Spermidine Anti-Hypertension Study
Phase: PHASE3
Conditions: Hypertension
Sponsor: Medical University of Graz

 
 
i 
 
Protovol (Version 3.1 – updated 23MAY2020) 
Spermidine Anti-Hypertension Study (SMARTEST) 
 
Effect of spermidine on arterial blood pressure in hypertensive patients 
EK-ID: ID: 30-468 ex 17/18 
 
Principal Investigator: 
Assoc. Prof. Dirk von Lewinski, MD  
 
Medical University of Graz  
 
Department of Cardiology 
 
Auenbruggerplatz 15, A-8010 Graz                  
 
Tel.: +43 316 385 80684 
    
 
 
 
Fax: +43 316 385 13733 
 
Co-investigators: 
Assoc. Prof. Simon Sedej, PhD 
 
Medical University of Graz  
 
Department of Cardiology 
 
Auenbruggerplatz 15, A-8010 Graz                 
 
 
 
Ass. Prof. Tobias Eisenberg, PhD 
 
Institute of Molecular Biosciences 
 
Humboldtstrasse 50/EG 
 
8010 Graz, Austria 
 
 
 
 
 
 
Mahmoud Abdellatif, MD  
 
Medical University of Graz  
 
Department of Cardiology 
 
 
 
 
 
Auenbruggerplatz 15, A-8010 Graz      
 
 
 
 
 
 
Markus Wallner. MD 
Medical University of Graz  
 
Department of Cardiology 
 
 
 
 
 
Auenbruggerplatz 15, A-8010 Graz      

 
 
1 
Contents 
1 
Background ........................................................................................................ 3 
 Rationale of the study ......................................................................................... 3 
1.1
 Objectives ........................................................................................................... 7 
1.2
 Outcomes............................................................................................................ 7 
1.3
1.3.1 24h-BPM .................................................................................................. 7 
1.3.2 Laboratory parameters ............................................................................ 7 
1.3.3 ECG ......................................................................................................... 8 
1.3.4 Echocardiography .................................................................................... 8 
1.3.5 Quality of Life (QoL) ................................................................................. 8 
2 
Trial design ......................................................................................................... 8 
 Trial design ......................................................................................................... 8 
2.1
 Schedule of evaluations ...................................................................................... 9 
2.2
 Trial population ................................................................................................... 9 
2.3
 Inclusion and exclusion criteria ......................................................................... 10 
2.4
 Data management / documentation .................................................................. 11 
2.5
 Sample size considerations .............................................................................. 11 
2.6
 Follow-up .......................................................................................................... 11 
2.7
 Project duration and milestones ........................................................................ 11 
2.8
3 
Methods............................................................................................................ 12 
 24h BPM ........................................................................................................... 12 
3.1
 Lab analysis ...................................................................................................... 12 
3.2
 Electrocardiography .......................................................................................... 13 
3.3
 Echocardiography ............................................................................................. 13 
3.4
 QoL assessment ............................................................................................... 13 
3.5
4 
Safety and risk management ............................................................................ 13 
 Adverse events and reactions ........................................................................... 14 
4.1

  
  
2
 Study discontinuation ........................................................................................ 14 
4.2
 Potential risks within the study design .............................................................. 14 
4.3
5 
Statistical considerations and data analysis ..................................................... 15 
6 
Ethical considerations and informed consent ................................................... 15 
7 
Data collection and quality assurance, Data management, Monitoring ............ 16 
 
 
 

  
  
