Drug Design and Discovery in Alzheimer’s Disease

By Atta-ur Rahman and Muhammad Iqbal Choudhary

Drug Design and Discovery in Alzheimer’s Disease includes expert reviews of recent developments in Alzheimer's disease (AD) and neurodegenerative disease research. Originally published by Bentham as Frontiers in Drug Design and Discovery, Volume 6and now distributed by Elsevier, this compilation of the sixteen articles, written by leading global researchers, focuses on key developments in the understanding of the disease at molecular levels, identification and validation of molecular targets, as well as innovative approaches towards drug discovery, development, and delivery. Beginning with an overview of AD pharmacotherapy and existing blockbuster drugs, the reviews cover the potential of both natural and synthetic small molecules; the role of cholinesterases in the on-set and progression of AD and their inhibition; the role of beta-site APP clearing enzyme-1 (BACE-1) in the production of ß-amyloid proteins, one of the key reasons of the progression of AD; and other targets identified for AD drug discovery.

• Edited and written by leading experts in Alzheimer’s disease (AD) and other neurodegenerative disease drug development
• Describes existing drugs for AD and current molecular understanding of the condition
• Reviews recent advances in the field, including coverage of cholinesterases, BACE-1, and other drug development targets

• Language: English
• Publisher: Elsevier Science
• Release date: Jun 27, 2015
• ISBN: 9780128039601

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Chapter 1

Pharmacotherapy of Alzheimer’s Disease: Current State and Future Perspectives

Jan Korabecnya,b; Filip Zemeka; Ondrej Soukupb; Katarina Spilovskaa,b; Kamil Musilekb,c; Daniel Junb,d; Eugenie Nepovimovaa,b; Kamil Kucab,d,*    a Department of Toxicology, Faculty of Military Health Sciences, Trebesska 1575, 500 01 Hradec Kralove, Czech Republic

b University Hospital Hradec Kralove, Sokolska 581, 500 05 Hradec Kralove, Czech Republic

c University of Hradec Kralove, Faculty of Science, Department of Chemistry, Rokitanskeho 62, 50003 Hradec Kralove, Czech Republic

d Centre of Advanced Studies, Faculty of Military Health Sciences, Trebesska 1575, 500 01 Hradec Kralove, Czech Republic

* Address correspondence to Kamil Kuca: Centre of Advanced Studies, Faculty of Military Health Sciences, Trebesska 1575, 500 01 Hradec Kralove, Czech Republic; Tel: [phone]; E-mail: [e-mail]

Abstract

Alzheimer’s disease (AD) is a multifactorial disorder and apparently involves several different etiopathogenetic mechanisms. Up-to-date, there are no curative treatments or effective disease modifying therapies for AD. A strategy to enhance the cholinergic transmission by using acetylcholinesterase inhibitors (AChEIs) has been proposed more than two decades ago. Food and Drug Administration (FDA) gradually marketed these AChEIs: tacrine (1993), donepezil (1997), rivastigmine (2000) and galantamine (2001); tacrine is no longer used because of its high prevalence of hepatotoxicity. In addition to the AD cholinergic hypothesis, there is great evidence that voltage-gated, uncompetitive, N-methyl-D-aspartate (NMDA) antagonist memantine with moderate affinity can protect neurons from excitotoxicity. It was approved by FDA for treatment of moderate to severe stages of AD in 2003. Beyond symptomatic approaches there are anti-amyloid, neuroprotective and neuron-restorative strategies that hold promise of redefining the course of the disease as it is known. This contribution summarizes the main symptomatic strategies available for treating AD and future perspectives of pharmacotherapy for improving the AD course.

Keywords

Acetylcholinesterase

Alzheimer’s disease

butyrylcholinesterase

donepezil

galantamine

GSK-3β

inhibitors

memantine

metal chelators

modulators of secretases

M1 agonists

rivastigmine

statins

tacrine

1 Alzheimer’s Disease - Historic Overview

In 1906, German psychiatrist Alois Alzheimer firstly diagnosed and defined clinical-pathological syndrome that was later named Alzheimer’s disease (AD). When diagnosing the disease on one of his forty years old patient, Alois Alzheimer described observed symptoms as a rare pre-senile dementia occurring before 65 years of age. Nor did he or his colleagues distinguish this new disease from well-known and described senile dementia. Nevertheless, several symptoms which had Alzheimer described are common for majority of patients with diagnosed AD e.g. progressive memory loss, impaired cognitive function, behavioral changes, disruption of the integrity of the individual, hallucinations, impaired self-control and loss to the decline of spoken and written speech [1, 2].

