Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder and the most common cause of dementia in the elderly all over the world [1]. According to statistical data, the number of patients with AD in Russia is 1.5–2 million [2].

The presence of extracellular fibrillary aggregates, or amyloid plaques, showing a characteristic signal when Congo red stained is one of the main pathological features of AD [3]. The aggregated amyloid-beta (Aβ) species are the main components of amyloid plaques [4]. Amyloid-beta is a short polypeptide (37–43 amino acid residues), the low nanomolar concentrations of which are found in human blood and cerebrospinal fluid [5]. However, during the AD pathogenesis cerebral amyloidosis starts to develop in the brain for unknown reasons, due to which monomeric Aβ molecules are first converted into soluble oligomers and then accumulate in various parts of the brain as amyloid plaques, but especially and primarily in the hippocampus [6]. The first amyloid plaques in brain tissues are formed 10–20 years before the onset of AD clinical manifestations, but it is Aβ aggregation that initiates further AD-associated disease processes, including tau-protein hyperphosphorylation and neurodegeneration [4]. Therefore, the search for candidate drugs for amyloid plaque disruption and/or amyloid plaque formation prevention represents the key strategy of the development of disease-modifying therapy for AD [7].

To date, three anti-Aβ antibodies, aducanumab, lecanemab, and donanemab, have been legally registered in the USA [8–10] for initial treatment of the early-stage AD, including patients having mild cognitive impairment or mild dementia due to Alzheimer's disease with the diagnosis confirmed based on Aβ using positron emission tomography of amyloid or cerebrospinal fluid biomarkers. These novel disease-modifying treatment methods act by decreasing the amount of amyloid plaques in the brain and demonstrate clinical benefits of antiamyloid therapy [11].

However, due to dangerous side effects of antibodies [12], the development of drug candidates from the class of low molecular weight compounds, involving peptides and peptidomimetics, as molecular agents that target specific binding to various regions of the Aβ amino acid sequence, is being continued [13].

The Ac-His-Ala-Glu-Glu-NH2 (HAEE) synthetic peptide (PubChem CID: 56971578) has been developed as a potential disease-modifying drug for AD therapy [14], which effectively passes from blood into the brain tissue crossing the bloodbrain barrier [15]. The drug target of HAEE is the 11-Glu-ValHis-His-14 (EVHH) Aβ fragment that forms an intermolecular complex stabilized by complementary ionic interactions with HAEE (review in [16]). The EVHH fragment was selected as a drug target due to the fact that it represents an Aβ molecular determinant, which has a rigid conformation of the main polypeptide chain and controls the Aβ binding to the α4β2 subtype nicotinic acetylcholine receptor, as well as all the Aβ zinc-dependent interactions [17, 18]. Considering crucial role of all the above interactions in the AD pathogenesis, it was expected that blocking the EVHH fragment by HAEE should inhibit amyloid plaque formation in vivo. Indeed, high antiamyloid efficacy of HAEE was reported in pilot experiments involving the use of transgenic animals (APP/PS1 mice and CL2120 nematodes) as AD models (review in [18]).

Thus, the mechanism of the HAEE anti-amyloid effect is based on disruption of the zinc-dependent intermolecular interfaces in the amyloid-beta oligomers and aggregates, as well as in the complexes formed by Aβ and subtype α4β2 nicotinic acetylcholine receptors via selective HAEE binding with the 11-Glu-Val-His-His-14 amino acid sequence fragment of amyloid-beta, leading to reduction of the amount of amyloidbeta aggregates in the model animal’s body.

The study aimed to determine the HAEE optimal therapeutic dose for effective inhibition of amyloid plaque formation in the hippocampus of APP/PS1 mice within the framework of the pre-clinical trial of the HAEE-based drug as a potential antiamyloid drug for treatment of AD.

METHODS

Laboratory animals

The experiments involved APPswe/PS1dE9/Blg transgenic mice [19]. This lineage was obtained by crossing the B6;C3Tg(APPswe,PSEN1dE9)85Dbo/Mmjax transgenic mice (#034829-JAX, JAX, Maine, USA) also referred to as APP/PS1 with the C57Bl6J wild type mice of the C57Bl6J/ChG lineage (Institute of Physiologically Active Compounds, Chernogolovka, Russia). The APP/PS1 transgenic mice are acknowledged AD models [18]. Since the age of 4–6 months these animals show typical cognitive signs of the AD-like disorder; there is a considerable amount of congophilic amyloid plaques in specific brain regions, including hippocampus and brain cortex [20]. Experimental animals were kept in the pathogen-free vivarium (Engelhardt Institute of Molecular Biology, Moscow, Russia) under the conditions, including standard diet, ad libitum access to food and water, daylight lasting 12 h, ambient temperature between +22 and +24 °С, and relative humidity 50–65%. Breeding and control genotyping of bloodstock were performed as previously reported [19].

