A Cross-Sectional Analysis of Registered Studies on the Promising Dipeptide Carnosine

Carnosine (β-Alanyl-L-Histidine) is a naturally occurring endogenous dipeptide and over-the-counter dietary supplement with a multimodal mechanism of action. The use of carnosine and its analogues in is as diverse as its mode of action and application. Carnosine’s ready availability and protective properties make it an interesting candidate for clinical use. We have now examined the mode of use in registered clinical studies. In a cross-sectional study, we evaluated the status of clinical studies on carnosine and carnosine analogues. We searched all 16 primary clinical trials registries listed in the WHO Clinical Trials Registry. Registered studies to published studies were identified and the ratio of published/unpublished studies as well as the time to publication and thematic focus were evaluated. The 16 selected registries listed 70 studies on carnosine, of which 34 have been completed and 25 have been published to date, with an average time to publication of 28 months. Carnosine/carnosine analogues were used as dietary supplements in 56% of the studies. Twelve studies were clinical trials in healthy volunteers that focused on dietary changes and underlying physiology. The other 22 studies deal with various clinical pictures, in particular metabolic and psychological disorders. This structured evaluation shows that the applications of carnosine are very versatile, and the registration in one of the clinical registries and the timely publication would facilitate the planning of further studies.


Introduction
Carnosine (ß-alanyl-L-histidine), an over-the-counter food supplement, belongs to the group of naturally occurring histidine-containing dipeptides. In mammals and humans, carnosine is one of the most abundant dipeptides with the highest concentration in muscle (Boldyrev et al. 2013). In experimental settings, carnosine has been shown to exert a variety of cytoprotective functions, such as anti-glycation and antioxidant effects (Hou 2003), HNE quenching (Aldini et al. 2021), methylglyoxal polymerisation (Brings et al. 2017;Weigand et al. 2018) and contributes to intramuscular buffer capacity (de Brandt et al. 2022). Carnosine supplementation has been suggested as a possible strategy to prevent and treat various diseases in animals (Hajimoradi et al. 2023;Peters et al. 2020;Rašković et al. 2023;Spaas et al. 2021) and humans (Cesak et al. 2023;de Camargo et al. 2023;de Jager et al. 2022;Schön et al. 2023;Solana-Manrique et al. 2022;Spaas et al. 2021). The ready availability of carnosine and its protective properties make it an interesting candidate for clinical use (Caruso 2022;Feehan et al. 2022;Menon et al. 2022).
The registration of clinical trials contributes significantly to improving the transparency of clinical trials. This reduces publication bias and increases selective reporting (Aslam et al. 2013), and has been considered an ethical and scientific obligation (Venugopal and Saberwal 2021). Since a positive intervention result is as twice as likely to be published as a negative one (Song et al. 2010 which carnosine was studied and the proportion of studies in which carnosine (or carnosine analogues) was administered. The therapeutic potential of carnosine is increasingly being investigated in in vivo studies. Therefore, we examined the clinical studies on carnosine in terms of registration, publication status/time to publication, study site and subject areas. The aim was to validate and structure the therapeutic potential of this promising molecule to provide a basis for systematic future targeted studies.

Identification and Characterization of Studies
To identify clinical trials on carnosine, on March 3, 2022 we performed a search of all 16 primary clinical trials in the WHO clinical trial registry (Juneja et al. 2019), with the keyword "carnosine"-with no other restrictions, e.g., study status, study type or study phase (Suppl. Table 1). Publications accessible after March 3, 2022 were counted as unpublished for this study. To analyze the registered trials, we screened all available data from named registries (Table 1). Studies were divided in completed and ongoing/unfinished studies, with only completed studies being considered for further analysis. In addition, studies were analyzed in terms of their clinical background and clustered in terms of their reported description in health and disease. Subgroups were formed (e.g., metabolic disorders, dietary modification) for clustering when there was a scientific or medical connection between diseases or topics of interest. Study country of origin and publication status and time-to-publication were considered along with enrolment size, study phase, sponsor, and level of masking.

Search for Publication of Completed Trials
To verify that the results of the registered and completed studies were published, the NCBI data base and Google scholar were checked for the registered study identification number, principal investigator, subject and title of the study in the clinical trial registries. Where no publication was found, the responsible investigators were contacted via email for further information. Time-to-publication was calculated from actual study completion date to the timepoint when the results were published in online or printed version (the timespan is given in months). Trial completion date is defined by ClinicalTrials.gov as "the date on which the last participant in a clinical study was examined or received an intervention/treatment to collect final data for the primary outcome measures, secondary outcome measures, and adverse events (that is, the last participant's last visit). The "estimated" study completion date is the date that the researchers think will be the study completion date".

