Original Article
Acetyl-l-carnitine versus placebo for migraine prophylaxis: A randomized, triple-blind, crossover study
Cephalalgia 2015, Vol. 35(11) 987–995 ! International Headache Society 2015 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0333102414566817 cep.sagepub.com
Knut Hagen1,2, Eiliv Brenner1, Mattias Linde1,2, Gøril Bruvik Gravdahl2, Erling Andreas Tronvik1,2, Morten Engstrøm1,2, Ursula Sonnewald1, Grethe Helde1, Lars Jacob Stovner1,2 and Trond Sand1,2 Abstract Background: Preventive medication is indicated for many migraine patients, but is used in relatively few. The aim of the present study was to evaluate the efficacy of acetyl-l-carnitine as a prophylactic drug in migraine patients. Methods: A single-center, randomized, triple-blind, placebo-controlled, crossover study was carried out. Men and women, age 18–65 years, with episodic migraine but otherwise healthy, were recruited mostly through advertisements. After a four-week run-in-phase, 72 participants were randomized to receive either placebo or 3 g acetyl-l-carnitine for 12 weeks. After a four-week washout, treatment was switched. The primary outcome was days with moderate or severe headache per four weeks. Secondary outcomes were days with headache, hours with headache, proportion of responders (>50% reduction in migraine days from baseline) and adverse events. Results: In the complete case analyses, no statistically significant differences were found between acetyl-l-carnitine and placebo in severe or moderate headache days per month (3.0 versus 3.1, p ¼ 0.80), headache days per month (5.1 versus 5.2, p ¼ 0.73) or for the other secondary outcome measures. Conclusion: In this triple-blind crossover study no differences were found in headache outcomes between acetyl-lcarnitine and placebo. Our results do not provide evidence of benefit for efficacy of acetyl-l-carnitine as prophylactic treatment for migraine. Trial registration: EUDRACT (2012-001624-36), ClinicalTrials.gov (NCT01695317). Keywords Migraine, triple-blind, crossover, placebo-controlled, single-center Date received: 16 October 2014; revised: 25 November 2014; accepted: 6 December 2014
Introduction Migraine is ranked seventh highest among specific causes of disability globally (1). Prophylactic medication is indicated for many migraine patients, but is used in relatively few, e.g. because of limited therapeutic effects, side effects or contraindications of existing drugs (2,3). There is thus an urgent need for new prophylactic drugs with few side effects that can be used by most migraine sufferers (3). One substance that may fulfill these requirements is acetyl-l-carnitine, which is available as a dietary supplement, and which also occurs naturally in plants and animals. Acetyl-l-carnitine stimulates lipid-mediated energy production in the central nervous system (4),
and has an impact on several neurotransmitter pathways, including serotonergic, noradrenergic and
1
Department of Neuroscience, Norwegian University of Science and Technology, Norway 2 Norwegian Advisory Unit on Headache, St. Olav’s University Hospital, Norway Corresponding author: Knut Hagen, Norwegian Advisory Unit on Headache, Department of Neuroscience, Norwegian University of Science and Technology and St. Olav’s Hospital, N-7491 Trondheim, Norway. Email: [email protected]
988 dopaminergic pathways important in migraine pathophysiology (5). Over the last few decades, acetyl-l-carnitine has been evaluated in placebo-controlled trials for a large number of diseases (6–12), including neurological disorders such as Alzheimer’s dementia and amyotrophic lateral sclerosis (13–15). Several clinical studies have indicated analgesic effects of acetyl-l-carnitine in different types of painful neuropathies (16). Interestingly, acetyl-l-carnitine has been shown to be effective as a migraine prophylactic in one single-blind, randomized, parallel trial in children. Unfortunately, this study has been published only in an abstract (17). The aim of the present study was to evaluate the efficacy of acetyl-l-carnitine as a prophylactic drug in adult migraine patients.
