Safety Assessment of H2 Inhalation in Patients With Mild Cognitive Impairment

Justification for Molecular Hydrogen as a Potential Intervention Molecular hydrogen (H₂) has emerged as a promising therapeutic agent due to its selective antioxidant and anti-inflammatory properties. Studies suggest that H₂ inhalation may provide neuroprotection by targeting key pathways implicated in cognitive decline.

Key scientific justifications include:

• Oxidative stress reduction: H₂ selectively neutralizes hydroxyl radicals and peroxynitrite, mitigating cellular damage in neural tissues.
• Neuroinflammation modulation: Preclinical studies indicate that H₂ reduces neuroinflammatory markers and cytokine expression, potentially delaying cognitive impairment progression.
• Mitochondrial function improvement: H₂ has been shown to enhance mitochondrial efficiency, supporting neuronal energy metabolism and function.

Despite promising preclinical data, the safety profile of H₂ inhalation in MCD patients remains insufficiently characterized. This study aims to establish safety and tolerability parameters, serving as a foundation for future efficacy trials.

Description of the device H2-Air Blend Inhaler is a medical device designed to generate and deliver molecular hydrogen (H₂) gas for therapeutic inhalation. It is manufactured by H2 Medical Technologies s.r.o. and utilizes SPE/PEM electrolysis technology to ensure high-purity hydrogen production.

The device is intended for use in clinical and research settings, where it can provide controlled hydrogen inhalation therapy to patients. It is equipped with multiple safety features and operational modes, making it suitable for a variety of applications.

The device consists of several key components:

• main unit - the core hydrogen generation system, including the electrolysis chamber
• water reservoir - a compartment for purified water, essential for hydrogen production
• inhalation delivery system - includes nasal cannulas, facial masks, and additional adapters to facilitate patient-specific usage
• control panel and display - provides operational controls, status indicators, and timer settings
• power supply unit - an external power adapter ensuring stable operation Each device is individually identified by a serial number (SN), which is displayed on the label and included in clinical documentation for tracking and regulatory purposes.

Intended use The H2-Air Blend Inhaler is designed to produce molecular hydrogen gas for controlled inhalation therapy. It aims to serve as a potential neuroprotective treatment, especially for those with mild cognitive impairment (MCI).

This device is meant for clinical research settings, where it can be utilized under medical oversight. Studies are underway to evaluate the safety and tolerability of hydrogen inhalation among patients at risk of neurodegenerative disorders.

This device is not for self-use by untrained individuals and should be operated in accordance with the instructions for use (IFU).

Regulatory status H2-Air Blend Inhaler is a medical device currently undergoing clinical evaluation. Its regulatory classification is under assessment, and the device is being tested to ensure compliance with safety and performance standards.

The device:

• meets IEC 60601-1 standards for medical electrical equipment
• complies with ISO 13485 for medical device quality management
• is under evaluation for conformity with EU MDR (2017/745) This clinical investigation will generate essential safety data, supporting potential regulatory submissions for CE certification.

Risk and safety considerations H2-Air Blend Inhaler has been designed with multiple safety features, but potential risks must be considered.

Potential risks and adverse effects:

• mild respiratory irritation, particularly in individuals with pre-existing respiratory conditions such as asthma or COPD
• dizziness or lightheadedness, especially with prolonged inhalation
• dry throat or mild throat discomfort, which may occur during extended use
• allergic reactions, though rare, due to sensitivity to hydrogen exposure
• transient headaches, occasionally reported in previous studies

Safety measures and risk mitigation:

• the device is equipped with automatic shut-off and pressure control to prevent overuse
• only purified water must be used, preventing damage to the electrolysis membrane
• proper ventilation is required to minimize the risk of hydrogen accumulation
• vital signs must be monitored during clinical use to detect any adverse effects Based on risk analysis according to ISO 14971, the device is classified as low risk, with appropriate mitigation strategies in place. All identified risks have been evaluated, and the benefits of hydrogen therapy outweigh the potential risks.

Study procedures description Demographics and baseline characteristics The subject's date of birth, gender, race, height, weight will be recorded at baseline visit.

For body weight measurement, subjects must be undressed with the exception of underwear. For height measurement, subjects must be without shoes; subject's height will be measured by site staff by local standard.

Medical history A detailed medical history for each subject will be obtained at baseline visit. All relevant past or present conditions including concomitant medication, as well as prior surgical interventions will be recorded. In addition, subject's history of tobacco, alcohol and caffeine use will be recorded.

MCI - Mild cognitive impairment Mild cognitive impairment (MCI) is a prevalent condition characterized by cognitive decline that exceeds normal aging but does not meet the criteria for dementia.