Statistical Analysis
The ratio of published to unpublished studies in completed clinical trials in the selected clinical study registries was determined, time-to-publication (reported as median) and number of participants per year of completion of the clinical study were calculated, thematic focus was assessed. The following continuous or categorial variables were considered: Study identification numbers, study title, study type, intervention, completion date, availability of study results, enrolment, publication date and study location and sponsors/ collaborators. The variable "enrolment" displayed the size of the study (included patients) and it was used for study size analysis. The variables `publication date´ and `availability of study results´ refer to publication status and were used for inclusion of the trial in the analysis of time-to-publication as well as publication status. The variable 'study location' refers to the geographical localisation the study was conducted and was used for respective analysis. The variable "sponsors/collaborators" refers to the monetary sponsors and were categorized as industry or university. The purpose of the clinical studies or the intervention was analysed. Statistical calculations, were performed with Graphpad prism 9. Missing values were not imputed. Sensitivity and confounders analysis were not conducted. The geographical distribution of registered clinical trials was visualized by MapChart.net.

Results
We searched all 16 WHO-listed primary clinical trial registries (Table 1) for the keyword "carnosine" and identified 70 clinical trials (Fig. 1).
Most clinical trials were registered on ClinicalTrials. gov (45) Table 1). Carnosine or a corresponding carnosine analogue or precursor was supplemented in 19 studies (56%; Suppl. Table 2). Median time to publication of studies with carnosine/carnosine analogue supplementation is lower (19 months) than those without supplementation (27 months). The cohort size ranged from 3 to 516 patients, the median number of enrollments per year after graduation was 43 in the years 2007-2022 (Fig. 2B). Of the completed studies, 28 were funded by academia, 5 by industry, and 1 with no listed sponsor (Fig. 2C).
Clinical studies on carnosine exist for various diseases (22) and in healthy subjects (12) (Fig. 3). In the disease areas, the main areas of interest were metabolic disorders (7 studies), mental disorders (4 studies), cancer (3 studies) and multiple sclerosis (3 studies). The studies on carnosine metabolism in health examined effects on dietary modification (5 studies), physiology (5 studies), aging (1 study) and biomarkers (1 study).
Most registered clinical trials were conducted in the US (n = 9), followed by Belgium and Italy (n = 5). A total of 11 studies were conducted or sponsored in North and South America, compared to 19 in Europe and 4 in Africa/Asia (Fig. 4).

Discussion
Registering clinical trial offers benefits in terms of transparency, comparability, reducing publication bias, and selective reporting (Aslam et al 2013;Venugopal and Saberwal 2021). In a cross-sectional study, we evaluated the proportion of registered studies on carnosine and their publication status.
About one third of all studies focused on aging, its role as a putative biomarker or the alterations of carnosine metabolism through dietary intervention. Therapeutic use has been studied in metabolic diseases/diabetes, cancer or brain/mental disorders and nutritional interventions on muscle exercise performance have been studied in healthy people. The publication rate for the registered studies is 71%, and thus higher than previously reported

IdenƟfied studies n=70
Unfinished/ongoing studies n=36 Completed studies n=34 Unpublished studies n=10 Published studies n=24 No response from invesƟgator n=6  (Anderson et al. 2015) where only 38% of all completed or early terminated trials registered at ClinicalTrials.gov were published. Studies on paediatric emergent delirium (45%) (Meyburg and Ries 2020), paediatric appendicitis (63%) (Breil et al. 2018) or diabetic nephropathy (63%) (Modafferi et al. 2019) found a somewhat higher publication status. The median time to publication of results was 24 months and more than 8 months beyond the time recommended by Federal Drug Administration Amendments Act of 2007 (FDAAA). With only 5 clinical trials with at least 100 participants, the enrolment number was lower compared to many other trials, such as cancer (about 300 participants (Tran et al. 2020) or paediatric appendicitis (around 230 participants) (Breil et al. 2018).
There is an urgent need to perform pharmacokinetic studies to evaluate bioavailability considering the specific routes of administration, dosages and duration of treatment. Caruso's results (Caruso 2022) and the current study will help plan future studies of the promising dipeptide carnosine. Finally, there is also a need for coordinated preclinical studies that lay the foundation for clearly defined future clinical studies. In view of the published results, the number of registered studies appears to be very small. This makes it much more difficult to make a statement about the current status. Clinical registries can only be as good as the people who use them.

Strength and Limitations
No statements of quality or outcome of the registered and in this cross-sectional study named clinical trial were performed. The heterogeneity of the studies, the different doses and durations of carnosine supplementation, the lack of gold-standard methodology to measure outcomes and the general low number of participants makes a comparison or statements about the potential of carnosine of these studies impossible.

Conclusion
This study shows the broad variety of application areas and studies with the dipeptide "carnosine". Even though this study represents the clinical trials registered in clinical databases, the broad spectrum of study interests will be read from this study and projected to the non-registered ones. It is certainly desirable that all studies are registered so that further targeted studies and their evaluations can be carried out. Funding Open Access funding enabled and organized by Projekt DEAL. This study was funded by grants from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)-project number 236360313-SFB 1118.
Data Availability All data are in the manuscript.

Declarations
Competing interests None.

Ethical Approval
No human material was used in this study.

Patient Consent Not required.
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