Protective Properties Of GLP-1 And Associated Peptide Hormones in Neurodegenerative Disorders Part 3

12 | CLINICAL TRIALS IN ALZHEIMER'S DISEASE AND PARKINSON'S DISEASE PATIENTS

As there are several GLP-1 receptor agonists on the market to treat type 2 diabetes mellitus, it is relatively straightforward to test these drugs in the clinic in patients with Alzheimer's disease or Parkinson's disease. 

Diabetes ist eine chronische Krankheit, die sich negativ auf unsere Gesundheit auswirkt. Viele Menschen wissen jedoch nicht, dass Diabetes auch unser Gedächtnis beeinträchtigen kann. Untersuchungen zufolge leiden Menschen mit Diabetes häufig unter Symptomen von Gedächtnisverlust.

Dies bedeutet jedoch nicht, dass Diabetes für den Gedächtnisverlust verantwortlich ist. Im Gegenteil: Wenn wir rechtzeitig geeignete Vorsorgemaßnahmen ergreifen, wird Diabetes nicht zu einem gedächtnisbeschränkenden Hindernis. Beispielsweise kann die Aufrechterhaltung eines guten Blutzuckerspiegels dazu beitragen, den Gedächtnisverlust zu verlangsamen. Gleichzeitig können wir auch unsere Gehirnfunktion schützen, indem wir einen gesunden Lebensstil und Essgewohnheiten praktizieren und beibehalten.

Es ist wichtig, dass wir uns daran erinnern, dass wir, selbst wenn bei uns Diabetes diagnostiziert wird, unser Selbstvertrauen und unseren Lebensmut nicht verlieren sollten. Wir können immer noch Veränderungen vornehmen, um unseren Körper und unser Gehirn gesund zu halten. Gehen wir also mit einer positiven Einstellung an Diabetes heran, übernehmen wir die Kontrolle über unsere Gesundheit und führen wir weiterhin ein gesundes, glückliches und erfülltes Leben. Es ist ersichtlich, dass wir das Gedächtnis verbessern müssen, und Cistanche deserticola kann das Gedächtnis erheblich verbessern, da Cistanche deserticola auch das Gleichgewicht von Neurotransmittern regulieren kann, beispielsweise durch die Erhöhung des Acetylcholinspiegels und der Wachstumsfaktoren. Diese Substanzen sind für das Gedächtnis und das Lernen sehr wichtig . Darüber hinaus kann Cistanche deserticola auch die Durchblutung verbessern und die Sauerstoffversorgung fördern, wodurch sichergestellt werden kann, dass das Gehirn ausreichend Nährstoffe und Energie erhält, wodurch die Vitalität und Ausdauer des Gehirns verbessert werden.

Click Know to improve short-term memory

Based on the encouraging results of preclinical studies, clinical trials have been conducted or are on the way to investigate the neuroprotective effects of exendin-4, liraglutide, liraglutide, or other GLP-1 mimetics in Parkinson's disease or Alzheimer's disease patients. First results from clinical trials demonstrate that the preclinical results translate to the clinic.

13 | PARKINSON'S DISEASE

A pilot trial testing exendin-4 (exenatide, Byetta, Bydureon) in Parkinson's disease patients had been conducted. This clinical trial tested exenatide in an open-label trial in 45 non-diabetic patients. 

The average time since diagnosis was 10 years, which means that Parkinson's disease had already progressed. Exendin-4 was administered for 12 months and patients were retested 2 months after the trial had stopped. Drug-treated patients showed an improvement of 2.7 points on the Movement Disorder Society (MDS)-UPDRS test battery of motor activity, whereas control patients declined 2.2 points. 

In addition, patients were assessed in the Mattis DRS-2 cognitive test battery, as late-stage Parkinson's disease patients often develop cognitive impairments, too. There was a clear improvement in the exendin-4 group, whereas the control group deteriorated rapidly (Aviles-Olmos et al., 2013). After the trial had finished, patients were tested again 12 months later. 

The drug group had not deteriorated in motor skill tests or the cognitive assessment since the beginning of the trial 24 months ago, whereas the control group had deteriorated continuously as expected for Parkinson's disease patients. This demonstrates that the exendin-4 effect was not as short-lived as it is with LDOPA treatment, but stopped disease progression, particularly in cognitive measures (Aviles-Olmos et al., 2014). 

