Methylene blue MMX® tablets for chromoendoscopy. Safety tolerability and bioavailability in healthy volunteers

Contemporary Clinical Trials 33 (2012) 260–267

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Methylene blue MMX® tablets for chromoendoscopy. Safety tolerability and bioavailability in healthy volunteers A. Repici a, A.F.D. Di Stefano b,⁎, M.M. Radicioni b, V. Jas c, L. Moro d, S. Danese a a b c d

Department of Gastreonterology, IRCCS Istituto Clinico Humanitas, Milan, Italy Cross Research S.A., Via F. A. Giorgioli, 14, CH-6864 Arzo, Switzerland Analytisch Biochemisch Laboratorium B.V. (ABL), W. A. Scholtenstraat 7 9403 AJ Assen, The Netherlands Cosmo Technologies Ltd., 42–43 Amiens Street, Dublin 1, D1, Ireland

a r t i c l e

i n f o

Article history: Received 27 June 2011 Received in revised form 14 October 2011 Accepted 5 November 2011 Available online 11 November 2011 Keywords: Methylene blue Pharmacokinetics Healthy subjects MMX Chromoendoscopy

a b s t r a c t Methylene blue-MMX® tablets are proposed as colonic diagnostic staining. Methylene blue taken prior to colonoscopy is expected to provide an effective staining of colonic and rectal mucosa leaving unstained the dysplastic or polypoid areas. The present single dose, open-label study investigated the safety of methylene blue after single oral doses of 200 and 400 mg in healthy volunteers. The absolute bioavailability was also investigated after the intake of 2 L of bowel cleansing preparation in 2 h and by comparing the dose of 200 mg with a single iv dose of 100 mg in the same subjects. Only non-serious adverse events occurred. Related events occurred to 8/22 subjects. Most of the events were mild and transient. Abnormal transaminases, gastrointestinal disorders and dysuria frequency were 13.6%. After intake of the laxative and the oral dose of 200 mg, systemic exposure to methylene blue was shown in all subjects with concentrations increasing for 12 h. The peak was reached in a median of 16 h. Peak blood concentration did not increase proportionally with the dose. AUC0–t was 32.94 μg/mL × h after 200 mg and 38.08 μg/mL × h after 400 mg. Half life ranged between 14 and 27 h after the lower dose and between 6 and 26 h after the higher dose. The cumulative excretion was about 40% of the injected dose, 39.67% after 200 mg and 23.48% after 400 mg. Absolute bioavailability of methylene blue calculated as ratio between AUC0–t oral/iv corrected for the dose was on average Fabs = 139.19 ± 52.00%. © 2011 Elsevier Inc. All rights reserved.

1. Introduction Methylene blue is able to stain tissues without impairing their vital functions. Currently, methylene blue is used in human medicine in a number of therapies and diagnoses. Methylene blue MMX® tablets are a new oral modified release formulation manufactured using a multimatrix structure (MMX®, Cosmo Technologies, Ireland). MMX is a modified release technology that ensures a colonic drug delivery. In the MMX structure, microparticles of active ingredient are dispersed in a sequence of lipophilic and hydrophilic matrices. The multi⁎ Corresponding author. Tel.: + 41 91 6300510; fax: + 41 91 6300511. E-mail address: [email protected] (A.F.D. Di Stefano). 1551-7144/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.cct.2011.11.006

matrix creates a partially hydrophobic environment which slows and controls the drug dissolution process. The outer tablet gastroresistant polymer film begins to disintegrate when the suitable intestinal pH is reached. Thus, the tablets arrive unaltered to the terminal ileum. When the colon fluids interact with the tablet after coating dissolution, the matrix structure forms an outer, viscous gel mass that controls the diffusion of the active ingredient. Whilst the tablet progresses in the colon towards the rectum, debris of the gel mass disaggregate and release the active ingredient in proximity to the mucosa. MMX formulations' gastrointestinal transit and colonic delivery was demonstrated by pharmaco-scintigraphic investigations of various drugs in healthy male volunteers [1,2]. In the study of gastrointestinal transit and distribution of budesonide MMX® tablets, 153 Sm-budesonide radioactivity incorporated into tablets was