The invention of the electron microscope in the 20th century led to the clarification of the histological changes in the brain characteristic for the AD particularly neuritic plaques and neurofibrillary tangles [3]. The existence of the neuritic plaques has been described no sooner than in the seventies of the twentieth century. Furthermore, the adverse effect of neuritic plaques on the neurons producing and releasing acetylcholine (ACh) were also observed. Further research led to the conclusion that these adverse effects are related directly to the enzymes associated with ACh. In particular, decrease of concentration and activity of cholineacetyltransferase (ChAT, EC 2.3.1.6) concentration and acetylcholinesterase (AChE, EC 3.1.1.7) in the limbic system and cerebral cortex were associated with the loss of cholinergic neurons in subcortical areas [4]. These findings opened a novel chapter of pharmacological research in an effort to increase ACh brain levels in the synaptic gaps. First experiments concentrated on the inhibition of AChE responsible for the degradation of ACh. The research was successful and it led to the introduction of several novel compounds into clinical practice: tacrine (Cognex®), donepezil (Aricept®), rivastigmine (Exelon®) and galantamine (Reminyl®) [5, 6].

2 Current Status and Prevalence of AD

AD is one of the most common forms of dementia. The unpredictability and yet unknown etiology makes it increasingly disturbing problem for humanity, not just in terms of health, but also in terms of social and economic parameters. Furthermore, AD is a fatal illness as every person dies after 3-10 years from being diagnosed [1, 2].

Globally, AD is the fifth cause of death among people over 65 years. Cases of death caused by AD are increasing dramatically. Between 2000 and 2008 there were 66% of deaths caused by AD alone. However, cases of death due to heart failure, strokes and prostate cancer were only 13%, 20%, 8% respectively [2, 7].

In United States of America, it is estimated that 5.4 million people have been diagnosed with AD and 200000 of them are under 65 years of age (early AD onset). The total number of patients with dementia in the world is currently estimated to be around 35.6 million people. By 2050, this number is expected to increase up to 115.4 million patients [2]. The most vulnerable population are people over 85 years of age (more than 50% of AD patients). In the European Union it is estimated that from AD suffers approximately 6 million people and this figure is likely to double by 2050 [8-11]. The main reason is principally the aging population and the absence of an effective therapy. The limited understanding of the basic AD pathophysiology was the major drawback in the research of potential therapeutics. Though the tremendous progress was made in the past 30 years in the biological, biochemical, toxicological and pharmaceutical research, the considered therapeutic procedures failed or came too late in the last stage of the disease [2].

3 Risk Factors for AD

The trigger mechanism of AD is not yet fully understood, although certain risk factors contributing to the development of AD were determined. Aging population is the most threatened by AD, while it is important to note that AD is not a normal component of the aging process. People over 65 years of age display the greatest increase in the AD incidence, however even people below 65 years of age may develop AD known as early-onset AD. [5].

An important role is played by the genetic factors and heredity. It was scientifically proven that individuals with a close relative (brother, sister, parents), who were AD diagnosed are more likely to develop AD also than patients with only distant relatives suffering from AD [12, 13].

AD is genetically linked to the presence of allele ε4 of apolipoprotein E (ApoE ε4). Allele ε4 can occur in three isoforms, ε2, ε3 and ε4 however only ε4 increases the risk of AD later in life. On the other hand the presence of ε2 allele decreases the risk of developing AD [13-16].

Other risk factors include cardiovascular disease, brain injury, trauma, depression, low education level, smoking, low concentrations of folate and vitamin B12, elevated homocysteine concentration in plasma, high cholesterol, diabetes mellitus type 2, high blood pressure, physical inactivity and obesity. These factors not only promote the formation and development of AD but are also involved in the etiopathogenesis of different dementia subtypes and other diseases [17-29].

Former research has also identified protective factors, which in turn reduce the risk of AD development. These include a higher education level, regular use of anti-inflammatory agents and cholesterol lowering agents (statins), estrogen replacement therapy in post-menopausal period, antihypertensive therapy and a diet rich in fish. The big unknown is the use of compounds with antioxidant properties e.g. vitamins C and E [30-33].

4 Cholinergic Theory

The cholinergic hypothesis is the first and still the only widely accepted theory explaining the nature of AD [34, 35]. Moreover, almost all currently used drugs for relieving symptomatic effects of moderate to severe AD forms are based on this hypothesis [36-38]. The cholinergic theory works with the assumption that the loss of cholinergic activity observed in AD patients confirms a close relationship between the neuromediator ACh and learning plus remembering [39]. The blockade of central cholinergic system with scopolamine in young adults produces similar symptoms as observed in individuals affected by AD. Normal function of the central nervous system can be restored by reversible cholinesterase inhibitors such as physostigmine [40]. Based on these experimental studies, clinical trials were conducted for other type of compounds (reversible cholinesterase inhibitors), which hold the most promise for the treatment of deficient memory function in AD patients (see chapter 6.1 Drugs used in clinical practice).

