Role of the Blood-Brain Barrier in Stress Resilience: Investigating New Pathways Towards Pharmacological Augmentation of Stress Resilience (a PHASR-PP Project Study)
Phase: NA
Conditions: Stress Resilience, Blood-Brain Barrier Integrity, Mental Health
Sponsor: Leibniz-Institut für Resilienzforschung (LIR) gGmbH

 
Role of the blood-brain barrier in stress resilience: 
investigating new pathways towards pharmacological 
augmentation of stress resilience 
(a PHASR-PP project study) 
 
 
Study Protocol (including Statistical Analysis Plan) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Date: 12-03-2025 
NCT  number: T.B.D. 

1 
 
 
 
 
APPLICANT / CO- 
INVESTIGATORS 
 Oliver Tüscher, Univ.-Prof. Dr. med., Applicant and Investigator, 
University Medical Center of the Johannes Gutenberg University 
Mainz (UM), Dept. of Psychiatry, Untere Zahlbacher Str. 8, 55131 
Mainz, Germany; phone: +49 6131 17 2920; Fax +49 6131 17 6690; 
tuescher@uni-mainz.de 
 Raffael Kalisch, Prof. Dr., Co-Investigator, Leibniz Institute for 
Resilience Research gGmbH (LIR), Wallstraße 7, 55122 Mainz, 
Germany; phone: +49 (0)6131 178419, raffael.kalisch@lir-mainz.de 
 Flurin Cathomas, Dr., Co-Investigator, University Zurich (UZH), 
Psychiatric University Hospital, Dept. of Psychiatry, Psychotherapy 
and 
Psychosomatics, 
Lenggstrasse 
31, 
CH-8032 
Zürich, 
Switzerland; phone: +41 44 384 34 35, flurin.cathomas@pukzh.ch 
 Dorota 
Kobylińska, 
Dr., 
Co-Investigator, 
University 
Warsaw 
(UNIWARSAW), Faculty of Psychology, Krakowskie Przedmieście 
26/28, 
00-927 
Warsaw, 
Poland; 
phone: 
+48225549700, 
dorotak@psych.uw.edu.pl 
 Signe Mezinska, Prof. Dr., Co-Investigator, University of Latvia (LU), 
Faculty of Medicine and Life Sciences, Institute of Clinical and 
Preventive Medicine, Jelgavas Str. 3, Riga, LV-1048, Latvia; phone: 
+37129146661, signe.mezinska@lu.lv 
TITLE OF STUDY
Role of the blood-brain barrier in stress resilience: investigating new 
pathways towards pharmacological augmentation of stress resilience (a 
PHASR-PP project study) 
CONDITION
Healthy university students aged 18-25 years 
Role of the blood-brain barrier in stress resilience: investigating new 
pathways towards pharmacological augmentation of stress resilience 
(a PHASR-PP project study)  
Die Rolle der Blut-Hirn-Schranke in der Stressresilienz: Untersuchung neuer Wege zur 
pharmakologischen Förderung der Stressresilienz (eine Studie des PHASR-PP-Projekts) 
3.1 
STUDY SYNOPSIS 

2 
 
OBJECTIVE(S)
Overarching aim is to better understand the relationship between blood-brain 
barrier (BBB) function and stress resilience and to investigate a potential 
causal role for BBB function in stress resilience in humans 
 
Primary objective (O1): 
O1a) testing whether better BBB integrity, assessed with neuroimaging at 
whole-brain level, is prospectively associated with better resilience in the 
short term (reduced average stressor reactivity at time points T4-T6 during 
early follow-up). 
O1b) testing whether an experimental pharmacological manipulation, 
consisting of the administration of metformin from time points T0-T3, 
improves BBB integrity at time point T3 at end of experimental manipulation 
relative to time point T0 at baseline. 
O1c) testing whether the effect of the experimental manipulation on BBB 
integrity at time point T3 mediates a potential effect of the experimental 
manipulation on short-term resilience (reduced average stressor reactivity at 
time points T4-T6 during early follow-up). 
Secondary objective (O2): 
O2a) testing whether better BBB integrity, assessed with neuroimaging at 
whole-brain level, is prospectively associated with better resilience in the 
long term (reduced average stressor reactivity at time points T4-T9 during 
whole follow-up). 
O2b) testing whether the effect of the experimental manipulation on BBB 
integrity at time point T3 mediates a potential effect of the experimental 
manipulation on long-term resilience (reduced average stressor reactivity at 
time points T4-T9 during whole follow-up). 

3 
 
INTERVENTION(S)
Experimental intervention: Metformin 850 mg twice daily 
Control intervention: Placebo 
Duration of intervention per participant: 12 weeks  
Follow-up per participant: 24 weeks 
Experimental and / or control off label or on label in Germany: off-label 
experimental intervention 
 
KEY 
INCLUSION 
AND EXCLUSION 
CRITERIA 
Key inclusion criteria (participants meeting all of the following criteria will 
be considered for enrolment in the study): 
 
Absence of mental disorder diagnosis  
 
University students 
 
GHQ-28 score ≥ 20 
 
Three or more adverse life events (LEs) acc. to LE list in their past 
 
Beck Depression Inventory (BDI) ≤ 14 & Columbia-Suicide Severity 
Rating Scale (C-SSRS) ≤ 1 (thereby excluding concurrent 
depression and suicidality) 
 
Age 18 - 25 years 
 
Able to understand character and individual consequences of the 
study (MMSE Folstein > 28) 
 
Key exclusion criteria (participants presenting 1 of the following criteria 
will not be enroled in the study): 
 
Life-time and current diagnosis of any severe mental disorder 
determined by M.I.N.I. diagnostic interview 
 
Known history of brain injuries or neurodevelopmental disorder 
 
Evidence of neurodegenerative disorder (e.g., Parkinson) 
 
Multimorbidity or significant organ (esp. liver or renal) 
dysfunction or manifest diabetes or substance abuse (esp. 
alcohol) 
 
Contraindication to metformin such as renal insufficiency (Creatinin- 
Clearance <60ml/min), recent (<3 months) ischemic events (e.g., 
myocardial infarction or stroke) 
 
Women of childbearing age, who do not practice a medically 
accepted contraception (i.e., systematic contraceptives, diaphragm, 
condoms with spermicide, sexual abstinence) during the study and 
during a 2 years post-study period and who do not present a negative 
pregnancy test (serum or urine) 
 
History of hypersensitivity to the study drug, to any drug with similar 
chemical structure, or to any excipient present in the pharmaceutical 
form of the study drug 
 
Diabetes type 2 (would result in interference with the experimental 
manipulation) 
 
Participation in other studies employing a drug during the present 
study or within the last three months 
 
Current use of antidiabetic, weight-loss, or psychoactive medication or 
substances 
 
Pacemaker, implanted medical pumps, implanted cardiac catheters 
or acute or unstable heart disease (angina pectoris). 
 
Intracranial implant (aneurysm clips, shunts, stimulators, cochlear 
implants or electrodes) or other metallic objects inside or near the 
head (mouth excluded) that cannot be removed. 
 
Claustrophobia or another contraindication to MRI. 
 
Insufficient language skills 

4 
 
OUTCOME(S) 
Primary endpoint:
 
Relating to O1a): Prospective association between participants’ 
whole-brain water exchange rate (kw) from blood to brain tissue 
assessed with arterial spin labelling in neuroimaging (i.e., whole-brain BBB 
permeability as an inverse marker of BBB integrity) at time points T0 at 
baseline and T3 at end of experimental manipulation and participants’ 
average stressor reactivity (SR score) at time points T4-T6 during early 
follow-up (as an inverse marker of short-term resilience), taking into account 
the experimental manipulation. 
 
Key secondary endpoints: 
 
Relating to O1b): Effect of the experimental manipulation between 
time points T0 and T3 (groups verum and placebo) on participants’ 
whole-brain BBB permeability at time point T3 at end of experimental 
manipulation, taking into account BBB permeability at time point T0 at 
baseline. 
 
Relating to O1c): Mediation by the effect of the experimental 
manipulation on participants’ whole-brain BBB permeability at time 
point T3 at end of experimental manipulation on participants’ average 
stressor reactivity at time points T4-T6 during early follow-up. 
 
Further secondary endpoints: 
 
Relating to O2a): Prospective association between participants’ 
whole-brain water exchange rate (kw) from blood to brain tissue 
assessed with arterial spin labelling in neuroimaging (i.e., whole-brain BBB 
permeability as an inverse marker of BBB integrity) at time points T0 at 
baseline and T3 at end of experimental manipulation and participants’ 
average stressor reactivity (SR score) at time points T4-T9 during whole 
follow-up (as an inverse marker of long-term resilience), taking into account 
the experimental manipulation. 
 
Relating to O2b): Mediation by the effect of the experimental 
manipulation on participants’ whole-brain BBB permeability at time 
point T3 at end of experimental manipulation on participants’ average 
stressor reactivity at time points T4-T9 during whole follow-up. 
 
Assessment of safety:  
 
Frequencies of participants experiencing at least one adverse event (AE) 
will be displayed by body system and preferred term according to 
MedDRA terminology. 
 