A. Repici et al. / Contemporary Clinical Trials 33 (2012) 260–267

followed by means of pharmaco-scintigraphic imaging [2]. The relative percentage of budesonide absorption in the time during which radioactivity was detectable in the target region (i.e. the region comprised between the ascending and the descending-sigmoid colon) was 95.9±4.2%. Budesonide tablets were detected in the ascending colon between 6 and >24 h. The descending colon was left 12 to >24 h post-dosing. Initial tablet disintegration started, on average, 9.48±5.11 h after administration either in the small intestine (n =2), the ileum (n=5), the ascending (n =2), transverse (n=2) or sigmoid colon (n=1). Times of tablet residence in the gastroenteric regions were 17–117 min (stomach), 37 min–9.95 h (small intestine), 0.5–12 h (ileum), 3 to >15.5 h (ascending colon), 4 to >17 h (transverse colon), and 12 to >17 h (descending colon) [2]. Methylene blue has the property to be selectively absorbed and stain the intestinal columnar epithelium [3–5]. Normal, non-dysplastic mucosa generally exhibits a diffuse, homogeneous, dark blue staining, after methylene blue is absorbed into the columnar cytoplasm and abundant goblet cells. In contrast, severely dysplastic, inflamed or malignant epithelium exhibits decreased cytoplasm and reduced to absent goblet cells. These alterations result in decreased uptake of methylene blue and endoscopic appearance of focal light blue or pink (unstained) or heterogeneously stained (specked) mucosa within the diffusely stained epithelium. This property is applied to the detection of abnormalities of colonic mucosa. During the chromoendoscopy, different dyes, like methylene blue and indigo carmine, are topically applied by a spraying probe [4,6]. Sprayed methylene blue has been used to screen colonic neoplasias at concentrations of 0.1–0.5% [7]. The new tablets would represent a potential alternative to the topical spray. When taken before the colonoscopy, methylene blue tablets are expected to provide an accurate staining in the colon and rectum. The colonic release would increase the contrast of the mucosal cells, lower the polyp detection failure rate and reduce the total time of the colonoscopy by ensuring the mucosal staining before initiating the endoscopy. The excessive prolongation of the endoscopy due to the dye spraying, absorption into the epithelium and excess removal would also be avoided. The present study aimed at preliminarily investigating safety and absolute bioavailability of methylene blue administered as MMX tablets at doses of 200 and 400 mg to healthy male and female volunteers after the intake of a bowel cleansing preparation. The investigation of the colonic mucosa staining after a single dose of methylene blue tablets is the objective of ongoing trials. 2. Methods 2.1. Study design The present study was a single ascending dose, open-label, randomised, safety, bioavailability study. Primary objective was the investigation of the safety of methylene blue after single oral doses of 200 and 400 mg as Methylene blue MMX tablets in healthy volunteers undergoing a standardised bowel cleansing preparation. The new formulation was administered for the first time. The study was also aimed at investigating the absolute bioavailability of methylene blue after a single oral dose of 200 mg as compared with a single iv dose of 100 mg

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of methylene blue 1% and at investigating the PK profiles of methylene blue in the main biological fluids blood and urine. The study consisted of a 1st stage including 2 periods and a 2nd stage including only one period. Subjects enrolled for the 1st stage received a single oral dose of 200 mg and a single iv dose of 100 mg according to a randomised two-way crossover design to investigate the absolute bioavailability of methylene blue. In the 2nd stage, a 2nd cohort of volunteers was enrolled and administered a single oral dose of 400 mg of methylene blue for the investigation of safety, tolerability and PK profile of the active substance. The blood sampling and the urine collection schedule were planned up to 72 h post-dose considering the recently published data about the PK of methylene blue [8,9] and considering the peculiar delivery profile of the previously studied MMX® formulations [1,2]. Since methylene blue had never been administered orally for the mucosal staining, the selection of doses was made by considering the dose regimen indicated for the marketed injectable and oral products of methylene blue for the treatment of methaemoglobinaemia and urinary infections. Maximal doses for these two indications are 120 mg/day iv and 200 mg/day orally. In the peculiar case of treatment of ifosfamide-induced encephalopathy daily iv injections of up to 300 mg of methylene blue are recommended [10]. On the basis of this safety consideration, single ascending doses of 200 and 400 mg were expected to be safe and tolerable to the healthy subjects. The previously published PK trial of methylene blue in healthy subjects by Walter-Sack et al. was also taken into account. In that study, single doses of 500 mg of methylene blue as a 2.5% aqueous solution were administered without raise of safety concerns [9]. According to Walter-Sack et al., the most frequent untoward effects were nausea and vomiting on one side and temporary blue staining of tissues on the other side. In particular, nausea and vomiting were due to the particular oral solution dosage form used in that study, which had a poor palatability. Less frequent untoward effects were diarrhoea, dysuria and headache. According to this relevant previous experience, the safety assessments of the present study were focussed on the recording of adverse events and on the clinical laboratory assays. A continuous monitoring of ECG and vital signs was regarded as superfluous, since this measurement had been already performed in the Walter-Sack's trial and no significant change had been observed. In the study design, the review of methylene blue tolerability by Clifton and Leikin was also taken into account [11]. Indeed, the expected untoward effect of a dose of 2–4 mg/kg of methylene blue was haemolytic anaemia. Furthermore, the authors reported the untoward effect of blue-green discolouration of urine and faeces after a 4 mg/kg dose. The known expected untoward effects of 7 mg/kg of methylene blue were nausea, vomiting, abdominal pain, chest pain, fever, haemolysis, profuse sweating, dizziness, headache and mental confusion according to the same authors. It is also known that untoward effects may be exacerbated in case of glucose-6-phosphate dehydrogenase deficiency. Therefore, the enzyme was assayed specifically in the present study.