Experimental groups of animals

In this study, the experiments involved the total of 40 male and 40 female APP/PS1 transgenic mice. The animals were randomized into five groups, 8 males and 8 females per group (table). The age of each animal at the time of the first injection and euthanasia was 5 and 7 months, respectively. Mice of group 1 (controls) were subcutaneously administered the isotonic 0.9% sodium chloride solution (normal saline). Mice of groups 2–5 were administered the HAEE synthetic peptide solution at a dose of 0.18 mg/kg, 0.30 mg/kg, 1.50 mg/kg, and 3.00 mg/kg, respectively. The 7-months-old mice were euthanized.

Reagents

All the chemical compounds and solvents used in the study were of and HPLC or higher grade, these were purchased from Sigma-Aldrich (St. Louis, Missouri, USA), unless otherwise specified. Medicinal product “Ac-His-Ala-Glu-Glu-NH2 Tetrapeptide, solution for subcutaneous administration”, 3.5 mg/mL (Pharmsynthez, Russia). The HAEE peptide structure was confirmed by NMR spectroscopy and tandem mass spectrometry.

Preparation of the synthetic HAEE-based drugs for injection

The HAEE-based drug with the baseline concentration of 3.5 mg/mL was diluted with the sterile isotonic 0.9% sodium chloride solution to achieve the working peptide concentration ensuring the desired HAEE-based drug dose (for example, 0.30 mg/kg) per 125 µL of the diluted solution. The total volume of a single injection sample was 150 µL for all the HAEE doses used in the study. Each animal was administered 125 µL of the sample.

Administration of test drugs to experimental animals

Transgenic mice of five experimental groups were administered test drugs (normal saline or samples with different HAEE peptide doses) twice a week throughout 9 weeks via subcutaneous injection of appropriate drugs in accordance with the existing requirements for and approaches to drug dose adjustment in laboratory animals [21]. The frequency and total number of injections administered to mice of the experimental groups throughout the study, as well as information on the HAEE dosage in the injection samples for each experimental group are provided in the table.

Preparing histological sections of the brain

Transcardial perfusion with the 4% paraformaldehyde solution in phosphate-buffered saline was performed in animals after the terminal anesthesia with Avertin (Sigma Aldrich, Germany). The dissected brain was fixed in the 4% paraformaldehyde solution in phosphate-buffered saline for 12–16 h. Tissues were dehydrated in the solutions with increasing ethanol concentrations: 75%  — 1 h, three 95% changes  5 min each, 100% — 15 min, 100% — 45 min, ethanol-chloroform (1 : 1) — 30 min, chloroform — 1 h, chloroform — 12–16 h. Brain specimens were paraffin soaked at 60 °С in three consecutive paraffin changes, with 1 h incubation in each. Embedding tissues in paraffin blocks was accomplished using the Leica EG1160 system (Leica Microsystems, Germany). The 8 µm serial brain sections were obtained using the Leica RM2265 microtome (Leica Microsystems, Germany) and mounted on the poly-L-lysine coated glass slides. Sections was deparaffinized in xylene — two changes 10 min each, rehydrated in ethanol: 100% — 5 min, 90%  — 5 min, 75%  — 5 min, water — 10 min, stained with 0.5% alcohol solution of Congo red dye — 5 min, differentiated in the 0.2% KOH solution in 80% ethanol; slices were washed with deionized water for 10 min. The Immu-Mount medium (Thermo Scientific) was used to mount coverslips.

Identification of congofilic amyloid plaques in the hyppocampal areas СА1, СА2, СА3 and dentate gyrus by histochemistry method

The sections obtained covering the area of the brain from 0.48 to 1.92 mm relative to the midline in lateral stereotaxic coordinates were used for congophilic amyloid plaque quantification in the hyppocampus [22]. Every 15th section was assessed, which resulted in 10 sections per animal. Since the Congo red dye is capable of forming a fluorescent complex with amyloid fibrils being part of amyloid plaques, detection of congophilic amyloid plaques was performed using the LSM880 confocal microscope (Carl Zeiss, Germany) by mosaic scanning of individual hyppocampal areas at 10× magnification in the spectral range with peak excitation at 596 nm and emission at 620 nm (figureА). To identify anatomic structures of the brain, the resulting fluorescence image was combined with the transmitted light microscopy image (figureB). All visible congophilic amyloid plaques of any size were counted manually. Mean values and standard deviations of congophilic amyloid plaque counts per section were calculated for each group of mice (table).