In a comprehensive examination of the deficient cholinergic system in AD impaired signaling from a population of neurons based in the basal forebrain nuclei and in the Meynert nucleus basalis to the cerebral cortex and hippocampus was determined. Furthermore, the concentration and ChAT activity responsible for the synthesis of ACh during AD is significantly reduced, particularly in the areas of the cerebral cortex and hippocampus [41-43]. The release of ACh in the same sections of the brain induced by depolarization and choline uptake into the presynaptic neurons was found to be significantly reduced. If impaired memory function is considered as the primary AD indicator, the above-mentioned role of ACh in cholinergic transmission and its importance for cognitive function supports these findings in AD [44]. All these observations launched the the cholinergic theory, which was published by Bartus et al. (1982) [36]. Bartus in his work fully reflects the relationship between the cholinergic hypothesis, age-dependent cholinergic dysfunction and AD-type dementia.

5 Cholinesterases

Cholinesterases (ChE) are a group of essential enzymes that can be divided into two subgroups based on their catalytic properties.

An enzyme that favors decomposition (hydrolysis) of small substrates, such as ACh (Fig. (1)) is called acetylcholinesterase (AChE, EC 3.1.1.7).

Figure 1 Substrates of cholinesterases.

The enzyme, which is able to adapt to the bulkier substrates such as benzoyl- or butyryl-choline (Fig. (1)) and catalyze their decomposition, is termed as butyrylcholinesterase (BChE, EC 3.1.1.8). AChE and BChE can both catalyze the hydrolysis of ACh (Fig. (2)) [45, 46].

Figure 2 Hydrolysis of ACh catalyzed by AChE or BChE.

In the determination process of the amino acid sequence of both ChE, it was concluded that they belong to a large group of enzymes containing α/β-hydrolase clusters, which are also common for the different lipase, peptidase, haloalkane dehalogenases and even adhesion proteins. AChE and BChE are so similar that it is almost inherent to describe the structure of one without referral to the other. Both have three entrenched disulfide bridges and the amino acid sequence for human BChE (hBChE) and eel AChE isolated from the electrical eel (lat. Electrophorus electricus) is equivalent by 54%. The main difference between AChE and BChE is in the level of N-glycosylation as AChE is less glycosylated than BChE. Glycosylation affects the stability and pharmacokinetic properties, but not the catalytic ability of the enzyme. Differences in the binding affinity of the individual substrates are given by the spatial arrangement of the active site of both enzymes [47].

Active site is located at approximately 20 nm deep straight cavity and is composed of three main clusters - acylation, cation-π and peripheral anionic (aromatic) sites [48].

Hydrolysis of the substrate occurs in the acylation site, which consists of the catalytic triad with amino acids Ser203, Glu202, His447 for human AChE (hAChE) and Ser198, Glu325, His438 for hBChE respectively [48]. Furthermore, entrance to the active site is lined by numerous aromatic residues.

Cation-π site in hAChE is formed by Trp86 and Phe338, which are responsible for weak type interactions such as Columbic forces between the aromatic system and the positively charged quaternary nitrogen moieties. An important role plays Tyr133, which directly interacts with Trp86 and contributes to the stabilization of the cation-π conformation upon binding of the substrate. In hBChE, the stabilization function is represented by Trp82 and Ala328. The absence of phenylalanine in BChE affects affinity to certain inhibitors [49, 50]. Moreover, cation-π site is also capable to interact with ligands without a positive charge [51].

Particularly important are amino acid residues at the peripheral anionic site of hAChE (Tyr72, Tyr124, Trp286), which are all situated at the cavity entrance. However, on hBChE these aromatic residues are missing, what explains the weaker affinity of specific AChE substrates, such as fasciculin (snake venom) or propidium (selective inhibitor of AChE peripheral anionic site). Further studies revealed that the anionic site on BChE consists of Asp70 and Tyr332. (Fig. (3)) and (Fig. (4)) for the amino acid residues at the peripheral anionic site of hAChE and hBChE, respectively) [49-51].

Figure 3 Spatial distribution of amino acid residues of human AChE (in magenta), the enzyme is shown in ribbon formation (in grey). Figure was created using PyMol viewer (v. 1.3).

