Leucine and ACE inhibitors as therapies for sarcopenia (LACE trial): study protocol for a randomised controlled trial

LACE is a multicentre, placebo-controlled, 2 × 2 factorial randomised controlled trial analysed as randomised. Participants will be randomised to receive either perindopril or placebo, plus leucine or placebo. The intervention and follow up will be for 1 year. Participants, clinical teams, outcomes assessors and trial analysts are masked to treatment allocation.

The study will recruit 440 community-dwelling participants aged 70 and over with sarcopenia according to the EWGSOP definition [4]. Inclusion and exclusion criteria are presented in Table 1, and the criteria for diagnosing sarcopenia in the trial are presented in Table 2.

Potential participants are recruited from both primary care and secondary care clinics, including Medicine for the Elderly clinics, falls clinics and general medicine clinics, at each site. Where local patient registries are kept these are exploited to search for potentially suitable participants. High numbers of patients need to be screened, as patients cannot be identified by diagnosis (sarcopenia is only rarely diagnosed in clinical practice at present) and primary and secondary care electronic systems in the UK do not hold data on physical function that would facilitate identification of those who are more likely to have sarcopenia. A telephone pre-screening phase is therefore included (Fig. 1). This allows rapid, efficient selection of participants most likely to pass a screening visit, and represents the best available strategy for practical screening in sarcopenia trials. Pre-screening is based on a brief telephone conversation conducted by the research nurse exploring any contraindications (e.g. heart failure, taking ACEi) and on administration of the SARC-F tool [27]. The SARC-F tool consists of five questions about physical function, giving a score between 0 (best) and 10 (worst); the score has been developed to assist with screening for sarcopenia and functional impairment. A threshold score is used to denote a high probability of having sarcopenia, and prompts progression to a screening visit. The threshold score was initially set at 4 points or above (out of 10) but is reviewed regularly in the light of screening information, and is adjusted up or down to maximise efficiency of recruitment.

At the screening visit, muscle mass is determined using bioimpedance analysis. This approach minimises the need to perform large numbers of DEXA scans for trial screening, thus reducing costs, enhancing participant convenience and improving recruitment efficiency. The Akern BIA (Bioimpedance Analyser) 101 machine is used at all sites, and appendicular muscle mass is calculated using the equation developed by Sergi et al. [28]; this equation was developed in a white, older European population using the BIA 101 machine. Thresholds for muscle mass are derived from the UK Biobank cohort [29] and are stratified by body mass index for each sex; such an approach ensures that participants with sarcopenic obesity are represented in the trial and is consistent with the more recent Foundation for the National Institutes of Health (FNIH) consensus guideline on diagnosing sarcopenia [30]

Primary care recruitment is carried out with the assistance of the primary care research networks in Scotland (SPCRN) and England (CRN). Network involvement via the Ageing UKCRN specialty group assists with recruitment, access to patients and access to network research nurse time. Written informed consent is obtained from all participants prior to enrolment; the full participation information and consent form are accessible via Additional files 1 and 2. Research ethics committee approval has been given by the East of Scotland Research Ethics Committee (reference 14/ES/1099). The trial is approved by the UK Medicines and Healthcare Regulatory Agency (EudraCT number 2014-003455-61) and the trial is registered online (www.​isrctn.​com, ISRCTN90094835). The trial is funded by the UK National Institute of Healthcare Research (NIHR) and Medical Research Council (MRC) Efficacy and Mechanisms Evaluation programme (reference 13/53/03). The Sponsor is Tayside Academic Health Sciences Centre, a joint initiative of the University of Dundee and NHS Tayside, and the Sponsor project number for this trial is 2013GR06. Trial management is provided by Tayside Clinical Trials Unit (UKCTU number 49). The protocol on which this report is based is version 7.0, dated 27 April 2017.

Participants are randomised to receive either perindopril 4 mg once daily or matching placebo, and to receive leucine 2.5 g three times per day or matching placebo. The perindopril/placebo is administered as oral capsules. Placebo and active capsules are identical in external appearance. Perindopril tablets are over-encapsulated with a hard gelatin capsule and backfilled with lactose. The placebo capsules are identical in appearance and packed with lactose. The starting dose of perindopril is 2 mg daily which is up-titrated to 4 mg after 2 weeks if tolerated and renal function, serum potassium levels and blood pressure remain within defined limits (potassium < 5.0 mmol/L, creatinine < 180 μmol/L and < 30 μmol/L rise from baseline, systolic blood pressure > 90 mmHg). Participants in the placebo group also undergo a ‘mock’ up-titration. If the 4 mg daily dose is not tolerated, participants are down-titrated to the original 2 mg daily. If 2 mg is also not tolerated, medication is withdrawn.