STUDY TYPE
Prospective,
multi-center,
double-blind,
randomized,
parallel-group
placebo-controlled experimental study 
STATISTICAL 
ANALYSIS 
Population:  
 
The primary population for the analyses of efficacy is the intention to treat 
(ITT) population. Complementary analyses will be conducted in the per-
protocol population (ITT population without major protocol violations).  
 
Significance testing:  
 
All hypotheses will be tested on a two-sided level of significance α=0.05. 

5 
 
The primary hypothesis (primary analysis relating to the primary objective 
O1a) will be tested separately and without correction for multiple testing. 
Correction for multiple testing will be applied family-wise to the key 
secondary hypotheses (two key secondary analyses relating to the 
primary objectives O1b and O1c), provided that the conditions to test the 
key secondary hypothesis relating to objective O1c are met. 
 
Primary analysis: 
 
Relating to O1a): To test for a prospective association of whole-brain BBB 
permeability with average SR at T4-T6 during early follow-up (SR(T4-
T6)), a linear mixed model will be used to regress BBB permeability at 
baseline (BBB(T0)), BBB permeability at end of experimental 
manipulation (BBB(T3)), and experimental manipulation (EM; groups 
metformin and placebo) onto (SR(T4-T6)). 
 
Key secondary analyses: 
 
Relating to O1b): To test for an effect of the experimental manipulation 
between time points T0 and T3 (groups metformin and placebo) on 
participants’ whole-brain BBB permeability at time point T3 at end of 
experimental manipulation (BBB(T3)), a linear mixed model will be used 
to regress EM and BBB(T0) onto BBB(T3). 
 
Relating to O1c): If BBB(T3) shows a significant association with 
SR(T4-T6) (see O1a) and if EM shows a significant association with 
BBB(T3) (see O1b): To test for mediation by the effect of the 
experimental 
manipulation 
on 
participants’ 
whole-brain 
BBB 
permeability at time point T3 at end of experimental manipulation on 
participants’ average stressor reactivity at time points T4-T6 during early 
follow-up, mediation analysis will be conducted using a Baron-Kenny 
approach with EM as predictor, BBB(T3)  as mediator, SR(T4-T6) as 
outcome, and , BBB(T0) and earlier covariates as covariates. 
 
Further secondary analyses: 
 
Relating to O2a): To test for a prospective association of whole-brain BBB 
permeability with average SR at T4-T9 during whole follow-up (SR(T3-
T9)), a linear mixed model will be used to regress BBB(T0), BBB(T3), 
and EM onto SR(T4-T9). 
 
Relating to O2b): If BBB(T3) shows a significant association with 
SR(T4-T9) (see O2a) and if EM shows a significant association with 
BBB(T3) (see O1b): To test for mediation by the effect of the 
experimental 
manipulation 
on 
participants’ 
whole-brain 
BBB 
permeability at time point T3 at end of experimental manipulation on 
participants’ average stressor reactivity at time points T4-T9 during whole 
follow-up, mediation analysis will be conducted using a Baron-Kenny 
approach with EM as predictor, BBB(T3)  as mediator, SR(T4-T9) as 
outcome, and , BBB(T0) and earlier covariates as covariates. 
 
Safety:  
 
All summaries and listings of safety data will be performed for the safety 
population. Summary tables of AEs will present the number of 
participants observed with AEs and corresponding percentages. 
Laboratory values and vital signs will be analyzed by descriptive methods 

6 
 
and exploratory p-values.
 
SAMPLE SIZE
To be assessed for eligibility: n = 122 (61 per group)  
To be allocated to study: n = 36-46 per study site 
To be analyzed: n = 109 
STUDY DURATION Time for preparation of the study (months): 5 months (M01-M05) 
Recruitment period (months): 10 months (M03-M12) 
First participant-in to last participant-out (months): 18 months (M06-
M023)  
Time for data clearance and analysis (months): 9 months (M024-M032) 
Duration of the entire study (months): 32 (M01-M032) 
 
PARTICIPATING 
CENTERS 
To be involved (n): 3 actively recruiting centers (UM, UZH, UNIWARSAW)

7 
 
 
 
N.A. 
 
 
3.2.1 SUMMARY 
 
The project builds on findings from animal research indicating that the integrity of the Blood-Brain 
Barrier (BBB) is an important resilience factor. Stress and maladaptive stress-related behavioral 
alterations have been associated with low-grade peripheral inflammation and inflammation-
associated impairments in BBB function that allow inflammation mediators to enter the brain 
parenchyma and adversely affect neural processes. BBB vulnerability to stress-related 
impairment, however, varies between individual animals, and individuals with a less vulnerable 
BBB also exhibit fewer stress-related behavioral alterations, that is, more stress resilience. 
Further, animal studies indicate that the mTOR pathway contributes to BBB vulnerability and that 
pharmacological inhibition of this pathway improves stress resilience. This effect is likely achieved 
via improvements in BBB function.  
Translating these results to the human, the study aims to establish a prospective association 
between BBB integrity and stress resilience in stressor-exposed individuals at risk to develop 
stress-related mental health problems. It primarily builds on assumed natural inter-individual 
variation in BBB integrity, assessed with neuroimaging, which it attempts to relate to inter-
individual variation in stress resilience in an observational approach. It is hypothesized that better 
BBB integrity predicts better stress resilience. Because the animal literature does not allow us to 
estimate the duration of the hypothesized effect, both a short-term and a long-term resilience 
outcome are employed. As a further measure to maximize sensitivity, we attempt to enhance 
inter-individual variation in BBB integrity in the study sample by administering the indirect mTOR 
pathway inhibitor metformin to half of the participants, using a randomized parallel-group placebo-
controlled double-blind multi-center multimodal pre-registered experimental design. This 
experimental-interventional approach allows us to ask whether metformin improves BBB integrity 
and whether this in turn improves stress resilience.  
In additional analyses, blood-based biomarkers are used as measures of BBB integrity, and the 
influence of immunological, psychological, and socio-demographic covariates on BBB function 
and stress resilience is also considered.  
The study has the potential to establish for the first time a relationship between BBB function and 
stress resilience in humans and to yield hints at a potential causal role of BBB function in 
resilience. In the longer term, it may open the path towards a pharmacological method to augment 
BBB function and resilience, to be investigated in follow-up projects. 
 
3.2.2 KEY WORDS 
 
BBB integrity, stress resilience, at-risk, prospective, metformin, placebo-controlled 
 
3.2.3 INTERVENTION SCHEME / STUDY FLOW 
 
Design: Prospective, randomized, parallel-group, placebo-controlled, double-blind,  multi-center  
experimental study to investigate the relationship between individual variation in BBB integrity and 
individual variation in stress resilience, making use of a 12-week treatment with orally 
administered metformin (850 mg twice daily) as an experimental tool in participants at risk for 
stress-related mental health problems in order to enhance individual variation in BBB integrity. 
A.3.2. RESULTS OF THE PREVIOUS FUNDING PERIOD 
3.2 
SUMMARY OF THE STUDY 

8 
 
 
 
Figure 1. Schematic overview of study design. Online monitoring of stressor exposure (E) and mental 
health problems (P) as basis for the calculation of individual stressor reactivity (SR; endpoint) are conducted 
every four weeks (T0, T1, …). Neuroimaging-based assessments of whole-brain BBB integrity are 
conducted at time points T0 and T3. They bracket the experimental manipulation (metformin vs. placebo in 
a parallel-group design), expected to affect BBB integrity. Inter-individual variation in BBB integrity, both 
naturally occurring (spontaneous) and experimentally induced, can thus be related to SR. Additional 
laboratory-based assessments are shown below the time axis. Bottom: nomenclature of study phases and 
key time points. 
 
 
Screening phase: Participants will be assessed at three different study sites (i.e. Germany, 
Switzerland, Poland). Potential participants will be screened and assessed for eligibility. If eligible, 
informed consent will be obtained. 
Time point T0 (Baseline; week 1): Medical exam, blood sampling, neuroimaging, randomization, 
distribution of study drug starting dose (500mg once daily), start of monthly online monitoring. 
Time points T0 to T3 (Experimental manipulation phase): Four weeks of dose increase (T0 to T1), 
i.e., first and second week 500 mg once daily, third and fourth week 500mg twice daily. Full-dose 
administration (850 mg twice daily) for eight weeks (T1 to T3). Monthly online monitoring. 
Time point T3 (End of experimental manipulation; week 12): Medical exam, blood sampling, 
neuroimaging, end of full-dose administration. AE monitoring. 
Time points T4 to T9 (Follow-up): separated into early (T4 to T6) and late (T7 to T9) follow-up 
period. Monthly online monitoring. 
Time point T9 (End of follow-up; week 36): Medical exam, blood sampling, AE monitoring. 
 