3
1 
Background 
 
Rationale of the study 
1.1
Hypertension is the leading risk factor for cardiovascular disease and mortality1. Standard 
pharmacotherapy, albeit fairly effective, is far from being optimized and adequate blood pressure 
control can be maintained only in a limited number of hypertensive patients. In line with this clear 
medical need, we recently discovered that supplementation of the natural polyamine spermidine 
effectively delays the development of experimental hypertension and the associated heart failure2. 
Importantly, higher dietary intake of spermidine correlates with reduced blood pressure2, as well as 
decreased overall mortality3 in human cohort studies. That said, randomized clinical trials 
examining the anti-hypertensive effect of spermidine in humans are still lacking.  
Relevance to the international status of research in the field 
Despite the widely recognized benefits of reducing elevated blood pressure, hypertension is still 
the leading cause of many life-threatening cardiovascular diseases, such as stroke, coronary 
artery disease as well as heart and renal failure4, 5. The current prevalence of hypertension is 
devastating as the number of hypertensive individuals, i.e., systolic blood pressure (SBP) of 140 
mmHg or higher, has almost doubled between 1990 and 2015, and now impacts 874 million 
patients5. In fact, non-optimal systolic blood pressure is the major contributing risk factor to 
morbidity and all-cause mortality worldwide, causing a loss of 212 million healthy life years and 9.4 
million deaths annually6. Thus, hypertension poses a massive burden on both the public health and 
the economy, as it is the costliest cardiovascular disease7.  
This unprecedented prevalence of hypertension is influenced by the growing elderly population 
due to increased life expectancy. In addition, habitual risk factors, such as increased caloric and 
salt intake combined with a sedentary life-style, have facilitated=fostered the age-related surge in 
blood pressures. That being said, very few interventions have been successful in achieving 
extended and long-lasting control with any of these risk factors. For decades, studies have 
extensively searched for more effective interventions, but anti-hypertensive pharmacotherapy 
remains the most important means (I would rather say tool) for reducing high blood pressures. 
However, anti-hypertensive pharmaceuticals, even though being fairly effective and accessible at 
an affordable price, are far from ideal. Indeed, although a great majority (87.5%) of those 
diagnosed with hypertension receive regular pharmacological therapies, only a minority (32.5%) 
show controlled blood pressures (SBP < 140 mmHg), despite the fact that nearly one third of them 
already receive 2 or more drugs8. Using Austria as an example, controlled blood pressure is 
achieved in only 17% of hypertensive patients9 and data from other European countries reveals 

  
  
4
comparable results.  Therefore, further investigation is necessary to develop better therapies 
for managing hypertension and avoiding hypertension-related complications. 
In this regard, spermidine ─ a natural caloric restriction mimetic ─ delays the onset of hypertension 
in a strain of rats sensitive to salt intake2. Our initial goal of preventing these animals from 
developing fatal heart failure induced by severe hypertension led us to discover a direct effect of 
spermidine on hypertension2. This finding was further corroborated by an epidemiological study in 
humans (BRUNECK study), where we observed that higher dietary intake of spermidine correlated 
with reduced blood pressure and decreased cardiovascular events2. Interestingly, spermidine 
supplementation in elderly subjects coincided with a trend towards reduced blood pressure (a 
phase I safety trial)10. Even in the absence of hypertension, preclinical evidence supports vascular 
benefits of spermidine treatment, which was shown to reduce age-related vascular stiffness and 
endothelial dysfunction11.  
In general, the beneficial actions of spermidine are strongly linked to its ability to induce 
autophagy2, 11, 12, a key process that is indispensable for tissue homeostasis in both health and 
disease13. Based on the existing evidence, it is reasonable to hypothesize that inducing endothelial 
autophagy enhances vascular function and, thus, contributes to the reduction in blood pressure. 
Indeed, a causal implication of autophagy in the blood pressure-reducing effect of spermidine is 
currently being tested in our ongoing experimental studies.  
Besides boosting autophagy, spermidine has been shown to stimulate and improve immune cell 
functions in several pathophysiological settings12, 14-16. Given the accumulating evidence supporting 
a critical role of immune cells in the development of hypertension17 and cardiovascular disease 
(CVD)18, it is possible that spermidine also modulates elevated blood pressures via anti-
inflammatory and immune cell-specific functions.  
Another plausible and autophagy-independent mechanism through which exogenous spermidine 
may reduce high blood pressures is through increasing the bioavailability of L-arginine2, 19, a nitric 
oxide (NO) precursor. An increase in spermidine intake subsequently results in an increase in 
circulating and tissue levels of polyamines, which in turn decreases the de novo polyamine 
synthesis from their precursor L-arginine. Therefore, L-arginine can be primarily directed for 
increased production of the vasodilator NO.  
Inspite of the initial epidemiological indication that spermidine effect may differ between men and 
women2, spermidine – like caloric restriction and other caloric restriction mimetics (CRMs) – 
extends life span of several animal species in a sex-independent manner (for details – see Table 
S1 in this review12). More importantly, the first prospective trial administering a spermidine-rich 

