Patent Application
20210196799
Alzheimer's disease (AD) is a chronic, degenerative disease of the brain which currently affects 44 million people worldwide, with one new case being diagnosed every 3.2 seconds. The disease is a neurodegenerative condition characterised by the accumulation of amyloid-β plaques, significant neuroinflammation, deterioration of synapse structure and function and cognitive decline. The incidence of disease and other forms of dementia is expected to continue to grow significantly with an estimated 100 million people impacted worldwide by 2050.
A recent study published in the medical journal of the American Academy of Neurology has pinned AD as the third leading cause of death in the US and the global cost of the disease is estimated to be 1% of the world's GDP (USD605 billion).
Development of treatments for AD has been notoriously slow and only 5 drugs are currently marketed that offer only symptomatic relief. Treatment for AD is currently limited to these five drugs, which are from two different classes (cholinesterase inhibitors and NMDA receptor antagonists). The complex, multifactorial pathophysiology of AD means that even currently marketed drugs possess limited and inconsistent efficacy. Cholinesterase inhibitors, such as donepezil, offer only symptomatic relief and clinical trials in mild cognitive impairment (MCI) patients have shown that cholinesterase inhibitors fail to halt the progression to AD.
Clinical trials aimed at developing treatments based on the predictions of the amyloid cascade hypothesis have also been largely unsuccessful. Amyloid-β (Aβ) immunisation is thought to activate microglial phagocytosis of Aβ or directly neutralise toxic Aβ oligomers and reduces or prevents development of amyloid plaques in preclinical models. Clinical trials however have suggested that active or passive immunisation has limited therapeutic efficacy in AD patients and may also produce harmful side effects.
While immunotherapy enhances the immune system in order to clear Aβ, it is well known that neuroinflammation contributes to the pathophysiology of AD. Therefore, suppression of the immune system has been considered as an alternative therapeutic approach in AD. It has been observed that non-steroidal anti-inflammatory drug (NSAID) use considerably reduces the likelihood of developing AD, but debate is ongoing as to the precise nature of the relationship between NSAID use and AD risk and although observational and longitudinal, population-based studies continue to suggest a protective effect, randomised controlled trials testing NSAIDs as an intervention in AD patients have mostly been unsuccessful.
Inhibitors of γ-secretase have been shown to influence APP processing and reduce Aβ production in vivo and in vitro, along with restoration of memory function in a cognitively impaired mouse model of AD. However, several clinical trials testing γ-secretase inhibitors in AD patients have either been halted due to serious adverse effects, shown limited efficacy, or both. Similarly, while inhibition of β-secretase shows promise as a modulator of AD pathology in preclinical models, clinical trial success for β-secretase inhibitors is relatively scarce. In any case, β- and γ-secretase enzymes are essential for normal physiological functioning of the CNS, and amyloid precursor protein (APP) is only one of a number of secretase substrates and it is clear that these enzymes have several important physiological roles. This could explain the disappointing results of clinical trials of secretase inhibitors. The serious adverse effects and limited efficacy demonstrated in these trials suggests that β- and γ-secretase inhibitors may have limited clinical application. Although there is evidence that indicates Aβ production can be suppressed in the CNS through modulation of secretase enzymes, inhibiting β- and γ-secretase may not be a feasible therapeutic approach.
Taken together, existing therapies only provide for symptomatic treatment and there are few drugs in development which offer promise. As such, there is a clear and unmet need for development of new therapies possessing disease-modifying abilities and differentiated mechanisms of action to combat this devastating disease.
Biological peptides are one of the most attractive and emerging candidates currently being developed for a host of therapeutic indications due to their numerous advantages over small molecules and/or biologics including high potency, low toxicity, high specificity, low risk of organ accumulation and drug-drug interaction.
According to a first aspect of the present invention, there is provided a peptide analogue of xenin for use in the treatment of neurological disorders.
Optionally, the peptide analogue is a peptide analogue of human xenin.
Optionally, the peptide analogue is an analogue of xenin having the GenBank Accession No AAB23907.
Further optionally, the peptide analogue is an analogue of xenin having the GenBank Accession No AAB23907.1.
Optionally, the peptide analogue is an analogue of xenin having the amino acid sequence MLTKFETKSARVKGLSFHPKRPWIL.
Optionally, the peptide analogue comprises xenin and at least one amino acid substitution or modification.
Optionally, the peptide analogue comprises xenin and at least one amino acid modification.
Optionally, the at least one amino acid modification comprises addition of a fatty acid selected from the group comprising a C-8 octanoyl group, a C-10 decanoyl group, a C-12 lauroyl group, a C-14 myristoyl group, a C-16 palmitoyl group, a C-18 stearoyl group, and a C-20 acyl group.
Further optionally, the at least one amino acid modification comprises the addition of a C-16 palmitoyl group.
Optionally, the peptide analogue comprises xenin and a C-16 palmitoyl group.
Optionally, the or each fatty acid is attached adjacent or at the N-terminal amino acid residue of the peptide analogue; adjacent or at the C-terminal amino acid residue of the peptide analogue; or at any other amino acid residue of the amino acid sequence of the peptide analogue.
Optionally, the or each fatty acid is attached at a lysine residue.
Further optionally, the or each fatty acid is attached at an alpha or epsilon amino group of a lysine residue.
Optionally, the peptide analogue comprises xenin and a C-16 palmitoyl group attached at a lysine residue.
Further optionally, the peptide analogue comprises xenin and a C-16 palmitoyl group attached at the lysine residue at position 13 of the peptide analogue.
According to a second aspect of the present invention, there is provided a peptide analogue of gastric inhibitory polypeptide, or a fragment thereof, for use in the treatment of neurological disorders.
Optionally, the peptide analogue is a peptide analogue of human gastric inhibitory polypeptide, or a fragment thereof.
Optionally, the peptide analogue is an analogue of gastric inhibitory polypeptide having the GenBank Accession No AAA88043, or a fragment thereof.
Further optionally, the peptide analogue is an analogue of gastric inhibitory polypeptide having the GenBank Accession No AAA88043.1, or a fragment thereof.
Optionally, the peptide analogue is an analogue of gastric inhibitory polypeptide having the amino acid sequence MVATKTFALLLLSLFLAVGLGEKKEGHFSALPSLPVGSHAKVSSPQPRGPRYAEGTFISDYSIAMDKI HQQDFVNWLLAQKGKKNDWKHNITQREARALELASQANRKEEEAVEPQSSPAKNPSDEDLLRDLLI QELLACLLDQTNLCRLRSR, or a fragment thereof.
Optionally, the peptide analogue is an analogue of a fragment of gastric inhibitory polypeptide comprising amino acid residues at positions 52-93 of the amino acid sequence
Further optionally, the peptide analogue is an analogue of a fragment of gastric inhibitory polypeptide, the fragment comprising the amino acid sequence
Further optionally, the peptide analogue is an analogue comprising at least 12 amino acid residues from the N-terminal end of gastric inhibitory polypeptide having the amino acid sequence
Optionally, the peptide analogue is an analogue of gastric inhibitory polypeptide having the amino acid sequence YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ.