 

9 
 
3.2.4 FREQUENCY AND SCOPE OF STUDY VISITS 
 
Action / Visit 
Screening 
Baseline/ 
Visit 1 (at T0)
Visit 2 (at T3)
Visit 3 (at T9)
Study week
0
12
36
Participant information and written informed consent
x
 
 
 
Demographics (e.g. sex, age, race)
x
 
 
 
Vital signs (BP, pulse, temperature)
x
 
 
 
Clinical signs of infection
x
 
 
 
Physical and psychiatric examination
x
 
 
 
Medical history
x
 
 
 
Inclusion/exclusion criteria
x
 
 
 
MMSE (according to Folstein)
x
 
 
 
BDI
x
 
 
 
CSSR-I
x
 
 
 
M.I.N.I.
x
 
 
x 
Medical examination, incl. blood samples
x
x 
x 
x 
Online: life events, daily hassles, internalizing symptoms (every four weeks)
 
x
x
x
Neuroimaging: BBB assessment, anatomy and diffusion (T1, T2), resting-state fMRI
 
x 
x
Assessment of online battery of psycho-social resilience and risk factors
 
x
x
x
Distribution of study drug
 
x 
 
Randomization
 
x
 
 
Adverse events
 
x
x
x
End of study (final visit)
 
 
 
x 

10 
 
 
 
3.3.1 EVIDENCE 
 
Blood-brain barrier function, inflammation, and stress 
The central nervous system has traditionally been viewed as an absolute immune-privileged site1. 
However, there is increasing evidence that there are ways by which the brain extensively interacts 
with other organ systems2. The blood-brain barrier (BBB) tightly controls the bidirectional 
communication between the central nervous system and the circulation and is therefore vital for 
brain protection and function. This complex selective interface – the neurovascular unit – consists 
of several specialized cell types: non-fenestrated brain endothelial cells that are characterized by 
highly specific tight junctions sealing the para-cellular space, pericytes and smooth muscle cells 
that play a major role in controlling the cerebral blood flow, and astrocytic end-feet covering most 
of the vasculature3. The immune system and the BBB are tightly intertwined3. While under 
physiological conditions, most peripheral cytokines or immune cells cannot penetrate the BBB or 
depend on specialized transporters regulating their passage, pathological conditions such as 
acute or chronic inflammatory states can lead to increased BBB permeability3,4, involving 
the influx of potentially neurotoxic proteins or factors, such as peripheral IL-6 into the brain 
parenchmya5. Given that stress is associated with profound changes in the immune system6, 
one could hypothesize that BBB integrity and function might be also impaired in stress-related 
conditions. A recent seminal pre-clinical study showed that upon exposure to chronic social defeat 
(CSD), which is one of the best-validated stress models for the mouse, stress-reactive (but not 
stress-resilient) mice showed increased BBB permeability. This was caused by a stress-
induced downregulation of the endothelial tight junction gene/protein Claudin-5 resulting in the 
influx of potentially neurotoxic proteins, e.g., peripheral IL-67. In the same study, the Claudin-5 
gene was also shown to be downregulated in postmortem tissue from patients with major 
depressive disorder (MDD). The link between stress and BBB permeability was further 
substantiated in a study showing that hippocampal BBB permeability was increased in mice that 
underwent the learned helplessness paradigm and that BBB permeability and behavioral 
abnormalities could be reversed after pharmacologically blocking the cytokine TNFα7. Areas of the 
brain where BBB function appears to be particularly sensitive to stress include the hippocampus 
(HPC), the prefrontal cortex (PFC), and the ventral striatum (VS), which are also important nodes 
in brain networks regulating stress and emotion5,8. 
 
Measurement of BBB function in humans 
In humans, BBB permeability can be assessed using invasive methods, specifically contrast 
agent-enhanced magnetic resonance imaging (MRI) of the brain as a direct indicator9 and 
measurement of plasma or cerebrospinal fluid (CSF) proteins as indirect indicators10,11. Recently, 
a non-invasive MRI-based neuroimaging method without contrast agent has been 
developed12,13. To date, however, the evidence linking BBB dysfunction and stress-related 
disorders exclusively stems from studies that have used indirect measures like vascular 
markers in circulation or ratios between CSF and blood proteins, such as the CSF/serum albumin 
quotient14. Because albumin is not synthesized in the CSF, albumin measured there stems from 
the circulation and can be used as a proxy to assess blood-CSF leakiness. In a study performed 
on elderly women, those with MDD had a higher CSF/serum albumin ratio15. In addition, an early 
study showed increased markers of permeability in MDD16. Another peripheral marker of BBB 
dysfunction is S100β. This calcium-binding protein, which is mainly expressed in glial cells, is 
normally not detectable in serum; however, it is elevated in the presence of BBB damage17. 
Several studies have reported increased levels of S100β in patients with MDD18. In addition, low-
grade inflammation (assessed by CRP, SAA, ICAM-1, IL-6, IL-8, TNF-α) and endothelial 
dysfunction (assessed by VCAM-1, E-selectin, VWF, ICAM-1) were both associated with 
depression, while endothelial dysfunction was further associated with chronicity of depressive 
symptoms19,20. The anatomical localization of stress-related BBB leaks in the human brain cannot 
be established with these indirect marker methods. 
3.3 
THE MEDICAL PROBLEM 

11 
 
In summary, in the context of a close interaction between peripheral inflammation and 
neurovascular dysfunction, there is increasing evidence that both contribute to the detrimental 
effects of stressor exposure on mental health, and improved methodology now allows for a more 
detailed and non-invasive investigation of the role of the human BBB in this interplay. 
 
Stress-dependent regulation of behavioral and BBB function via the mTOR pathway 
Recent animal work from LIR and UM (with PHASR-PP project partner Prof. S. Schweiger, Dept. 
Human Genetics) has identified the mTOR (mechanistic target of rapamycin) pathway as a 
likely mediator of the detrimental effects of exposure to a chronic social stressor on normal 
adaptive behavior in mice21. Specifically, pathway analyses of differentially expressed genes after 
CSD in hippocampal cells indicated mTOR pathway upregulation in stressor-reactive mice, 
whereas mice that maintained behavioral functioning despite CSD (stress-resilient mice) 
showed relatively reduced expression of downstream mTOR effectors and enhanced 
expression of negative mTOR regulators (MAPK and PI3K-Akt pathways). Importantly, 
administration of the mTOR inhibitor (and MAPK and PI3K-Akt upregulator) rapamycin22 over 
several days following CSD prevented CSD-induced behavioral impairments, that is, made 
animals more stress resilient21. 
Interestingly, mTOR-related differential expression was observed not in neuronal, but mainly in 
glial, mural, and endothelial cells, pointing towards a role for the BBB in stress vulnerability vs. 
resilience. The mTOR signaling pathway is an evolutionarily conserved pathway that senses and 
integrates a broad range of environmental cues, e.g., growth factors or immune modulators, to 
regulate several homeostatic processes, including in the vasculature23. In various mouse models 
of neurological and inflammatory diseases that have been associated with impairments in the 
neurovascular unit and reduced BBB integrity, rapamycin had a positive effect on BBB and 
vascular function24–27. An additional BBB-protective effect of rapamycin beyond mTOR inhibition 
may be achieved by its anti-inflammatory actions28. 
Taken together, pharmacological mTOR pathway inhibition and immunosuppression, as 
effectuated for instance by rapamycin, presumably enhance resilience via promotion of BBB 
integrity and reduced exposure of the brain to mediators of inflammation. 
 
Metformin as an experimental tool to enhance BBB function in humans 
These combined insights from human and animal studies give rise to the hypothesis that better 
BBB integrity in stressor-exposed humans reduces their risk to develop stress-related mental 
health problems, that is, BBB integrity in the face of stress is an inter-individual resilience factor. 
Further, next to naturally occurring (spontaneous) inter-individual variability in BBB integrity, inter-
individual differences may also be generated pharmacologically via mTOR inhibition (relative to a 
control pharmacological intervention). Improvement of BBB integrity as a result of mTOR inhibition 
should enhance resilience (mediation). Therefore, an experimental design where natural and 
pharmacologically induced inter-individual variance in BBB function is combined should 
be highly sensitive to demonstrate effects of BBB integrity on stress resilience. 
To pharmacologically manipulate BBB function, rather than using the mTOR inhibitor and 
immunosuppressor rapamycin, it is likely advantageous to use the indirect mTOR inhibitor and 
immunosuppressor metformin. Rapamycin has already been employed in humans (as sirolimus), 
however, with an unfavorable side effects profile. Metformin inhibits mTOR via PI3K-Akt29–31. 
Orally administered metformin has been used for decades in millions of patients in the prevention 
and treatment of type 2 diabetes and has recently been suggested to have anti-aging and anti-
cancer effects, among others29–31. Metformin has a highly favorable safety profile and can be 
administered over extended times; adverse effects can be avoided by excluding individuals with 
kidney dysfunction, liver disease, or vitamin B12 deficiency or in pregnancy and by gradual 
dosing29,30. The possibility to administer metformin over longer time periods is important since the 
beneficial effect on resilience (and presumably BBB function) of rapamycin in stressed mice was 
achieved through chronic treatment21. Metformin is off-patent and relatively inexpensive. 
Importantly, there are indications that metformin also has anti-inflammatory effects31,32 and may 
protect against cardiovascular disease29–31, and recent work in mice has shown that metformin 
attenuates BBB disruption following middle cerebral artery occlusion33, suggesting that the 
protection of BBB function is a plausible working mechanism of metformin. 