  
  
5
wheat-germ extract to elderly people with subjective cognitive decline revealed no sex-based 
differences in any of the tested parameters12.  
In summary, growing experimental and epidemiological evidence suggests that spermidine 
preserves vascular health and delays the onset of hypertension as a result of common risk 
factors, including aging and salt sensitivity. However, whether spermidine can be used for 
managing hypertension as an adjuvant to the existing pharmacotherapy in humans is still 
unknown. In fact, human data obtained from interventional spermidine trials are sparse. Similarly, 
the mechanisms ─ whether they are autophagy-dependent or independent ─ that underlie 
cardiovascular benefit of spermidine still require further investigation in humans. In the proposed 
project, we are aiming to define an optimal blood pressure management strategy by using a 
spermidine-rich plant extract in patients who are not responsive to first-line anti-
hypertensive drugs. This extract has an established safety profile as observed in a randomized, 
placebo-controlled phase I trial in elderly subjects10. In addition, we will explore potential 
mechanisms of action for the blood pressure-lowering effect of spermidine.  
Work leading up to this project (preliminary results) 
In an experimental pilot study (not published), we examined the therapeutic effect of spermidine 
supplementation on hypertension using male Dahl salt-sensitive rats. After four weeks of a high-
salt diet (8% NaCl), 12-week-old Dahl rats developed severe hypertension (190-200 mmHg, Fig. 
1A) associated with hypertrophy (not shown). All rats then received ‘standard’ treatment in the form 
of a diuretic (furosemide, 10 mg/kg body weight, single intraperitoneal injection), as well as a low-
salt diet (0.3% NaCl) combined with or without spermidine supplementation (3 mM) in drinking 
water for the following six weeks. Induced diuresis combined with a change from a high- to low-salt 
diet resulted in a transient and relatively small reduction in blood pressure, which ultimately 
returned to the same level it had been before the intraperitoneal administration of the diuretic (Fig. 
1A). However, spermidine supplementation resulted in a long-lasting reduction in blood pressure 
(Fig. 1A) and restored vascular elasticity (Fig. 1B) to levels comparable with those from healthy 
controls (rats fed only a low-salt diet). In addition, spermidine reduced cardiac hypertrophy and 
end-diastolic pressure, while enhancing exercise tolerance as indicated by improved running 
distance and maximum oxygen consumption (Fig. 1C-F). Taken together, this indicates – for the 
first time – a therapeutic, rather than preventive, effect of spermidine supplementation on salt-
induced hypertension. 
More importantly, initial data obtained from humans also indicate a potential blood pressure-
lowering effect of spermidine, as shown in the SMARTAGE trial in elderly patients with subjective 
decline in cognitive function10. In this trial, spermidine supplementation resulted in a prominent 

  
  
6
trend towards decreased systolic blood pressure, despite the moderate dose and low number of 
subjects involved.   
 
 
 
 
 
 
Figure 1. Spermidine potentiates the effects of standard hypertension therapy. Excluding the 
healthy control group (blue), all animals received a high-salt diet and developed hypertension and 
cardiac hypertrophy before the onset of therapy, which consisted of the diuretic furosemide and 
reduced salt intake with or without 3 mM spermidine supplementation in drinking water. Depicted 
are the mean±S.E.M of (A) non-invasive (tail-cuff-based) systolic blood pressure measurements, 
invasively (hemodynamically) measured (B) arterial elastance (i.e. stiffness) and (C) end-diastolic 
pressure, (D) heart weight-to-left tibia length ratio as a measure for cardiac hypertrophy, as well as 
fatigue and exercise tolerance as determined by (E) treadmill run distance and (F) maximum 
oxygen consumption (VO2max), respectively. n=5-6 rats/group. *p<0.05, **p<0.01, ***p<0.001 
(Bonferroni-corrected ANOVA). Abbreviations: SPD, spermidine; LS, low-salt. 
B 
C 
D 
E 
F 
Diuretic 
injection±SPD 
A 
Healthy Control 
 Hypertension Standard Therapy (Diuretic + LS diet) 
 Hypertension Standard Therapy + SPD 
 