Materials and methods The study was designed as a placebo-controlled, tripleblind, crossover trial, with a four-week run-in-phase (baseline period), and two 12-week treatment periods with a four-week washout period in between. It was conducted at the neurological outpatient clinic of St. Olav’s University Hospital between April 2013 and July 2014. Participants were recruited either from patients referred to the clinic or among those who contacted the study nurse in response to advertisements in newspapers or on websites. The study followed the International Headache Society (IHS) guidelines for trials on prophylactic medication in migraine (18,19). The primary efficacy variable was number of days with moderate or severe headache, lasting 4 hours or being treated with the patient’s usual medication (‘‘migraine days’’) per four weeks (19). Secondary measures were: days with headache; hours with headache; headache intensity on days with headache; doses of analgesics; doses of triptans; number of days of sick leave; number of responders ( 50% decrease in migraine days compared with baseline) (18). Adverse event (AE) measures were the occurrence of AEs (yes/no), the level of discomfort due to AEs (scale 1–5), and the number of AEs occurring during treatment periods. Inclusion criteria were: age 18–65 years; signed informed consent; migraine with or without aura (18) or chronic migraine (19); 2 migraine attacks per month during the last three months before inclusion, and 2 migraine attacks during the four-week baseline period documented in the diary, debut of migraine 1 year prior to inclusion, and before the age of 50; body mass index (BMI) between 18 kg/m2 and 35 kg/m2, and no medication-overuse headache during the last three months, defined as headache >14 days/month combined with overuse of simple analgesics in monotherapy
Cephalalgia 35(11) >14 days/month or triptans or combined medications 10 days/month. Exclusion criteria were: headache other than migraine not distinguishable from the migraine attacks; chronic tension-type headache or other headache occurring on 15 days/month; pregnancy, nursing or no use of contraceptives in fertile women; history of angioneurotic edema; diabetes or previous or present history of asthma or vascular disease, including arterial claudication; decreased hepatic or renal function; psychiatric illness and/or Hospital Anxiety and Depression Scale (HADS) anxiety score 11 or HADS depression score 11; use of daily migraine prophylactics within four weeks prior to start of study; use of 3 prophylactic drugs against migraine during the last five years; consistent failure to respond to any acute migraine medication; alcohol or illicit drug dependence; previous use of acetyl-l-carnitine; BMI 35 kg/m2; and individuals requiring detoxification from acute medication. During a screening examination by a neurologist, a thorough history was taken to ascertain compliance with inclusion and exclusion criteria. All participants received a neurological examination that also included measurements of height, weight, blood pressure (BP) and pulse. Throughout the whole study, patients were instructed to keep a headache diary recording relevant attack variables, as well as AEs or other new healthrelated conditions. At inclusion and all follow-up visits, the patients were asked to fill in the HADS questionnaire. The four-week baseline period without prophylactic medication before randomization was used to ascertain whether patients had 2 attacks per month. If not, the baseline period was extended by four weeks, and if there were 4 attacks during the whole eight-week period, the patient was included. After the baseline period, there was a further visit to evaluate whether the patient could be included, before randomization and dispensing of the study drug for the first period. Seventy-two patients were randomized in blocks of 18 to one of two possible treatment sequences. The study drug consisted of tablets of either acetyl-l-carnitine 500 mg or placebo. Because the optimal daily doses for investigating acetyl-l-carnitine for adult migraine patients were unknown, the selection of daily dose in the present study was based on a review of doubleblind, randomized trials evaluating acetyl-l-carnitine versus placebo in the treatment of mild cognitive impairment and mild Alzheimer’s disease (varied between 1.5 g and 3 g/day) (20). In week 1, the patient took three tablets each day (1.5 g), and during weeks 2–12, six tablets (3 g). The active drug and placebo were prepared by an external producer (Kragerø Tablettproduksjon AS). The placebo tablets (500 mg
989
Hagen et al. Prerandomization
Double-blind phase
Treatment period 1
Baseline
Wash-out
Telephone by nurse
4 (or 8) weeks
Screening visit by doctor
Telephone by nurse
12 weeks
4 weeks Evaluation by doctor
Randomization visit by doctor
Treatment period 2
Telephone by nurse
12 weeks
Final evaluation by doctor
Figure 1. Study design.
dextrose and citric acid) were made with a similarly acidic taste to that of active drug and were packaged in identical bottles in order to ensure a blinded design. After two weeks of each treatment period, all patients were contacted by telephone by the study nurse to check for AEs and compliance with the protocol (Figure 1). If there were AEs after increasing the dose, the dose could be reduced to three tablets per day for the rest of the period. A few days after finishing the first treatment period of 12 weeks, the patients visited the doctor for evaluation (Figure 1). Adherence was checked again, and study drugs for the next period were also handed out. Approximately one week after the second treatment period, there was a final visit (Figure 1). AEs were recorded in the headache diary (free text, one line for each day), and enquired about during telephone interviews and visits using an open-ended question about side effects and other health-related complaints. Adherence (completion per protocol; PP) was defined as having taken the tablets 8 weeks in both periods, and was measured using self-reports and pill counts. For individuals to be included in the complete case (CC) analyses, drugs had to be used 4 weeks in each period. The remaining participants were defined as dropouts. Intention to treat (ITT) analyses (not specified in the protocol) were also performed. AEs were reported in all patients who had taken at least one tablet.