2.1.1. Study population and criteria for inclusion The study was performed at the Phase I Unit of Cross Research S.A., Arzo, Switzerland.

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Healthy males and post-menopausal females were included into the trial according to the following main inclusion criteria: (i) age of 18 to 65 y, (ii) a BW ≥ 60 kg, (iii) good health based on medical history, physical examination, a 12-lead electrocardiogram (ECG) and routine haematology and blood chemistry tests, (iv) use of highly effective contraceptive methods for at least 2 months prior to the study start [12], (v) willingness to provide written informed consent. Main exclusion criteria were the standard criteria for bioavailability estimation of new drugs, namely (i) intake of any medication, (ii) a history of drug, caffeine (>5 cups coffee/ tea/day) or tobacco (≥10 cigarettes/day) abuse, (iii) history of alcohol consumption in excess of two drinks per day in males and one drink per day in females, as defined by the U.S.D.A. dietary guidelines [13]. The study was descriptive and non comparative. The bioavailability of methylene blue after administration of MMX® modified release tablets was unknown and investigated for the first time in the present study. Therefore, the planned sample size was not derived from a statistical power calculation. A total of 22 subjects were included and completed the study as per protocol.

2.1.2. Investigational treatments and dose regimen Besides the investigational product Methylene blue MMX® 200 mg tablets manufactured by Cosmo S.p.A. Italy, the reference marketed product Methylene blue 1% injectable solution by Akorn Inc., USA was used for the iv dosing. Moviprep® laxative solution by Norgine AG, Switzerland was also used to mimic exactly the potential administration conditions of MMX® tablets in case of colonoscopy. The 1st study stage consisted of periods 1 and 2. The 1st enrolled 10 subjects received one single oral dose of 200 mg of test product and a slow intravenous injection of reference according to the randomised, cross-over design. Between administrations, a wash-out interval of at least 7 days elapsed. For the injection of reference product, the whole content (10 mL) of one 1% vial was injected as a single slow iv injection lasting 6 min into an arm vein. Concomitantly with the administration of Methylene blue MMX® tablets, the subjects received also a full dose regimen of 2 L of Moviprep® solution. Moviprep® was administered following the instructions enclosed with the product. All the subjects received a full dose regimen of laxative. They drank 200 mL of solution every 15 min. The subjects received methylene blue 30 min of the end of intake of bowel cleansing preparation, which lasted 2 h. During the intake of laxative the subjects were recommended to drink additional non gaseous water according to the manufacturer specifications. In the 2nd study stage, the 2nd cohort of 12 subjects received a single oral dose of 400 mg of methylene blue MMX® (2 × 200 mg tablets) 30 min of the end of intake of a full dose regimen of of Moviprep®. Subjects were confined in the Phase I Unit during the whole study periods. The sequence of treatments in the two study Periods of the 1st stage was assigned to each randomised subject according to a computer generated randomisation list, starting from the lowest number to be assigned to the subject in

chronological order. Randomisation number was given to the subjects on study day 1 of Period 1. In the 2nd study stage, no randomisation was applied. On study day 1, all the subjects received the same treatment. 2.2. Ethical procedures The study CRO-PK-10-236 — Sponsor code CB-17-01/01 was approved by the independent ethics committee of Canton Ticino on 22JUN10. Ref. nr. 2330. The Swiss Federal Health Authorities (Swissmedic) authorised the study on 22SEP10 and assigned the reference number 2010DR1184. Both studies were conducted in compliance with the Swiss ordinance on clinical trials of therapeutic agents and in accordance with the Declaration of Helsinki and the general principles of ICH Harmonised Tripartite Guidelines for GCP. Subjects did not undergo any study procedure before signing the written informed consent form. The first subject was enrolled on 04OCT10 and the last subject completed the trial on 07NOV10. 2.3. Pharmacokinetic variables and data analysis The following PK parameters were measured and/or calculated for methylene blue according to a non-compartmental model using the validated software WinNonLin® 5.2 (Pharsight Corporation): Whole blood C0, Cmax, Tmax, AUC0–t, AUC0–∞, t1/2, MRT for the iv route; Whole blood Cmax, Tmax, AUC0–t, AUC0–∞, t1/2, MRT, Fabs for the oral route. Urine Xu0–t ΣXu and %ΣXu. The listed parameters were defined as follows: Cmax C0 Tmax t1/2 AUC0–t