However, the pilot study did not incorporate a placebo control group, questioning the validity of the results. Hence, a follow-up Phase II double-blind, placebo-controlled trial had been conducted. These patients were not as progressed in Parkinson's disease and the period since diagnosis was around 6 years. In the MDS-UPDRS Part 3 test battery, the drug group improved after 48 weeks compared with the placebo group. 

Twelve weeks after the trial had stopped, patients were retested and the difference between groups was still statistically significant. The outcome confirmed the first pilot study. It showed disease modification by exendin-4 treatment, as improvements were still visible even when the drug was no longer present in the body. 

Tests of cerebrospinal fluid (CSF) samples demonstrated that the drug can enter the brain and that when people were retested after the trial had finished, no drug remained in the CSF (Athauda et al., 2017). To investigate the underlying mechanism of action, exosomes were analyzed from blood plasma. These exosomes originate from the brain. 

The content of the exosomes showed that drug treatment normalized insulin signaling in neurons, as predicted from preclinical studies. When analyzing the levels of the insulin receptor-activated second messenger cascade by measuring phosphorylated IRS-1, Akt, and mTOR, it was shown that insulin desensitization was much reduced by the drug (Athauda et al., 2019). 

This is proof of concept that GLP-1 mimetics can normalize insulin signaling in the brain and modify disease progression. Several other clinical trials are currently ongoing, testing the drugs lixisenatide (clinical trials identifier NCT03439943), liraglutide (NCT02953665), liraglutide (NCT03659682), and the PEGylated version of exendin-4 (NLY01) in Parkinson's disease patients (NCT04154072). 

Further clinical trials are in planning. The group that tested exendin-4 in a pilot study and a Phase II trial will test exendin-4 in a larger Phase II trial, testing 200 patients over 2 years and extending the range of biomarkers to be measured (NCT04232969). The company Peptron developed a novel formulation for administering exendin-4 and plans a clinical trial, testing their product (NCT04269642). 

The trial will recruit 99 patients and will continue for 60 weeks. The range of different trials in Parkinson's disease demonstrates that the strategy of activating the GLP-1 receptor in the brain has evolved into a fully-fledged research and drug development area.

14 | ALZHEIMER'S DISEASE

A pilot study testing the effects of liraglutide had been conducted in Alzheimer's disease patients. This double-blind, randomized, placebo-controlled trial included memory tests 18FDG-PET brain imaging, and PIB-PET imaging to estimate amyloid plaque load (Egefjord et al., 2012). 

The low number of 38 patients in this trial meant that the trial was underpowered for the cognition tests, which require much higher numbers to reach the statistical power to show a drug effect. 

Additionally, drug treatment only lasted for 6 months, which is too short for the placebo control group to deteriorate sufficiently to allow a drug effect on disease progression to become visible. However, there was a clear drug effect in the 18FDG-PET brain scans. Whereas the placebo control group showed up to 20% reduced 18FDG-PET activity, the drug group showed a stable 18FDG-PET signal over time, with some brain regions even showing higher signals. 

This demonstrates that glucose utilization and neuronal activity in the cortex did not deteriorate in the drug group (Gejl et al., 2016). This result is what one would expect from a drug that re-sensitizes insulin signaling in the brain. 

Another double-blind placebo-controlled pilot study testing liraglutide in cognitively impaired patients showed a drug effect in fMRI brain scans after 1 year of treatment. Brain activity and the connection between different active brain areas were reduced in placebo-treated subjects, but not in the drug-treated patients, suggesting that the drug prevented disease progression (Watson et al., 2019). 

A placebo-controlled double-blind Phase II clinical trial has been conducted, testing liraglutide in over 200 MCI/Alzheimer's disease patients for 1 year (ELAD study). It analyzed the effects on cognition (ADAScog and ADASexec tests), 18FDG-PET activity, brain volume changes as measured by MRI brain scans, the content of exosomes that originate from the brain and microRNA harvested from blood plasma (Femminella et al., 2019). 

The first results were published at the CTAD conference in 2020. It was found that liraglutide reduces cognitive impairment in ADASexec tests (P < .001) and that brain temporal lobe volumes shrank less than in the placebo group (P < .001) and total grey matter cortical volume shrank a lot less, too (P = .002), indicating that neuronal loss has been reduced by the drug. 

Other biochemical marker results have not been published yet (Edison et al., 2020). The result is a proof of concept for the use of GLP-1 mimetics to treat Alzheimer's disease and demonstrates that liraglutide is neuroprotective in the brain and can reduce disease progression. In addition, the company Novo Nordisk announced in December 2020 that a Phase III clinical trial in Alzheimer's disease patients will be conducted, testing their GLP-1 analog semaglutide in its oral formulation (Rybelsus), which is currently on the market to treat type 2 diabetes mellitus. 

