Crossover randomized controlled trial of Bumetanide to rescue an attack of exercise induced hand weakness in Hypokalaemic Periodic Paralysis

To assess if bumetanide can abort an acute attack of weakness in patients with HypoPP. This was a randomised, double-blind, cross-over, placebo-controlled phase II clinical trial. Focal attack of weakness was induced by isometric exercise of ADM followed by rest (McManis protocol). Participants had two study visits and received either placebo or 2mg bumetanide at attack onset (defined as 40% decrement in the abductor digiti minimi CMAP amplitude from peak). CMAP measurements assessed attack severity and duration. Nine participants completed both visits. CMAP percentage of peak amplitudes in the bumetanide (40.6%) versus placebo (34.9%) group at 1hr following treatment did not differ significantly (estimated effect difference 5.9% (95% CI: (-5.7%; 17.5%), p=0.27, primary outcome). CMAP amplitudes assessed by the area under the curve for early (0-2hr post-treatment) and late (2-4hr post-treatment) efficacy were not statistically different between bumetanide and placebo (early effect estimate 0.043, p=0.3; late effect estimate 0.085, p=0.1). Two participants recovered from the attack following bumetanide intake; none recovered following placebo. Bumetanide was well tolerated but not efficacious to rescue a focal attack in an immobilised hand in the majority of patients, although data supports further studies of this agent.


Highlights
• Patients with HypoPP experience disabling attacks of weakness • Animal models showed promising data in the use of bumetanide in HypoPP • Bumetanide was not efficacious in reversing an attack of hand weakness in HypoPP • This study supports further investigation of bumetanide as a therapeutic agent

ABSTRACT
To assess if bumetanide can abort an acute attack of weakness in patients with HypoPP.This was a randomised, double-blind, cross-over, placebo-controlled phase II clinical trial.Focal attack of weakness was induced by isometric exercise of ADM followed by rest (McManis protocol).Participants had two study visits and received either placebo or 2mg bumetanide at attack onset (defined as 40% decrement in the abductor digiti minimi CMAP amplitude from peak).CMAP measurements assessed attack severity and duration.Nine participants completed both visits.CMAP percentage of peak amplitudes in the bumetanide (40.6%) versus placebo (34.9%) group at 1hr following treatment did not differ significantly (estimated effect difference 5.9% (95% CI: (-5.7%; 17.5%), p=0.27, primary outcome).
CMAP amplitudes assessed by the area under the curve for early (0-2hr post-treatment) and late (2-4hr post-treatment) efficacy were not statistically different between bumetanide and placebo (early effect estimate 0.043, p=0.3; late effect estimate 0.085, p=0.1).Two participants recovered from the attack following bumetanide intake; none recovered following placebo.Bumetanide was well tolerated but not efficacious to rescue a focal attack in an immobilised hand in the majority of patients, although data supports further studies of this agent.

Introduction
Hypokalaemic periodic paralysis (HypoPP) is a neuromuscular disorder caused by mutations in the voltage-gated skeletal muscle channel genes CACNA1S and SCN4A.The genetic changes result in an aberrant inward leak current across the sarcolemma, which increases the susceptibility of the muscle membrane to sustained depolarisation at low serum potassium levels.Clinically this manifests as recurrent paralysis. 1The only pharmacological option to abort acute symptoms in HypoPP is potassium supplementation. 2 Experimental data in two mouse models of HypoPP (SCN4A R669H and CACNA1S R528H mutations) have indicated that the inhibiting effect of bumetanide on the Na-K-Cl cotransporter stabilised muscle fibre membranes by limiting the sarcoplasmic chloride ion concentration when exposed to low potassium levels.This not only prevented the loss of muscle force but also induced recovery of force during an established attack. 3,4 date there have been no clinical trials of bumetanide in patients with HypoPP.We have assessed for the first time if bumetanide can abort an episode of induced hand weakness in patients with HypoPP.

Standard protocol approvals, registrations, and patient consents.
The study protocol and related documents were approved by ethical and appropriate regulatory bodies (REC reference 14/LO/0774).Signed consent was obtained from all participants.This trial was registered at ClinicalTrials.gov(NCT02582476).An independent UCL Steering Committee monitored study safety.