AUC0–∞

AUCextra

MRT Fabs

Maximum observed blood concentration Back-extrapolated theoretical blood concentration at the start of iv injection Time to achieve Cmax Half life, calculated, as ln2/λz where λz is the terminal elimination rate Area under the concentration curve from administration to the last observed concentration time t, calculated with the linear trapezoidal method Area under the concentration/time curve extrapolated to infinity, calculated, as AUC0–t + Ct/λz, where Ct is the last measurable drug concentration (%) Percentage of the residual area (Ct/λz) extrapolated to infinity in relation to the total AUC0–∞, calculated, if feasible, as 100 × [(Ct/λz)/AUC0–∞] Mean residence time calculated, as AUMC0–∞/AUC0–∞, where AUMC is the area under the moment curve Absolute bioavailability, calculated as ratio: AUC0−∞ ðevÞ=DoseðevÞ AUC0−∞ ðivÞ=DoseðivÞ

Xu ΣXu

where ev refers to the extravascular route of administration and iv to the intravascular route of administration Urinary amount of excretion in a collection interval Cumulative urinary amount of excretion

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2.4. Sample collection, handling and analytics The concentration of methylene blue was determined in whole blood at the following times after the oral dosing: ➢ pre-dose (0), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 16, 20, 24, 36, 48, 60 and 72 h post-dose; and at the following times after the iv dosing: ➢ pre-dose (0), 6, 12, 18, 24, 36, 48 min, 1, 2, 4, 6, 8, 10, 12, 16, 24, 36, 48 and 72 h post-dose. The concentration of methylene blue was determined in urine in the following time intervals after the oral and the iv dosing: ➢ pre-dose (0), 0–4, 4–8, 8–16, 16–24, 24–48 and 48–72 h post-dose. Blood samples for PK analysis were collected using an indwelling catheter with switch valve. The cannula was rinsed, after each sampling, with about 1 mL of Na-heparin solution in sterile saline (20 I.U./mL) solution. The first 2 mL of blood was discarded at each collection time. The remaining 4 mL was collected from the catheter by using 5-mL needle Luer syringes (Injekt® Solo by B. Braun, USA) and then transferred in about 30 s into EDTA blood collection tubes, mixed and divided into two glass tubes (aliquots B1 and B2), stored protected from light on ice for no more than 20 min and stored frozen at ≤−70 °C until analysis. During each interval, urine was collected into glass containers, kept refrigerated at ≈4 °C and protected from light. At the end of each collection interval, urine volumes were measured and after thorough mixing, two aliquots (U1 and U2) of 10 mL each were stored protected from light in glass tubes at ≤−70 °C pending analysis. The concentration of methylene blue in whole blood and urine was determined at Analytisch Biochemisch Laboratorium ABL BV, the Netherlands, using fully validated LC-MS/ MS methods with a lower quantification limit (LQL) of 20 ng/mL for blood and 50 ng/mL for urine. A full validation of the methods was performed according to the current guidelines for bioanalytical method validation [14,15]. Long term stability tests indicated that methylene blue stored frozen at ≤−70 °C is stable up to 92 days in whole blood and up to 71 days in urine. The two LC-MS/MS methods for the determination of methylene blue in human EDTA whole blood and human urine samples produced accurate and precise results. The calibration range covered 20.0–20,000 ng/mL in human whole blood and 50.0–150,000 ng/mL in human urine, and included the determination of the parameters calibration, accuracy and precision, recovery, specificity, dilution and stability. The correlation coefficient r for the assay in whole blood was r (mean) = 0.9996, corresponding to r2

