Improvement of synaptic plasticity and cognitive function in RASopathies—a monocentre, randomized, double-blind, parallel-group, placebo-controlled, cross-over clinical trial (SynCoRAS)

The aim of this study is to investigate changes in synaptic plasticity and alertness in patients with RASopathies, namely NS and NF1, after the application of lovastatin (to NS patients) and lamotrigine (to NS and NF1 patients).

The SynCoRAS study is a monocentre, randomized, double-blind, parallel-group, placebo-controlled, cross-over clinical trial phase IIa. The trial design is shown in Figs. 1 and 2. The clinical trial has been approved before trial commencement by the local ethics committee (vote 461/17 Af dated 13–02-2018, with the last amendment No. 3 dated 12–11-2020) and by the German medical regulatory authorities (Federal Institute for Drugs and Medical Devices (Bundesinstitut für Arzneimittel und Medizinprodukte (BfArM)). Prior start of recruitment the clinical trial was registered in ClinicalTrials.gov (https://​www.​clinicaltrials.​gov) as well as in the European Union (EU) Clinical Trials Register (EudraCT-Nr.: 2016–005022-10).

Within the monocentric setting, the trial site is located at the Department of Social Pediatrics, School of Medicine, Technical University of Munich. Sponsor-Delegated Person is Prof. Dr. med. Volker Mall.

Inclusion criteria are defined as follows: (1) Group 1: NS, Group 2: NF1 (both genetically assured); (2) age ≥ 16 years; (3) signed informed consent of the adolescent (16–17 years of age) who is capable to give his consent and understand the aim and rationale of the study and of the legal guardian; (4) signed informed consent of persons who are ≥ 18 years old and capable to give their consent. In case of doubts, an independent medical practitioner will evaluate the capacity to consent; (5) male and female participants who are not capable of bearing children or who use a method of contraception that is medically approved by the health authority of the respective country.

Exclusion criteria are (1) epilepsy, (2) participants taking medication with known central nervous system (CNS) effects, (3) severe mental retardation, (4) side effects during previous medication with and contraindications for LTG and/or LOV and/or transcranial magnetic stimulation (TMS), (5) psychiatric diseases, (6) previous history of allergic reactions with LTG and LOV medications, (7) potentially unreliable patients, (8) patients who are not suitable for the study in the opinion of the investigator, (9) pregnancy (incl. positive urine pregnancy test) and (10) persons who are incapable of giving consent or do not understand the aim or rationale of the study.

Participants are randomized into either one out of the three approaches (exp. I–III). Three randomizations take place—one for each experiment. Patients are randomly assigned to receive either first verum then placebo, or vice versa. The randomization lists were created using RANCODE professional 2015. Randomization is performed block-wise. The randomization list was provided to the pharmacy for blinding, labelling and distribution of trial medication. For the case of emergency unblinding, a second set of sealed envelopes includes the information on the type of medication for each randomization number and is stored at the trial site. The integrity of the envelopes is monitored until end of study.

Transcranial magnetic stimulation (TMS) is a standard method used in medical diagnostics and research in humans [13, 14]. The planned investigations promise progressing basic findings regarding the neurophysiological mechanisms of the TMS and of neuronal plasticity in patients with NF1 and NS. Planned neurophysiological investigations with TMS delivered by the PowerMAG quadri-pulse stimulation (QPS) device (Mag & More GmbH, Munich) bear only very little risks and have been used by the investigators before [15]. An increased susceptibility to seizures, which can be observed with high-frequency, above-threshold TMS, was not seen so far using the planned stimulation protocols [15]. The clicking noise, which appears using transcranial magnetic stimulation, does not cause any adverse effect. The examination is not painful. Some participants are not used to the involuntary muscle contraction, which appears under magnetic stimulation, and sometimes this is felt as an awkward sensation [14]. However, experience has shown a fast adaption to these contractions. Participants are informed about this beforehand.