Vital signs Vital signs include blood pressure and pulse rate.

• Subject should avoid food immediately before the measurement
• Subject should go to the toilet before BP measurement. A full bladder can increase blood pressure slightly
• Subject should sit for ten minutes in a comfortable position with his/her legs and ankles uncrossed and his/her back supported against a chair before and during a measurement
• Subject should be asked to sit quietly before and during monitoring
• When subject is ready to take his/her blood pressure, subject should be calm and not think about stressful things and not to talk at all
• Subject's arm must be positioned properly. The same arm must be always used when taking subject's blood pressure. Subject should rest his/her arm, raised the arm to the level of his/her heart, and put the arm on a table, desk or chair arm. If needed, a pillow or cushion should be placed under subject's arm to elevate the arm high enough
• The cuff must be placed on bare skin, not over clothing. The appropriately sized cuff should be always used
• One measurement should be performed. If the monitor doesn't automatically log blood pressure readings or heart rates, the measurement should be repeated until a valid reading is, given that the procedure is accepted by the subject.

Physical examination:

This includes examination of the main body systems.

Neurological examination:

The physical examination includes detailed neurological examination so that the eligibility of patients for this study can be evaluated and confirmed.

Patient questionary European Quality of Life 5 Dimensions questionnaire (EQ-5D-5L) This tool represents a standardized measure of health-related quality of life. It comprises the following five dimensions, each describing a different aspect of health: mobility, self-care, usual activities, pain/discomfort, and anxiety/depression. Each dimension has five response levels: no problems, slight problems, moderate problems, severe problems, unable to/extreme problems. The result is represented by the EQ-5D-5L index, which is derived by applying a formula that attaches values (weights) to each of the levels in each dimension. The higher the EQ-5D-5L index, the better is health-related quality of life.

In addition, the EQ Visual Analog Scale (EQ-5D VAS) provides a quantitative measure of the patient's perception of their overall health status. EQ-5D VAS is numbered from 0 ("the worst health status you can imagine") to 100 ("the best health status you can imagine").33 In this study, EQ-5D-5L version 3.0 will be used - see Appendix.

Concomitant medication All medications that are considered necessary for the subject's welfare may be used at the Investigator's discretion, however dose of any chronic medications, if applicable, must be stable for at least 90 days prior to study entry. Clinical trial reports will follow accurate and comprehensive documentation of concomitant medications to assess drug safety, efficacy, and potential interactions.

H2-Air Blend Inhaler treatment description The H2-Air Blend Inhaler is a medical-grade device crafted for the safe and controlled inhalation of molecular hydrogen (H₂) gas. It utilizes advanced solid polymer electrolyte (SPE)/proton exchange membrane (PEM) electrolysis technology, guaranteeing the generation of high-purity hydrogen gas with a stable and reliable output.

This device is specifically designed for clinical use and supervised home applications, offering a non-invasive method for cognitive support, neuroprotection, and reducing oxidative stress. Its compact, user-centric design, along with integrated safety features, ensures precision, reliability, and ease of use in both research and therapeutic settings.

Key features and functionalities

• high-purity hydrogen generation - utilizes SPE/PEM electrolysis technology to produce hydrogen with a purity of not exceeding 4 %, ensuring safe and efficient inhalation therapy
• fixed and controlled hydrogen flow rate - delivers a standardized concentration of hydrogen gas, ensuring consistency in therapeutic exposure while maintaining safety standards
• lightweight and portable design - compact and easy to handle, making it suitable for both clinical applications and supervised home-based therapy
• integrated safety mechanisms - features automatic shutoff, overpressure protection, and real-time hydrogen concentration monitoring, minimizing risks associated with prolonged or improper use
• interval-based inhalation mode - the device operates in predefined inhalation cycles, allowing for optimized exposure without risk of excessive hydrogen intake

Sample Size Estimation This study is an exploratory investigation in its early phase, focusing on collecting preliminary data regarding safety and feasibility rather than identifying statistically significant treatment effects. Due to these aims, no formal sample size calculation has been conducted. Instead, we selected a sample size of 30 participants in accordance with standard practices for pilot clinical studies. This number is deemed adequate to detect trends in safety, tolerability, and potential cognitive effects, while also allowing for variability estimation in outcome measures. The findings from this study will yield essential insights necessary for planning larger clinical trials that are more statistically robust. If significant trends emerge, they will guide the design of future studies with improved endpoints and stronger statistical frameworks.

Analysis Population The study will define multiple analysis populations to ensure a structured and meaningful evaluation of the collected data.