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of 0.999. The correlation coefficient r for the assay in urine was r (mean) = 0.9989, corresponding to r2 of 0.998. Whole blood samples were spiked with an internal standard (methylene violet 3 RAX). Subsequently, the samples were subjected to protein precipitation using acetonitrile. Urine samples were subjected to oxidation with HCl at a temperature of 70 °C. Subsequently, the samples were spiked with an internal standard (methylene violet 3 RAX) and diluted with injection solvent (ammonium formate buffer in acetonitrile/water). The processed whole blood and urine samples were injected into the LC-MS/MS system for quantification. The samples were chromatographed on a XBridge® C18 LC column. The mass-spectrometer was equipped with a Turbo Ion Spray interface and operated in the positive ion mode. 2.5. Safety variables The primary variables were safety variables including: ➢ the recording of adverse events during the whole study duration ➢ blood pressure, heart rate and body weight measurements as well as ECG recordings at the screening, at predose and 5 days post-dose ➢ physical examinations and routine blood chemistry, coagulation, haematology and urinalysis laboratory assays performed at screening and upon discharge after each dosing. 3. Results 3.1. Disposition of subjects Ten (10) male Caucasians and 12 female Caucasians, aged 24 to 61 y were enrolled. Baseline demographic data are summarised in Table 1. All the 22 enrolled subjects concluded the study as per protocol and were considered in the safety and PK analysis. 3.2. Methylene blue blood concentration after single oral and iv dose After single dose administration of either 200 or 400 mg of methylene blue tablets or single injection of 100 mg of methylene blue the blood methylene blue vs. time profiles were as in Fig. 1 in linear scale. The main kinetic whole blood parameters measured or calculated for all 3 doses are described in Table 2. After single doses of 200 and 400 mg, the dye became quantifiable in blood after 3–12 h and 5–12 h of the dosing, due to the peculiar modified release profile of MMX tablets

Table 1 Mean (± SD) baseline demographic data. Sex

Age

Height

BW

BMI

Males

Females

(y)

(cm)

(kg)

(kg/m2)

12 (54.5%)

10 (45.5%)

46.0 ± 12.4

168.7 ± 11.1

72.1 ± 7.2

25.5 ± 2.8

BW: body weight; BMI: body mass index.

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Fig. 1. Mean (± SD) blood methylene blue concentrations (ng/mL) vs. time profiles after single dose administration of 200 mg of Methylene blue MMX® tablets (■ solid line) or 400 mg of Methylene blue MMX® tablets (● solid line) or 100 mg of methylene blue 1% injectable solution (▲ solid line). Linear scale.

Table 2 Mean (± SD) of main PK parameters of blood methylene blue after single dose administration of either 200 or 400 mg of Methylene blue MMX® or single injection of 100 mg of methylene blue 1% injectable solution. For Tmax median and range are reported. Treatment

C0 (μg/mL)

(μg/mL)

200 mg of Methylene blue-MMX® 400 mg of Methylene blue-MMX® 100 mg of methylene blue 1% solution

– – 2.66 ± 1.35

1.66 ± 0.50 1.64 ± 0.73 2.07 ± 0.57

Cmax

Tmax

AUC0–t

AUC0–∞

T1/2

(h)

(μg/mL × h)

(μg/mL × h)

(h)

16.00 (9.00–20.00) 16.00 (12.00–24.00) 0.10 (0.10–0.20)

32.94 ± 14.30 38.08 ± 23.07 11.86 ± 2.80

36.82 ± 17.57 42.11 ± 26.22 13.47 ± 3.29

20.19 ± 4.68 17.25 ± 7.43 26.71 ± 8.12

C0: back-extrapolated theoretical blood concentration at the start of iv injection; Cmax: maximum observed blood concentration, Tmax: time to achieve Cmax, AUC0–t: area under the concentration curve from administration to the last observed concentration time t; AUC0–∞: area under the concentration/time curve extrapolated to infinity; t1/2: half life.

[1,2]. Systemic exposure to the active ingredient reached a peak in a median time of 16 h. Peak blood concentration did not increase proportionally with the dose. On average, Cmax was very similar after both oral doses. Consistently with the peak values, AUC was on average 32.94 μg/mL × h after the dose of 200 mg and 38.08 μg/mL × h after 400 mg. Elimination half life ranged between 14 and 27 h after 200 mg and between 6 and 26 h after the higher dose. 3.3. Methylene blue urinary excretion after single oral and iv dose The main kinetic urine parameters measured or calculated for all 3 doses are described in Table 3. Methylene blue quantifiable concentrations were found in urine of 4 subjects at the pre-dose collection interval only in Period 2 of the 1st study stage. These very low pre-dose concentrations were a negligible carry-over effect due to the administration of methylene blue in Period 1. In the 1st post-

dose collection interval, methylene blue was quantifiable for the totality of subjects after single iv injection, whilst the analyte was not quantifiable in urine after single oral dose. When methylene blue was quantifiable in this collection interval after single dose of 200 mg, it was only for the 3 subjects with carry-over values at pre-dose.