12 
 
3.3.2 THE NEED FOR A STUDY 
 
The medical problem: resilience factors 
Mental disorders are one of the leading causes of the global burden of disease34, with depression 
and anxiety being the most outstanding35. These disorders can have significant personal, 
economic, and societal costs and are often triggered by stressor exposure and the ensuing stress 
reactions during childhood and adulthood36. For various reasons (including the mostly arbitrary 
cut-off used to differentiate healthy from diseased persons in current diagnostic systems), 
research on stress-related and other mental health dysfunctions increasingly abandons diagnostic 
categories (such as Major Depressive Disorder) as research-relevant entities and instead focuses 
on continuous dimensions of psychopathology and impairments in the underlying functional 
systems, following a transdiagnostic approach37,38. In this logic, we here speak of stress-related 
mental health problems, which can be observed across different stress-related disorders but 
may also be found to a smaller or greater extent in individuals without a diagnosis, yet may cause 
significant burden. Accordingly, we understand the term mental health not as a binary construct, 
but in a continuous-multidimensional fashion. No good epidemiological information exists about 
the prevalence of the various known stress-related mental health problems, however, they can be 
safely considered a major societal challenge. It is assumed that factors or mechanisms that 
reduce the occurrence of stress-related mental health problems (resilience factors) will also 
reduce the occurrence of stress-related disorders. In the current project, we focus on factors 
conveying resilience to internalizing problems as psychopathological dimensions characteristic 
of depression and anxiety disorders. 
Despite enormous efforts made in the last decades to understand stress-related pathophysiology 
and improve treatments, there has been no concomitant decline in disease prevalence. 
Insufficient investments into prevention research and prevention efforts (‘prevention gap’) have 
been identified as one major cause39. An approach focusing on the maintenance of mental 
health despite stressor exposure (i.e., stress resilience) is therefore an attractive 
complementary strategy to disease-oriented research to combat stress consequences, with the 
potential to prevent much individual suffering and societal burden40. This reasoning motivates 
research into the mechanisms and factors underlying stress resilience (resilience factors). 
Better understanding resilience mechanisms may lead to new or improved prevention methods. 
 
The study population: emerging adults 
Stress-related mental disorders or problems often have their first onset during adolescence or 
early adulthood, where they tend to peak41. During the COVID-19 pandemic, youth and emerging 
adults were among the most strongly mentally affected groups35,42. The vulnerability of this age 
group may partly relate to the critical transition many of them undergo from life in a familiar 
environment (family, school, friends) into the unfamiliarity and challenges of professional life or 
higher education, often accompanied by geographical relocation. This is supported by reports of 
frequent stress-related problems specifically in university student populations43. These data and 
the observation that early-onset stress-related problems are often associated with life-long mental 
vulnerability, strongly suggest that investment in the mental health of emerging adults is likely 
to yield lasting gains and to be economically particularly efficient41. 
In the ongoing longitudinal study MARP (Mainz Resilience Project) of LIR (coordinated by 
PHASR-PP project coordinator R. Kalisch) and UM (PI PHASR-PP group leader O. Tüscher)44, 
we investigate young adults aged 18-19 years at study inclusion, who are faced with the 
challenges of transitioning into adulthood (>75% students). An additional inclusion criterion was a 
history of at least three significant adverse life events (LEs). A past or present formal mental 
disorder diagnosis was excluded. We observed an average 30-49% of participants at critical levels 
(above screening cut-off) of internalizing problems/symptoms (measured with the General Health 
Questionnaire, GHQ-2845), at any given study time point, and an average 4.8% symptom increase 
over the course of 3.5 years (unpubl.). In the EU H2020 project DynaMORE (Dynamic Modeling 
of Resilience, dynamore-project.eu), coordinated by LIR (R. Kalisch) and including partners UM, 
UZH (PI PHASR-PP project partner Prof. Birgit Kleim, Inst. of Psychology), and UNIWARSAW (PI 
PHASR-PP group leader D. Kobylinska), we have conducted a 9-months longitudinal 
observational study in 18 to 25 year-olds without current diagnosis, of which nearly all were 

13 
 
students and where we additionally required a GHQ score of 20 or higher and three past LEs as 
inclusion criteria. Participants were allowed to have a distant history of diagnosed stress-related 
disorders (multi-center study DynaM-OBS, incl. sites UM and UNIWARSAW46). In this study, we 
find 33-45% participants with critical GHQ levels at any time point and an average 2.5% increase 
over the study (unpubl.). 
In both cohorts, we have conducted extensive regular monitoring via self-report of both mental 
health problems and stressor exposure. Mental health problems in the past two weeks were 
measured with the GHQ. Stressor exposure measurement was divided into macrostressors 
(adverse LEs since the last monitoring) and microstressors (daily hassles, DHs, in the past week). 
For the purpose of stressor monitoring, we have used tools specifically developed by LIR47,48. We 
find that LE and DH measures are typically highly correlated, and using either a combined stressor 
exposure score (expressing the occurrence of both LEs and DHs48) or also only the DH exposure 
score results in correlations of approx. r=0.25-0.48 of exposure with concurrent mental health 
problems P (GHQ) across time points and cohorts, in line with the literature (unpubl.). This 
suggests the presence of specifically stress-related internalizing problems in this population. See 
Figure 2. 
These observations make an emerging adult population suitable from a viewpoint of 
resilience research, because such a population allows one to ask which factors underly the 
variance in internalizing problems or, more specifically, which factors and mechanisms are 
associated with fewer internalizing problems despite exposure (resilience factors). Further 
pragmatic advantages of working in this population are their easy recruitment and high compliance.  
 
 
 
 
 
 
Figure 2. Relationship between stressor exposure (E) and mental health problems (P) in MARP and DynaM- 
OBS. In MARP, E is calculated as a z-score combining the occurrence (counts) of LEs and DHs, as reported each 
three months via online monitoring and averaged across the first three monitorings (first nine study months). N=133. In 
DynaM-OBS, E is a z-score of DH occurrence only, as reported at each bi-weekly monitoring and averaged across the 
whole 9-months study period. N=238. P is derived in both studies from the GHQ-28 over the respective monitorings. 
Blue line: regression with 95% confidence interval. 
 
 
Operationalization of stress resilience: inverse stressor reactivity 
Based on the observations in MARP and DynaM-OBS, we have defined a stressor reactivity 
(SR) score as a way to quantify resilience48.  SR is the residual deviation of an individual from the 
population mean of the relation between stressor exposure (E) and mental health problems (P) 
(Figure 2). A positive residual indicates more mental health problems than expected based on the 
normative E-P relationship in the study sample. A negative residual (negative SR score) indicates 
less mental health problems than expected (normal). Hence, SR is an inverse continuous 
expression of an individual’s resilience. To build SR, E is calculated as a z-score combining 
the average occurrence (counts) of LEs and DHs, as reported via online monitoring, over the study 
phase of interest. P, also obtained from online monitoring, is also averaged across a given study 

14 
 
phase. Hence, SR scores can be calculated for any observation time window, such as T4-T6 or 
T4-T9 in the current project. 
 
Longer-term perspective: towards a pharmacological augmentation of stress resilience 
If a role of the BBB in stress resilience can be shown, methods to enhance BBB function under 
stress could in principle be exploited for resilience promotion, that is, for the prevention of stress-
related mental health problems in at-risk groups. Such methods may include pharmacological 
augmentation, for instance with metformin. Another future way to exploit the anticipated results is 
to use an assessment of BBB function, via MRI or blood-based markers, as a precision medicine 
tool allowing for identifying individuals with heightened risk and/or for targeting BBB-protective 
interventions specifically at individuals with impaired BBB function. Future follow-up work 
exploiting the anticipated results will require clinical efficacy trials. 
3.3.3 PATIENT INVOLVEMENT 
 
The DynaM-OBS study, on which the current study design is based, has been designed with the 
participation of target group members (young student focus groups). In another sub-study of the 
ERA-NET Neuron project PHASR-PP (qualitative research project on ethical, legal, and societal 
aspects (ELSA) of pharmacological augmentation of stress resilience, guided by LU), we will 
conduct stakeholder interviews with representatives of European patient, patient family, and 
student organizations, in order to obtain a broader picture of needs of at-risk individuals and 
families, including from different national and cultural perspectives. For this purpose, we will 
collaborate with umbrella organizations representing organizations across Europe to recruit 
interviewees, such as EPF, MHE, ESU, EUFAMI. Further, participants of the current study will be 
asked to give consent to be contacted for qualitative interviews by LU and to make their study 
data available to the ELSA study team. 
 
3.3.4 STRATEGIES FOR DATA HANDLING AND THE DISSEMINATION OF RESULTS 
 
Data collection and data management will be conducted in compliance with the principles of the 
declaration of Helsinki (1996) and relevant national and regional regulations and will only follow 
study protocols approved by the regional ethics committee at each partner site/country. The 
investigator at each partner site will be responsible for data management in accordance with these 
principles. The PIs are further responsible for keeping records of experiments and data collection 
in line with good laboratory practice. 
We will collect and process MRI imaging data (digital: DICOM), biosamples (blood), biological 
data from the analysis of biosamples (digital: concentrations of plasma proteins/peptides, RNAs, 
metabolites, and other molecules), and computerized (digital: offline and online collection) and 
paper-and-pencil (analogue) questionnaire and interview data. Data will be made findable with a 
study description and metadata following fairsharing.org conventions. Data will be made 
interoperable by using common standardized formats (in particular, BIDS for MRI) and a detailed 
codebook. Open accessibility and data re-use are complicated by the sensitive personal nature 
of most data and the principled non-anonymizability esp. of MRI data. Participant consent forms 
will permit sharing of pseudonymized data and biosamples among the sites and with collaborators 
within the European Union and countries with an adequacy decision under Article 45 GDPR, 
under the supervision of a Data Use and Access Committee (DUAC). All anonymized data will be 
made openly accessible through public depositories (in particular, OSF) upon publication or at 
latest two years after the termination of the project and will remain accessible for at least ten years 
upon completion of the study. Data can be accessed and processed, for example, in R and 
Python, which are open-source tools that permit extensive documentation. 
In general, data storage will be locally for raw data and on central data storage facilities for 
processed and quality-assured data. Online monitoring data will be collected on the secured 
SoSci Survey platform and exported directly to a secured server located at, and administered by, 
LIR. Biosamples will be shipped by courier to UM (Biobank) for central analysis. Full data security 
is assured by the participating institutions. 
Results will be disseminated through publication in peer-reviewed English-language scientific 

15 
 
journals. 
Resources are covered by the LIR, UM, UZH, UNIWARSAW, and LU (PhD students, 
infrastructure, staff) after project end. 
 