Optionally, the peptide analogue comprises gastric inhibitory polypeptide and at least one amino acid substitution or modification.
Optionally, the peptide analogue comprises gastric inhibitory polypeptide and at least one amino acid modification.
Optionally, the at least one amino acid modification comprises acylation of an amino acid residue.
Optionally, the at least one amino acid modification comprises acetylation of an amino acid residue.
Further optionally, the at least one amino acid modification comprises acetylation of the N-terminal amino acid residue of the peptide analogue; acetylation of the C-terminal amino acid residue of the peptide analogue; or acetylation of any other amino acid residue of the amino acid sequence of the peptide analogue.
Still further optionally, the at least one amino acid modification comprises acetylation of the N-terminal amino acid residue of the peptide analogue.
Optionally, the peptide analogue comprises gastric inhibitory polypeptide, wherein the N-terminal amino acid residue of the peptide analogue is acetylated.
Optionally, the peptide analogue further comprises at least one amino acid modification comprising addition of a fatty acid selected from the group comprising a C-8 octanoyl group, a C-10 decanoyl group, a C-12 lauroyl group, a C-14 myristoyl group, a C-16 palmitoyl group, a C-18 stearoyl group, and a C-20 acyl group.
Further optionally, the at least one amino acid modification comprises the addition of a C-16 palmitoyl group.
Optionally, the peptide analogue comprises gastric inhibitory polypeptide and a C-16 palmitoyl group. Further optionally, the peptide analogue comprises gastric inhibitory polypeptide wherein the N-terminal amino acid residue of the peptide analogue is acetylated, and a C-16 palmitoyl group.
Optionally, the or each fatty acid is attached adjacent or at the N-terminal amino acid residue of the peptide analogue; adjacent or at the C-terminal amino acid residue of the peptide analogue; or at any other amino acid residue of the amino acid sequence of the peptide analogue.
Optionally, the or each fatty acid is attached to a lysine residue.
Further optionally, the or each fatty acid is attached to an alpha or epsilon amino group of a lysine residue.
Optionally, the peptide analogue comprises gastric inhibitory polypeptide and a C-16 palmitoyl group attached to a lysine residue. Further optionally, the peptide analogue comprises gastric inhibitory polypeptide wherein the N-terminal amino acid residue of the peptide analogue is acetylated, and a C-16 palmitoyl group attached to a lysine residue.
Further optionally, the peptide analogue comprises gastric inhibitory polypeptide and a C-16 palmitoyl group attached to the lysine residue at position 37 of the peptide analogue. Further optionally, the peptide analogue comprises gastric inhibitory polypeptide wherein the N-terminal amino acid residue of the peptide analogue is acetylated, and a C-16 palmitoyl group attached to the lysine residue at position 37 of the peptide analogue.
Further optionally, the peptide analogue comprises gastric inhibitory polypeptide and a C-16 palmitoyl group attached to the lysine residue at position 37 of the peptide analogue. Further optionally, the peptide analogue comprises gastric inhibitory polypeptide wherein the N-terminal amino acid residue of the peptide analogue is acetylated, and a C-16 palmitoyl group attached to the lysine residue at position 37 of the peptide analogue having the amino acid sequence YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ.
Optionally, the peptide analogue further comprises at least one amino acid modification comprising attachment of a polymer moiety of the general formula HO—(CH2-O—CH2)n-H, wherein n is an integer between 1 and 22.
Further optionally, the peptide analogue comprises xenin and at least one amino acid substitution or modification, and the peptide analogue further comprises at least one amino acid modification comprising attachment of a polymer moiety of the general formula HO—(CH2-O—CH2)n-H, wherein n is an integer between 1 and 22.
Alternatively, the peptide analogue comprises gastric inhibitory polypeptide or a fragment thereof and at least one amino acid substitution or modification, and the peptide analogue further comprises at least one amino acid modification comprising attachment of a polymer moiety of the general formula HO—(CH2-O—CH2)n-H, wherein n is an integer between 1 and 22.
Optionally, n is an integer between 1 and about 10.
Further optionally, n is an integer between about 2 and about 5.
Optionally, the polymer moiety has a branched structure. The branched structure may comprise the attachment of at least two polymer moieties of linear structure.
Alternatively, the branch point may be located within the structure of each polymer moiety.
Alternatively, the polymer moiety has a linear structure.
Optionally, the or each polymer moiety is attached adjacent or at the N-terminal amino acid residue of the peptide analogue; adjacent or at the C-terminal amino acid residue of the peptide analogue; or at any other amino acid residue of the amino acid sequence of the peptide analogue.
Optionally, the or each polymer moiety is attached adjacent or at the C-terminal amino acid residue.
Further optionally, the or each polymer moiety is attached at the C-terminal amino acid residue.
Optionally, the or each polymer moiety is attached at a lysine residue.
Further optionally, the or each polymer moiety is attached at an alpha or epsilon amino group of a lysine residue.
According to a third aspect of the present invention, there is provided a composition comprising a peptide analogue according to a first or second aspect of the invention and a pharmaceutically acceptable carrier.
Optionally, the composition comprises a pharmaceutically effective amount of the peptide analogue according to a first or second aspect of the invention and a pharmaceutically acceptable carrier.
Optionally, use comprises administration of a peptide analogue according to a first or second aspect of the invention or a composition according to a third aspect of the invention.
Further optionally, use comprises administration of a peptide analogue according to a first or second aspect of the invention or a composition according to a third aspect of the invention to a subject.
Further optionally, use comprises administration of a peptide analogue according to a first or second aspect of the invention or a composition according to a third aspect of the invention to a subject suffering from a neurological disorder.
Further optionally, use comprises administration of a pharmaceutically effective amount of a peptide analogue according to a first or second aspect of the invention or a composition according to a third aspect of the invention.
Still further optionally, use comprises administration of 0.25-25.00 nmol/kg body weight of a peptide analogue according to a first or second aspect of the invention or an equivalent amount of a composition according to a third aspect of the invention.
Still further optionally, use comprises administration of 2.50-25.00 nmol/kg body weight of a peptide analogue according to a first or second aspect of the invention or an equivalent amount of a composition according to a third aspect of the invention.
Still further optionally, use comprises administration of 25.00 nmol/kg body weight of a peptide analogue according to a first or second aspect of the invention or an equivalent amount of a composition according to a third aspect of the invention.
According to a fourth aspect of the present invention, there is provided a method for treating neurological disorders, the method comprising administering a peptide analogue according to a first or second aspect of the invention or a composition according to a third aspect of the invention.
According to a fifth aspect of the present invention, there is provided use of a peptide analogue according to a first or second aspect of the invention in the manufacture of a medicament for treating neurological disorders.
Optionally, the neurological disorder is a disorder selected from the group of disorders affecting cognitive function; and dysfunctional cognitive processes.
Optionally, disorders negatively affecting cognitive function include, but are not limited to: dementia, stroke, schizophrenia, bipolar disorder, and neurodegenerative diseases.
Optionally, disorders positively affecting cognitive function include, but are not limited to: post-traumatic stress disorder, epilepsy, Tourette's syndrome, and hallucinations.