The leucine/placebo is administered as a powder. A 1.5-ml scoop is provided and participants are instructed to take three scoops (4.5 ml equivalent to 2.5 g of leucine/placebo) three times per day. The placebo is matching lactose powder. Participants are instructed that they should mix the powder with food or drink. Exploratory work with our local Older People’s Advisory Group indicated that mixing the leucine powder with food or drink was palatable, particularly if mixed with foods such as yoghurt or orange juice.

Randomisation is conducted via a centrally controlled, web-based Good Clinical Practice-compliant randomisation system, run by Tayside Clinical Trials Unit. Investigators at each site access the web-based system to randomise each participant at the end of the baseline visit. Randomisation is stratified by site. To ensure balanced assignment across critical variables, a minimisation algorithm is employed, using baseline age (> 80 vs ≤ 80 years), sex, SPPB score (> 8 vs ≤ 8), grip strength (> 25 vs ≤ 25 kg for males; > 15 vs ≤ 15 kg for females) and Charlson co-morbidity score (> 3 vs ≤ 3) to balance allocation across trial arms.

Participants are asked to return all unused study medication including empty bottles/containers. Adherence to medication will be recorded by capsule count of the perindopril/placebo capsules and by the weight of the container for the leucine/placebo powder. Serum angiotensin converting enzyme (ACE) is measured to assess adherence to the perindopril/placebo capsules. To ensure that medication adherence is maximised, we employ a combination of written information about the study medication and why taking it is important, together with aide memoirs including LACE-branded mugs and fridge magnets. At each visit, participants are reminded about the importance of medication adherence. Interventions incorporating these components have been shown in a Cochrane review to enhance adherence [31]. If one of the study drugs needs to be stopped or the participant wishes to stop, they are encouraged to continue with the other study drug. Participants stopping both study drugs are encouraged to remain in the study in order to facilitate analysis as randomised.

The primary outcome for the trial is the Short Physical Performance Battery, measured at baseline, 3, 6, 9 and 12 months. The between-group difference in the SPPB across the whole follow up will be reported as the primary outcome, calculated using generalised estimating equations to incorporate repeated measures and missing data. The SPPB is a validated measure of lower limb function that reflects everyday activity. It is a powerful predictor of subsequent disability, institutionalisation and death in older people [32‐34], and has more data validating its use in older people than any other measure of physical function. Secondary outcomes include between-group differences in muscle mass measured by dual X-ray absorptiometry, other measures of physical function, instrumental activities of daily living and quality of life. We also measure blood pressure, insulin resistance, hip bone mineral density and a panel of biomarkers to explore both mechanisms of disease and predictors of response to treatment. Key outcomes and times of collection are listed as part of the SPIRIT figure (Fig. 2).

In addition, we are collecting data on adverse events, falls, hospitalisation, death and admission to institutional care. Consent is obtained for open-label follow up of participants for these outcomes collected from routine clinical data following the end of the 1-year study period. We are collecting data on usual physical activity and diet at baseline and 12 months using step counts measured by an OMRON pedometer and the Scottish Collaborative Group Food Frequency Questionnaire (FFQ) respectively; this will allow the interaction between treatment and protein intake (total, plant vs animal protein) to be ascertained.

Analyses will be by group as randomised and will comply with the International Conference on Harmonisation (ICH) E9 ‘Statistical Principles for Clinical Trials’ [35]. Two-sided p < 0.05 will be taken as significant for all analyses. The primary analysis will be a repeated-measures, mixed-model between-group comparison of SPPB utilising all available data points during follow up. Initially, a test for treatment interaction between perindopril and leucine will be carried out and if not significant the main analysis will proceed using the full power of the factorial design; each intervention will be assessed separately as an independent hypothesis. Treatment effect estimates for all primary and secondary outcomes will be adjusted for baseline values, age, sex and minimisation variables.

Secondary outcomes will be analysed with similar methodology using repeated-measures, mixed-model, between-group comparisons. Unadjusted and adjusted analyses will be presented as already discussed. Sensitivity analyses will be performed to further test the effect of missing data both by multiple imputation and by assigning worst-possible result status to missing data points. A supplementary per-protocol analysis will be performed to examine adverse events in those taking at least 80% of their study medication.

The analytes that will be investigated in the biomarker sub-study can be divided into three main groups; those that reflect the activity of the renin–angiotensin system or its targets, those that are markers of protein turnover and those potential novel markers of regenerative capacity that may reflect the capacity to respond to treatment. To look at the activity of the renin–angiotensin system we will measure circulating levels of ACE and renin as well as two truncated forms of angiotensin (ang(1–7) and ang(1–9)) that have been shown to alter muscle mass and are likely to be modified by an ACEi [36, 37]. There are a number of different processes through which activation of the renin–angiotensin system is thought to contribute to muscle loss, including increased inflammatory cytokine production, reduced IGF-1 production and increased myostatin production in signalling [38]. We will therefore quantify IL-6, TNF-α and myostatin. We will also quantify circulating levels of GDF-15, another member of the TGF-β family of cytokines, as it is a marker of all-cause mortality and is associated with muscle mass in patients with COPD [39] and with muscle loss following surgery [40]. The influence of specific polymorphic variation in the renin–angiotensin system will also be analysed.