3.3.5 GENDER ASPECTS 
 
Because of the potential impact of gender, a comparable number of persons of the two dominating 
genders (male and female) in the study sample is desirable. Further, with the proportion of 
persons identifying as non-binary among younger individuals in European countries now reaching 
an 
approximate 
4% 
(https://www.ipsos.com/sites/default/files/LGBT%20Pride%202021%20Global%20Survey%20R 
eport%20%20US%20Version.pdf), representation of persons of non-binary gender appears 
necessary. We therefore plan to recruit at least 45% of participants of either male or female 
gender, leaving the remaining 10% to any gender, while explicitly also addressing non-binary 
persons in the recruitment campaign. 
 
 

16 
 
 
 
3.4.1 CONTROL(S) / COMPARATOR(S) 
 
Since the pharmacological experimental manipulation is not employed in order to test or achieve 
a clinical effect, placebo comparison is ethically acceptable. 
 
3.4.2 DOSE, MODE AND SCHEME OF INTERVENTION 
 
We have no specific information available on the optimal dose of metformin to achieve BBB 
effects. We therefore rely on experiences with the use of metformin for current indications (esp. 
diabetes), where dosages range from 250 to 3000 mg/day over months or years36,37. 850 mg twice 
daily, administered daily, is an average dose used for the treatment of Diabetes mellitus type 2. In 
a comparable setting (safety and tolerability of metformin for treatment of amnestic mild cognitive 
impairment), most participants tolerated dosages between 1000 mg and 1500 mg a day15. Similar 
regimens are used in trials of metformin in aging-related diseases. These data define a safe range, 
which to exploit enhances chances of finding an effect (sensitivity criterion). At the same time, it 
needs to be considered that our study participants are formally mentally healthy and that the 
motivation to participate and the adherence during the study may be negatively affected by a high 
dose (feasibility criterion). Minimization of participant burden is a further relevant aspect (safety 
criterion). In this trade-off, we choose a daily oral dose of 1700 mg (850 mg BID with meals) during 
the full-dose treatment (weeks 5 to 12) that is still well in the range of current dosage regimens 
and also well below the maximum recommended dose of 3000 mg, and we restrict administration 
to three months overall. We further opt for an initial titration starting comparably low at 500 mg 
daily the first two weeks, followed by 1000 mg daily during the third and fourth weeks.  
Metformin is readily available as a generic drug. 
 
3.4.3 ADDITIONAL TREATMENTS 
 
Symptomatic treatment is allowed as long as there are no known interactions with metformin as 
documented in PSIAC. For other, especially for excluded, treatments see in-/exclusion criteria. 
3.4.4 INCLUSION / EXCLUSION CRITERIA 
 
Inclusion criteria: (participants meeting all of the following criteria will be considered for enrolment 
in the study) 
[1] 
Absence of mental disorder diagnosis. 
[2] 
University students. 
[3] 
GHQ-28 > 20 
[4] 
Three or more adverse life events acc. to LE list in the past 
[5]   Beck Depression Inventory (BDI) ≤ 14 & Columbia-Suicide Severity Rating Scale 
(C-SSRS) ≤ 1. Thereby concurrent depression and suicidality are excluded. 
[6] 
Age 18 to 25 years 
[7] 
Ability of participant to understand character and individual consequences of the study 
(MMSE Folstein > 28) 
[8] 
Signed and dated informed consent of participant 
 
Exclusion criteria: (participants presenting 1 of the following criteria will not be enroled in the study) 
[1]   Life-time and current diagnosis of any severe mental disorder determined by M.I.N.I. diagnostic 
interview. 
[2]   Known history of brain injuries or neurodevelopmental disorder. 
[3]   Evidence of neurodegenerative disorder (e.g., Parkinson). 
[4]   Multimorbidity or significant organ (esp. liver or renal) dysfunction or manifest diabetes or 
substance abuse (esp. alcohol). 
3.4 
JUSTIFICATION OF DESIGN ASPECTS 

17 
 
[5]   
Contraindication to metformin such as renal insufficiency (Creatinin-Clearance< 60ml/min), 
recent (<3 month) ischemic events (e.g. myocardial infarction or stroke). 
[6]   
Women of childbearing age, who do not practice a medically accepted contraception (i.e., 
systematic contraceptives, diaphragm, condoms with spermicide, sexual abstinence) 
during the study and during a 2 years post-study period and who do not present a negative 
pregnancy test (serum or urine). 
[7]   
History of hypersensitivity to the study drug, to any drug with similar chemical structure, or 
to any excipient present in the pharmaceutical form of the study drug. 
[8]   Diabetes type 2 (would result in interference with the experimental manipulation) 
[9]   Participation in other studies employing a drug during the present study or within the last 
three months. 
[10] Current use of antidiabetic, weight-loss, or psychoactive medication or substances. 
[11] Pacemaker, implanted medical pumps, implanted cardiac catheters or acute or unstable 
heart disease (angina pectoris). 
[12] Intracranial implant (aneurysm clips, shunts, stimulators, cochlear implants or electrodes) 
or other metallic objects inside or near the head (mouth excluded) that cannot be removed. 
[13] Claustrophobia or another contraindication to MRI. 
[14] Insufficient German language skills. 
3.4.5 MEASURES 
 
Determination of primary, secondary, and tertiary measures 
Primary endpoint/ outcome measure 
 Relating to O1a): Average stressor reactivity (SR) score in study phase ‘early follow-up’ 
(time points T4-T6): SR(T4-T6) 
Secondary endpoints/ outcome measures 
 Relating to O1b): Whole-brain BBB permeability, assessed with neuroimaging, at time point 
T3 at end of experimental manipulation: BBB(T3). 
 Relating to O1c): SR(T4-T6) 
 
Tertiary endpoints/ outcome measures 
 Relating to O2a): Average stressor reactivity (SR) score in study phase ‘whole follow-up’ 
(time points T4-T9): SR(T4-T9) 
 Relating to O2b): SR(T4-T9) 
 
Calculation of SR score 
To build the SR score, first, the stressor exposure score E is calculated for every four-weekly 
monitoring time point T1 to T9 as the z-scored sum of occurrences of all daily hassles (DHs), 
assessed with the MIMIS48. Life event (LE) occurrence, assessed with a life-events list48, is 
expected to be less frequent than DH occurrence but, if reported, to correlate with DH occurrence 
(see above). This will be used to corroborate the validity of the DH-based E score. For each 
monitoring time point, a mental health problem score P is also calculated. Unlike in the previous 
studies, we replace the measurement of internalizing symptoms with the GHQ-28 with the shorter 
(16 item) Patient Health Questionnaire – Anxiety and Depression Scale (PHQ-ADS)49. The PHQ-
ADS is better suited for repeated measurements than the GHQ. It has been successfully used by 
us in four studies of the EU RESPOND consortium50–53. Its sum score provides a composite 
measure of depression and anxiety. P i the z-standardized sum score. 
To quantify how strongly participants’ mental health problems relate to stressor exposure, we then 
calculate the normative relationship between E and P in the per-protocol analysis sample for each 
endpoint by fitting a linear mixed model to predict P by E over all included participants and 
monitoring time points Tx, with random slopes and intercepts for participants. The E-P regression 
line is then determined by the fixed effect estimates for the E~P slope and intercept and serves 
as the normative E-P relationship for the analysis sample over the study phase of interest. The 
form of regression that explains most variance in P (linear or quadratic) will be used, as in prior 
work54–56. See also Figure 2. Based on experiences in DynaM-OBS, which has a comparable 
monitoring frequency and study duration in a comparable sample (see above), we expect an E-P 

18 
 
correlation of about r=0.27 to 0.43. Notably, exclusive reliance on a DH-based E score has been 
feasible in DynaM-OBS, with adding a measure of the (rare) LE occurrence not relevantly 
improving variance explanation (unpubl.).  
On this basis, at each Tx, we enter participants' individual E scores into the normative E~P line 
equation, giving us their expected P score when assuming normal stressor reactivity. The SR 
score is the individual’s average residual onto the regression line for the study phase of interest. 
 