Optionally, neurodegenerative diseases are selected from, but not limited to: Alzheimer's disease (AD), Creutzfeldt-Jacob disease (CJD), Huntington's disease, and Parkinson's disease.
Optionally, dysfunctional cognitive processes include, but are not limited to: attention, calculation, memory, judgment, insight, learning, and reasoning.
Optionally, the neurological disorder is Alzheimer's disease.
Optionally, the peptide analogue is for use in improving cognitive function.
Optionally or additionally, the peptide analogue is for use in improving memory. Further optionally or additionally, the peptide analogue is for use in improving recognition or spatial memory.
Optionally, the peptide analogue is for use in reducing amyloid beta, optionally for use in reducing amyloid beta dense core plaque number.
Optionally, the peptide analogue is for use in reducing inflammation. Further optionally, the peptide analogue is for use in reducing inflammation in the brain.
Optionally, the peptide analogue is for use in increasing synapse density or number.
Optionally, the peptide analogue is for use in increasing neurogenesis.
Embodiments of the present invention will now be described with reference to the accompanying drawings in which:
Embodiments of the present invention will now be described by way of non-limiting examples.
Materials & Methods
Animals
APPswe/PS1AE9 mice with a C57Bl/6 background were bred at the animal unit of Ulster University. Heterozygous males were bred with wild-type C57/Bl6 females bought locally (Harlan, UK). Offspring were ear punched and genotyped using PCR with primers specific for the APP sequence (Forward “GAATTCCGACATGACTCAGG”, Reverse: “GTTCTGCTGCATCTTGGACA”). Mice not expressing the transgene were used as wild-type controls. Male animals were used in all studies. Animals were caged individually and maintained on a 12/12 light-dark cycle (lights on at 08 h00, off at 20 h00), in a temperature-controlled room (T:21.5° C.±1). Food and water were available ad libitum. Animals were handled daily for two weeks prior to commencement of the study. APP/PS1 and wild-type animals were 7 months of age when treatment began. They were randomized and were administered their designated treatment, liraglutide (2.5 nm/kg bw), N-AcGIP(Lys37PAL37), xenin25(Lys13PAL) or saline (0.9% w/v), intraperitoneally (i.p.) once daily (at 15:00 h). Treatment groups comprised n=12-14. All experiments were licensed by the UK home office in accordance with the Animal (scientific procedures) Act of 1986.
Peptides
N-AcGIP(Lys37PAL), xenin25(Lys13PAL) and liraglutide were purchased from EZ Biolabs (Indiana, USA). The purity of peptides was analysed by reversed-phase HPLC and characterised using matrix assisted laser desorption/ionisation time of flight (MALDI-TOF) mass spectrometry, with a purity >99%. Peptides were reconstituted in Ultrapure® water to a concentration of 1 mg/ml in polypropylene tubes and frozen in aliquots to permit fresh preparation of doses required for injection.
Experimental Design and Treatments
APP/PS1 and wild-type animals were 7 months of age when treatment began. They were randomised and assigned to 4 treatment groups per genotype, such that there were 8 groups in total. Wild-type and APP/PS1 mice were respectively divided into four groups that received either liraglutide (2.5 nmol/kg bw), NAcGIP[Lys(37)PAL] (2.5 nmol/kg bw) Xenin-25[Lys(13)PAL] (25 nmol/kg bw) or saline (0.9% w/v), i.p. once daily (at 15 h00) for 8-10 weeks. Treatment groups comprised n=12-14. Liraglutide, NAcGIP[Lys(37)PAL] and Xenin-25[Lys(13)PAL] were purchased from EZ Biolabs (Indiana, USA). The purity of peptides was analysed by reversed-phase HPLC and characterised using matrix assisted laser desorption/ionization—time of flight (MALDI-TOF) mass spectrometry, with a purity >99%. Peptides were reconstituted in Ultrapure® water to a concentration of 1 mg/ml in polypropylene tubes and frozen in aliquots to permit fresh preparation of doses required for injection. Peptides were then prepared in 0.9% NaCl prior to administration in a volume of 10 ml/kg.
Open Field Task
Mice were subjected to the open field task (OFT) in order to assess anxiety and locomotor activity. In short, mice were placed into a circular arena (58 cm in diameter and 31 cm in height) and allowed to explore freely for 5 minutes. The arena was illuminated by a 60 W light positioned 2 m above the arena, directly adjacent to the position of the camera. Mice movements were recorded using a computerised tracking system (Biosignals, New York, USA), with which OFT performance was also analysed. Grooming events, rearing events and defecation were recorded manually to indicate levels of anxiety. Grooming was defined as behaviour in which the mouse stopped running and started licking, chewing or scratching its fur. Rearing was defined as behaviour in which the mouse stopped running and lifted both of its front paws off the floor and reared up on its hind legs. Defecation was measured by counting the number of fecal pellets in the arena after each trial. Path length, speed and linearity were recorded digitally as an indication of locomotor activity. The OFT was conducted 24 hours prior to the novel object recognition task, in order to acclimatize the mice to the apparatus and diminish the natural stress and anxiety that an unfamiliar environment might cause. The arena was thoroughly cleaned with ethanol (70%) between each animal to prevent build up of olfactory cues.
Novel Object Recognition Task
After 9 days (group 1), 25 days (group 2), 8 weeks (group 3) or 19 weeks (group 4) mice were assessed in the novel object recognition task (ORT). This task exploits the natural preference for novelty of rodents and is used as a measure of working memory in mice. In short, mice were placed into the arena for an initial habituation phase, along with two identical objects; either two red cubes (1.8 cm wide) or two white balls (2 cm wide) which were secured to the floor of the arena using Blu tack, 15 cm from the wall. The arena was dimly lit with a 60 W light bulb, positioned 2 m above the arena. Mice were allowed to freely explore the arena, with the familiar objects for 10 minutes. Their movements were recorded with an overhead camera and the time spent exploring the two familiar objects was calculated using Tracker software (Biosignals, New York, USA) within a 2 cm radius of the objects. The mice were then removed and returned to their home cage for 3 hours. After the interval, mice were then placed back into the arena for the test phase, along with one familiar object from the habituation phase and one novel object (either a red cube or a white ball). The familiar object that was used and the position of the novel object (left or right) were randomised to reduce the influence of any confounding factors. Previous studies show that neither object nor place preference is apparent with the use of the red cube and white ball. Again, mice were allowed to freely explore for 10 minutes and their movements recorded. The arena was cleaned with 70% ethanol between each trial to remove potentially confounding olfactory cues. Data are expressed as a recognition index (RI), which is calculated for each object in both the acquisition and test phases. RI is defined as the amount of time spent exploring object A or B over the total time spent exploring both objects multiplied by one hundred (tA or tB/(tA+tB)×100).