Figure 4 Spatial distribution of amino acid residues of hBChE (in magenta), the enzyme is shown in ribbon formation (in grey). Figure was created using PyMol viewer(v. 1.3).

Since cholinesterases have a catalytic function, they are the main target of various reversible, irreversible or pseudo-irreversible inhibitors. Among reversible inhibitors belong aromatic tertiary amines such as tacrine or donepezil. Pseudo-irreversible binding may interact strongly with the catalytic serine (e.g. physostigmine, rivastigmine) and a group of irreversible inhibitors is represented by organophosphate compounds, whose effect on ChE and the entire body is life-threatening (e.g. nerve agents sarin, soman, tabun or VX; pesticide paraoxon) [45].

6 Current Therapeutic Approaches to AD

6.1 Drugs Used in Clinical Practice

6.1.1 Donepezil

Donepezil ((RS)-2-[(1-benzyl-4-piperidyl) methyl] -5,6-dimethoxy-2,3-dihydroinden-1 one, Table 1) is still considered as one of the most effective and safest drugs established to combat AD. It is a selective inhibitor of AChE used to treat mild to moderate AD forms. Nevertheless, donepezil like other AChE inhibitors only slows the progression of the disease to a certain extent, but it cannot completely stop the progression of AD or even cure it [52, 53]. In addition, donepezil improves symptoms of Aβ induced neurotoxicity and positively influences the process of APP cleavage [54]. Donepezil enters cortical neurons and interrupts a mechanism, which leads to the Aβ formation [55]. Moreover, donepezil increases the expression of nicotinic receptors in the cortex and at least partially prevents the AD progression. At the same time, donepezil also reduces the concentration of glutamate, an important neurotransmitter that under AD pathological conditions has neurotoxic effect [56]. Donepezil increases the production of AChE-S isoform, while the synthesis of AChE-R isoform is suppressed, which again is another example of neuroprotective ability [57]. Donepezil has also a significant antioxidant activity, as it is capable to neutralize reactive oxygen species (ROS) and improve blood rheological properties [58-61]. Donepezil is marketed under the commercial brand name Aricept® since 1997. Other features concerning donepezil are summarized in Table 1 and its spatial orientation in AChE from Torpedo californica (pdb code: 1e3q) is shown in Fig. (5) [62].

Table 1

AChE Inhibitors in Clinical Practice and their Properties

Figure 5 Spatial orientation of donepezil on AChE from Torpedo californica and significant interactions with amino acid residues (pdb code: 1e3q). Interactions via π-π bonds are predominant. Figure was created using PyMol viewer (v. 1.3) [ 62 ].

6.1.2 Galantamine

Galantamine ((4aS,6R,8aS)-5,6,9,10,11,12-hexahydro-3-methoxy-11-methyl-4aH-[1]-benzofuro-[3a,3,2-ef][2]benzazepin-6-ol, Table 1) is a selective, reversible inhibitor of AChE. It is a natural alkaloid that was firstly isolated from a plant commonly known as snowdrop (Galanthus woronowii) and its presence was confirmed in the bulbs of other various species in the family Amaryllidaceae [63]. In addition to its inhibitory activity against AChE galantamine also acts as an allosteric modulator of the N-receptors [64]. Galantamine binds to the opposite side of the receptor than ACh and causes a conformational change of the receptor [65]. The whole process potentiates N-receptors, which results in the enhanced postsynaptic response. Presynaptic N-receptors play an important role in the release of ACh and also regulate concentration of other transmitters such as γ - aminobutyric acid, glutamic acid, serotonin or norepinephrine. All these neuromediators are important for the memory functions and also influence mood and emotions [66, 67]. In the course of AD, pathologically increased levels of glutamate may lead to learning and memory impairment, while pathologically decreased levels of serotonin contributes to the emotional misbalance (note: most patients with AD suffer from depressive episodes) [68-70]. Galantamine is currently the most preferred drug for the treatment of AD mainly because of the above-mentioned advantages. Absorption after oral administration of galantamine is around 100%, but concomitant food intake decreases the rate of absorption. Galantamine has a large distribution volume and thus it has a tendency to form depots in the human tissues. Similarly to donepezil, galantamine has also gastrointestinal and anorectic side effects. However, rarely do these side effects prevent long-termed therapeutic use [71]. In Fig. (6), spatial orientation of the AChE active site isolated from Torpedo californica (pdb code: 1dx6) is displayed [72]. Galantamine is marketed under the commercial brand name Reminyl® since 2001.