Participants.
Genetically confirmed male/female HypoPP patients (18-64 years) with symptomatic disease (at least one attack in the last 12 months) were recruited. 5Supplement 1 describes inclusion and exclusion criteria.

Study design.
We conducted a randomised, double-blind, placebo-controlled phase II clinical trial with a cross-over design of bumetanide in patients with HypoPP at the Queen Square Centre for Neuromuscular Diseases in the UK (Figure 1).

Randomization and blinding.
Each participant received one capsule containing either placebo or 2mg bumetanide on two different occasions.Subjects were randomly assigned to either sequence B-P (treatment with bumetanide followed by placebo), or sequence P-B (placebo followed by bumetanide).The randomisation algorithm was defined using a computer-based pseudo-random numbers generator, and was defined and performed by an independent statistician from the UCL Statistical Science Department (FR).
Blocked randomisation, with allocation ratio 1:1, was used to ensure a balance to the treatment orders throughout the study.Participants and all investigators enrolling participants and assessing outcomes (RSS and DF) were blinded to group assignment.Over-encapsulated bumetanide and matching placebo were provided by the Royal Free Hospital, UK.

Intervention.
Study eligibility was assessed at a screening visit.Each participant then undertook two assessment visits 2-8 weeks apart following an identical protocol.Participants withheld carbonic anhydrase inhibitor medications for 72hrs prior to assessment visits as is standard for McManis protocol at our centre and restarted their routine treatment immediately after each visit.Anti-inflammatory medication was withheld for 72hrs prior to assessment visits to prevent renal toxicity.Other pharmacological treatments to include diuretics and oral potassium were allowed to continue if previously prescribed.The assigned treatment was taken by mouth at the onset of a focal attack, defined as 40% decrement in the abductor digit minimi (ADM) compound muscle action potential (CMAP) amplitude from the maximum response.
Participants completed a food and activity diary 24hrs prior to visit 1 (V1) and were encouraged to follow the same dietary intake and similar physical activity pattern during the 24hrs prior to visit 2 (V2) in order to ensure similar baseline conditions for both visits.
2.6.Procedures.Baseline assessments included urine pregnancy test, vital signs, MRC muscle strength score, serum potassium levels and ADM CMAP amplitude in both hands.A localised attack of weakness was induced by 5min of isometric ADM exercise followed by rest in the non-dominant hand as per McManis protocol. 6The exercised hand was then immobilised with a splint and the ADM CMAP amplitude was recorded every minute following the exercise until Investigational Medicinal Product (IMP) intake, every two minutes for the first hour after IMP intake and every 5-10min during the next three hours.In the dominant hand, the ADM CMAP amplitude was recorded every hour as a safety procedure to detect early stages of a generalised attack of weakness.Participants were encouraged to mobilise during study visits to minimise the risk of a major attack of paralysis.MRC muscle strength score was re-assessed at the study visit end.Serum potassium levels and vital signs were assessed regularly (Supplement 2).

Attack duration:
Onset of a focal attack was defined as 40% CMAP amplitude decrement compared to the peak CMAP amplitude during or after the exercise.Attack recovery was defined as 65% of peak CMAP amplitude.

Extended McManis.
If 40% CMAP decrement was not achieved within 50min after exertion, the exercise was repeated once extending it to 10min using the same protocol.

Outcome measures. The primary outcome measure was the focal attack severity one
hour after treatment intake measured as CMAP amplitude expressed as a percent of peak CMAP.Secondary outcome measures were a) focal attack duration measured as the time between treatment administration until CMAP returned to 65% of peak within four hours following IMP intake, b) the initial effect of treatment on severity of a focal attack measured as area under the curve (AUC) of the CMAP amplitude (in percent compared to peak) from treatment administration until two hours post-treatment, c) the late effect of treatment on severity of a focal attack measured as AUC of the CMAP amplitude (in percent compared to peak) from treatment administration during the third and the fourth hours post-treatment, and d) safety of 2mg bumetanide measured by changes in: vital signs, physical exam (MRC muscle strength score), potassium levels and self-reported adverse events. 5Adverse events recorded in a home diary were also assessed by phone call one week following each study visit.

Compliance.
The same study investigator (RSS) gave the IMP to all participants ensuring 100% compliance.