Morris Water Maze
After 10 days (group 1), 26 days (group 2), 8 weeks (group 3) or 19 weeks (group 4) mice were assessed in the Morris water maze (MWM). The maze was 40 cm in height, with a diameter of 120 cm and was filled with water maintained at 25° C.±1° C. A small escape platform (10×6.5×21.5 cm) was placed on the centre of one quadrant, 25 cm from the edge and hidden 1 cm below the surface of the water. Visual cues were placed on the walls of the room. The acquisition training phase of the MWM consisted of four 90 second trials per day, for 4 consecutive days. Mice were placed into a water-filled tank and were allowed to swim freely. The point at which the each mouse was placed into pool was randomised across the trials. Using extra-maze visual cues mice had to learn the location of an escape platform, which was hidden 1 cm below the surface of the water in the centre of the south-west quadrant of the pool. If a mouse failed to find the platform within 90 seconds, it was guided there, and encouraged to remain there for 20 seconds. Mice were then returned to their home cage for an inter-trial period of 20 minutes. Twenty-four hours following the final training day mice were placed back into the water, with the escape platform removed for a single probe trial. Mice were allowed to swim freely for 90 seconds and the time spent in the quadrant that previously contained the escape platform was measured as an indication of functional working memory. Time spent in the exact location of the escape platform was recorded as a measure of spatial acuity.
Reversal Water Maze
After 15 days (group 1), 31 days (group 2), 9.5 weeks (group 3) or 20.5 weeks (group 4) mice were subjected to the reversal water maze (RWM), in which the escape platform was moved from the south-west to the north-west quadrant. This modification is designed to assess cognitive flexibility, or the ability to relearn the new location of the platform. There were 4 trials per day, for ¾ consecutive days, followed by a final reversal probe trial.
Surgery and LTP Recording in the Hippocampus Area CA1
Mice were used for in vivo electrophysiological experiments in order to assess long-term potentiation. Mice were anesthetized with urethane (ethyl carbamate, 1.8 g/kg ip.) for the duration of all experiments. The skull was exposed and 3 holes with 0.8 mm diameter were drilled. Electrodes (tungsten with Teflon coating, Bilaney, Kent, United Kingdom) were implanted, 1.5 mm posterior and 1.0 mm lateral to the midline for the recording electrode, and 2.0 mm posterior to bregma and 1.5 mm lateral to the midline for the stimulating electrode. The electrodes were slowly lowered through the cortex and the upper layers of the hippocampus and into the CA1 region until the appearance of a negative deflecting excitatory postsynaptic potential (EPSP) that had a latency of ca 10 msec. Recordings of field excitatory postsynaptic potentials (fEPSPs) were made from the stratum radiatum in the CA1 region of the right hippocampal hemisphere in response to stimulation of the Schaffer collateral/commissural pathway. fEPSPs were recorded on a computerized stimulating and recording unit (PowerLab, ADI Instruments) in which the trigger threshold was adjustable. The triggered unit activated a constant current stimulus isolation unit (Neurolog, United Kingdom). The data acquisition system was triggered simultaneously to record all events. Sampling speed was at 20 kHz for recordings of fEPSPs. In order to assess the excitability and determine an appropriate voltage for high frequency stimulation and recording, the response of each of the mouse to increasing voltage was measured in an input/output curve at intervals of 0.25 V from 2.0 V to a maximum of 5 V (or until a maximal response was elicited). Stimulation voltage was then adjusted to generate approximately 60% of the maximum fEPSP, and the recording of the baseline was measured at this value. The high frequency stimulation protocol for inducing LTP consisted of 4 trains of 100 stimuli with an interstimulus interval of 5 msec (200 Hz) and an inter-train interval of 2 sec. This LTP induction protocol was chosen to prevent saturation of LTP and thus allow the possibility to detect facilitation of LTP. LTP was measured as a percentage of baseline fEPSP slope recorded over a 15-minute period before application of high frequency stimulation. This value was taken as 100% of the excitatory postsynaptic potential slope and all recorded values were normalized to this baseline value. The post high frequency stimulation LTP data were then analysed by a two-way repeated measures ANOVA with treatment group as the between subject factor and time as the within subject factor.
Histology
Animals were perfused trans-cardially with PBS buffer followed by ice-cold 4% paraformaldehyde in PBS. Brains were removed and hemispheres separated. One hemisphere was fixed in 4% paraformaldehyde (PFA) until processing and the other was snap frozen in liquid nitrogen and stored at −80° C. for future analysis. Hemi-brains were then transferred from PFA to 30% sucrose for at least 24 hours to prevent the formation of ice crystals upon freezing. Brains were then set with freezing spray (Envirotech Freezing Spray, Thermoscientific, UK; Cat. No. 6769038) and left in holders within the cryostat (Leica Microsystems, Milton Keynes, UK), which was set at −25° C. Brains were then cut in 40 μm coronal sections at a depth of −2 to −3 Bregma using a Leica cryostat, and collection began on appearance of the hippocampus (−0.94 μm to bregma) until approximately 3.28 μm to bregma. For stereological purposes the first section to be kept was selected at random, and placed in a 24 well plate, and one section in every 7 were collected sequentially, such that 7 sets of sections, with 10-13 sections each were collected from each mouse brain. Plates were stored at −20° C. until required.
Sections were chosen according to stereological rules with the first section taken at random and every 6th section afterwards. Staining was carried out for beta-amyloid plaques, and for congophilic dense-core amyloid plaques. Staining was also carried out for synaptophysin, in order to determine synapse numbers, and Doublecortin (DCX), a marker for immature neurons, as a measure of neurogenesis. Staining was carried out for GFAP; a marker of astrocytes, Iba1; a marker of microglia, 8-oxoguanine; a marker of oxidative stress, IRS-1 pSer616 and synaptophysin, which was used to determine synapse density. All sections were incubated in 0.08%-5% H2O2 to quench endogenous peroxidase activity and permeabilised using 0.01%-0.03% Triton X.
The Iba1 protocol required incubation of sections in 0.05 M trisodium citrate (pH 9) at 90° C. for 30 minutes to enhance antigen recognition. Non-specific binding was then blocked using 5%-10% normal serum prior to incubation with mouse, anti-GFAP (1:250, in PBS with 0.3% Triton X; Merck Millipore, Feltham, UK; Cat. No. MAB3402) polyclonal, rabbit anti-Iba1 (1:200 in PBS with 2% goat serum and 0.3% Triton X; Wako Chemicals, Neuss, Germany; Cat. No. 019-19791), polyclonal, rabbit anti-Aβ antibody (1:200 in TBS; Invitrogen, Paisley, UK; Cat. No. 71-5800), goat antimouse doublecortin (1:200 in PBS with 2% rabbit serum; Santa Cruz Biotechnology Inc. Dallas, Tex., USA; Cat. No. Sc-8066) or synaptophysin (1:200; Abcam, Cambridge, UK; Cat. No. ab7837) overnight at 4° C. After blocking the sections in 5% normal serum to avoid non-specific antibody binding, they were incubated with rabbit polyclonal anti amyloid beta peptide (1:250, Invitrogen, UK, 71-5800) or goat polyclonal anti doublecortin (1:200, Santacruz, USA, sc-710), or rabbit anti-synaptophysin (1:200, Abcam, Cambridge, Mass., 7837-500). After overnight incubation at 4° C., the sections were incubated in respective secondary antibodies. The following day, sections were incubated with respective secondary antibodies and visualisation was achieved using Vectastain Elite and SG substrate (Vectamount AQ, Vector Laboratories Ltd, Peterborough, UK; Cat. No. H5501). Dense-core amyloid plaques were detected using Congo red. Sections were washed with PBS and were then mounted onto salinised slides and allowed to air dry, immersed in distilled water for 30 seconds and then a solution of saturated sodium chloride (NaCl) for 20 minutes. Prior to the addition of the slides, sodium hydroxide (NaOH) was added to the saturated NaCl. Slides were then transferred from the NaCl solution to 0.2% alkalised Congo red (Sigma Aldrich, St Louis, Mo., USA; Cat. No. 60910) diluted in saturated NaCl solution for 30 minutes. Slides were then dehydrated in graded ethanol and immersed in xylene 3 times for 5 minutes, before cover slips were mounted using Vectamount permanent mounting medium (Vector laboratories, Burlingame, Calif.; Cat. No. H-5000). Images were obtained of stained sections using a Zeiss Axio microscope (Axio scope A1, Zeiss, Germany) and the percentage area stained in each image (2 images per section, approximately 8-10 sections per mouse, 16-20 images total) was quantified using a multi threshold plug-in within Image J (ImageJ, U. S. National Institutes of Health, Bethesda, Md., USA). Analysis of percentage stained area was conducted for all markers, except synaptophysin, for which analysis of OD was performed. Images were converted to grayscale, a threshold was set and the average percentage area positively stained was calculated for each brain. All immunohistochemistry analysis was conducted in a blinded manner, whereby codes were applied to microscope slides by colleagues not involved in the studies. Codes were only broken upon completion of analysis.