Statistical analysis methods

The data were presented as mean values for at least three independent amyloid plaque counts ± standard error of the mean (SEM). The Shapiro–Wilk test was used to test the distribution for normality. Pairwise comparison of the studied groups was performed using the Mann–Whitney U-test. The significance level was 99.9% (р < 0.001). Statistical analysis was performed using the STATISTICA 8.0 software package (StatSoft Inc., USA).

RESULTS

Fig. figure presents a typical image of the Congo red stained brain section from an intact 5-months-old APP/PS1 mouse. Congophilic amyloid plaques (CAP) that could be seen in the brain of the 7-months-old experimental animals of all five experimental groups after injections showed similar localization and size distribution in the brain parenchyma. However, plaque quantification revealed significant intergroup differences.

The data on the CAP counts in the hippocampal areas СА1, СА2, СА3 and the dentate gyrus of the 7-months-old APP/PS1 transgenic mice of experimental groups 1–5 are provided in the table. In control animals of group 1 not exposed to the HAEE peptide, the CAP count is 15.7 ± 4.6, which is consistent with the literature data on the intact 7-months-old APP/PS1 transgenic mice [23, 24].

In mice of group 2, which were repeatedly administered the HAEE initial dose (0.18 mg/kg), the CAP count was 7.5 ± 2.1, i.е. it decreased twice compared to control mice. In mice of group 3, which received HAEE at a dose of 0.30 mg/kg, the CAP counts decreased considerably (more than twice) compared to mice of group 2 and were almost 5 times lower compared to controls. The further 5- and 10-fold HAEE dose increase relative to the dose of 0.30 mg/kg did not lead to any changes in the CAP counts in animals of groups 4 and 5 compared to mice of group 3.

Thus, in this study it was found for the first time that the dose of 0.30 mg/kg was optimal for CAP formation inhibition in male and female APP/PS1 transgenic mice with the repeated subcutaneous administration of the HAEE synthetic peptide.

DISCUSSION

In this study, we provide and analyze the experimental data on the congophilic amyloid plaque counts in the sections of the isolated brain areas of transgenic mice, i.e. amyloid plaques identified using Congo red stain only. This classic visualization method, that is specific for fibrillary amyloid plaques representing the main neuro-morphological sign of amyloidosis associated with the Alzheimer's disease pathogenesis, does not take into account the possible presence of soluble amyloidbeta aggregates and oligomers in brain tissues.

The HAEE peptide capability of suppressing amyloid plaque formation in APP/PS1 transgenic mice, that are widely used as the AD animal model (review in [18]), has been already shown in a few papers. Such a capability was first demonstrated in 2015 [24] on an example of a limited sample of male and female animals (n = 6 and n = 7 in the control and experimental groups, respectively). Experimental animals received intravenous injections of the HAEE peptide at a dose of 1.13 mg/kg twice a month throughout 5 months, since the age of 2 months (each animal received a total of nine injections). As a result, the 7-months-old transgenic mice exposed to the HAEE peptide showed the decrease in congophilic amyloid plaque counts in the hyppocampal areas СА1, СА2, СА3 and the dentate gyrus by 60% compared to control animals. Then, male APP/PS1 transgenic mice received intravenous injections of a dose of 0.05 mg/kg monthly throughout 5 months, since the age of 2 months (each animal received a total of six injections) [25]. In the hippocampus of 8-months-old mice administered the

HAEE-based drugs, the congophilic amyloid plaque count (24.7 ± 3.4 per section) turned out to be 22% lower compared to control animals (31.7 ± 4.9 per section). Finally, it has recently been shown that both intraperitoneal and intranasal injections of the synthetic HAEE at a dose of 50 mg/kg to male APP/PS1 transgenic mice every 48 h throughout 1.5 months, since the age of 6 months, resulted in the decrease in the amyloid load on brain tissues in the injected animals compared to control animals. The load assessed based on the area of the apparent surface of amyloid plaques in the hyppocapus decreased 4.7- and 3.5-fold with the use of intraperitoneal and intranasal administration routes, respectively [26].