High-salt diet 
Low-salt diet 

 
 
7
 
Objectives 
1.2
The primary objective of this single-centre, placebo-controlled, double-blind and randomized trial 
with a crossover design and washout period between treatment arms is to assess the effect of oral 
spermidine supplementation (4 mg/day) for 8 weeks on systolic blood pressure, as measured by 
24h-blood pressure measurement (BPM).  
The secondary objective of the study is to examine the effect of spermidine on pulse-wave velocity, 
ambulatory blood pressure and 6MWT. 
As exploratory objectives of the trial, we aim to investigate if certain amino acids and metabolites, 
as well as markers of autophagy and immune cell profiles, correlate with blood pressure-lowering 
effects. 
 
Outcomes 
1.3
1.3.1 24h-BPM 
- Systolic, diastolic, mean, and central blood pressure 
- Pulse wave velocity (PWV) 
- Augmentation @75 Index (AIx@75) 
- Stroke volume 
- Peripheral resistance 
1.3.2 Laboratory parameters 
Spermidine and its metabolites (polyamine profiles will be determined using HPLC-MS/MS from 
plasma, whole blood, peripheral blood mononuclear cells (PBMCs) and saliva samples), NT-pro-
BNP, liver and kidney function, tryptophan, kynurenine, kynurenine acid, 3-hydroxykynurenine, 
quinolic acid, global arginine bioavailability ratio (GABR: L-Arginin/[Citrulline+Ornithin]), arginase 
activity 
(L-Arginine/Ornithine 
ratio), 
asymmetric 
dimethylarginine 
(ADMA), 
symmetric 
dimethylarginine (SDMA), L-Arginine/ADMA ratio, C-reactive protein (hs-CRP), homoarginine, 
amino acids and their metabolites, ACTH, (nor-)epinephrine, electrolytes (K+, Na+), aldosterone, 
plasma renin activity, immuno cell profile (WBC, specific leukocyte populations), circulatory 
cytokines (e.g., IL-6, TNF-alpha, IL-17), markers of autophagy including the lipidation of LC3 
(assessed using an ex vivo leupeptine-based autophagy flux assay20), and p62 protein levels in 
PBMCs. A total of 30 ml of whole blood will be collected from each patient at every visit. 

 
 
8
1.3.3 ECG 
Frequency, rhythm, repolarisation times. 
1.3.4 Echocardiography 
Systolic and diastolic function, left ventricular mass index (LVMI), left atrial volume index (LAVI), 
flow velocities (E- and A-wave, LVOT); myocardial velocities (e’) and strain analysis (longitudinal, 
circumferential and radial). Also see the protocol for Echo-CRF (Appendix). 
1.3.5 Quality of Life (QoL) 
Quality of life will be assessed at all visits using the established SF-36 questionnaire. 
2 
Trial design 
 