The study was approved by the Regional Committee for Ethics in Medical Research (REK-Midt) (from which the full trial protocol can be accessed on request: [email protected]), the Norwegian Data Inspectorate, and the Norwegian Medicines Agency.
Randomization, blinding and data handling Participants fulfilling criteria for randomization were consecutively given a randomization number (1–72) assigning them to one of the two treatment sequences (Figure 2) according to a computer-generated list, premade by the company producing the drugs, but unknown to participants, clinicians and the statistician. Randomization numbers were preprinted on study medication labels, and information about the medication in each period for each participant was placed in a sealed envelope. Hence, in the event of serious AEs (SAEs), it was possible to unblind. The envelope was kept in a limited-access area. Data entry was performed by the study nurse. After completion of the study, the data file, together with the unopened envelopes containing randomization codes, were handed over to personnel at the Unit for Applied Clinical Research at the Norwegian University of Science and Technology who decoded the data and returned the file with each treatment type having a code (D or S). A predetermined statistical protocol had been written for the analysis of the
990
Cephalalgia 35(11)
220 Assessed for eligibility by nurse 135 Cause for exclusion or not fulfilling inclusion criteria 85 Screened by doctor 4 Did not fulfill inclusion criteria 81 Signed consent and entered baseline period 1 Withdrew consent 8 Had too few attacks
72 Randomised to treatment
Dropouts
Dropouts
Period 1 (12 weeks)
PLACEBO
1
ALCAR
2
Period 2 (12 weeks)
ALCAR
1
PLACEBO
4
72 ITT-completers
36
36
64 CC-completers
34
30
61 PP-completers
33
28
Figure 2. Flow of participants through the study (figures refer to number of patients). ALCAR: acetyl-l-carnitine; ITT: intent-to-treat; CC: complete course; PP: per protocol.
primary and secondary efficacy variables, and the statistician (TS) performed analysis of this file without knowing the treatment type. This was not revealed until final tables with coded efficacy data had been completed (triple-blind study). A few data entry errors were detected during the blinded analysis, and these were corrected before the final unblinding. The study was monitored by personnel at the Unit for Applied Clinical Research with experience and formal training in monitoring clinical trials.
Statistical methods The predetermined hypotheses were: H1: Acetyl-lcarnitine is better than placebo (superiority analysis). H2: Placebo has fewer AEs than acetyl-l-carnitine (superiority analysis). A power analysis before study start indicated that 60 evaluable patients would give >80% power to detect a difference between acetyl-l-carnitine and placebo of 0.5 SD (approximately 1.3–1.7 headache days/month based on our previous data) for the main efficacy variable
(3,21). Expecting a dropout rate of 15%–20%, 72 patients would have to be included. All statistical tests were between treatment periods, and did not include baseline data (except for the analyses of responder rate). In accordance with the predetermined statistical protocol, H1 was tested with Wilcoxon’s paired signed rank test in the CC group. A secondary analysis was performed in the PP group. In order to perform the ITT analyses for the main outcome, cases with no data or insufficient data for period 2 were also imputed with period 1 data, if available, or baseline data. A sensitivity analysis for number of responders, replacing missing observations with either ‘‘non-responder’’ or ‘‘responder’’ status, was also performed. An AE reported several times during one treatment period was counted only once. The differences in responder rate and AE proportions were tested with a standardized normal deviate. Analysis of period effects was not included in the protocol since a long washout was used to minimize any crossover effects and the study was not powered
991
Hagen et al. to provide precise estimates of periodicity. However, a post-hoc period analysis was performed with repeated-measures analysis of variance (ANOVA) for imputed ITT and CC data sets, while mixedmodel ANOVA was used for complete non-imputed data. SYSTAT v11 (Systat Software Inc, Chicago, IL, USA) was used with two-sided level of significance