No other undesirable side effects are expected during stimulation. The investigators used TMS in patients with NF1 [6] as well as with NS [7] reviewed in [8] to demonstrate deficits in synaptic plasticity which were associated with attention deficits (NF1: [6]). The investigators have a long time experience with TMS and published several manuscripts in international journals [10, 15‐17]. The study procedure contains pre-measurements before interventional, repetitive TMS using the quadri-pulse theta burst stimulation protocol (qTBS) [15] and three (Post 1–3) follow-up measurements (Fig. 3). Changes in motor-evoked potentials (MEP), referring to the model of long-term potentiation (LTP)-like plasticity, as primary outcome measure, are monitored until 1 h after interventional TMS, conducted by the qTBS protocol. Changes in local excitability are investigated by the resting motor threshold.

The computerized Test for Attentional Performance (TAP) battery is a widely used tool in the systematic assessment of attention [6]. Attention skills of the participants are evaluated by conducting the Alertness, Visual Scanning, GoNogo and Incompatibility task. These four subtests of the TAP were selected because of a strong analogy to attention deficit responsive testing in patients with NF1. The Alertness test is a visual reaction time task to a presented visual target stimulus (cross) with or without previous acoustic warning. The Visual Scanning test requires accuracy and performing speed while detecting the target in a field of similar figures. The GoNogo test assesses the ability to discriminate between a target and a similar but irrelevant non-target, where participants have to respond to the target and inhibit their response to the non-target. In the Incompatibility test, participants have to detect the direction of a simple arrow (right/left) by pressing a button on the left or right side regardless on which side of the screen (right/left) the arrow appears.

Changes in MEP are a well-established and safe method to probe changes in cortico-spinal excitability in humans [18]. MEPs are elicited by TMS using a figure-of-eight coil with an outer diameter of 100 mm centred tangentially on the scalp over the primary motor cortex (M1) of the nondominant hand with its handle pointing in a posterior direction and laterally at an angle of approximately 45° away from the midline. MEPs are recorded from the abductor pollicis brevis (APB) muscle at rest by surface electromyography (EMG) using silver/silver chloride electrodes with a surface area of 263 mm² (AMBU, Ballerup, Denmark) mounted in belly-tendon recording technique. Data are band-pass filtered (20–2000 Hz) and amplified using an Ekida DC universal amplifier (EKIDA GmbH, Helmstadt, Germany), digitized at 5 kHz sampling rate using a MICRO1401mkII data acquisition unit (Cambridge Electronic Design Ltd, Cambridge, UK) and stored on a standard personal computer for online visual display and later offline analysis using Signal Software version 5 (CED Ltd, UK). MEP size is determined by measuring the two highest peaks of opposite polarity. Twenty trials will be recorded and then averaged for each point of investigation as described elsewhere [15, 16]. Resting motor threshold is recorded to probe changes in local cortical excitability [19].

The secondary endpoints for each experiment include the difference between the neuropsychological testing of attention by the TAP and differences in short interval cortical inhibition (SICI) after placebo and after medication (LTG and LOV) [6, 20]. Another endpoint is the comparison of LTG and LOV effects on synaptic plasticity and attentional performance in the NS group. The TAP is a well-established tool and used to measure the influence of LTG and LOV on attention [7, 20]. We previously demonstrated that LOV leads to an amelioration of attentional performance in humans with NF1 [6]. Consequently, we hypothesized that this would also lead to better attention in patients with NS and that LTG ameliorates attention in NF1 and NS.

For SICI, a subthreshold conditioning stimulation is delivered 2, 3 and 5 ms before a test stimulus [6]. It has been demonstrated before that an increased inhibition is the most prominent factor in mice with NF1 to prevent synaptic plasticity [5]. The authors have previously demonstrated that LTG is a potent modulator of synaptic plasticity in healthy humans [10]. Decreasing intracortical inhibition by LOV was associated with an increase in synaptic plasticity in patients with NF1 [6]. The central role of intracortical inhibition regulating synaptic plasticity in NF1 has recently been certified by animal studies revealing a new option of pharmacological intervention with LTG [5]. We have previously shown that LOV leads to a disinhibition in patients with NF1 and, therefore, hypothesized that this would be a key factor to normalize cortico-spinal excitability in patients with NF1 and NS.