Trial design 
2.1
The study is designed as a single-centre, placebo-controlled, double-blind and randomized 
controlled trial with a balanced 2x2 crossover design and a washout period between treatment 
arms. Specifically, half of the recruited patients will be treated with spermidine for eight weeks, 
followed by placebo for eight weeks. The other half of patients will first receive placebo and then 
spermidine. There will be a washout period of four weeks between the first period of eight weeks 
and second period of eight weeks to separate the two interventions in both arms, which will avert 
any carryover or residual effects. A balanced 2x2 crossover trial design is ideal for this study for 
the following two reasons: 
(i) Physiological reason: spermidine levels in the body are substantially affected by the diet. Thus, 
patients with different dietary habits could already have different spermidine levels at baseline (i.e. 
before starting the trial). Even if patients were randomized into 2 groups correcting for baseline 
measured circulating spermidine, this wouldn't guarantee that spermidine levels in the body tissues 
are comparable without confirming via biopsy, not to mention that food intake would need to be 
strictly controlled and be identical in both arms throughout the trial. For this reason, 
crossover/repeated measurements are not only more practical, but rather essential for our 
proposed study. The effect of any other confounding factors (e.g. body mass index, age, smoking, 
baseline blood pressure, LDL, etc.) will be also reduced to a minimum as every patient will serve 
as his/her own control. 

 
 
9
(ii) Statistical reason: a crossover design is particularly efficient as it provides the highest power-to-
sample size ratio. Thus, the study endpoints can be examined with as few patients as possible.  
 
Schedule of evaluations 
2.2
The assessments planned for during the study are shown below (Table 1). 
Table 1: 
Visit number 
1 
2 
3 
4 
5 
Week number  
 
-4 
(+/-3 days) 
0 
(+/-3 days) 
8 
(+/-3 days) 
12 
(+/-3 days) 
20 
(+/-3 days) 
Screening 
x 
 
 
 
 
Informed consent 
x 
 
 
 
 
24h-BPM 
x 
x 
x 
X 
Laboratory analysis 
x 
x 
x 
X 
ECG 
x 
x 
x 
X 
Echocardiography 
 
x 
x 
x 
X 
SF-36 questionaire 
 
x 
x 
x 
X 
 
 
Trial population 
2.3
The trial population consists of mixed adults from both genders screened during hospitalisation at a 
medical clinic. Patients selected must present with elevated blood pressure despite being treated 
with at least two guideline-recommended drugs. This approach secures a trial population with well-
established arterial hypertension and ensures that patients will have received baseline treatment 
based on the guideline recommendations before being enrolled in the proposed study. Therefore, 

 
 
10
our study offers an additional novel and innovative strategy for patients following their initial 
baseline treatment which failed to fully manage their hypertension. Patients who need to be 
excluded due to systolic blood pressures > 180mmHg will directly be advised to get further 
treatment at the outpatients clinic and the educational program herz.leben. 
Recruitment 
Patients with systolic arterial hypertension of more than 150 mmHg during hospitalisation despite 
being treated with at least two guideline-recommended drugs will be informed of the possibility to 
participate in the trial. If they consent, patients will be contacted four weeks later by telephone to 
check for unchanged medication and blood pressure. Patients with persistent arterial hypertension 
(>150 mmHg), despite being on a consistent medication routine with at least two guideline-
recommended anti-hypertensive drugs within the last four weeks, will be invited for a screening 
(Visit 1) at our hospital. Upon confirmation of sustained high blood pressure (>150 ≤180mmHg) 
and fulfilment of inclusion and exclusion criteria, patients will again be informed that participation in 
the trial is voluntary before signing the informed consent form. After signing the ICF, patients will 
be randomized in a 1:1 ratio and a block size of 46 using the web-based randomizer 
(https://randomizer.at/) provided by Prof. Andrea Berghold (Institute for Medical Informatics, 
Statistics and Documentation – IMI, Medical University of Graz). We aim to have both genders 
equally represented within the trial population. We will perform a gender-specific analysis of clinical 
and biochemical assessments. 
 
Inclusion and exclusion criteria 
2.4
Inclusion criteria 
- Able to provide signed and dated informed consent form 
- Male or female, 19 to 99 years of age 
- Persistant arterial hypertension with systolic blood pressure above 150 mmHg during 
hospitalisation and the day of randomisation  
- Stable anti-hypertensive medication with at least two guideline-recommended anti-
hypertensive drugs 
Exclusion criteria  
  - Systolic blood pressure ≥180mmHg on the day of randomisation 
- Spermidine intolerance 
- Significant renal impairment defined as glomerular filtration rate < 45ml/min 
- Insulin-dependent diabetes mellitus (IDDM) 
- Wheat allergy or gluten intolerance 
- Life expectancy of less than 12 months 

 
 
11
- Participation in another clinical trial  
 
Data management / documentation 
 
 
 
 
2.5
Data will be collected on paper-based CRFs. CRFs will remain the property of the study center 
(Medical University of Graz). Data analysis will be performed using SPSS on the institutional 
computers of the investigators. 
 