To identify changes in muscle protein turnover in the absence of direct measurements, which are expensive and invasive, we will quantify pro-collagen III N-terminal peptide as a measure of new collagen synthesis [41] and levels of circulating muscle restricted miRNAs (myomiRs) that are increased in the circulation of patients with diseases associated with muscle wasting including Duchenne muscular dystrophy [42] and in chronic obstructive pulmonary disease [43].

Finally, we will examine circulating levels of a set of imprinted miRNAs that may reflect the regenerative capacity of the individual. These miRNAs include miR-518e from a paternally expressed cluster on chromosome 19 and miR-485-3p from a maternally expressed cluster on chromosome 14 which we have shown to be associated with muscle phenotype in patients with COPD [44, 45].

Univariate analyses will be conducted examining the interaction between individual baseline biomarker values and treatment effect for muscle mass and the SPPB at 12 months, and between biomarker change between baseline and 3 months and treatment effect for muscle mass and the SPPB at 12 months. In addition, multivariable analysis for baseline values including age, sex, SPPB, grip, muscle mass, albumin, creatinine, all measured biomarkers and treatment group will be conducted to examine whether baseline values or change at 3 months predicts the SPPB change and muscle mass change at 12 months.

We have taken a deliberately conservative approach and used the minimum clinically important difference (MCID) for the SPPB to ensure we have proof of efficacy. In order to detect a MCID in the SPPB of 0.5 points (anticipated SPPB of 8 in the placebo group and 8.5 in the intervention group, SD = 2.7 points [46]) with a power of 90% at α = 0.05, and assuming a correlation between time points of 0.7 as seen from previous work [26], we would require a total of 352 participants (88 for each of the four groups). Assuming 20% dropout at 12 months (a rate of attrition consistent with that seen in previous trials of ACEi in frail older people [26]), we therefore need to recruit 440 patients. This sample size will also have 90% power to detect a 5% difference in muscle mass at 12 months, assuming a baseline value of 19 kg (SD = 2.8) for total skeletal muscle mass [47].

Trial oversight is provided by an independent Trial Steering Committee (TSC), which meets at least every 6 months. Members include academic geriatricians with trials experience and three lay members, together with the chief investigator. The TSC reviews trial progress, approves any significant protocol changes, advises on trial conduct and approves analysis plans and dissemination plans. The TSC reports to the funder (NIHR), who has final decision-making authority for the trial. An independent Data Monitoring Committee (DMC) meets at least every 6 months; this committee comprises three independent members, chaired by an academic geriatrician with trials experience, and contains a minimum of one independent statistician. The DMC reviews trial adverse events and safety data, and makes recommendations to the TSC. Charters for conduct of the TSC and the DMC are available from the study team and from the funder. Routine management of the trial is provided by the Trial Management Group (TMG), which comprises the grant co-applicants, local site investigators, trials unit personnel from project management, data management and statistics teams, Sponsor representatives and Sponsor monitors.

Monitoring of the trial conduct at each site is provided by the Sponsor. Monitoring takes place according to a pre-specified plan, with site visits conducted according to the recruitment rate and frequency of problems identified by remote monitoring of study data. Both the Sponsor and the Clinical Trials Unit are subject to periodic audit by the UK Medicines and Healthcare products Regulatory Agency. The funder provides monitoring of overall trial progress, with additional input from the independent TSC. The final decision to publish lies with the funder (NIHR) and the Sponsor; the funder and the Sponsor have both reviewed the study design. The Sponsor will have no input into data collection, analysis and interpretation; the final report will be peer reviewed by the Funder prior to finalisation.

Significant protocol amendments are implemented across all sites after approval by the Sponsor, the independent TSC, the funder, the ethics review board, the UK Medicines and Healthcare products Regulatory Agency and the UK Health Research Authority. The current protocol is available via the NIHR website and via the trial website (www.​lacetrial.​org.​uk).

Trial data are held separately from personal identifiers, on password-protected secure servers at the University of Dundee. Identifiable data will not be shared outside the trial team or Sponsor without written participant consent. De-identified trial data will be stored for 15 years after the end of the trial. Supplies of trial medication will not be made available to participants after the end of the trial, but both interventions are available via either prescription or via health shops. Compensation for harm suffered as a result of trial interventions or processes will be available via UK National Health Service compensation or via University of Dundee trial indemnity.

Authorship for the main trial results will be based on the International Committee of Medical Journal Editors authorship criteria, and will include, but not be limited to, grant co-applicants, local investigators, trial managers, statisticians and data analysts fulfilling these criteria. Dissemination of results will take place via multiple routes: presentation at scientific conferences; publication in scientific journals and via the NIHR library; presentations to participants and their families; written summaries of results to participants; and presentations to local healthcare teams. Results will be uploaded to the EudraCT and ISRCTN databases.