Thus, the efficacy of using the synthetic HAEE to suppress amyloid plaque formation in the hippocampus of APP/PS1 transgenic mice has been earlier reported for both males and females with the use of intravenous, intraperitoneal, and intranasal administration routes. Treatment of mice with HAEE was started at the age of both 2 months, when these animals had no amyloid inclusions in the brain, and 6 months, when there were at least five congophilic amyloid plaques in the hyppocampus [23]; the administration period changed from 1.5 to five months, and the intervals between injections were 2–30 days.

In the above studies, the dose also varied between 0.05 and 50 mg/kg with various administration rates. All these differences in experimental procedures made it impossible to draw a conclusion, which dose (0.05 mg/kg, 1.13 mg/kg or 50.00 mg/kg) was the most appropriate as a therapeutic dose for clinical trials. It is clear that the dose of 50.00 mg/kg is safe, but redundant. At the same time, the dose of 0.05 mg/kg seems to be less effective, than the dose of 1.13 mg/kg.

Despite variability of the above experimental protocols, it seems entirely reasonable to use the data on the changes in congophilic amyloid plaque counts in the hyppocampal areas СА1, СА2, СА3 and the dentate gyrus of APP/PS1 transgenic mice counted by the method [24], previously reported as reliable criterion for assessment of the HAEE peptide antiamyloid efficacy.

It is well known, that up to the age of seven months there are no significant sex differences in the development of cerebral amyloidosis in APP/PS1 transgenic mice [27], therefore, in this study, subcutaneous injections were administered to 5-months-old animals of both sexes, and brain harvesting for histochemical analysis was performed in 7-months-old mice. Injections were administered twice a week for nine weeks. The following four synthetic HAEE doses were selected for testing: 0.18 mg/kg; 0.30 mg/kg; 1.50 mg/kg; 3.00 mg/kg. Testing showed that under exposure to HAEE the CAP counts of the injected mice decreased considerably compared to control animals with all the studied doses. However, the CAP counts were roughly the same when using the doses of 0.30 mg/kg, 1.50 mg/kg, and 3.00 mg/kg. The fact that the 5- and 10-fold increase of the dose of 0.30 mg/kg caused no therapeutic effect enhancement (i.e. no CAP count decrease) suggests that when other experimental conditions are the same (period and rate of injections, age of mice at the time of the first injection), the dose of 0.30 mg/kg is necessary and sufficient for optimal suppression of amyloid plaque formation in the studied animal model of AD. The dose of 0.30 mg/kg is roughly equivalent to the dose of 1.75 mg in terms of an adult human. 

It is well known, that in 5-months-old APP/PS1 transgenic mice, amyloid plaques are formed within 24 h [28], after which the plaques are in dynamic equilibrium with soluble Aβ oligomers, and some of these oligomers initiate formation of new amyloid plaques due to their structural and functional features [29]. Recently, it has been found in animal models that the HAEE anti-amyloid effect mechanism is based on the peptide capability of effectively passing from blood to brain tissues through the blood-brain barrier and binding the Aβ molecules that are present both in the form of soluble monomers and oligomers, and as part of insoluble amyloid plaques, which ensures rapid removal of excess Aβ molecules from the brain [18, 30].

The findings that the dose increase over 0.30 mg/kg does not result in the HAEE peptide anti-amyloid effect enhancement suggest the limited number of drug targets for HAEE, i.e. this dose is enough for the HAEE molecules to “brush” the already formed amyloid plaques from the neuronal surface and prevent the soluble Aβ oligomer formation.

CONCLUSIONS

The study made it possible to define the value of 0.30 mg/kg as the optimal Ac-His-Ala-Glu-Glu-NH2 (HAEE) synthetic peptide dose for cerebral amyloidosis inhibition in male and female APP/ PS1 transgenic mice. This mouse lineage is widely used as model animals to study the Alzheimer's disease pathogenesis all over the world. That is why the research results obtained using this model are considered as highly reliable in terms of translation into clinical practice. Thus, the findings represent a scientific foundation for the use of repeated subcutaneous injections of the HAEE synthetic peptide prepared in 0.5 mL of normal saline at a dose of 1.75 mg for phase 1 clinical trial. Certainly, the efficacy of the HAEE peptide as an anti-amyloid agent for disease-modifying therapy of AD can be proven in further clinical trials only, and the results obtained represent an essential element of successful clinical trials.