Table 3 Mean (± SD) amount of excretion of methylene blue after single dose administration of either 200 or 400 mg of Methylene blue MMX® or single injection of 100 mg of methylene blue 1% injectable solution. Treatment

200 mg of Methylene blue-MMX® 400 mg of Methylene blue-MMX® 100 mg of methylene blue 1% solution

ΣXU0–t

ΣXU0–t

(mg)

(%)

69.67 ± 79.34 93.93 ± 61.52 40.15 ± 7.63

39.67 ± 19.19 23.48 ± 15.38 40.15 ± 7.63

ΣXu: cumulative urinary amount of excretion.

A. Repici et al. / Contemporary Clinical Trials 33 (2012) 260–267

In the following collection interval of 4–8 h post-dose, methylene blue was still quantifiable after single iv injection. In the same collection interval, methylene blue was quantifiable in the samples of 6 subjects after 200 mg and of 7 subjects after 400 mg. The totality of the subjects had quantifiable excreted amounts of methylene blue in the 8–72 h post-dose interval after single oral dose of both 200 and 400 mg. The cumulative excretion of methylene blue after single iv injection was about 40% of the injected dose. After single oral dose of 200 mg, the cumulative excretion was almost identical by accounting for 39.67%. After single oral dose of 400 mg the cumulative excretion was 23.48% of the administered dose. 3.4. Absolute bioavailability and comparison between administration routes Absolute bioavailability of methylene blue was calculated for the 10 subjects included in the 1st cross-over study stage. The ratio between AUC0-t after single oral administration and AUC0–t after single iv injection corrected for the administered dose was on average Fabs = 139.19 ± 52.00%. No formal calculation of the absolute bioavailability could be preformed for the single oral dose of 400 mg, because no cross-over design was applied to the 2nd study stage. In detail, 4 subjects had individual bioavailability values lower or very close to 100%. Two other subjects had values of 155 and 120% respectively. Finally, 4 subjects had values close to 200% of bioavailability. These higher values of bioavailability were due to values of AUC0–t remarkably higher than the average. Interestingly, not all 4 subjects with higher systemic bioavailability showed a higher excretion. Two of them had a cumulative amount of excretion >40%, whilst the other 2 subjects had values closer to the average of 40%. After single injection of 100 mg, the same 4 subjects had individual values of cumulative excretion, which were not far from the mean value of 40%.

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3.5. Safety AEs occurred at a frequency of 45.5% (10/22 subjects). None of these AEs was serious or required specific treatment. The majority of reported AEs was judged in relationship with methylene blue intake and was considered of mild severity. The related AEs occurred at a frequency of 36% (8/22 subjects). The reported related AEs are summarised by system organ class and low level term in Table 4, where the frequency of subjects with AEs is presented. Overall, 3 subjects complained of alteration of transaminases, which returned to normal values at the end of the 15-day observation period. For one subject only, the increase of alanine aminotransferase was of grade 3 up to 6 days of the start of the AE. Afterwards, the severity decreased to grade 1. The elevation of aspartate aminotransferare was of grade 2 at the beginning and then it decreased to grade 1 in 2 days. Dysuria and gastrointestinal disorders occurred to a frequency of 13.6% (3/22 subjects). Dysuria was of grade 2 for one subject and of grade 1 for the others. All other AEs were of grade 1 according to the Common Terminology Criteria for Adverse Events, version 0.4. 4. Discussion For the investigation of the safety and tolerability of methylene blue formulated as MMX® modified release tablets, single oral doses of 200 and 400 mg were administered for the first time to healthy male and post-menopausal female subjects after the intake of a 2 L bowel cleansing preparation. The bowel cleansing preparation was administered with the aim of reproducing conditions similar to those of the new product proposed use. Ten (10) subjects were exposed to 200 mg of methylene blue MMX tablets and to additional 100 mg of methylene blue 1% solution according to a randomised cross-over design. Other 12 subjects were exposed to 400 mg of methylene blue tablets to check the linearity and the dose-proportionality of methylene blue kinetics by increasing the dose.