Sample size considerations 
2.6
As outlined in 2 and 2.1., the blood pressure-lowering effect of spermidine is well established in 
animal models. Furthermore, 28 elderly patients with subjective declining cognitive function within 
the SMARTAGE trial revealed a drop in systolic blood pressure of 10 mmHg (4.5 mmHg more than 
in the placebo group) upon spermidine (1.2 mg/day) supplementation for three months. In the 
proposed trial, we will use a three-fold higher dose of spermidine and we will enroll patients with a 
higher baseline blood pressure (≥150 and ≤180 mmHg systolic BP). Therefore, we expect a 
reduction of systolic blood pressure of at least 5 mmHg. To this end, we calculated that a total of 
38 patients would be sufficient to detect that effect at a power of 80%, assuming a two-tailed α 
value (type-I error) of 5%, blood pressure variance (SD) of 13 mmHg and within-subject correlation 
coefficient (r) of 0.65. We expect a drop-out rate of 15% due to the 20-week-long duration of the 
trial, thus, a total number of 46 patients will be enrolled. 
 
Follow-up 
2.7
Study follow-up will be twenty weeks, comprised of two treatment periods of eight weeks and a 
washout period of four weeks between both treatments. 
 
Project duration and milestones 
2.8
We envision that a period of 21 months will be needed to successfully accomplish this study. The 
recruitment (18 months) is planned as follows: 
First patient visit: 
 
  
 
 
February 2020 
Last patient enrolled:  
 
 
 
June 2021 

 
 
12
3 
Methods 
 
24h BPM 
3.1
During each of the four visits patients will be supplied with an ambulatory blood pressure 
measurement (ABPM) device (Mobil-O-Graph® PWA, I.E.M. GmbH, Stolberg, Germany). After 24 
hours of continuous BPM at their homes, patients will remove the device and return it to the study 
center via postal service. Mobil-O-Graph® PWA is a well established device to measure central 
blood pressure and pulse wave velocity used and validated in multiple clinical trials and settings. 
Despite being a non-invasive technique good accuracy compared to intra-aortic readings has been 
proven (linear correlation R=0.81, p <0.0001) in 120 patients (see validation data on 
www.iem.de/en/health-management/studies-validations.html 
and 
a 
bibliography 
at 
www.iem.de/files/content/DE/IEMintern/Support/Hintergründe/Bibliography%20Mobil-O-
Graph%20Blood%20Pressure%20PWA%202.9.pdf) During the visit on-site blood pressure 
measurements and a standardized 6MWT will be conducted at every visit. 
 
Lab analysis 
3.2
Blood withdrawal will be performed using: 
- 
4x Lithium-heparinate: 8+7+2+2 ml=19 ml blood 
- 
1x Serum: 4 ml blood for approx. 2 ml serum 
- 
1x EDTA: 7 ml blood for approx. 3.5 ml plasma 
Blood draws will be performed at each of the four visits (total blood volume taken during the trial 
will be 120 ml).  
In addition to standard clinical assessments (see 2.3.2), the isolation of PBMC will be performed 
from the whole blood samples and analysed to assess alterations in immune cell populations using 
state-of-the-art multi-parameter flow cytometry and/or Cy-TOF. In several multi-systemic 
pathologies, possibly rare, differentiated or non-differentiated hematopoietic cell subpopulations, 
which can be detected in circulation, have been increasingly recognized as a read-out to 
determine/distinguish between health and disease status. Advances in single cell-resolved 
molecular measurement technologies enabled the description of cell population heterogeneity, 
including rare disease-associated subpopulations. However, the identification of such 
subpopulations remains a difficult computational task. Therefore, our collaborator Prof. Claassen 
(ETH Zurich, Switzerland) has developed21 an  artificial intelligence approach that detects disease-
associated subpopulations of cells from single-cell proteomic measurements using this CyTOF 