The trial will recruit 3700 patients and drug treatment will go on for 2 years.

15 | GLUCOSE-DEPENDENT INSULINOTROPIC POLYPEPTIDE/GASTRIC INHIBITORY POLYPEPTIDE (GIP)

GIP is the 'sister' incretin hormone of GLP-1 and their physiological roles are closely related (see Figure 1) (Baggio & Drucker, 2007; Finan et al., 2016). It is a 42-amino acid long peptide hormone that is expressed in a range of cells, including neurons (Nyberg et al., 2007). The GIP receptor is a seven membrane-spanning Gprotein-coupled receptor of the glucagon-type family that enhances cAMP levels when activated (Park et al., 2013). 

GIP receptor expression has been observed on large neurons such as the pyramidal neurons in the cortex and hippocampus, granule neurons in the dentate gyrus, Purkinje cells in the cerebellum and basal brain areas (Kaplan & Vigna, 1994; Nyberg et al., 2005; Usdin et al., 1993). 16 | GIP ANALOGUES ARE PROTECTIVE IN ANIMAL MODELS OF ALZHEIMER'S DISEASE We have tested protease-resistant long-acting GIP analogs in the APP/PS1 mouse model of Alzheimer's disease. D-Ala2 GIP protected learning and memory in 12-month-old APP/PS1 mice. 

Synapse loss was reduced and synaptic plasticity in the hippocampus was protected in electrophysiology studies, whereas saline-treated mice showed extensive loss of synapses and impaired synaptic plasticity. 

The amyloid plaque load was reduced by the GIP analog, too. The activation of microglia and astrocytes in the chronic inflammation response in the brain was diminished by drug treatment, as were oxidative stress and DNA damage (Duffy & Holscher, 2013; Faivre & Holscher, 2013b). In 19-month-old APP/PS1 mice, D-Ala2 GIP was still able to reduce synaptic loss and inflammation in APP/PS1 mice and even in wild-type control animals. 

Furthermore, the drug was able to enhance synaptic plasticity in the hippocampus of aged APP/PS1 and wild-type mice, suggesting that the loss of synapses can be reversed by the drug (Faivre & Holscher, 2013a). In addition, oxidative stress and DNA damage were reduced also (Duffy & Holscher, 2013). 

Direct infusion of native GIP into the brain was found to be effective in preventing memory impairments induced by i.c.v. injection of amyloid (Figueiredo et al., 2010). These and other results demonstrate that GIP receptor agonists have similar protective properties as GLP-1 receptor agonists and that improving GIP signaling in the brain may be protective against Alzheimer's disease, too (Ji, Xue, Li, et al., 2016).

17 | GIP ANALOGUES SHOW NEUROPROTECTIVE EFFECTS IN ANIMAL MODELS OF PARKINSON'S DISEASE

Because GLP-1 and GIP analogs showed good effects in Alzheimer's disease models and GLP-1 receptor agonists showed protective effects in Parkinson's disease also, we tested long-acting GIP analogs in animal models of Parkinson's disease. 

D-Ala2-GIP-gluPAL showed good neuroprotective effects in the MPTP mouse model of Parkinson's disease. Motor coordination and grip strength were normalized by the drug, as was tyrosine hydroxylase expression in dopamine neurons in the SN. Synapse numbers were protected from MPTP toxicity, too. MPTP treatment induced a chronic inflammation response activated microglia and astrocytes and increased levels of pro-inflammatory cytokines in the brain. 

Drug treatment reduced the inflammation response and normalized cAMP/PKA/CREB second messenger signaling in the SN, indicating that growth factor signaling had been restored (Li, Liu, Li, & Holscher, 2016). After chronic MPTP treatment, a model of Parkinson's disease that is considered to be more realistic, D-Ala2-GIP-glu-PAL, was able to improve motor activity, protect dopamine neurons, and additionally reduce the increased α-synuclein levels in the brain. 

MPTP treatment leads to a much-increased expression of this protein. Moreover, drug treatment reduced the chronic inflammation response in the brain, lowered oxidative stress and lipid peroxidation, and increased the levels of BDNF (Li et al., 2017). BDNF can protect synapses in a range of neurodegenerative disorders (Allen et al., 2013; Blurton-Jones et al., 2009; Nagahara & Tuszynski, 2011). 

