INTRODUCTION

The natural and ubiquitously occurring polyamines (spermidine, spermine) and the diamine putrescine, result from amino acid metabolism and comprise essential cellular functions, including regulation of cell growth, proliferation and autophagy [1]. Concentration of polyamines in the body is sustained by endogenous biosynthesis, microbial activity in the intestines, and exogenous food intake. However, it has been shown that intracellular polyamine concentrations of several organs decline with age in animals [2] and humans [3-6]. Previous studies indicate that higher intake of polyamines could represent a feasible approach to restore endogenous polyamine concentrations, enhance autophagy rates, and possibly improve health in aging organisms. A translational study, for example, showed that higher external supply of dietary polyamines, administered via a polyamine-enriched diet in mice (26 weeks) and humans (two months), increased blood spermine concentration in both rodents and healthy middle aged males [7]. In aging fruit flies, spermidinerich feeding inhibited the development of agedependent memory impairment by restoring polyamine levels in the brain of aging flies and enhancing autophagy [8]. Moreover, increased external administration of polyamines (spermidine in particular) is suggested to promote longevity and autophagy in worms, flies, yeast, and mice [9, 10]. The above-described benefits suggest dietary supplementation with spermidine as a promising prevention strategy in older adults with an elevated risk of developing dementia. However, before conducting larger studies with cognitive outcomes in human trials, safety and tolerability of polyamine supplementation need to be established.

Food and water intake were not influenced by the supplementation with the polyamine-rich wheat germ extract (Table 1). While bodyweight of animals of all treatment groups did not differentiate from the control group, relative kidney weight to bodyweight ratio was significantly increased in female mice treated with the extreme overdose of 50 g/kg bodyweight. Relative kidney weight was increased by 12% in this group compared to control animals. At lower concentration, this effect was not observed (Supplementary Figure 1). Weights of other examined organs were unaffected by the treatment at all concentrations. 

Polyamine levels in selected murine tissues 

Analysis of polyamine levels showed no significant differences in accumulation of polyamines in several tissues after the oral spermidine supplementation in both sexes with few exceptions (Figure 1). While spermidine levels in whole blood of female mice supplemented with highest concentration of spermidinerich extract in chow was significantly increased compared to the control group after 4 weeks, spermidine blood level in female mice at lower extract concentrations and all male mice remained unaffected by the treatment. Levels of other polyamines in the whole blood remained comparable to the control group in both sexes. Cardiac increase in spermidine and putrescine concentrations after the treatment was observed only in female mice with 50 g/kg bodyweight supplementation, but not at lower concentrations in female or at any supplement concentration in male mice. Concentration of various polyamines in brain tissue showed no correlation to provided treatment. LOrnithine concentration was not changed in any examined tissues.

Study enrollment and baseline characteristics of human cohort

One-hundred-seventy-one adults were interviewed by telephone for study eligibility based on inclusion and exclusion criteria (Figure 2). One-hundred-thirty-eight subjects were excluded, because they were not eligible (n = 100) or declined participation (n = 38). In total, 33 participants were invited from January 2016 to March 2016 for on-site screening. Here, three more individuals had to be excluded because they did not meet inclusion and exclusion criteria. Thirty participants (target sample size of the trial, see clinicaltrials.gov NCT ID: NCT02755246) completed baseline assessment, were subsequently randomized to either spermidine or placebo intervention, and started with the 3-month intervention. During this time, two participants (spermidine group: n = 1; placebo group: n = 1) dropped out due to missing motivation. Twenty-eight participants completed the intervention and were included in the present analysis. 

DISCUSSION

Spermidine supplementation is known to positively influence various age-related health parameters in model organisms, including memory function [8, 17], cardiac and renal function [12] as well as autophagy rates [8], thereby promoting longevity [9]. However, safety and tolerability of spermidine supplementation, as a prerequisite for larger clinical trials, have not been established yet in mice and humans. Here we demonstrate for the first time that spermidine supplementation using a spermidine-rich plant extract was safe and well tolerated in mice and in older adults with SCD

In conclusion, this study showed that the applied spermidine-rich plant extract was safe and well tolerated in mice and older adults with SCD. These findings open the possibility to investigate the impact of spermidine supplementation on functional and structural brain health as well as on cognition in larger studies with longer intervention times in older adults at risk for dementia. 

METHODS

Preclinical trial setup and animal housing 

In order to examine the safety of the used extracts, a standardized OECD repeated dose oral toxicity study was conducted. [SOURCE: OECD: Guidelines for the testing of Chemicals - “Test 407: Repeated Dose 28-day Oral Toxicity Study in Rodents”] In brief, groups of 10 male and 10 female BALB/cAnNRj mice (Janvier Labs S.A.S.) were provided with either control chow or chow supplemented with a polyamine-rich extract corresponding to 0.5 g/kg, 5 g/kg or 50 g/kg animal bodyweight daily intake over the period of 28 days. An additional group of 5 male and 5 female mice was fed with control chow diet for additional 14 days (two weeks washout) after receiving the highest dosage of supplemented diet for 28 days. All experiments were started at the age of 8 weeks. Animals were housed in a 12h light/dark cycle in type 3 IVC cages (Tecniplast, Model 1284 L) in groups of 5 individuals. Autoclaved nest material and paper houses served as cage enrichment, while access to food and water was provided ad libitum. 