Statistics.
The sample size was calculated on the primary outcome of the CMAP amplitude one hour after treatment was administered.After one hour, the CMAP amplitude was expected to remain at a mean of 40% with placebo administration.An increase in CMAP amplitude of 15% with treatment was regarded as clinically important, so the study was powered to identify this difference.Based on clinical experience it was estimated that the standard deviation of the difference in CMAP amplitude between the two groups will be 15%.Using a 5% significance level and 90% power it was calculated that 11 patients were required for the study.To ensure an even number of patients in each randomisation arm, the number of patients to be enrolled into the study was 12 patients.It was not anticipated that there would be any study drop-outs.
We summarised baseline covariates using descriptive statistics.To analyse the primary outcome, the CMAP amplitude one hour after the administration of treatment relative to the peak value, a multilevel random effects model was fitted to the data to estimate the effect of the treatment, adjusting for time period and treatment group.
Using a separate model carryover from the first to the second period was examined by fitting the treatment by time interaction.In both cases, point estimates, 95% confidence intervals and p-values for the parameter associated with treatment assignment were obtained.A pvalue equal to 0.05 or lower was indicative of statistical significance.Initial and late treatment effects were analysed using the same regression model as per the primary outcome.
Isolated missing CMAP values were estimated as the mean of the previous and next value.
Focal attack duration was analysed descriptively, calculating means and SDs by treatment received.The nature of adverse events was summarized giving their frequencies by treatment received.Descriptive statistics for potassium levels, blood pressure and pulse were calculated, by treatment received, before and after McManis test.To evaluate mild serum potassium reduction a multilevel regression model for potassium levels following McManis test, controlling for their baseline values, was fitted to the data.As with the primary outcome analysis, the parameter associated with treatment assignment was the quantity of interest.

HypoPP?
2.13.Data Availability.Anonymized data and the study protocol are available to qualified investigators upon submission of an acceptable analysis plan.Requests for data access should be directed to the corresponding author.The data will be available immediately from publication of the manuscript for a minimum of 7 years.To gain access, data requestors will need to sign a data access agreement.

Results
The first patient was enrolled in February 2015.In May 2017 the study was terminated early due to slow recruitment and expired funding (Figure 2).Three female and seven male participants were recruited; the median age was 45 years (18-55) (Supplement 3).All participants had mutations in the CACNA1S gene.Three participants had taken 500mg or 250mg acetazolamide 12hr prior to both study visits to treat acute attacks of weakness (protocol deviation).

Primary outcome.
Statistical analysis showed the primary outcome to be normally distributed (data not shown).The mean CMAP percentage of peak amplitudes for bumetanide and placebo groups were 40.6% and 34.9%, respectively.The estimated effect difference for CMAP percentage of peak amplitude in the bumetanide group compared to the placebo group, adjusting for time period and treatment group, was 5.9% (95% CI: (-5.7%; 17.5%); pvalue: 0.27).

Secondary outcomes: attack duration. None of the participants recovered following
placebo intake, thus it was not possible to analyse this outcome.Two participants recovered from the attack of weakness following bumetanide intake: at 26 and 240min, one of them had also received potassium supplement for an attack of atypical weakness, which was later not thought to be secondary to periodic paralysis and was not associated with low potassium.

Initial and late treatment effect.
From the two regression models, coefficients for the effect of bumetanide, P-values and 95% confidence intervals are reported in Table 1.Mean CMAP amplitude for all participants during four hours following treatment intake is illustrated in Figure 3.

Safety of Treatment.
There was no severe adverse event following 2mg bumetanide intake.Table 2 illustrates adverse events according to IMP intake.None of the participants had symptomatic hypokalaemia following IMP intake.Rescue treatment, consistent of two tablets of oral potassium, was given on four occasions to three participants, mostly following an instant potassium level lower than 3.5 mEq/L as per study protocol to ensure safety (bumetanide n=2; placebo n=1: one participant received rescue treatment twice at 30 and 120min following placebo intake).One participant received oral potassium for management of muscle weakness, which improved after a few minutes (bumetanide n=1).Mild serum potassium reduction was seen following bumetanide intake as compared to placebo intake (coeff: -0.238); this difference was not statistically significant (p=0.18;95% CI=[-0.675 ;
Descriptive statistics for serum potassium levels measured at the laboratory before and after each study visit, blood pressure and pulse recorded before and following IMP intake and visual representation of the potassium levels over time, separately for each participant, is given in a supplementary file (Supplement 4).due to an unexpected pregnancy.The participant reported good compliance with oral contraception, and urine pregnancy tests were negative on screening visit and V1 but it is possible that the participant was at the early stages of her pregnancy when she received bumetanide.She delivered a healthy newborn (2976g) at 38 weeks.