• full analysis set (FAS): This population will include all enrolled participants who have received at least one hydrogen inhalation session and have undergone at least one post-baseline assessment. The FAS will be used for descriptive safety and exploratory efficacy analyses to provide insights into potential treatment effects.
• per-protocol population (PP): This population will consist of participants who complete the study without significant protocol deviations, such as non-compliance with treatment schedules or major data inconsistencies. The PP will be used for secondary and exploratory efficacy analyses.
• safety evaluation set (SES): This group will include all participants who have received at least one hydrogen inhalation session. The SES will be the primary population for safety analyses, ensuring that all potential treatment-related adverse events are documented and analyzed.

Statistical Analysis General Descriptive Analysis Descriptive statistical methods suitable for each variable's nature will present all study variables. Unless noted otherwise, data will be summarized individually for each study visit and for changes noted throughout the treatment period.

Continuous Variables Descriptive statistics for continuous variables will include at least number of non-missing observations, mean, standard deviation, minimum, maximum, median and quartiles per study arm.

Categorical Variables In case of categorical items, at least counts and percentages are presented as well as the number of missing and of non-missing observations per study arm and site.

Demographic and Other Baseline Characteristics Demographic information and baseline traits will be summarized descriptively to give an overview of the study group. Continuous variables like age, weight, height, and initial cognitive scores will be summarized in tables detailing central tendency and variability. Categorical variables, such as sex, race, and medical history, will be displayed in frequency tables.

Primary Analysis The main analysis will examine the occurrence and seriousness of adverse events (AEs) and serious adverse events (SAEs) during the intervention period. Descriptive statistics will be used to analyze the distribution of adverse events, comparing event rates across relevant subgroups. Adverse event severity will be categorized based on the Common Terminology Criteria for Adverse Events (CTCAE), while trends in event frequencies over time will also be assessed. The analysis will encompass an evaluation of treatment-related adverse effects and any unforeseen safety issues.

Secondary Analysis The secondary analysis will examine shifts in cognitive function and quality of life from baseline to post-treatment visits. Cognitive scores, evaluated through the ALBA and POBAV tests ,will be compared using paired t-tests or alternative non-parametric methods, based on the data distribution. Quality of life evaluations, assessed via the EQ-5D-5L, will be analyzed using comparable approaches.

Additionally, exploratory analyses may assess relationships between cognitive changes and other variables, including baseline health characteristics or adherence to the intervention.

Safety Analysis Safety analyses will utilize the safety evaluation set (SES). Descriptive statistics will be presented for all treatment-emergent adverse events (TEAEs), covering both the frequency and severity of reported symptoms. The analysis will distinguish among events related to the investigational device, those with an uncertain relationship, and those assessed as unrelated.

Detailed reporting will cover serious adverse events (SAEs), serious adverse device effects (SADEs), anticipated serious adverse device effects (ASADEs), and unanticipated serious adverse device effects (USADEs). The analysis will also evaluate adverse events that led to study discontinuation or were classified as life-threatening.

For continuous safety parameters like vital signs, changes from baseline will be assessed using summary statistics and visualized when applicable. If noteworthy trends in safety parameters arise, additional sensitivity analyses may be conducted to evaluate potential risk factors.

Efficacy analysis The selection of the statistical test will be based on the scope and characteristics of the data to meet the relevant criteria and provide statistically significant results. Efficacy outcomes will be evaluated by comparing changes in cognitive function and quality of life scores from baseline to post-treatment visits. A paired t-test will be used for the primary comparison if the data distributions satisfy normality assumptions; otherwise, a Wilcoxon signed-rank test will be utilized. For categorical efficacy measures, such as cognitive status classification or subjective patient-reported outcomes, the differences between pre- and post-treatment assessments will be assessed using the chi-square test. If any of the criteria for the mentioned tests, such as data normality or sample size, are not met, an appropriate alternative test will be used, such as the Friedman test, the Kolmogorov-Smirnov test, or another suitable test. As this is a pilot study, formal testing of statistical significance will not be conducted, but probability values will be reported to reflect the strength of the observed effects.

Interim Analysis There are no formal interim analyses planned because of the study's exploratory nature. Nevertheless, ongoing safety monitoring will occur during the study, and if any unexpected trends arise, adjustments to the study protocol may be made.

Handling of Missing Data/ Dropouts

Participants identified as ineligible during screening or who do not meet the inclusion criteria will be replaced to sustain the target sample size. However, those who withdraw from the study after the baseline assessment will not be substituted. In regard to reporting adverse events, any missing values will be filled in using conservative assumptions:

• if causality is missing, the adverse event will be assumed to be related to the investigational device
• if severity is missing, the adverse event will be classified at the highest intensity reported for similar events in the study For missing efficacy data, multiple imputation methods will be applied where appropriate. Sensitivity analyses will be conducted to assess the potential impact of missing data on study conclusions.