Human APP ELISA Brain Extraction
Cell extraction buffer (Invitrogen, Camarillo, Calif., USA; Cat. No. FNN0011) composed of 10 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na4P2O7, 2 mM Na3VO4, 1% Triton X, 10% glycerol, 0.1% SDS, 0.5% deoxycholate, was supplemented with 250 μl protease inhibitor cocktail per 5 ml (Sigma-Aldrich, St Louis, Mo., USA; Cat. No. P8340) immediately prior to use. A volume of 4 ml of cell extraction buffer was added per 100 mg brain tissue that had been homogenised under liquid nitrogen. Samples were lysed for 30 minutes on ice and vortexed at 10 minute intervals, before being centrifuged at 13,000 rpm for 10 minutes at 4° C. The resulting supernatant was aliquoted into a new eppendorf and kept on ice for immediate analysis of APP levels.
Human APP ELISA Brain Extraction
Cell extraction buffer (Invitrogen, Camarillo, Calif., USA; Cat. No. FNN0011) composed of 10 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na4P2O7, 2 mM Na3VO4, 1% Triton X, 10% glycerol, 0.1% SDS, 0.5% deoxycholate, was supplemented with 250 μl protease inhibitor cocktail per 5 ml (Sigma-Aldrich, St Louis, Mo., USA; Cat. No. P8340) immediately prior to use. A volume of 4 ml of cell extraction buffer was added per 100 mg brain tissue that had been homogenised under liquid nitrogen. Samples were lysed for 30 minutes on ice and vortexed at 10 minute intervals, before being centrifuged at 13,000 rpm for 10 minutes at 4° C. The resulting supernatant was aliquoted into a new eppendorf and kept on ice for immediate analysis of APP levels.
Aggregated Aβ Oligomer Sandwich ELISA
Each test sample was diluted 1:10 in Standard Diluent Buffer provided by the Aggregated Aβ ELISA Kit (Invitrogen, Camarillo, Calif., USA; Cat. No. KHB3491). Aggregated Aβ standards were prepared in duplicate from Human Aggregated Aβ Standard solution (Invitrogen, Camarillo, Calif., USA; Cat. No. KHB3491) from 5.7 ng/ml to 0.0195 ng/ml, diluted in Standard Diluent Buffer. Standard and test samples (100 μl) were pipetted into wells coated with monoclonal antibody specific for the N-terminus of Aβ using a 96-well plate (Invitrogen, Camarillo, Calif., USA; Cat. No. KHB3491). The first 2 wells were left empty, as chromogen blanks while 100 μl standard diluent buffer was added to zero wells. Wells were covered with an adhesive plate cover and samples were incubated for 2 hours at room temperature. Using a multi wash III tricontinent machine (Tricontinent, Grass Valley, Calif., USA), wells were washed 4 times with wash buffer (Invitrogen, Camarillo, Calif., USA; Cat. No. KHB3491) diluted 1:25 in distilled water beforehand. After washing, 100 μl of biotinylated detection antibody (Invitrogen, Camarillo, Calif., USA; Cat. No. KHB3491) was added to each well except chromogen blank wells and incubated for 1 hour at room temperature. After 4 washes in wash buffer, wells were incubated for 30 minutes at room temperature with 100 μl of horseradish peroxidase-labeled streptavidin (SAV-HRP) (Invitrogen, Camarillo, Calif., USA; Cat. No. KHB3491) except chromogen blank wells. Excess SAV-HRP was removed and wells were washed 4 times with wash buffer. Each well was then incubated with 100 μl of stabilised chromogen (Invitrogen, Camarillo, Calif., USA; Cat. No. KHB3491) for 20-30 minutes at room temperature in the dark until a blue colour developed. Finally, 100 μl of stop solution (Invitrogen, Camarillo, Calif., USA; Cat. No. KHB3491) was added, eliciting a blue to yellow colour change in the solution. The plate was introduced to a spectrophotometer (Flexstation 3 Benchtop multimode microplate reader, Molecular Devices Inc, Sunnyvale, Calif., USA) immediately after adding stop solution. Absorbance of each well was performed at 450 nm having blanked the plate reader against a chromogen blank. Absorbance readings of the standards were plotted against the standard concentration and Human Aggregated Aβ standard curve generated was used to determinate aggregated Aβ concentrations in each sample. Aggregated Aβ concentrations of each test sample were then divided by the corresponding brain protein concentrations, as determined by the Bradford assay.
Aβ1-40 and Aβ1-42 ELISA Brain Extraction
Brain tissue, previously homogenised under liquid nitrogen was mixed with 8 volumes of ice-cold guanidine buffer (5.0 M guanidine HCl (Sigma-Aldrich, St Louis, Mo., USA; Cat. No. G3272)/50 mM Tris HCl (Sigma-Aldrich, St Louis, Mo., USA; Cat. No. T5941, pH 8.0) at 4° C. overnight. The homogenates were then further diluted (1:10) with ice-cold reaction buffer BSAT-DPBS (Dulbecco's PBS (Sigma-Aldrich, St Louis, Mo., USA; Cat. No. D1408) with 5% BSA and 0.03% Tween 20 (Sigma-Aldrich, St Louis, Mo., USA; Cat. No. P9416), supplemented with 250 μl protease inhibitor cocktail per 5 ml (Sigma-Aldrich, St Louis, Mo., USA; Cat. No. P8340). Samples were then centrifuged for 20 minutes at 4° C. The supernatant was transferred into a new eppendorf and stored on ice until use.