Table 4 Display of adverse events. Number of subjects reporting treatment-related adverse events by system organ class and low level term. SOC and PT/LLT

200 mg of oral methylene blue (N = 10)

100 mg of iv methylene blue (N = 10)

400 mg of oral methylene blue (N = 12)

Overall (N = 22)

Investigations ALT increased AST increased γ-GT increased Gastrointestinal disorders Nausea Vomiting Musculoskeletal and connective tissue disorders Back pain Renal and urinary disorders Dysuria Nervous system disorders Dizziness General disorders and administration site conditions Asthenia Subjects with at least one related AE

1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 1

0 0 0 0 1 0 1 0 0 0 0 1 1 0 0 2

2 2 0 1 2 2 1 1 1 3 3 1 1 1 1 6

3 (13.6%) 3 (13.6%) 1 (4.5%) 1 (4.5%) 3 (13.6%) 2 (9.1%) 2 (9.1%) 1 (4.5%) 1 (4.5%) 3 (13.6%) 3 (13.6%) 2 (9.1%) 2 (9.1%) 1 (4.5%) 1 (4.5%) 8 (36.4%)

(10.0%) (10.0%) (10.0%) (0) (0) (0) (0) (0) (0) (0) (0) (0) (0) (0) (0) (10.0%)

(0) (0) (0) (0) (10.0%) (0) (10.0%) (0) (0) (0) (0) (10.0%) (10.0%) (0) (0) (20.0%)

Frequencies of events reported as SOCs and number of subjects with at least one related AE are in bold characters.

(16.7%) (16.7%) (0) (8.3%) (16.7%) (16.7%) (8.3%) (8.3%) (8.3%) (25.0%) (25.0%) (8.3%) (8.3%) (8.3%) (8.3%) (50.0%)

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After intake of methylene blue throughout the study, 10/ 22 subjects lamented AEs. Related AEs occurred to 8/22 subjects. None of these AEs was serious or required specific treatment. Overall, 3 subjects complained of a mild and transient alteration of transaminases. Dysuria, back pain and gastrointestinal disorders occurred to a frequency of 3/22 each. All AEs resolved spontaneously. In particular, the hepatic enzyme levels increasing after dosing in 3 subjects returned to the individual baseline after the end of the study. The longest follow-up was of 15 days. Methylene blue administered orally as MMX tablets at the ascending doses of 200 and 400 mg was absorbed from the colonic epithelium and became available systemically between 5 and 12 h post-dose. This peculiar colonic release of methylene blue from the MMX® modified release tablets is consistent with the results of other active substances formulated with the MMX technology and previously studied [1,2]. The time of drug appearance in the blood is consistent with a mainly colonic absorption. According to the cited studies of MMX formulations, 4 to 5 h is the typical transit time through the small intestine, generally independent from the product unit size and the subjective conditions, when the drug is administered in fast conditions. A comparison of the mean AUC values after single oral dose of 400 mg and after single iv injection of 100 mg shows that the extent of absorption increased with the dose. On average, AUC after single oral dose of 400 mg was nearly 4-fold higher than AUC after injection of 100 mg. The mean AUC value after single dose of 200 mg as obtained in the 1st study stage was nearly 2.7 times higher than after single iv injection. It is interesting to stress that, if the mean AUC after single dose of 200 mg is re-calculated without the subjects who had an absolute bioavailability close to 200% in comparison with iv injection, the ratio to the mean AUC after single iv injection was on average 0.9. On the contrary, the rate of absorption did not vary with the dose allegedly. The mean Cmax after both 200 and 400 mg did not show any increase. After both oral doses, the mean Cmax was lower than after iv injection of 100 mg. However, 2 out of 12 subjects receiving the oral dose of 400 mg had AUC values which were from 4.4 to 16.5 times lower than the other subjects’ values. On average, half life of methylene blue was somewhat shorter on average after the oral dose of 400 mg than after the doses of 100 and 200 mg. The 2 subjects with lower peak concentrations also had a half life of less than 6 h. Systemic exposure as measured and calculated in the present study is consistent with the AUC mean values found by Walter-Sack [9], where AUC0–t of methylene blue (4.89 ± 2.89 μg/mL × h) after single iv injection of 50 mg was approximately half the AUC0–t obtained in the present study with 100 mg (11.86 ± 2.80 μg/mL× h). Elimination half life and AUC values found by Walter-Sack after the administration of 500 mg oral solution were not far from those of the oral dose of 400 mg. Absolute bioavailability of methylene blue when administered as 200 mg of modified release tablets was far higher than 100%. Such a high percentage was mostly due to 4 out of 10 subjects who had individual bioavailability values close to 200%. On the other hand, if the bioavailability is roughly calculated as the ratio of AUC0–∞ values after 400 mg oral vs. 100 mg iv the obtained figure is about 78%.