 
 
13
techniques22. These data will be examined for correlations with systolic and diastolic blood 
pressures as well as with markers of inflammation employing flow cytometry-based assessment of 
cytokines. In addition, autophagic flux will be measured using LC3-lipidation quantification in ex 
vivo leupeptin-treated PBMCs20) in human leukocytes. Altogether, this will allow us to describe, for 
the first time, in vivo effects of spermidine intervention on human immune cells, inflammatory 
cytokines, and markers of autophagy. 
 
Electrocardiography 
3.3
Conventional 12-lead ECG.  
 
Echocardiography 
3.4
Standardized 2D-echocardiography will be performed at every visit. Loops will be recorded and 
stored as outlined in the echo manual (parasternal long and short axis, 2-, 3- and 4-chamber view, 
subcostal view). For details, see the attached protocol for Echo-CRF. 
 
QoL assessment 
3.5
The standardized and well-established SF-36 questionnaire will be used to evaluate a subjective 
quality of life of the patients. 
4 
Safety and risk management 
The planned daily dose of spermidine is an approved dietary supplement extracted from 
spermidine-rich 
wheat 
germ 
(see 
http://eur-lex.europa.eu/legal-
content/EN/TXT/PDF/?uri=CELEX:32017R2470&from=EN). Additionally, spermidine is part of a 
standard healthy diet, present in a wide variety of foods. Therefore, the dose of 4 mg spermidine 
per day is unlikely to cause any harm to the patients participating in the study. The reduction in 
blood pressure would be a beneficial therapeutic effect considering the hypertensive baseline. 
However, a NO/arginine-mediated blood pressure-lowering effect may also cause headaches, 
dizziness or tachycardia. The study protocol does not interfere with standard baseline therapy. 
Upon study completion, patients will be invited to participate in the educational program 
“herz.leben” (ADD link to the webpage!) and, therefore also receive optimal lifestyle modification 
training.  

 
 
14
 
Adverse events and reactions 
4.1
Adverse Events (AE), Adverse Reactions (AR), Unexpected Adverse Reactions (UAR) as well as 
Serious Adverse Events (SAE), and Serious Adverse Reactions (SAR) will be recorded and 
reported to the institutional ethics committee as outlined in the ethics approval. 
 
Study discontinuation  
4.2
One or more of the following incidences would lead to a discontinuation of the study for a single 
patient (drop-out):  
• Withdrawal of consent 
• Intolerable investigational drug-related effects 
• Severe violation of the study protocol 
• New onset of an exclusion criterion during the treatment period 
• Decrease in blood pressure of more than 40 mmHg with symptomatic hypotension 
Early discontinuation of the study will be taken into a consideration if the risk-benefit relationship 
worsens:  
• Intolerable investigational drug-related effects in more than 10 patients 
• Decrease in blood pressure of more than 40 mmHg with symptomatic hypotension in more than 
10 patients 
 
Potential risks within the study design 
4.3
All clinical assessments in this trial are standard methods and well established within the 
Department of Cardiology (MedUniGraz) and its clinical trial section. Thus, the applicant does not 
expect any methodological risk with respect to the trial. The recruitment of patients into this clinical 
trial will be based on the cooperation between the applicant and the herz.leben project, in which 
120 highly motivated patients per year have been prospectively enrolled since 2007. Therefore, we 
do not anticipate having problems with recruiting 42 patients from this educational program within 
12 months and expect these patients to fully adhere to the study protocol.  
Saliva collection by swabbing is a very convenient and non-invasive method for collecting cells in 
clinical practice. However, saliva samples might not contain enough cells to perform all analyses, 
including the assessment of autophagic activities. Therefore, whole blood and blood cells (e.g. 
PBMCs) will be used to detect differences in autophagy, immune cell, and polyamine metabolite 
profiles. We work under the hypothesis that spermidine induces autophagy in hypertensive 
patients. If we fail to detect increased autophagy markers upon spermidine supplement, we will use 