Aβ 1-40 Sandwich ELISA
Each test sample was diluted 1:3 in Standard Diluent Buffer provided by the Aβ1-40 ELISA Kit (Invitrogen, Camarillo, Calif., USA; Cat. No. KHB3481). Aβ1-40 standards were prepared in duplicate from Human Aβ1-40 Standard solution (Invitrogen, Camarillo, Calif., USA; Cat. No. KHB3481) from 0.5 ng/ml to 0.00781 ng/ml, diluted in Standard Diluent Buffer. Standard and test samples (50 μl) were pipetted into wells coated with monoclonal antibody specific for the N-terminus of Aβ1-40 using a 96-well plate (Invitrogen, Camarillo, Calif., USA; Cat. No. KHB3481). The first 2 wells were left empty, as chromogen blanks, while 50 μl standard diluent buffer was added to zero wells. A rabbit detection antibody (50 μl) that recognised the C-terminus of the 1-40 Aβ sequence was then added to each well, except for the chromogen blanks. Wells were then covered with an adhesive plate cover and samples were incubated for 3 hours at room temperature with shaking. Using a multi wash III tricontinent machine (Tricontinent, Grass Valley, Calif., USA), wells were washed 4 times with wash buffer (Invitrogen, Camarillo, Calif., USA; Cat. No. KHB3481) diluted 1:25 in distilled water beforehand. After washing, 100 μl of anti-rabbit IgG HRP was added to each well, except the chromogen blanks. The wells were covered with a plate cover and the samples were incubated for 30 minutes at room temperature. Excess anti-rabbit IgG HRP was removed and wells were washed 4 times with wash buffer. Each well was then incubated with 100 μl of stabilised chromogen (Invitrogen, Camarillo, Calif., USA; Cat. No. KHB3481) for 20-30 minutes at room temperature in the dark until a blue colour developed. Finally, 100 μl of stop solution (Invitrogen, Camarillo, Calif., USA; Cat. No. KHB3481) was added, eliciting a blue to yellow colour change in the solution. The plate was introduced to a spectrophotometer (Flexstation 3 Benchtop multimode microplate reader, Molecular Devices Inc, Sunnyvale, Calif., USA) immediately after adding stop solution. Absorbance of each well was read at 450 nm having blanked the plate reader against a chromogen blank. Absorbance readings of the standards were plotted against the standard concentration and Human Aβ1-40 standard curve generated was used to determinate Aβ1-40 concentrations in each sample. Aβ1-40 concentrations of each test sample were then divided by the corresponding brain protein concentrations, as determined by the Bradford assay.
Aβ 1-42 Sandwich ELISA
Each test sample was diluted 1:3 in Standard Diluent Buffer provided by the Aβ1-42 ELISA Kit (Invitrogen, Camarillo, Calif., USA; Cat. No. KHB3441). Aβ1-42 standards were prepared in duplicate from Human Aβ1-42 Standard solution (Invitrogen, Camarillo, Calif., USA; Cat. No. KHB3441) from 1 ng/ml to 0.01563 ng/ml, diluted in Standard Diluent Buffer. Standard and test samples (50 μl) were pipetted into wells coated with monoclonal antibody specific for the N-terminus of Aβ using a 96-well plate (Invitrogen, Camarillo, Calif., USA; Cat. No. KHB3441). The first 2 wells were left empty, as chromogen blanks while 50 μl standard diluent buffer was added to zero wells. A rabbit detection antibody (50 μl) that recognised the C-terminus of the 1-42 Aβ sequence was then added to each well, except for the chromogen blanks. Wells were then covered with an adhesive plate cover and samples were incubated for 3 hours at room temperature with shaking. Using a multi wash III tricontinent machine (Tricontinent, Grass Valley, Calif., USA), wells were washed 4 times with wash buffer (Invitrogen, Camarillo, Calif., USA; Cat. No. KHB3441) diluted 1:25 in distilled water beforehand. After washing, 100 μl of anti-rabbit IgG HRP was added to each well, except the chromogen blanks. The wells were covered with a plate cover and the samples were incubated for 30 minutes at room temperature. Excess anti-rabbit IgG HRP was removed and wells were washed 4 times with wash buffer. Each well was then incubated with 100 μl of stabilised chromogen (Invitrogen, Camarillo, Calif., USA; Cat. No. KHB3441) for 20-30 minutes at room temperature in the dark until a blue colour developed. Finally, 100 μl of stop solution (Invitrogen, Camarillo, Calif., USA; Cat. No. KHB3441) was added, eliciting a blue to yellow colour change in the solution. The plate was introduced to a spectrophotometer (Flexstation 3 Benchtop multimode microplate reader, Molecular Devices Inc, Sunnyvale, Calif., USA) immediately after adding stop solution. Absorbance of each well was performed at 450 nm having blanked the plate reader against a chromogen blank. Absorbance readings of the standards were plotted against the standard concentration and Human Aβ1-42 standard curve generated was used to determinate Aβ1-42 concentrations in each sample. Aβ1-42 concentrations of each test sample were then divided by the corresponding brain protein concentrations, as determined by the Bradford assay.
Statistics
Data were analysed using the program Prism (Graphpad software Inc., USA), with the level of probability set at 95%. Results are expressed as means±SEM. Data were analyzed by 1-way or 2-way ANOVA, followed by post hoc tests, or student t-tests.
In Vivo Studies Establishing Effects of Peptide Analogues of the Invention on Early Cognitive Decline in APP/PS1 Mice.
APP/PS1 mice were injected with either saline, liraglutide, or peptide analogue of the invention (Xenin-25[Lys(13)PAL]) once daily for 10 weeks. Xenin-25[Lys(13)PAL] and liraglutide were assessed for their ability to preserve levels of synaptophysin (a presynaptic vesicle protein), a marker of synaptic density. Synaptic loss is associated with cognitive decline. Therefore, the ability to preserve synapses is indicative of neuroprotective properties, with increased synaptic integrity associated with improved cognitive function.
As illustrated in
Liraglutide-treated APP/PS1 animals had significantly higher synaptophysin levels than saline-treated APP/PS1 mice in the polymorphic layer (A, p<0.001), the molecular layer (C, p<0.05), the stratum pyramidale (E, p<0.0001) and the interior (G, p<0.0001) and exterior (H, p<0.0001) cortex. Xenin-25[Lys(13)PAL] treated APP/PS1 mice had significantly higher synaptophysin levels than saline-treated APP/PS1 mice in the polymorphic layer (A, p<0.01), the stratum pyramidale (E, p<0.01) the interior (G, p<0.0001) and exterior (H, p<0.0001) cortex, indicating the protective ability of both liraglutide and Xenin-25[Lys(13)PAL] on synapse numbers. Synaptic loss is associated with the progression of Alzheimer's disease.
These data show both Xenin-25[Lys(13)PAL] and liraglutide to preserve synapses in the hippocampus and cortex (both of which are important brain regions in the context of learning and memory), further demonstrating their use in treating neurological disorders.
Analysis of Synaptic Density Following Treatment with Peptide Analogues of the Invention in APP/PS1 Mice
APP/PS1 mice were injected with saline, liraglutide and NAcGIP[Lys(37)PAL]once daily for 10 weeks; and NAcGIP[Lys(37)PAL] and liraglutide were assessed for their ability to preserve levels of synaptophysin.