Safety measures include documentation of blood pressure and heart frequency prior to the intervention as well as monitoring of EMG activity during TMS intervention and documentation of adverse events (AE), severe adverse events (SAE) and suspected unexpected serious adverse reactions (SUSAR) following established definitions and legal requirements. The intensity of AEs is defined according to the common terminology criteria for adverse events (CTCAE Version 4.0, https://​www.​eortc.​be/​services/​doc/​ctc/​CTCAE_​4.​03_​2010-06-14_​QuickReference_​5x7.​pdf).

This is a series of three experiments in a double-blind, placebo-controlled, randomized, cross-over design. Exp. I: LTG vs. placebo on group 1, Exp. II: LOV vs. placebo on group 1, Exp. III: LTG vs. placebo on group 2.

Sample size: Sample size calculation was done using the primary endpoints, nQuery Advisor, and expected means of MEP amplitudes and standard deviation (SD) for the different measurements from Mainberger et al. [7].

An increase in MEP amplitudes over time is considered to refer to the concept of long-term potentiation (LTP)-like plasticity as it has been demonstrated in Mainberger et al. [7] as well. Twelve patients per disease entity will be enough to provide appropriate power to all planned primary endpoint tests (two-sided paired samples t-tests) with a global significance level of 5% and adjusted local significance levels using the Bonferroni-Holm procedure. In order to account for some unobtainable data and drop outs, the sample size will be increased to 14 patients per disease (28 trial participants in total).

Analysis sets: For each experiment, there will be a separate analysis set defined, as it is possible that a patient is suitable for analysis in only one of the first two experiments. All analyses will be performed on the full analysis set (FAS-I, FAS-II, FAS-III), consisting of all patients who delivered a full set of MEP measurements within the corresponding experiment. Since all measurements will take place within a very short time per patient, it is expected that the data will be either complete or entirely missing.

Primary endpoint: The primary endpoint analyses will be performed in three separate testing procedures. The global significance level will be 5%. Since experiments I and II are done on the same set of patients, the significance level for those two experiments will each be 2.5%. The significance level of experiment III will be 5%. The primary endpoint analysis will consist of three series of three paired samples two-sided t-tests, comparing MEP under verum vs. placebo at the three measurement time points. The local significance level will be adjusted using the Bonferroni-Holm procedure and will be as follows for the ordered by p-value tests:

Secondary endpoints and safety: Analyses of baseline and safety data and secondary endpoints will be done using appropriate descriptive statistics and paired sample tests for difference between the two study groups. All tests will be two-sided with an exploratory significance level of 5%. No adjustment for multiple comparisons will be done.

Organizational/regulatory project management, safety management, monitoring and data management are performed by the Münchner Studienzentrum (MSZ), Technical University of Munich, School of Medicine. An independent safety monitoring board (SMB) is established. The underlying principles for the SMB are ethical and safety aspects for the patients. The SMB examines whether the conducting of the study is still ethically justifiable, whether safety of the patients is ensured and whether the process of the study is acceptable. For this, the SMB is informed regularly about patient recruitment and observed safety advents. Serious adverse events (SAEs) are recorded in a study-specific safety form and transferred into a safety database by the safety management, which processes further SAE documentation in written form to the SMB, the Investigator, the ethics committee and regulatory authority (BfArM) according to applicable law.

All study procedures agree with the guidelines of Good Clinical Practice (GCP) of the International Council on Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) and the principles of the Declaration of Helsinki. All participating investigators agreed to adhere to the instructions and procedures described in the study protocol and thereby to adhere to the principles of ICH-GCP. All members of the safety board are independent from the sponsor. The current approved protocol version is 5.0 (amendment 3, 21.09.2020). All protocol versions are available in German language at nikolai.jung@tum.de.

