biopsy only for multifocal HGPIN. Several studies, indeed, have ascertained that the number of cores with HGPIN at a sextant [START_REF] Kronz | Predicting cancer following a diagnosis of high grade PIN on needle biopsy[END_REF] or extended biopsy [START_REF] De Nunzio | The number of cores positive for high grade prostatic intraepithelial neoplasia on initial biopsy is associated with prostate cancer on second biopsy[END_REF][START_REF] Roscigno | Monofocal and pluri-focal high-grade prostatic intraepithelial neoplasia on extended prostate biopsies: factors predicting cancer detection on extended repeat biopsy[END_REF][START_REF] Akhavan | The proportion of cores with high-grade prostatic intraepithelial neoplasia on extended-pattern needle biopsy is significantly associated with prostate cancer on site-directed repeat biopsy[END_REF] correlates with the risk of finding PCa at re-biopsy, although there are also data against this conclusion [START_REF] Naya | Can the number of cores with high-grade prostate intraepithelial neoplasia predict cancer in men who undergo repeat biopsy?[END_REF][START_REF] Brimo | Outcome for repeated biopsy of the prostate: roles of serum PSA, small atypical glands, and prostatic intraepithelial neoplasia[END_REF]. Among the studies supporting the correlation between multifocality of HGPIN and the risk of PCa, different cut-off values in the number of cores with HGPIN have been reported: 2 cores for Merrimen et. al [START_REF] Merrimen | Multifocal high grade prostatic intraepithelial neoplasia is a significant risk factor for prostatic adenocarcinoma[END_REF] in a population study on 564 cases, 4 cores for Netto et al. in an institutional study on 41 cases in which such a condition, worsened by a risk reaching approximately 40%, was formally defined as "widespread PIN" [START_REF] Netto | Widespread high-grade prostatic intraepithelial neoplasia on prostatic needle biopsy: a significant likelihood of subsequently diagnosed adenocarcinoma[END_REF]. Again, also concerning the timing of re-biopsy, the literature reports heterogeneous opinions from controls every 6 months for two years and then yearly [START_REF] Montironi | Mechanisms of disease: high-grade prostatic intraepithelial neoplasia and other proposed preneoplastic lesions in the prostate[END_REF] to one single re-biopsy after 36 months [START_REF] Lefkowitz | Follow-up interval prostate biopsy 3 years after diagnosis of high grade prostatic intraepithelial neoplasia is associated with high likelihood of prostate cancer, independent of change in prostate specific antigen levels[END_REF].

From a single-institution population of approximately 5,500 patients submitted to a fine needle prostatic biopsy during a period of 9 years evaluated by a single experienced pathologist, the present study retrospectively reviews the data of 546 patients with isolated HGPIN, all subjected to at least one re-biopsy after a short interval of time (6 months) and, in a proportion of 65% (278/430) of cases, monitored with at least 2 re-biopsies.

Unlike what has been previously reported [START_REF] De Nunzio | The number of cores positive for high grade prostatic intraepithelial neoplasia on initial biopsy is associated with prostate cancer on second biopsy[END_REF][START_REF] Roscigno | Monofocal and pluri-focal high-grade prostatic intraepithelial neoplasia on extended prostate biopsies: factors predicting cancer detection on extended repeat biopsy[END_REF][START_REF] Akhavan | The proportion of cores with high-grade prostatic intraepithelial neoplasia on extended-pattern needle biopsy is significantly associated with prostate cancer on site-directed repeat biopsy[END_REF][START_REF] Naya | Can the number of cores with high-grade prostate intraepithelial neoplasia predict cancer in men who undergo repeat biopsy?[END_REF][START_REF] Brimo | Outcome for repeated biopsy of the prostate: roles of serum PSA, small atypical glands, and prostatic intraepithelial neoplasia[END_REF], we found a significative correlation between the cancer detection rate at the first re-biopsy and PSA value and DRE at the time of the initial biopsy: considering the short interval between biopsies and that HGPIN can justify neither the increased values of PSA nor the anomalous DRE [START_REF] Zynger | High-grade prostatic intrapeithelial neoplasia of the prostate: the precursor lesion of prostate cancer[END_REF], it is reasonable to suspect that in these cases a PCa already present was missed by the first sampling. On the contrary, in the cases in which the diagnosis of PCa has occurred at a subsequent re-biopsy, a statistical significant correlation was identified with the number of cores positive for HGPIN and with the PIN density of the initial biopsy. In these patients, the fact that the diagnosis of PCa was reached after a longer time and at least 2 re-biopsies, makes more reasonable that PCa wasn't present at the time of the initial biopsy and that a true transition from HGPIN to PCa happened, as it should be indirectly confirmed by the lower aggressiveness of these cancers. An accurate risk stratification was possible on the basis of cut-off values (see table 3). In out cohort of patients a PSA cut-off of 7 ng/ml and an anomalous prostatic DRE defined a group with a twofold risk (28.0% vs 13.9%) of PCa finding at the first re-biopsy. Conversely, 4 or more cores with HGPIN and 50% or more in PIN density doubled the risk (30.9% vs 14.6%) of the diagnosis at a further re-biopsy.

Our experience confirms also that the rate of PCa diagnosis after HGPIN is approximately 30%, but, once excluded the diagnoses probably missed by the first biopsy as discussed above, the actual risk of diagnosis of PCa after HGPIN would be reduced to 10%. Such an exiguous rate can support the criticisms of several authors about the real need of an indiscriminate bioptic control in all HGPIN cases.

The main limitation of the study is its retrospective design: indeed, even if bioptic controls followed a well-codified internal protocol some biases, especially in the number of cores/biopsy -there is a proportion of 13% of patients that weren't initially submitted to an extended biopsy -and intervals between biopsies, come from the fact that the patients weren't formally enrolled in a perspective study.

To conclude, 3 conditions deserving different control modalities can be identified: a) higher PSA value and/or anomalous DRE, in which an extended or saturation re-biopsy is indicated after approximately 2/4 months, i.e. the time required for the recovery from the bioptic damage in order not to invalidate pathological evaluation; b) number of cores with HGPIN>=4 and/or PIN density>=50%, in which a re-biopsy is indicated after at least 24 months; the finding of less aggressive cancers in such conditions allows to delay the control in order to increase the rate of diagnosis without loosing the chance of a radical therapy; c) no risk factors, in which it is advisable to carry out just a clinical and PSA monitoring to eventually indicate the repetition of the biopsy.

The authors declare that there is no competing financial interests in relation with this work.

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