As illustrated in
Specifically, NAcGIP[Lys(37)PAL]treated APP/PS1 mice had significantly higher expression levels than saline-treated APP/PS1 mice in the polymorphic layer (A, p<0.0001), the stratum pyramidale (E, p<0.0001), the stratum oriens (F, p<0.05) and the interior (G, p<0.0001) and exterior (H, p<0.0001) cortex, indicating the protective ability of NAcGIP[Lys(37)PAL] on synapse numbers. The effects were comparable to those observed in liraglutide treated APP/PS1 mice. Preservation of synapses in the hippocampus and cortex, which are important brain regions in the context of learning and memory, by NAcGIP[Lys(37)PAL] and liraglutide further demonstrate use in treating neurological disorders.
Effects of Peptide Analogues of the Invention on Key Biomarkers of AD
Studies were conducted in the APP/PS1 mouse model to assess the chronic effect of Xenin-25[Lys(13)PAL] in comparison to liraglutide and saline controls, on beta amyloid and dense core congophilic plaque load as well as neurogenesis and inflammation. Histological markers were analysed following 10 weeks of treatment.
As illustrated in
When assessed for its effects on inflammation, IBA-1 levels of Xenin-25[Lys(13)PAL] were comparable to saline-treated APP/PS1 mice (see
In addition to its effect on beta amyloid and dense core plaque load, Xenin-25[Lys(13)PAL] demonstrated an ability to significantly increase neurogenesis in APP/PS1 mice (
Effects of Peptide Analogues of the Invention on Key Biomarkers of AD
Studies were conducted in the APP/PS1 mouse model to assess the effect of NAcGIP[Lys(37)PAL] in comparison to liraglutide and saline controls, on beta amyloid and dense core congophilic plaque load, inflammation and neurogenesis.
As illustrated in
When assessed for its effects on inflammation, IBA-1 levels of NAcGIP(Lys37PAL)-treated mice were comparable to saline-treated APP/PS1 mice (see
In addition, NAcGIP[Lys(37)PAL] demonstrated an ability to partially restore neurogenesis in treated APP/PS1 mice (
Peptide Analogues of the Invention Reduce Soluble Oligomer Levels
As illustrated in
Xenin-25[Lys(13)PAL]-treatment resulted in a highly significant 80% reduction in aggregated beta amyloid oligomers compared with saline treated APP/PS1 mice (5B, p<0.001). This was greater than the reduction of 54% observed in liraglutde-treated mice compared with controls (5B, p<0.01). Such a significant reduction would be expected to improve neuronal communication in vivo.
Peptide Analogues of the Invention Reduce Soluble Oligomer Levels
As illustrated in
Dose Response Study
Male C57131/6 mice were bred at Ulster University and aged to 8-10 weeks before commencement of treatment. Animals were caged individually and maintained on a 12/12 light-dark cycle (lights on at 08 h00, off at 20 h00), in temperature-controlled room (T:21.5° C.±1). Food and water were available ad libitum. Mice were injected with liraglutide, N-AcGIP[Lys(37)PAL], or Xenin-25[Lys(13)PAL] at 3 doses; 0.25 nmol/kg, 2.5 nmol/kg, and 25 nmol/kg. Another group were injected with saline as a control. Animals were injected with their respective treatment once daily (at 15:00) for 21 days before being subjected to in vivo LTP. All experiments were licensed by the UK home office in accordance with the Animal (scientific procedures) Act of 1986.
When testing the inducibility of LTP in the hippocampus in vivo, using a weak stimulation protocol, saline-treated wild-type mice induced weak LTP (see
On analysis of post high frequency stimulation baselines liraglutide treatment was associated with enhanced LTP at lower concentrations (
On analysis of post high frequency stimulation baselines NAcGIP[Lys(37)PAL] treatment was associated with enhanced LTP at higher concentrations (
On analysis of post high frequency stimulation baselines Xenin-25[Lys(13)PAL] treatment was associated with enhanced LTP at higher concentrations (
Acute Effects of Treatment on Locomotor Function and Anxiety.
Wild-type and APP/PS1 mice were injected with saline, liraglutide, NAcGIP[Lys(37)PAL] or Xenin-25[Lys(13)PAL] 30 minutes prior to being introduced to the open field arena in order to assess acute effects of drugs on spontaneous locomotor behaviour and anxiety. Path length (
Acute Effects of Treatment on Recognition Memory
As illustrated in
Chronic Effects of Treatment on Spatial Learning and Memory
Morris water maze training began after 8 weeks treatment. Animals continued to receive treatment throughout behavioural testing. Training began at 09:00 daily, and drugs were administered at 15:00 to exclude the potential for acute drug effects on performance. During the acquisition phase of the Morris water maze task, animals completed 4 trials per day over 4 consecutive days. Average daily performance of wild-type mice is illustrated in
Spatial memory was assessed in a probe trial conducted on day 5, 24 hours following the final training session (
Spatial acuity was assessed by measuring the amount of time spent swimming at the precise location where the escape platform had been located during training (
Average daily performance of APP/PS1 mice in the acquisition phase of the Morris water maze task is illustrated in
Spatial memory of APP/PS1 mice was assessed in a probe trial conducted on day 5, 24 hours following the final training session. The escape platform was removed and spatial memory was assessed using the percentage of time spent swimming in the target quadrant (
Chronic Effects of Treatment on Reversal Learning and Memory
Average daily performance of wild-type mice during the acquisition phase of the reversal water maze task is illustrated in
Spatial memory was assessed in a reversal probe trial conducted 24 hours after the final training session on day 5 (
Spatial acuity was assessed by measuring the amount of time spent swimming at the precise location where the escape platform had been located (
Average daily performance of APP/PS1 mice during the acquisition phase of the reversal water maze task is illustrated in
Chronic Effects of Treatment on Locomotor Function and Anxiety
Path length (
Chronic Effects of Treatment on Recognition Memory
Object recognition memory in response to chronic administration of saline, liraglutide, NAcGIP[Lys(37)PAL] and Xenin-25[Lys(13)PAL] is illustrated in
Immunohistochemistry
Aβ Plaque Load
As illustrated in
Microglia
Representative micrographs illustrating Iba1 staining in the cerebral cortex (
Astrocytes
Representative micrographs illustrating GFAP staining in the cerebral cortex (
Neurogenesis
There were significantly enhanced levels of neurogenesis in wild-type Xenin-25[Lys(13)PAL]-treated mice (
Synapse Density
Representative micrographs of synaptophysin staining in wild-type (
Soluble Aβ
As illustrated in
The present disclosure demonstrates the effects of 8-10 weeks treatment with liraglutide, NAcGIP[Lys(37)PAL] and Xenin-25[Lys(13)PAL], long-lasting analogues of gut hormones glucagon-like peptide-1, glucose-dependent insulinotropic peptide and xenin-25, respectively, in the APP/PS1 mouse model of Alzheimer's disease. Cognitive function was measured using novel object recognition and Morris water maze tasks. Effects of treatment on soluble and insoluble amyloid-β burden, inflammation, synapse density and neurogenesis was assessed in the brains of APP/PS1 and wild-type mice. Liraglutide and NAcGIP[Lys(37)PAL] improved cognitive performance in APP/PS1 mice and, along with Xenin-25[Lys(13)PAL], reduced amyloid-β burden in the brain. Liraglutide ameliorated inflammation in the brain and synapse density was restored in the hippocampus and cortex of APP/PS1 mice following 10 weeks treatment with liraglutide, NAcGIP[Lys(37)PAL] and Xenin-25[Lys(13)PAL]. Additionally, Xenin-25[Lys(13)PAL] treatment increased numbers of doublecortin-positive neurons in the dentate gyrus of wild-type and APP/PS1 mice, suggesting that this peptide augmented neurogenesis. These results underscore the neuroprotective effects of liraglutide and also provide novel evidence that NAcGIP[Lys(37)PAL] and Xenin-25[Lys(13)PAL] possess neuroprotective properties.