There are known side effects of lovastatin (LOV) and lamotrigine (LTG). LOV and LTG are used in an off-label context and in a higher dosage as recommended in the Summary of Product Characteristics (SMPC) of the medicinal products. However, all side effects of the medication (LOV and LTG) have been described after longer application whereas in the present study patients will receive LOV only four times and LTG only one time. In one patient receiving LTG, a mild rash following the administration reported has been reported [21]. In an own placebo-controlled, randomized, double-blind study using the same single dose of LTG (300 mg), no side effects, apart from mild sedation/dizziness and lacrimation have been reported in 26 young healthy adults [10]. LTG has been well tolerated in another TMS study using dosages up to 325 mg [11]. Severe skin reactions such as Stevens-Johnson syndrome, toxic epidermal necrolysis, drug rash with Eosinophilia and systemic symptoms (DRESS) syndrome or aseptic meningitis occurring 8 weeks after long-term application of LTG have never been mentioned after single-dose application. These adverse events can be excluded to the actual state of knowledge.

A 4-day course of LOV has been used in an own study in n = 11 patients with NF1 and is not expected to provoke any side effects [6]. It has been shown that doses of 200 mg of LOV were proven to be save in adult human volunteers [22]. Moreover, accidental overdoses up to 6 g have been tolerated with no specific symptoms and recovery without sequelae. Adverse events of LOV occur after longer application (> 1–2 weeks). Overall, the treatment with LTG and LOV in the context of the present study is not expected to provoke any side effects. A randomized, placebo-controlled long-term study investigated the effect of simvastatin, another 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitor, on cognition in children with NF1 [23]. This study did not find effects of simvastatin on cognition in children with NF1 [23]. In the present study, LOV will be used to investigate its effects on synaptic plasticity and attentional performance in patients with NS. We previously demonstrated in a randomized, placebo-controlled trial effects of LOV on synaptic plasticity and attentional performance in patients with NF1 in short-term use [6]. Moreover, another study demonstrated effects of lovastatin in children with NF1 undergoing a dose escalation protocol for 3 months on verbal and nonverbal memory [24]. This may highlight potential pharmacological differences of LOV and simvastatin.

The results of the present study may help to design long-term studies focusing on the therapeutic effects of LTG on synaptic plasticity and cognition in patients with NF1. It is intended to publish the results in an international scientific journal. The inclusion of minors from the age of 16 years is not considered to increase the risk of potential side effects since persons are considered to be grown-up at this age from a medical point of view. We do not expect significant differences in body weight or in the metabolism of both medications (LTG and LOV) in adolescents aged 16 years why an adaption of the dosage is not necessary. LTG is a safe and commonly used medication in the treatment of seizures/epilepsy in children and adolescents. For medications (LTG and LOV), the risk of side effects after a single dosage (LTG) and a 4-day course of LOV is not considered to be increased in adolescents. Moreover, we estimate the benefit of an early treatment of attention deficits with the medication to be higher than the risk of the treatment in this study. Side effects associated with LTG or LOV are closely monitored and handled as described above. In case of acute threatening of the patients, treatment will be handled as described in the emergency plan of the hospital.

The current protocol version number is “Final 5.0 AM 3.0”, date 9/21/2020. Recruitment finished on 3/30/2023 prematurely due to unforeseen difficulties in recruitment of patients. Recruitment was still open on initial submission of the manuscript. Recruitment began in March 2018, and an actual number of 16 participants have been enrolled. A large panel including the sponsor-delegated person, the head of German Network of RASopathy Research (GeNeRARe) and the Sponsors representative for risk-based quality management decided to prematurely close the recruiting and to terminate the study in retrospect on 2/20/2023 due to the last patient last visit (LPLV) taking place on 2/9/2023.