Classification of evidence.
This proof-of-concept study provides evidence that oral intake of 2mg bumetanide was not efficacious to abort a focal attack of weakness at 60 minutes in an immobilised hand in HypoPP participants.

Discussion
This trial aimed to evaluate the efficacy of bumetanide as a potential new treatment option for acute attacks of weakness in HypoPP.Bumetanide is an established diuretic with few adverse events.Animal model studies provided sufficient evidence that bumetanide prevented and aborted acute attacks of weakness in two knock-in mouse models of HypoPP to warrant investigation in humans with the condition. 3,4 n this drug repurposing study we assessed firstly the efficacy of 2mg bumetanide in reducing severity and duration of a focal attack of weakness and secondly the safety in patients with HypoPP.
CMAP amplitudes between the bumetanide and placebo groups at 1hr (primary outcome), and the early and late effect of the drug assessed by the AUC (secondary outcomes) were not statistically significant.We could not perform statistical analysis of one of the secondary outcomes (reduction in the duration of a localised attack between both groups) as none of the participants recovered from an attack following placebo intake.Interestingly, however, two out of ten participants did recover after taking bumetanide.Our findings indicate that 2mg bumetanide was not efficacious to abort a focal attack at one hour in an immobilised hand in the majority of the study participants.
The dramatic benefit of bumetanide reported in mouse model studies was not replicated by this RCT.The discrepancy may be multifactorial.In the animal studies both a preventative as well as an abortive effect was demonstrated, but the most significant benefit was derived when the drug was used preventatively.Nevertheless, we assessed the abortive action of the drug in humans as in clinical practice the short half-life and the associated risk of hypokalaemia with chronic use would probably prohibit the use of bumetanide as a regular prophylactic agent.It is also likely that hypokalaemia induced attacks of weakness in the animal models differ from exercise induced focal paralysis with normal serum potassium.
During exercise chloride accumulates in muscle fibres facilitated by inhibition of the CLC1 channel due to acidosis.In the post-exercise recovery phase from acidosis this has a depolarising effect leading to paralysis in the presence of HypoPP mutations.Administering a Na-K-2Cl cotransporter inhibitor such as bumetanide after exercise may therefore be too late to influence membrane excitability as excessive chloride accumulation has already occurred. 7Another point to consider is the dose and duration of drug application.In vitro experiments designed to assess an abortive treatment strategy following a hypokalaemic challenge used a 30 min bath with a continuous exposure to bumetanide 3,4 which is very different to the relative brief period of time a peak serum dose is achieved in vivo due to the very short half-life of bumetanide.There has been no published in vivo study testing of an abortive treatment strategy for HypoPP.The only in vivo study of bumetanide in an R528Hm/m HypoPP mouse tested a single IV dose of 0.08mg/kg bumetanide, which prevented loss of muscle excitability following a continuous IV infusion of glucose with insulin. 4This is the equivalent of just over 5mg bumetanide for a 70kg person.As a first study of bumetanide in patients with HypoPP, we chose to test 2mg (equivalent to 80mg furosemide), a dose which was equivalent to some of the initial in vitro experiments and a level we felt would be easily tolerated in subjects naïve to this drug.It is possible that this dose was too low for attack recovery in an immobilised limb.
To minimise variability between study visits and between different participants a hand/forearm splint was used to avoid muscle mobilisation.Reasons for patients to decline participation were disease severity (e.g.too symptomatic to suspend acetazolamide treatment for 72hrs prior to each study visit), discomfort related to neurophysiology assessments and working commitments.We also acknowledge that patients in a specialist centre such as ours can be approached for multiple studies.As we are a national referral service many also live a significant distance from the hospital and work or family commitments can prevent participation.These difficulties should be considered when planning future clinical trials in patients with rare conditions.
Although efficacy could not be determined the trial did demonstrate that 2mg bumetanide was well tolerated by the study participants when serum potassium levels are within normal limits at the time of administration.No symptomatic hypokalaemia or generalised attacks of weakness were precipitated, and there were no serious adverse events.Asymptomatic hypokalaemia was seen following both bumetanide and placebo intake.A mild reduction in potassium levels was noted with bumetanide at the end of the visit, but the impact of this finding is unknown as the study was not powered for such an outcome.We would recommend further monitoring and the use of oral potassium supplementation with bumetanide in future studies, especially if the drug is administered in patients with likely concomitant low levels of serum potassium (e.g.: during a generalised attack of weakness).The McManis protocol used here as an objective outcome measure in a clinical trial for the first time was well tolerated by the enrolled participants.This protocol is utilised as a diagnostic test and is thought to have a high sensitivity in genetically confirmed HypoPP 8,9 .In five out of 19 visits a significant CMAP decrement did not occur with the standard protocol.Carbonic anhydrase inhibitors were discontinued before each visit but it is possible that the use of other regular HypoPP treatments (eg: amiloride, oral potassium) or non-compliance with the protocol in regards to treatment discontinuation was a factor.
Measurement of serum bicarbonate may have allowed assessment of acidosis level secondary to treatment as a confounder.The repeated test with a prolonged duration of isometric exercise (10min) produced a minimum of 40% decrement in all assessed participants, suggesting a prolonged exercise period may increase the test sensitivity.
Although this is a negative trial on the pre-specified outcome measures, there are some indications of the potential for bumetanide in this condition.Figure 3 of the mean CMAP amplitudes following placebo and bumetanide, shows separation, particularly in the late phase, during which there was a trend in the pre-specified secondary outcome measure of higher CMAP AUC in the treated group (p=0.1) and there was a statistically significant difference at two individual time points (200 and 240 minutes) in post-hoc analysis.It is also of note that two participants in the bumetanide group recovered from their attack of weakness (with CMAP amplitudes reaching more than 65% of peak) while none did in the placebo group although numbers overall are of course small.We believe further studies are warranted to assess whether 2mg bumetanide in combination with gentle movement particularly during a generalised attack of weakness may be sufficient to kick-start recovery and lead to faster symptom resolution.Future research may include the development of an n-of-1 trial to assess the efficacy of bumetanide in HypoPP patients with induced generalised attacks in a controlled environment (e.g.: an elective hospital admission).A dose-ranging study may provide further information on safety and efficacy, guiding the development of a multi-centre randomised controlled trial in a larger cohort in an outpatient setting.