Calculation of whole-brain BBB permeability 
A diffusion-prepared pseudo-continuous ASL (pCASL) MRI sequence will be used to 
estimate BBB permeability. By reading out pCASL signals at different post-labeling delays 
(PLD) under different diffusion gradients, perfusion signals in capillary and brain tissues 
can be reliably estimated and subsequently fitted into a model to estimate the water 
exchange rate (kw) from blood to brain tissue, an index quantifying BBB permeability. 
Analysis will use a toolbox provided by the sequence developer (http://loft-lab.org/index-
5.html) and includes image reconstruction, preprocessing (motion-correction, co-
registration, skull-stripping), and kw modeling. Whole-brain averaged kw values quantifying 
overall BBB permeability will be calculated. 
 
Additional measures 
For inclusion of covariates, socio-demographic and psycho-social variables will be 
assessed via questionnaire and scored acc. to standard procedures. For additional 
exploratory analyses, blood samples will be taken and used to determine blood-based 
molecular and cellular markers of immune system, BBB, and metabolic function (including 
proteins, peptides, RNAs, metabolites, HBA1C levels). 
 
3.4.6 METHODS AGAINST BIAS 
 
Randomization and blinding: Experimental manipulation groups are completely masked (double-
blind study). A randomization list will be generated by the respective study center. The 
randomization ratio will be 1:1. One copy of the randomization list will be sent to the pharmacy of 
the respective site. At the randomization visit (T0), each participant eligible for study participation 
will receive the next consecutive randomization number from a block of randomization numbers 
per site. The randomization list will be kept in safe and confidential custody at each study center 
site. In addition to the study drug, the investigator will receive a set of sealed envelopes, one for 
each randomization number. These envelopes contain information on each participant’s study 
drug and are to be opened only under circumstances in which it is medically imperative for 
diagnostic or therapeutic decisions to know what the participant is receiving. 
 
3.4.7 PROPOSED SAMPLE SIZE / POWER CALCULATIONS 
 
Because the association of BBB function in stressor-exposed individuals at risk for stress-related 
mental health problems with stress resilience (inverse SR score) has never been investigated, we 
base our power calculation for the primary endpoint on the sensitivity to detect a medium effect 
size of Cohen’s d = 0.3. With a power of 0.8, a two-sided alpha-level of 0.05, and a linear mixed 
model with eight predictor variables (3 variables of interest and 5 covariates), we need n=109 
participants. 
 
Compliance / Rate of loss to follow up 
Taking into account 10% attrition, we aim to recruit a total number of 122 participants (61 in each 
group). Adherence to online monitoring at a given online monitoring time point (T0-T9) will be 
based on a criterion of full completion of the DH and PHQ-ADS lists with reasonable scores (e.g., 
not all item scores in a list identical or zero) at that time point. Responses at online monitoring 
time points not fulfilling this criterion will be considered incomplete (missing). Adherence to the 
experimental manipulation will be based on a participant diary (paper and pencil) and the rendition 
of study drug blisters after the end of the experimental manipulation at study visit 2 (T3). 
In order to promote adherence, a step-wise reimbursement scheme is implemented. Participants 
receive a first reimbursement of max. 90 € after study visit 1 (50 € for participation in the medical 

19 
 
exam and online questionnaires, 10 € specifically for blood draws, 30 € for neuroimaging), max. 
290 € after study visit 2 (50 € for medical exam and online questionnaires, 10 € for blood draws, 
30 € for neuroimaging, 200 € for study drug administration, where 10% missed administrations 
are allowed), and max. 240 € after study visit 3 (50 € for medical exam and online questionnaires, 
10 € for blood draws, 180 € for online monitoring of stressor and internalizing symptoms, the latter 
proportional to completion). The total possible reimbursement is 620 €. 
3.4.8 FEASIBILITY OF RECRUITMENT 
 
We distribute participant recruitment across three different countries (D, CH, PL), since this will 
increase the heterogeneity of the study sample compared to a study in only one country and, 
consequentially, increase the likelihood that the results are generalizable (not only valid in a 
specific population). The recruiting centers UZH, UM, UNIWARSAW are all part of the former 
DynaMORE consortium and have proven to recruit participants from the target population 
(university students) in a variety of studies such as DynaM-OBS (UM n=39 participants; 
UNIWARSAW n=53 participants), DynaM-INT (UM n=27 participants; UNIWARSAW n=62 
participants), and ecological momentary intervention trials (UZH many participants), each within 
periods of 12-18 months, including despite complications related to the COVID-19 pandemic. 
3.4.9 INTERNATIONAL COLLABORATIONS 
 
University Warsaw, Poland; University Zurich, Switzerland; University of Latvia, Latvia.  
 
It is possible that the participant will be contacted during or after the study by the cooperation partner 
University of Latvia (LU) with the request to participate in interviews in which they will be asked 
about their opinions and attitudes towards the study topic. This will take place as part of another, 
separate study of the PHASR-PP project (Ethical, Legal, and Societal Aspects project part). 
Participation in the current study does not obligate participants to take part in this separate study. 
 
3.4.10 STOPPING RULES 
 
Discontinuation criteria are 
a) participant may discontinue participation due to any of the following reasons: 
- 
at their own request or at request of the legal representative 
- 
any medical condition demanding the immediate discontinuation of (potential) 
metformin treatment, e.g., need for urgent anaesthesia, renal failure etc. 
- 
for safety reasons at the request of the investigator or request of a regulatory agency 
- 
significant AEs related to the experimental manipulation (participants will be followed 
up for manipulation response and safety) 
- 
participant is non-compliant or not sufficiently compliant with the study procedures / 
study protocol 
- 
if, in the investigator’s opinion, continuation of the study would be detrimental to the 
participant’s well-being. 
- 
participant needs a medication not allowed in the protocol during the study 
- 
any clinically significant change in participant’s pre-study medical condition 
b) for the following reasons, a study site may be closed at the discretion of the investigator: 
- 
medical or ethical reasons that are detrimental to the continued performance of the 
study 
- 
difficulties in the recruitment of participants (i.e., which fail to include at least a third of 
estimated participants to be included by mid of the recruitment period (6.5 months after 
last participant-in)) 
- 
critical protocol violations 
- 
violations of legal and ethical regulations 
- 
non-compliance of the study site investigators 
c) for the whole study at the discretion of the investigator: 
- 
new risks for participants become known 

20 
 
- 
occurrence of AEs unknown to date in respect of their nature, severity, and duration 
or the unexpected increase in the incidence of known AEs 
- 
medical or ethical reasons that are detrimental to the continued performance of the 
study 
- 
difficulties in the recruitment of participants 
 
 

21 
 
 
Analysis populations 
The primary analysis population will be the intention-to-treat (ITT) population (all participants who 
signed informed consent and were randomized). Secondary analyses will be conducted in the 
per-protocol population (ITT population without major protocol violations, not further described).  
No interim analysis will be performed. 
There is no replication sample. 
 
Significance testing 
All hypotheses will be tested on a two-sided level of significance α=0.05. The primary hypothesis 
(primary analysis relating to the primary objective O1a) will be tested separately and without 
correction for multiple testing. Correction for multiple testing will be applied family-wise to the key 
secondary hypotheses (two key secondary analyses relating to the primary objectives O1b and 
O1c), provided that the conditions to test the key secondary hypothesis relating to objective O1c 
are met. 
 
Method 
All analyses will be conducted by means of linear mixed models. Depending on the pattern and 
extent of missing data, we will consider imputation approaches to ensure sufficient analysis 
power, especially in the analysis of SR at follow-up periods. We will perform autoregression-
informed imputation, given that sufficient autocorrelation is present and if there is no evidence for 
non- randomly missing data. 
Age, gender, site, and BMI will be used as covariates in all analyses. Selection of further 
covariates will follow a procedure used in earlier studies55–57 such that all potential covariates with 
a p value <0.2 in univariate regression models on the endpoint will be included. We expect this to 
result in the selection of one additional covariate, most likely childhood trauma. Hence, in total, 
we expect to use five covariates. 
 
Primary analysis 
Relating to O1a): To test for a prospective association of whole-brain BBB permeability with 
average SR at T4-T6 during early follow-up (SR(T4-T6)), a linear mixed model will be used to 
regress BBB permeability at baseline (BBB(T0)), BBB permeability at end of experimental 
manipulation (BBB(T3)), experimental manipulation (EM; groups metformin and placebo), and 
covariates onto (SR(T4-T6)). 
 
Key secondary analyses 
Relating to O1b): To test for an effect of the experimental manipulation between time points T0 
and T3 (groups metformin and placebo) on participants’ whole-brain BBB permeability at time 
point T3 at end of experimental manipulation (BBB(T3)), a linear mixed model will be used to 
regress EM, BBB(T0), and covariates onto BBB(T3). 
 
Relating to O1c): If BBB(T3) shows a significant association with SR(T4-T6) (see O1a) and if 
EM shows a significant association with BBB(T3) (see O1b): To test for mediation by the effect 
of the experimental manipulation on participants’ whole-brain BBB permeability at time point T3 
at end of experimental manipulation on participants’ average stressor reactivity at time points T4-
T6 during early follow-up, mediation analysis will be conducted using a Baron-Kenny approach 
with EM as predictor, BBB(T3) as mediator, SR(T4-T6) as outcome, and BBB(T0) and earlier 
covariates as covariates. 
 
Further secondary analyses 
Relating to O2a): To test for a prospective association of whole-brain BBB permeability with 
average SR at T4-T9 during whole follow-up (SR(T4-T9)), a linear mixed model will be used to 
regress BBB(T0), BBB(T3), EM, and covariates onto SR(T4-T9). 
 