Chronic liraglutide treatment in APP/PS1 mice improved spatial learning in the acquisition phase of the Morris water maze and spatial memory was also restored following liraglutide treatment. Chronic treatment with liraglutide, NAcGIP[Lys(37)PAL] and Xenin-25[Lys(13)PAL] also restored reversal learning in APP/PS1 mice, whereas reversal memory recall was improved by liraglutide only. These beneficial effects on cognitive function appear to be related to the positive effects of liraglutide on synaptic plasticity. The finding that liraglutide improves learning and memory is further supported by results depicted here, which demonstrate heightened performance in the reversal water maze task by liraglutide-treated APP/PS1 mice. In addition, time spent in the exact target area in the reversal probe trial, a measure of spatial acuity, was significantly elevated in liraglutide-treated APP/PS1 mice, highlighting the robust effect of liraglutide on cognitive performance. The present disclosure illustrates that reversal learning was also improved in APP/PS1 mice treated with NAcGIP[Lys(37)PAL] and Xenin-25[Lys(13)PAL].
The present disclosure provides further evidence that NAcGIP[Lys(37)PAL] also has a positive effect, albeit subtle, on cognitive function in APP/PS1 mice. Data illustrated here indicate that Xenin-25[Lys(13)PAL] enhanced reversal learning in cognitively impaired APP/PS1 mice. Since Xenin-25[Lys(13)PAL] exerts its effects through NTR1, the improvement in reversal learning depicted here, likely represents activation of central NTR1 by Xenin-25[Lys(13)PAL]. The cognitive flexibility that successful learning of the reversal water maze demands, relies on activity within several brain regions, including the entorhinal cortex, hippocampus and prefrontal cortex, areas in which NTR1 is expressed. Modulation of NTR1 in the hippocampus and cortical areas could explain the enhanced reversal learning illustrated here. Impaired recognition memory was apparent in saline-treated APP/PS1 mice and was restored following chronic treatment with liraglutide. Similarly, NAcGIP[Lys(37)PAL] treatment also restored recognition memory in APP/PS1 mice.
Ten weeks liraglutide treatment reduced Aβ immunopositivity and dense core plaque number in APP/PS1 mice. A significant reduction of Aβ deposition and plaque number was detected in NAcGIP[Lys(37)PAL] and Xenin-25[Lys(13)PAL]-treated APP/PS1 mice. Results depicted in the current disclosure may represent the culmination of a sequence of events initiated by activation of neuronal NTR1 in the brain by Xenin-25[Lys(13)PAL], enhanced intracellular calcium and stimulation of phospholipases C and A2, activation of PKC, enhancement of α-secretase APP cleavage and subsequent reduction of Aβ production and deposition.
The immunohistochemical data described above is supported by the biochemical data, which demonstrate reductions of soluble Aβ oligomers in the brains of APP/PS1 mice treated with liraglutide, NAcGIP[Lys(37)PAL] and Xenin-25[Lys(13)PAL]. Furthermore, treatment with liraglutide reduced the Aβ42/40 ratio. Since Aβ42 levels were no different between treatment groups, the altered 442/40 ratio is most likely due to a relative increase in non-toxic 440 in liraglutide-treated animals. Together, this suggests that liraglutide treatment reduced Aβ oligomers and plaque deposition by modifying APP processing, whereas NAcGIP[Lys(37)PAL] and Xenin-25[Lys(13)PAL] treatment reduced amyloid burden via alternative mechanisms. One possibility is that NAcGIP[Lys(37)PAL] enhanced Aβ clearance and degradation, a proposition that is supported by the finding that levels of microglia were increased in the brains of NAcGIP[Lys(37)PAL]-treated APP/PS1 mice. Given that microglia are known to actively engulf and degrade Aβ, it is not unreasonable to suggest that the reduction of Aβ deposition and soluble oligomer load shown here, is due to increased activation and mobilisation of phagocytic microglia.
Neurogenesis was increased in the dentate gyrus of wild-type and APP/PS1 mice treated with Xenin-25[Lys(13)PAL]. In the present disclosure, liraglutide and NAcGIP[Lys(37)PAL] moderately enhanced the number of doublecortin-positive neurons in the dentate gyrus, however the differences were not significant compared to saline-treated controls. This shows that while liraglutide and NAcGIP[Lys(37)PAL] increased the number of immature neurons in the dentate gyrus, hippocampal neurogenesis was robustly enhanced following Xenin-25[Lys(13)PAL] treatment in wild-type and APP/PS1 mice; a previously unreported finding.
The reduction in hippocampal and cortical synapse density that was apparent in APP/PS1 mice was ameliorated in the polymorphic layer and stratum pyramidale of the hippocampus and in the cerebral cortex of APP/PS1 mice treated with liraglutide, NAcGIP[Lys(37)PAL] and Xenin-25[Lys(13)PAL].
The current disclosure treated 7 month-old APP/PS1 mice for 10 weeks, which increased synaptophysin staining in the polymorphic layer, molecular layer and stratum pyramidale, as well as the cerebral cortex, suggesting that these particular areas of the brain contain synapses that are more readily modified by liraglutide. Since synaptophysin levels remained low in the granule cell layer, stratum radiatum and stratum oriens of treated APP/PS1 mice, these results might also reflect increased vulnerability of synapses in these areas to the synaptotoxic effects associated with Aβ pathology. NAcGIP[Lys(37)PAL] treatment in the present disclosure increased synapse density in the polymorphic layer, stratum pyramidale and the cortex of APP/PS1 mice. The present disclosure also demonstrates, for the first time, that Xenin-25[Lys(13)PAL] treatment in APP/PS1 mice increased synaptophysin staining in the polymorphic layer, stratum pyramidale and cerebral cortex. Results of the present disclosure demonstrate, for the first time, that Xenin-25[Lys(13)PAL] improves cognitive function, enhances neurogenesis and synapse density and ameliorates Aβ pathology in the APP/PS1 mouse model of AD. This disclosure has shown that liraglutide robustly ameliorates cognitive dysfunction and neuropathology in APP/PS1 mice. NAcGIP[Lys(37)PAL] and Xenin-25[Lys(13)PAL] were also shown to mitigate soluble and insoluble amyloid burden and modestly improved cognitive function in APP/PS1 mice.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1603510.7 | Feb 2016 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2017/054665 | 2/28/2017 | WO | 00 |