Other research groups found very similar effects of D-Ala2-GIP in this mouse model of Parkinson's disease. Again, motor activity and dopamine neurons were protected from MPTP toxicity. The drug effect was blocked by the GIP receptor partial antagonist (Pro3 )GIP. D-Ala2GIP furthermore reduced the levels of oxidative stress in the brain. 

D-Ala2-GIP was able to normalize dopamine levels in the striatum (Verma et al., 2017). Another animal model of Parkinson's disease is the 6-OHDA lesion rat model. Continuous infusion of GIP by an osmotic minipump reduced the 6-OHDA toxicity and motor impairments were brought back to normal levels (Yu et al., 2018). These findings show that GIP has similar neuroprotective properties as GLP-1 and is a promising research area for developing novel treatments for Parkinson's disease (Ji, Xue, Li, et al., 2016; Verma et al., 2018; Zhang & Holscher, 2020).

18 | NOVEL DUAL GLP-1/GIP RECEPTOR AGONISTS THAT CAN CROSS THE BLOOD-BRAIN BARRIER

As GIP and GLP-1 both have protective effects and work together on a cell signaling level and physiological levels in a synergistic fashion, novel GLP-1, and GIP receptor dual agonists have been developed as drug treatment for type 2 diabetes mellitus (Finan et al., 2013). GIP and GLP-1 receptors are expressed on the same cells and, when activated, can form receptor dimers that show enhanced second messenger signaling or even activate different second messenger cascades (see Figure 1) (Finan et al., 2016; Wellman & Abizaid, 2015). 

Studies in animals with type 2 diabetes mellitus have shown an added benefit of GIP analogs when added to GLP-1 analogs to control blood glucose levels (Gault et al., 2011). Several novel dual agonists have been tested in clinical trials in patients with type 2 diabetes mellitus and some show better effects when compared with single GLP-1 receptor agonists (Finan et al., 2013; Frias et al., 2017, 2018). 

We previously tested five different GLP-1/GIP dual agonists that we have named DA1–DA5 (Hölscher, 2018, 2020a). DA1-JC (NNC0090-2746) is a dual agonist that has been acetylated with a C16 fatty acid to enhance the biological half-life in the blood (Finan et al., 2013; Frias et al., 2017). 

DA2 is the same peptide that has been PEGylated with a 40-kDa PEGylation added to increase the biological half-life (Finan et al., 2013). DA3-CH is a peptide without any modifications (Panagaki et al., 2018). In addition, we have developed two dual agonist peptides with a CPP modification to enhance blood-brain barrier (BBB) penetration (DA4-JC and DA5-CH) (Hölscher, 2018, 2020a).

19 | IMPORTANCE OF THE BBB IN TREATING CNS DISEASES

The neuroprotective effects of these peptide drugs correlate directly with their ability to cross the BBB. The basis of the neuroprotective activity of these drugs is that they activate GLP-1 and GIP receptors on neurons and glia of the CNS. We tested the ability of these drugs to cross the BBB by using fluorescent-labeled peptides in rodents. 

When comparing all five dual agonists, DA4-JC and DA5-CH were the most effective, followed by DA3-CH, DA1-JC, and DA2, the PEGylated version, hardly crossed the BBB at all. Lipidated peptides such as liraglutide and DA1-JC showed lower penetration, whereas exendin-4 showed better BBB penetration that was on the level of DA3-CH, the unmodified dual agonist peptide (Li et al., 2020; Zhang et al., 2020). 

A recent study testing 125I radiolabelled peptides confirmed these results and demonstrated that lipidated peptides such as liraglutide, liraglutide, and DA1-JC crossed the BBB only in limited amounts, just like the PEGylated DA2 peptide. DA3-CH showed better BBB penetration, but DA4-JC with a poly-Lys modification crossed the BBB at the highest level (Salameh et al., 2020). 

The ability of drugs to protect mice from the effects of MPTP was directly correlated with their ability to cross the BBB. In a direct comparison, DA4-JC and DA5-CH were superior to DA3-CH and DA1-JC, which in turn were superior to liraglutide (Feng et al., 2018; Zhang et al., 2020). Unfortunately, few studies measure levels of such drugs in the CSF in humans to estimate the BBB penetrations. 

In the Phase II trial of Bydureon (exendin-4), CSF analysis showed that the drug does enter the brain readily (Athauda et al., 2017), confirming the rodent studies. In a study measuring liraglutide levels in the CSF of diabetic patients, only low levels were found, which matches the findings in rodent studies (Christensen et al., 2015). More studies will be needed to be able to make firm statements of how much peptide drugs can cross the BBB.

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