 
 
15
our data on plasma metabolites (such as L-arginine) and any detected changes in circulatory cell 
profiles to propose an alternative mode of action (potentially autophagy-independent). If 
spermidine levels are not increased in whole blood or plasma, our efforts will concentrate on the 
detection of spermidine-induced changes in polyamine metabolism by analyzing metabolites (i.e. 
precursors and derivatives of spermidine) in human plasma samples. Moreover, saliva samples 
offer an additional “tissue”-like sample that will prove the bioavailability of spermidine originating 
from the administered supplements. 
5 
Statistical considerations and data analysis 
Obtaining repeated measurements from the same patients in response to both treatments has 
fundamental physiological reasoning (see 3.1) and necessitates completing the study protocol. For 
this reason and because the trial is designed to measure the treatment efficacy, rather than 
adherence, patients who drop out before completing the protocol (4 visits) or their baseline 
treatment regimen changes due to medical reasons will be excluded to maintain a balanced 
crossover analysis of this trial.  
The effect of distinct treatments (spermidine or placebo) on a given parameter (e.g. SBP) will be 
expressed as a change score from baseline (Δ). The difference between treatment effects will then 
be evaluated using a mixed linear model, where Subjects will be included as a random factor, 
whereas Treatment (spermidine vs. placebo), Period (first vs. second), and Sequence (spermidine-
placebo vs. placebo-spermidine) will be fixed factors (i.e. predictors). Although we expect no 
carryover effects due to the washout period, including Period and Sequence in the model will help 
exclude any confounding effects of (or interaction with) these aspects, especially taking into 
account that the obtained data will be well-balanced due to the (2x2) study design. 
6 
Ethical considerations and informed consent 
The study shall be conducted in accordance with the ethical principles originating from the 
Declaration of Helsinki23 and in accordance with ICH-Good Clinical Practice (GCP) and applicable 
regulatory requirements. The study is registered at the local ethics committee with the number 30-
468 ex 17/18. 
Before randomisation, patients will be comprehensively informed about spermidine and its effects 
as a compound and the procedures within the trial, but they will be blinded to the treatment 
sequence (spermidine vs. placebo). Participants will be given the informed consent form and given 
enough time to consider voluntary participation. 

 
 
16
7 
Data collection and quality assurance, Data management, Monitoring 
A unique ID number will be attributed to every enrolled patient. Patients’ names will be protected 
and accessible only to the personnel/the applicant involved in the study. Blood/serum samples will 
be stored in an anonymous fashion and biologically destroyed 5 years after completion of the 
study.  
On-site monitoring will be conducted by the KKS (Koordinierungszentrum für Klinische Studien), 
MedUniGraz. Accuracy and completeness of the CRF entries, source documents, and other study-
related documents will be checked. After each monitoring visit, a report of any significant 
findings/facts, deviations, and deficiencies will be communicated to the investigator. 
Conflict of interest: 
Tobias Eisenberg and Frank Madeo declare a potential financial conflict of interest (equity in TLL). 
They will not analyze any clinical data and will be blinded throughout the lab analysis after  the trial. 
1. 
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Clinical Trial Synopsis  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Allocated to placebo (n= 23) 
 
Duration 8 weeks
Wash-out periode 
Duration 4 weeks 
Allocated to intervention (n= 23) 
 Spermidin 4g/day 
 
Wash-out periode 
Duration 4 weeks 
Allocated to placebo (n= 23) 
 
 
Allocated to intervention (n= 23) 
 Spermidin 4g/day 
Allocation Visit 1
Follow-Up Visit 3 
Follow-Up Visit 2 
Randomized (n=46 ) 
Enrollment 
Allocated to placebo (n= 23) 
 
Final analysis
Allocated to intervention (n= 23) 
 Spermidin 4g/day 
Follow-Up Visit 4