Acetyl-l-carnitine versus placebo for migraine prophylaxis: A randomized, triple-blind, crossover study
Cephalalgia 2015, Vol. 35(11) 987–995 ! International Headache Society 2015 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0333102414566817 cep.sagepub.com
Knut Hagen1,2, Eiliv Brenner1, Mattias Linde1,2, Gøril Bruvik Gravdahl2, Erling Andreas Tronvik1,2, Morten Engstrøm1,2, Ursula Sonnewald1, Grethe Helde1, Lars Jacob Stovner1,2 and Trond Sand1,2 Abstract Background: Preventive medication is indicated for many migraine patients, but is used in relatively few. The aim of the present study was to evaluate the efficacy of acetyl-l-carnitine as a prophylactic drug in migraine patients. Methods: A single-center, randomized, triple-blind, placebo-controlled, crossover study was carried out. Men and women, age 18–65 years, with episodic migraine but otherwise healthy, were recruited mostly through advertisements. After a four-week run-in-phase, 72 participants were randomized to receive either placebo or 3 g acetyl-l-carnitine for 12 weeks. After a four-week washout, treatment was switched. The primary outcome was days with moderate or severe headache per four weeks. Secondary outcomes were days with headache, hours with headache, proportion of responders (>50% reduction in migraine days from baseline) and adverse events. Results: In the complete case analyses, no statistically significant differences were found between acetyl-l-carnitine and placebo in severe or moderate headache days per month (3.0 versus 3.1, p ¼ 0.80), headache days per month (5.1 versus 5.2, p ¼ 0.73) or for the other secondary outcome measures. Conclusion: In this triple-blind crossover study no differences were found in headache outcomes between acetyl-lcarnitine and placebo. Our results do not provide evidence of benefit for efficacy of acetyl-l-carnitine as prophylactic treatment for migraine. Trial registration: EUDRACT (2012-001624-36), ClinicalTrials.gov (NCT01695317). Keywords Migraine, triple-blind, crossover, placebo-controlled, single-center Date received: 16 October 2014; revised: 25 November 2014; accepted: 6 December 2014
Introduction Migraine is ranked seventh highest among specific causes of disability globally (1). Prophylactic medication is indicated for many migraine patients, but is used in relatively few, e.g. because of limited therapeutic effects, side effects or contraindications of existing drugs (2,3). There is thus an urgent need for new prophylactic drugs with few side effects that can be used by most migraine sufferers (3). One substance that may fulfill these requirements is acetyl-l-carnitine, which is available as a dietary supplement, and which also occurs naturally in plants and animals. Acetyl-l-carnitine stimulates lipid-mediated energy production in the central nervous system (4),
and has an impact on several neurotransmitter pathways, including serotonergic, noradrenergic and
1
Department of Neuroscience, Norwegian University of Science and Technology, Norway 2 Norwegian Advisory Unit on Headache, St. Olav’s University Hospital, Norway Corresponding author: Knut Hagen, Norwegian Advisory Unit on Headache, Department of Neuroscience, Norwegian University of Science and Technology and St. Olav’s Hospital, N-7491 Trondheim, Norway. Email: [email protected]
988 dopaminergic pathways important in migraine pathophysiology (5). Over the last few decades, acetyl-l-carnitine has been evaluated in placebo-controlled trials for a large number of diseases (6–12), including neurological disorders such as Alzheimer’s dementia and amyotrophic lateral sclerosis (13–15). Several clinical studies have indicated analgesic effects of acetyl-l-carnitine in different types of painful neuropathies (16). Interestingly, acetyl-l-carnitine has been shown to be effective as a migraine prophylactic in one single-blind, randomized, parallel trial in children. Unfortunately, this study has been published only in an abstract (17). The aim of the present study was to evaluate the efficacy of acetyl-l-carnitine as a prophylactic drug in adult migraine patients.