To ensure that the food and water intake were comparable between all groups, drinking water and chow were weighed twice a week. Animals were examined weekly for their external appearance (i.e. examination of the fur, skin and tumors), interaction with cagemates and humans, behavior (i.e. aggressiveness, social interaction, fear, pain, lethargy based on standardized score sheets), and appearance of the fecal and urine excretions.  Bodyweight of all animals was recorded by weekly weighing. Animal monitoring was conducted in a blinded manner by an experienced professional. Pellets for both control and polyamine-enriched chow were prepared using a commercially available base formula (Ssniff, Product-Nr. DP110). All animal experiments were performed in accordance with national and European ethical regulation (Directive 2010/63/EU) and approved by the responsible government agencies (BMWFW-66.007/0012-WF/V/3b/2015). 

Post-mortem animal examination and tissue sampling

At the end of the 28-day long treatment, animals were anesthetized by inhalation of 3-4% isoflurane (Forane®, Baxter Healthcare Corporation) and euthanized by cervical dislocation. Whole blood was collected immediately after euthanasia by cardiac puncture in Ethylenediaminetetraacetic acid (EDTA) coated tubes and stored at -80 °C for polyamine analysis. Blood plasma was prepared from collected EDTA blood by centrifugation at 2000 xg at 4 °C for 15 min and stored at -80 °C for further investigation. 

After blood collection, animals were thoroughly examined for neoplasias and visible abnormalities. Further, various organs were weighed, snap frozen in liquid nitrogen and stored at -80 °C for further analysis. The post-mortem examination, sample collection and analysis were conducted in randomized and blinded manner.

Measurement of polyamine levels in biological samples 

A quantitative HPLC-MS/MS-based determination of polyamines in various tissues was performed using whole tissue lysates, as described previously [22]. In brief, polyamines were extracted on ice by incubating samples for 60 min in 5% trichloracetic acid solution. After centrifugation at 25,000 xg at 4 °C for 10 min, supernatant was neutralized with ammonium formiate and stored at -80 °C for subsequent quantification. Prior to the analysis, neutralized samples were derivatized using isobutyl chloroformate [23] and measured using standardized HPLC-MS/MS protocol [22].

Human study design 

A randomized, placebo-controlled, double-blind Phase II study was carried out at the NeuroCure Clinical Research Center, Charité University Hospital. Older adults with SCD and worries (see below for details) were recruited from the memory clinic of the Department of Neurology (Charité University Hospital), a neurology specialist practice (J.B.), and the general population through advertisements. Individuals who were interested in study participation underwent a telephone interview to assess their eligibility.

 The study was approved by the Ethics Committee of the Charité University Hospital Berlin, Germany (EA1/233/15), and was carried out in accordance with the declaration of Helsinki. Registration of the study was done in the public registry ClinicalTrials.gov (NCT ID: NCT02755246). 

Participants 

Enrolled participants were between 60 – 80 years of age, fluent German speakers, with the presence of SCD in accordance with existing guidelines [16, 24]. In detail, all participants had to express subjective cognitive complaints for at least 6 months and related worries, report no deficits in activities of daily living, and score ≤10 in the Geriatric Depression Scale [25]. Eligibility criteria for normal cognitive performance included a Mini-Mental State Examination score ≥26 [26], and performance within -1.5 standard deviation (SD) of age adjusted norms in the Logical Memory II subscale of the Wechsler Memory Scale–Revised [27] and the Trail Making Test A [28, 29]. Exclusion criteria encompassed major neurological, internal or psychiatric diseases, malignancies (current or on medical history), untreated thyroid dysfunction or untreated diabetes mellitus, anticoagulation therapy, platelet aggregation inhibitor, known allergy to wheat germs or gluten, histamine intolerance, drug abuse or alcohol dependency, disorders that impair attention, or intake of polyamine supplements before starting the trial. 

Preparation of capsules and dosing 

Based on the preclinical data, maximum safe dosage in humans was calculated using the NOAEL of 5 g/kg bodyweight from the murine model, mouse-to-human interspecies factor of 12.3 and factor 10 as safety distance between sub-chronic and chronic application [30].

 As a result, doses of up to 41 mg/kg bodyweight or 2.8 g extract containing 3.4 mg spermidine for the average person weighing 70 kg were set as the expected upper safety limit for the treatment. In total, 750 mg of polyamine-rich plant extract (1.2 mg spermidine, 0.6 mg spermine, 0.2 mg putrescine) and 510 mg cellulose were divided into three Type 00 capsules. This accounts for to an increase of approximately 10-20% of average spermidine intake in developed countries [31, 32]. Three placebo capsules per day were filled in sum with 750 mg potato starch and 510 mg cellulose. All capsules were identical in shape, color, taste and smell. Both placebo and spermidine capsules have been provided by TLL The Longevity Labs GmbH (Graz, Austria). Participants of both intervention groups were instructed to follow a regular intake of three capsules a day, one capsule at each main meal (breakfast, lunch, dinner), and not to change their dietary habits during the intervention time. Participants were asked to return capsule bottles at follow-up assessment and the number of remaining capsules was recorded as a measure of compliance and tolerability.

Examination of physical condition 

Physical condition of the participants was evaluated during the standardized medical examination at baseline and follow-up assessment. Following parameters were recorded for each participant at both time points after fasting overnight: weight, height, vital signs as well as any detectable abnormalities of physical condition. Vital signs, including systolic blood pressure, diastolic blood pressure, and heart rate, were assessed in sitting position.