3.5 
STATISTICAL ANALYSES 

22 
 
Relating to O2b): If BBB(T3) shows a significant association with SR(T4-T9) (see O2a) and 
if EM shows a significant association with BBB(T3) (see O1b): To test for mediation by the 
effect of the experimental manipulation on participants’ whole-brain BBB permeability at time point 
T3 at end of experimental manipulation on participants’ average stressor reactivity at time points 
T4-T9 during whole follow-up, mediation analysis will be conducted using a Baron-Kenny 
approach with EM as predictor, BBB(T3)  as mediator, SR(T4-T9) as outcome, and BBB(T0) and 
earlier covariates as covariates. 
 
Safety 
All summaries and listings of safety data will be performed for the safety population. Summary 
tables of AEs will present the number of participants observed with AEs and corresponding 
percentages. Laboratory values and vital signs will be analyzed by descriptive methods and 
exploratory p-values.

23 
 
 
 
Risks and benefits 
Individual participant risks are mainly associated with the potential adverse effects (AEs) of the 
study drug metformin, i.e., nausea, vomiting, stomach upset, diarrhea, weakness, or a metallic 
taste in the mouth may occur. In case of metformin accumulation lactic acidosis is possible as 
serious AE. There are no immediate benefits of this study for the individual participants aside from 
participant reimbursement. Participants will receive a reimbursement of 620€. 
 
Care and protection for research participants 
During the conduct of the study, the participant should not undergo clinical treatments except for 
cases of emergency. The participant is bound to inform the investigator immediately about any 
AEs and additionally drugs taken. According to Sect 40 AMG, the investigator is obliged to take 
out a participant insurance, which covers, in its terms and provisions, the legal liability for injuries 
caused to participating persons and arising out of this research performed strictly in accordance 
with the scientific protocol as well as with applicable law and professional standards. The terms 
and conditions of the insurance will be delivered to the participant at the time of informed consent. 
Insurance provisions for this study are given in separate agreements. 
 
Good clinical practice 
The procedures set out in this study protocol, pertaining to the conduct, evaluation, and 
documentation of this study, are designed to ensure that all persons involved in the study abide 
by good clinical practice (GCP) and the ethical principles described in the Declaration of Helsinki. 
The study will be carried out in keeping with local legal and regulatory requirements. The 
requirements of the AMG, the GCP regulation, and the Federal Data Protection Law (BDSG) will 
be kept. 
 
Patient information and informed consent 
Before being admitted to the study, the participant must consent to participate after being fully 
informed about the nature, scope, and possible consequences of the study. The documents must 
be in a language understandable to the participant and must specify who informed the participant. 
A copy of the signed informed consent document must be given to the participant. The original 
signed consent document will be retained by the investigator. The investigator will not undertake 
any measures specifically required only for the study until valid consent has been obtained. If the 
participant has a primary physician and if the participant agrees to the primary physician being 
informed, the investigator should inform the participant’s primary physician about the participant’s 
participation in the study. After reading the informed consent document, the participant must give 
consent in writing. The participant's consent must be confirmed by the personally dated signature 
of the participant and by the personally dated signature of the person conducting the informed 
consent discussions. 
 
Confidentiality 
The name of the participants and other confidential information are subject to medical professional 
secrecy and the regulations of the EU and German Data Protection Acts (EU-DSGVO and 
Bundesdatenschutzgesetz). During the study, participants will be identified solely by means of an 
individual identification code (e.g., participant number, randomization number). Study findings 
stored on a computer will be stored in accordance with local data protection law and will be 
handled in strictest confidence. For protection of these data, organizational procedures are 
implemented to prevent distribution of data to unauthorized persons. The appropriate regulations 
of data legislation will be fulfilled in its entirety. The participant will declare in the written consent 
to release the investigator from the medical professional secrecy to allow identification of 
participant’s name and/or inspection of original data for monitoring purposes by health authorities 
and authorized persons (monitors). The investigator will maintain a personal participant 
identification list (participant numbers with the corresponding participant names) to enable records 
to be identified. 
3.6 
ETHICAL CONSIDERATIONS 

24 
 
 
3.7.1 QUALITY ASSURANCE / MONITORING 
 
Requirements for investigational sites and staff 
The investigator should be able to demonstrate (e.g., based on retrospective data) a potential for 
recruiting the required number of suitable participants within the agreed recruitment period. The 
investigator should have sufficient time to properly conduct and complete the study within the 
agreed study period. The investigator should have available an adequate number of qualified staff 
and adequate facilities for the foreseen duration of the study to conduct the study properly and 
safely. The investigator should ensure that all persons assisting with the study are adequately 
qualified, informed about the protocol, any amendments to the protocol, the study drug, and their 
study-related duties and functions. 
 
Direct access to source data/documents 
The investigator/institution must permit study-related monitoring and auditing by the study-
external monitor, as well as inspections by the appropriate competent authorities and Ethics 
committees, providing direct access to source data/documents (confidentiality see 3.6). 
The participants will be informed that representatives of the investigator, independent ethics 
committees (IEC), or competent authorities may inspect their medical records to verify the 
information collected, and that all personal information made available for inspection will be 
handled in strictest confidence and in accordance with local data protection laws. 
 
Investigator site file and archiving 
The investigator will be provided with an investigator site file (ISF) at the start of the study. The 
investigator will archive all study data and relevant correspondence in the ISF. The ISF, all source 
data, and all documents will be kept filed according to the requirements of the ICH-GCP guidelines 
after termination of the study. It is the responsibility of the investigator to ensure that the participant  
identification sheets are stored for at least 15 years beyond the end of the study. All original 
participant files must be stored for the longest possible time permitted by the regulations at the 
hospital, research institute, or practice in question. 
 
Monitoring 
Monitoring will be done by personal visits from a study-external monitor. Pre-study visits will be 
conducted, and the reports will be reviewed by the investigator. To initiate the study, the 
monitor will visit all participating local study sites and study centers. The monitor shall ensure 
that the investigator and their staff understand all requirements of the protocol and their regulatory 
responsibilities. The monitor will ensure that the investigator will maintain a list of sub-investigators 
and other appropriately qualified persons to whom he or she has delegated significant study-
related duties (personnel log). Each site will be visited by the monitor at regular intervals to ensure 
compliance with the study protocol, GCP, and legal aspects. The monitor will review the entries 
into the CRFs for completeness and correctness and verify the entries on the basis of the source 
documents. The presence of correct informed consents will be checked for every participant. 
Details will be specified in the monitoring manual for this study. The investigator must allow the 
monitor to look at all relevant documents and must provide support at all times to the monitor. By 
frequent communications (letters, telephone, fax), the monitor will ensure that the study is 
conducted according to the protocol and regulatory requirements. 
 
Inspection by authorities and ethic committees 
Competent authorities and by the investigator authorized persons (auditor) may request access to 
all source documents, CRF, and other study documentation in case of an inspection or audit. 
Direct access to these documents must be guaranteed by the investigator who must provide 
support at all times for these activities. Source data documents can be copied during inspection 
or audit in case the identity of the participant have been made unrecognizable. 
 
 
3.7 
QUALITY ASSURANCE, SAFETY AND MANAGEMENT STRUCTURE 

25 
 
Audits 
No audits are planned for this study. 
3.7.2 SAFETY / PHARMACOVIGILANCE 
 
Assessment of safety 
Adverse Event (AE) definition will be used according to GCP: an AE is defined as any untoward 
medical occurrence in a participant treated with a pharmaceutical product and which does not 
necessarily have a causal relationship with this treatment. An AE can therefore be any unfavorable 
and unintended sign (including an abnormal laboratory finding), symptom, or disease temporally 
associated with the use of a study drug, whether or not related to that drug. 
 
Assessment of AEs by investigator 
Participants must be carefully monitored for AEs by the investigator at each study visit. The intensity 
of the AEs and the causal relation to the study drug and/or procedures are to be assessed. 
 
Period of observation 
In this study, the period of observation for collection of AEs extends from the time the participant 
has signed the informed consent document up to the end of the 24 weeks follow-up period. If the 
investigator detects a serious AE in a study participant after the end of the period of observation, 
and considers the event possibly related to the prior study, they should contact the data and 
safety monitoring board (DSMB) to determine how the AE should be documented and reported. 
 
Documentation of AEs and Follow-up 
All AEs reported by the participant or detected by the investigator will be documented on the 
appropriate pages of the case report form (CRF). All participants who have AEs, whether 
considered associated with the use of the study drug or not, must be monitored to determine the 
outcome. 
 
Immediate reporting of Serious AEs (SAEs) by investigator 
SAEs must immediately (within 24 hours of the investigator’s awareness) be reported to the DSMB. 
The initial SAE Report should be as complete as possible including the essential details of 
participant’s identification (screening number, random number), the SAE (medical term, 
diagnosis), the study drug, and the assessment of the causal relationship between the event and 
the study drug. The SAE report must be reviewed and signed by the investigator. The investigator 
should provide related additional information on the clinical course and the outcome of each SAE 
as soon as possible (Follow-up report). The “Serious Adverse Event Form” is provided in the 
investigator site file (ISF). The investigator should also inform the study monitor in all cases. 
 