Conclusions
In conclusion, 2mg bumetanide was not efficacious to rescue a focal attack in an immobilised hand in the majority of patients.The combination of prolonged limb immobilisation, a small study sample, abortive rather than preventive strategy and low dose in comparison to the animal experiments may have contributed to the negative results reported here, which is in contrast to the striking effect seen in animal models.This study does provide first evidence that 2mg bumetanide was well tolerated in a small population of normokalaemic HypoPP patients.Our data supports further studies of this agent in this disorder.

Declaration of interests
I certify that all the co-authors have seen and agreed with the contents of this manuscript, and none of the authors have any financial interest to disclose.This manuscript has not been submitted to any other journal.

3. 1 .
Neurophysiology: Extended McManis.In five out of 19 visits (26.3%) participants did not achieve 40% CMAP decrement within 50min of the standard McManis protocol involving five minutes of isometric exercise.All of those did show a decrement after repeating exercise for 10min (Extended McManis).40% CMAP decrement was seen following a median of 30.5 (range 12-49) and 28 (range 18-44) minutes following the standard and the extended McManis protocol respectively.

Figure 2 :
Figure 2: Diagram showing the flow of subjects through the trial.P-B: Allocated to Placebo

Figure 3 :
Figure 3: Mean CMAP values (presented as percentage of maximum during/after exercise)

events within 1 week of study visit
Prolonged immobilisation may have contributed to the lack of recovery in the majority of cases.Based on our clinical experience, patients with genetically confirmed HypoPP who have at least 40% CMAP amplitude decrement during a diagnostic McManis test do not show significant spontaneous improvement (e.g.: up to 65%) within 50min following exercise. 8This is the first time HypoPP patients were assessed for a prolonged time following the long exercise test (4hr) and data in the placebo group illustrates that little recovery takes place when physical activity is prevented.It is possible that a comparison of bumetanide versus placebo could have resulted in a different outcome if mobilisation had been permitted.