This percentage is consistent with the absolute bioavailability reported by Walter-Sack, i.e. 84.0 ± 16.7%, obtained by calculating the ratio of AUC0–∞ values after single oral dose of 500 mg and single iv injection of 50 mg. The apparent distribution volume of methylene blue after single iv injection of 100 mg was between 160 and 580 L. Indeed, mean volume values >100 L are rarely found and such very high values are among the maximal known values of distribution volume in pharmacology. Such high volumes indicate not only that methylene blue is deeply distributed in both highly and poorly perfused tissues and in both the interstitial and the intracellular fluids, but also that the compound binds some intracellular structure with a certain affinity. Trying to explain these unusual data it should be considered that interesting analogous cases are represented by other few compounds of pharmacological human use, which also exhibit bioavailability values varying with the dose. The existence of any saturable mechanism of absorption may not be excluded as a possible explanation of the lack of direct proportionality between the two tested doses and their bioavailability at the present state of the investigation. However, the bioavailability roughly calculated as the ratio of AUC0–∞ values after 400 mg oral vs. 100 mg iv accounting for a 78% does not seem to support the hypothesis of the saturation of an active transport mechanism. Should the hypothesis of the saturation be confirmed by further investigations, it would add a safety factor to the use of the new product even at higher doses. The results of the study demonstrated that methylene blue is released in the colonic tract of the bowel, becomes systemically bioavailable to a high extent and is well tolerated by healthy male and female subjects when given as MMX® tablets after the intake of 2 L of the bowel cleansing preparation. The proposed dose regimen for the dye tablets is expected to have no impact on the patients' bowel cleansing and preparation for the endoscopy. According to usual preparation procedure, the patients scheduled for endoscopies undergo the bowel cleansing in the evening before the day of endoscopy and follow a single-dose regimen for the intake of the bowel cleansing solution [16,17]. According to the dose regimen applied in the present study, the intake of methylene blue tablets was scheduled at the end of the intake of the bowel cleansing solution. This means that the study subjects drank the laxative in 2 h and took Methylene blue MMX® tablets 30 min after the end of the intake of the laxative. Therefore, the modified release tablet transited the gut after the laxative solution. The colonic release of methylene blue did not seem affected by previous transit of the laxative, since the appearance of the dye in the blood stream observed between 5 and 12 h after the dose was consistent with the results of previously investigated MMX formulations [1,2]. Hence, the uptake of methylene blue from epithelial cells should be improved in a colon cleansed from mucus and faecal debris [18]. Furthermore, the observed absorption profile suggests that methylene blue is delivered in the colon in a time frame suitable for the proposed use of the tablets. Study results gave preliminary knowledge about the safety, tolerability and pharmacokinetics of methylene blue in healthy subjects after intake of Methylene blue MMX® tablets concomitantly to the intake of the bowel cleansing preparation. The present study results may be used for the next

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clinical studies of methylene blue MMX tablets, which foresee the investigation of the colonic mucosa staining and the effective gain in intestinal lesion detection rate. Acknowledgements Cosmo Technologies Ltd., Ireland gave the financial support to the project. The relationships between the Sponsor, Cosmo Technologies, and Cross Research S.A. and ABL Laboratories were regulated by financial agreements. The Sponsor reviewed and approved the study design, was informed about the collection of data, reviewed and approved the analysis and the interpretation of data, reviewed and approved the manuscript for publication. MMR reviewed and approved the design of the study, was responsible for the clinical activities and collected the data, VJ was responsible for the bioanalysis, AR and SD proposed the study design and reviewed the draft manuscript, AFDD wrote the clinical trial report and drafted the manuscript. All authors read and approved the manuscript. References [1] Brunner M, Assandri R, Kletter K, Tschurlovits M, Corrado ME, Villa R, et al. Gastrointestinal transit and 5-ASA release from a new mesalazine extended-release formulation. Aliment Pharmacol Ther 2003;17(3): 395–402. [2] Brunner M, Ziegler S, Di Stefano AFD, Dehghanyar P, Kletter K, Tschurlovits M, et al. Gastrointestinal transit, release and plasma pharmacokinetics of a new oral budesonide formulation. Br J Clin Pharmacol 2006;61(1):31–8. [3] Canto MI, Setrakian S, Willis JE, Chak A, Petras RE, Sivak MV. Methylene blue staining of dysplastic and nondysplastic Barrett's esophagus: an in vivo and ex vivo study. Endoscopy 2001;33(5):391–400. [4] Bruno MJ. Magnification endoscopy, high resolution endoscopy, and chromoscopy; towards a better optical diagnosis. Gut 2003;52(Suppl. IV): iv7–iv11.

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