Immediate Reporting of pregnancy by investigator 
Any pregnancy diagnosed in a female participant or in the female partner of a male participant 
during the experimental manipulation must be reported immediately using the “Pregnancy 
Reporting Form” (provided in ISF) to the DSMB. 
 
Safety evaluation and Reporting by investigator 
The investigator will ensure that all legal reporting requirements are met. The investigator is 
responsible for the continuous safety evaluation of the study drug and the study. The investigator 
will conduct the management of SAEs and the expedited reporting as required by GCP regulation 
(GCP-V). Suspected unexpected serious adverse reactions (SUSARs) and safety issues as 
defined by GCP-V are determined for expedited reporting: The ethics committees should be 
notified as soon as possible but not later than 15 calendar days if the event is non-fatal and 7 
calendar days if it was fatal. All co-investigators and sub-investigators will be informed, too. Work 
flow and procedures concerning SAE management will be described in a separate document (e.g., 
Safety manual). During the study, the investigator will submit the annual safety report including a 
list of all serious adverse reactions to the ethics committee(s) once a year. 
 

26 
 
Emergency procedures 
During and following a participant’s participation in the study, the investigator should ensure that 
adequate medical care is provided to a participant for any AEs including clinically significant 
laboratory values. The investigator should inform a participant when medical care is needed for 
intercurrent illness(es) of which the investigator becomes aware. Emergency treatment for 
overdose will be described. Emergency Unblinding: If it is medically imperative to know what study 
drug the participant is receiving, the investigator or authorized person should open the 
randomization envelope. The investigator or the person who breaks the blind must record the 
date and the reasons for doing so in the CRF, in the participant’s medical record and on the 
randomization envelope. Unblinding within the scope of emergency treatment by third parties: For 
this double-blind pharmacological study, a 7 days/24 hours emergency unblinding will be 
established (for instance in case of an emergency hospitalization into an external hospital). It will 
be possible to contact the operator of the switchboard of UM Mainz and the on-call duty of the UM 
Pharmacy to organize such an emergency unblinding. 
 
Other safety data 
All observations pertinent to the safety of the study medication will be recorded on the CRF and 
included in the final report. Additionally to the registration of the AEs and serious AEs, we 
conduct further assessments of safety at every visit. 
 
Prior and concomitant illnesses 
Relevant additional illnesses present at the time of informed consent are regarded as concomitant 
illnesses and will be documented on the appropriate pages of the case report form (CRF). 
 
Prior and concomitant treatments 
Relevant treatments administered to the participants on entry to the study or at any time during the 
study are regarded as concomitant treatments and must be documented on the appropriate pages 
of the CRF. 
 
3.7.3 MANAGEMENT STRUCTURE AND PROCEDURES 
 
An independent data and safety monitoring board (DSMB; see list of proposed members) shall 
be established to supervise the progress of the study and to review relevant information from other 
sources to advise whether to continue, modify, or stop the study and provide the funding 
organizations with information and advice. The DSMB will meet twice: at mid of the recruitment 
period and end of the recruitment period. To monitor safety data and to ensure adherence to 
protocol will be responsibility of the study monitor. 
 
3.7.4 REGISTRATION 
 
The 
study 
will 
be 
registered 
with 
clinicaltrial.gov. 
 
 
 
 
 
 
 
 
 
 
 
 
 

27 
 
 
 
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55. Bögemann, S. A. et al. Psychological Resilience Factors and Their Association With Weekly 
Stressor Reactivity During the COVID-19 Outbreak in Europe: Prospective Longitudinal 
Study. JMIR Ment. Health 10, e46518 (2023). 
56. Zerban, M. et al. What helps the helpers? Resilience and risk factors for general and 
profession-specific mental health problems in psychotherapists during the COVID-19 
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lockdown. Transl. Psychiatry 11, 67 (2021). 

30 
 
Preparation
(incl. 
protocol, SOPs, 
site visits, 
registration), 
start 
recruitment 
M14:
last 
part. in
First part. in to last part. out: 18 months (M06-M23) 
 
 
 
Year 1 
 
 
 
Year 2 
 
 
 
Year 3 
 
1
4
7
10
1
4
7
10
1
4
7
10
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
3.9 
STUDY TIME FLOW 
Data acquisition and preprocessing (M06-M27)
Data analysis 
(M28-M32) 

31 
 
 
 
 
 
 
Study Management
#
Name
Affiliation
Responsibility / Role
Signature
1  Prof. Dr. Oliver Tüscher
 
Dept. of Psychiatry, 
UM, and LIR 
Applicant, 
Investigator 
 
 
Study Statistician
#
Name
Affiliation
Responsibility / Role Signature
2 
Dr. Kenneth Yuen 
Neuroimaging 
Center, UM 
Statistical advice 
 
Study Supporting Facilities (reference laboratories, pharmacies etc.)
#
Name
Affiliation
Responsibility / Role
3
Prof. Dr. Irene Krämer
Hospital Pharmacy, 
UM
Preparation of study drug
2
Dr. Kenneth Yuen
Neuroimaging 
Center, UM
Neuroimaging
4 
Dr. Susanne Englisch 
Studienzentrum 
Psychiatrie, Dept. of 
Psychiatry, UM 
Study center services 
5 
Prof. Dr. Philipp Wild 
CTH Biobank, UM 
Biobank services 
6 
Prof. Dr. Karl Lackner 
Inst. f. Laboratory 
Medicine, UM 
Blood sample analyses 
 
Recruiting centers
#
Name
Affiliation
No. of participants to be recruited 
1 
Prof. Dr. Oliver 
Tüscher 
Dept. of Psychiatry, UM, 
Mainz, Germany 
36-46 
7 
Dr. Flurin Cathomas 
Dept. of Psychiatry, 
Psychotherapy and 
Psychosomatics, UZH, 
Zurich, Switzerland 
36-46 
8 
Dr. Dorota Kobylinska
Faculty of Psychology, 
UNIWARSAW, Warsaw, 
Poland 
36-46 
Total sum of participants to be recruited                                     Σ = 122 
Data and Safety Monitoring Board (DSMB)
#
Name
Affiliation (only institution and city, no complete address)
9
Prof. Dr. Henrik Walter Dept. of Psychiatry and Psychotherapy, Charité Universitätsmedizin
Berlin
10 Prof. Dr. Dominik 
Bach
Dept. of Psychiatry and Psychotherapy, University Hospital Bonn
11 Prof. Dr. Helen 
Morrison
Leibniz Institute on Aging – Fritz Lippmann Institute, Jena
Other participating groups / bodies
#
Name
Affiliation
Responsibility / Role
12 
Prof. Dr. Raffael 
Kalisch 
LIR and Neuroimaging 
Center, UM 
Co-Investigator 
3.10 
LIST OF PARTNERS INVOLVED IN THE STUDY 

32 
 
13 
Prof. 
Dr. Signe 
Mezinska 
Faculty 
of Medicine 
and Life Sciences, LU 
Co-Investigator 
14 
Prof. 
Dr. Susann 
Schweiger 
Dept. of Human 
Genetics, UM, and LIR 
Project Partner 
15 
Prof. 
Dr. Birgit 
Kleim 
Inst. of Psychology, UZH Project Partner 
Review of study protocol (who will review and finalize the protocol? Please refer to numbers 
above and / or include others) 
#
Name
Affiliation (only institution and city, no complete address)
1 
Prof. Dr. Oliver 
Tüscher 
Dept. of Psychiatry, UM, and LIR 

33 
 
 
 
3.11.1 COMMERCIAL INTEREST 
 
Metformin is a generic drug and this study is not an efficacy trial. There is, hence, no commercial 
interest of a company to sponsor this study. 
 
3.11.2 FINANCIAL SUMMARY 
3.11 
FINANCIAL DETAILS OF THE STUDY 

34 
 
ERA-NET NEURON Call 2023 
Pre-proposal: Budget plan of the project 
 
Project Acronym: PHASR-PP 
 
Coordinator 
Partner 2 
Partner 3 
Partner 4 
Partner 5 
Partner 6*
 
Name (group leader) 
Kalisch 
Tüscher 
Cathomas 
Kobylinska 
Mezinska 
N.A. 
 
Institution 
LIR 
UM 
UZH 
UNIWARSAW 
LU 
 
 
Country 
Germany 
Germany 
Switzerland 
Poland 
Latvia 
 
 
Funding organisation 
BMBF (DFG) 
BMBF (DFG) 
SNSF 
NCBR 
LZP 
 
 
PROJECT COSTS (€) 
 
 
 
 
 
 
Total 
Personnel € 
169,600
0
224,458
(225,029 CHF)
53,617 (252,000
PLN)
139,800  
 
Consumables € 
0
28,600
24,905
(24,826 CHF)
10,000
(47,000 PLN)
0  
 
Equipment € 
0
0
0
0
3,000  
 
Travel €1 
10,800
2,400
10,800
(10,599 CHF)
10,800
(50,760 PLN)
10,800  
 
Other direct costs €2 
0
101,218
21,000
(20,609 CHF)
73,165 (343,876
PLN)
3,500  
 
Overheads €3 
16,960
26,444
0
23,104 (108,590
PLN)
39,275  
 
Total budget €4 
197,360
158,662
281,163
(281,064 CHF)
170,686
(802,225 PLN)
196,375  
1,004,246
Requested budget €4,5 
N.A.
N.A.
N.A.
N.A.
N.A.  
N.A.