Materials and methods The study was designed as a placebo-controlled, tripleblind, crossover trial, with a four-week run-in-phase (baseline period), and two 12-week treatment periods with a four-week washout period in between. It was conducted at the neurological outpatient clinic of St. Olav’s University Hospital between April 2013 and July 2014. Participants were recruited either from patients referred to the clinic or among those who contacted the study nurse in response to advertisements in newspapers or on websites. The study followed the International Headache Society (IHS) guidelines for trials on prophylactic medication in migraine (18,19). The primary efficacy variable was number of days with moderate or severe headache, lasting 4 hours or being treated with the patient’s usual medication (‘‘migraine days’’) per four weeks (19). Secondary measures were: days with headache; hours with headache; headache intensity on days with headache; doses of analgesics; doses of triptans; number of days of sick leave; number of responders ( 50% decrease in migraine days compared with baseline) (18). Adverse event (AE) measures were the occurrence of AEs (yes/no), the level of discomfort due to AEs (scale 1–5), and the number of AEs occurring during treatment periods. Inclusion criteria were: age 18–65 years; signed informed consent; migraine with or without aura (18) or chronic migraine (19); 2 migraine attacks per month during the last three months before inclusion, and 2 migraine attacks during the four-week baseline period documented in the diary, debut of migraine 1 year prior to inclusion, and before the age of 50; body mass index (BMI) between 18 kg/m2 and 35 kg/m2, and no medication-overuse headache during the last three months, defined as headache >14 days/month combined with overuse of simple analgesics in monotherapy
Cephalalgia 35(11) >14 days/month or triptans or combined medications 10 days/month. Exclusion criteria were: headache other than migraine not distinguishable from the migraine attacks; chronic tension-type headache or other headache occurring on 15 days/month; pregnancy, nursing or no use of contraceptives in fertile women; history of angioneurotic edema; diabetes or previous or present history of asthma or vascular disease, including arterial claudication; decreased hepatic or renal function; psychiatric illness and/or Hospital Anxiety and Depression Scale (HADS) anxiety score 11 or HADS depression score 11; use of daily migraine prophylactics within four weeks prior to start of study; use of 3 prophylactic drugs against migraine during the last five years; consistent failure to respond to any acute migraine medication; alcohol or illicit drug dependence; previous use of acetyl-l-carnitine; BMI 35 kg/m2; and individuals requiring detoxification from acute medication. During a screening examination by a neurologist, a thorough history was taken to ascertain compliance with inclusion and exclusion criteria. All participants received a neurological examination that also included measurements of height, weight, blood pressure (BP) and pulse. Throughout the whole study, patients were instructed to keep a headache diary recording relevant attack variables, as well as AEs or other new healthrelated conditions. At inclusion and all follow-up visits, the patients were asked to fill in the HADS questionnaire. The four-week baseline period without prophylactic medication before randomization was used to ascertain whether patients had 2 attacks per month. If not, the baseline period was extended by four weeks, and if there were 4 attacks during the whole eight-week period, the patient was included. After the baseline period, there was a further visit to evaluate whether the patient could be included, before randomization and dispensing of the study drug for the first period. Seventy-two patients were randomized in blocks of 18 to one of two possible treatment sequences. The study drug consisted of tablets of either acetyl-l-carnitine 500 mg or placebo. Because the optimal daily doses for investigating acetyl-l-carnitine for adult migraine patients were unknown, the selection of daily dose in the present study was based on a review of doubleblind, randomized trials evaluating acetyl-l-carnitine versus placebo in the treatment of mild cognitive impairment and mild Alzheimer’s disease (varied between 1.5 g and 3 g/day) (20). In week 1, the patient took three tablets each day (1.5 g), and during weeks 2–12, six tablets (3 g). The active drug and placebo were prepared by an external producer (Kragerø Tablettproduksjon AS). The placebo tablets (500 mg
989
Hagen et al. Prerandomization
Double-blind phase
Treatment period 1
Baseline
Wash-out
Telephone by nurse
4 (or 8) weeks
Screening visit by doctor
Telephone by nurse
12 weeks
4 weeks Evaluation by doctor
Randomization visit by doctor
Treatment period 2
Telephone by nurse
12 weeks
Final evaluation by doctor
Figure 1. Study design.
dextrose and citric acid) were made with a similarly acidic taste to that of active drug and were packaged in identical bottles in order to ensure a blinded design. After two weeks of each treatment period, all patients were contacted by telephone by the study nurse to check for AEs and compliance with the protocol (Figure 1). If there were AEs after increasing the dose, the dose could be reduced to three tablets per day for the rest of the period. A few days after finishing the first treatment period of 12 weeks, the patients visited the doctor for evaluation (Figure 1). Adherence was checked again, and study drugs for the next period were also handed out. Approximately one week after the second treatment period, there was a final visit (Figure 1). AEs were recorded in the headache diary (free text, one line for each day), and enquired about during telephone interviews and visits using an open-ended question about side effects and other health-related complaints. Adherence (completion per protocol; PP) was defined as having taken the tablets 8 weeks in both periods, and was measured using self-reports and pill counts. For individuals to be included in the complete case (CC) analyses, drugs had to be used 4 weeks in each period. The remaining participants were defined as dropouts. Intention to treat (ITT) analyses (not specified in the protocol) were also performed. AEs were reported in all patients who had taken at least one tablet.
The study was approved by the Regional Committee for Ethics in Medical Research (REK-Midt) (from which the full trial protocol can be accessed on request: [email protected]), the Norwegian Data Inspectorate, and the Norwegian Medicines Agency.
Randomization, blinding and data handling Participants fulfilling criteria for randomization were consecutively given a randomization number (1–72) assigning them to one of the two treatment sequences (Figure 2) according to a computer-generated list, premade by the company producing the drugs, but unknown to participants, clinicians and the statistician. Randomization numbers were preprinted on study medication labels, and information about the medication in each period for each participant was placed in a sealed envelope. Hence, in the event of serious AEs (SAEs), it was possible to unblind. The envelope was kept in a limited-access area. Data entry was performed by the study nurse. After completion of the study, the data file, together with the unopened envelopes containing randomization codes, were handed over to personnel at the Unit for Applied Clinical Research at the Norwegian University of Science and Technology who decoded the data and returned the file with each treatment type having a code (D or S). A predetermined statistical protocol had been written for the analysis of the
990
Cephalalgia 35(11)
220 Assessed for eligibility by nurse 135 Cause for exclusion or not fulfilling inclusion criteria 85 Screened by doctor 4 Did not fulfill inclusion criteria 81 Signed consent and entered baseline period 1 Withdrew consent 8 Had too few attacks
72 Randomised to treatment
Dropouts
Dropouts
Period 1 (12 weeks)
PLACEBO
1
ALCAR
2
Period 2 (12 weeks)
ALCAR
1
PLACEBO
4
72 ITT-completers
36
36
64 CC-completers
34
30
61 PP-completers
33
28
Figure 2. Flow of participants through the study (figures refer to number of patients). ALCAR: acetyl-l-carnitine; ITT: intent-to-treat; CC: complete course; PP: per protocol.
primary and secondary efficacy variables, and the statistician (TS) performed analysis of this file without knowing the treatment type. This was not revealed until final tables with coded efficacy data had been completed (triple-blind study). A few data entry errors were detected during the blinded analysis, and these were corrected before the final unblinding. The study was monitored by personnel at the Unit for Applied Clinical Research with experience and formal training in monitoring clinical trials.
Statistical methods The predetermined hypotheses were: H1: Acetyl-lcarnitine is better than placebo (superiority analysis). H2: Placebo has fewer AEs than acetyl-l-carnitine (superiority analysis). A power analysis before study start indicated that 60 evaluable patients would give >80% power to detect a difference between acetyl-l-carnitine and placebo of 0.5 SD (approximately 1.3–1.7 headache days/month based on our previous data) for the main efficacy variable
(3,21). Expecting a dropout rate of 15%–20%, 72 patients would have to be included. All statistical tests were between treatment periods, and did not include baseline data (except for the analyses of responder rate). In accordance with the predetermined statistical protocol, H1 was tested with Wilcoxon’s paired signed rank test in the CC group. A secondary analysis was performed in the PP group. In order to perform the ITT analyses for the main outcome, cases with no data or insufficient data for period 2 were also imputed with period 1 data, if available, or baseline data. A sensitivity analysis for number of responders, replacing missing observations with either ‘‘non-responder’’ or ‘‘responder’’ status, was also performed. An AE reported several times during one treatment period was counted only once. The differences in responder rate and AE proportions were tested with a standardized normal deviate. Analysis of period effects was not included in the protocol since a long washout was used to minimize any crossover effects and the study was not powered
991
Hagen et al. to provide precise estimates of periodicity. However, a post-hoc period analysis was performed with repeated-measures analysis of variance (ANOVA) for imputed ITT and CC data sets, while mixedmodel ANOVA was used for complete non-imputed data. SYSTAT v11 (Systat Software Inc, Chicago, IL, USA) was used with two-sided level of significance