Figure 6 Spatial orientation of galantamine bound to the AChE active site and significant interactions with amino acid residues (pdb code: 1dx6). Besides the π-π interaction (aromatic part of galantamine with Phe290), there is also hydrogen bridge between Ser200 and the hydroxyl group of galantamine and an aliphatic-π interaction with Phe330 and Trp84. Figure was created using PyMol viewer (v. 1.3) [ 72 ].

6.1.3 Rivastigmine

Rivastigmine ((S)-3-[1-(dimethylamino)ethyl]phenyl-N-ethyl-N-methylcarbamate, Table 1) is a reversible cholinesterase inhibitor intended for symptomatic treatment of moderate to severe AD stages. It is used in the form of a tartrate salt for its better solubility. In the contrary to all other AChE inhibitors, rivastigmine is selective towards both cholinesterases (AChE, BChE), which is particularly useful in later stages of AD. Generally, concentration of BChE increases, while the concentration of AChE decreases in the later stages of AD [73, 74]. Rivastigmine belongs into group of compounds called carbamates, which cause carbamoylation of the active site of the enzyme and impairs its function. The most common side effects are of gastrointestinal origin (e.g. vomiting, nausea, diarrhea, anorexia), however frequent dosing schedule can reduce these issues [75, 76]. Fig. (7) shows the spatial arrangement of rivastigmine on AChE from Torpedo californica (pdb code: 1gqr) [77]. Rivastigmine is marketed under the commercial brand name Exelon® since 2000.

Figure 7 Spatial orientation of rivastigmine on AChE from Torpedo californica and significant interactions with amino acid residues (pdb code: 1gqr). Ser200 is carbamoylated and the rest of the molecule inhibitor remains in the cavity stabilized by π-π interactions with Trp84 and Phe330. Figure was created using PyMol viewer (v. 1.3) [ 77 ].

6.1.4 Tacrine

Tacrine (1,2,3,4-tetrahydroakridin-9-amine, Table 1) is reversible, intermediate-acting cholinesterase inhibitor of AChE and BChE, which is known for more than 60 years [78]. It was originally developed as an antibacterial agent, but when it was thoroughly tested, it demonstrated to be very weak bactericide. However, other surprising characteristics were discovered [79]. The first study described the pharmacological effects of tacrine on analeptic animals sedated by morphine [80]. Nevertheless, the cholinergic properties have not been recognized until 1961 by Heilbronn et al. They concluded that tacrine is a stronger inhibitor of BChE than AChE. Previously, it was often used as an antidote for curare and for relieve of intractable pain. Moreover, other previously used or considered indications included Myasthenia gravis, antidepressant effects (similarity with tricyclic antidepressants), tardive dyskinesia or overdosing by anticholinergics [81]. Tacrine was introduced into clinical practice in 1993. It was often used in combination with lecithin [82, 83]. Crystallographic studies of tacrine bounded to AChE demonstrated that it binds to the catalytic AChE site [51]. Importantly, tacrine has the ability to interact with muscarinic (M) receptors and with nicotinic (N) receptors. However, it displays 100-fold higher affinity towards N-receptors. Moreover, tacrine also increases the release of ACh by stimulating M1-receptors and inhibits both isoforms of monoamine oxidase (MAO) [84, 85]. Tacrine further inhibits the re-uptake of dopamine and serotonin in the nerve endings, which contributes to its antidepressant effect [86].

The use of tacrine requires more frequent dosing schedule (Table 1), prolonged titration of blood concentration and monitoring of liver enzymes [87]. Tacrine is no longer used for its narrow therapeutic index, frequent cases of severe hepatotoxicity and gastrointestinal toxicity [88, 89]. In Fig. (8) tacrine is superimposed in the active site of AChE from Torpedo californica (pdb code: 1acj) [51]. Tacrine was marketed under the commercial brand name Cognex® in 1993.

Figure 8 Spatial orientation of tacrine in the active site of AChE from Torpedo californica and significant amino acid residues interactions. Tacrine is located in the peripheral anionic site between Trp84 and Phe330. The amino group in position 9 is stabilized by binding to the two water molecules (not shown). Figure was created using PyMol viewer (v. 1.3) (pdb code: 1acj) [51].

6.1.5 Memantine

Memantine (3,5-dimethyladamantan-1-amine, Fig. (9)) has mechanism of action completely different to other compounds mentioned in this chapter. It is a noncompetitive antagonist with medium affinity to the N-methyl-D-aspartate (NMDA) receptor. Potency of memantine is closely linked with glutamate. At normal concentration of glutamate, NMDA-receptor is inhibited only weakly, however elevated levels of glutamate increase the potency of memantine significantly and the NMDA-receptor is then strongly inhibited. Its mechanism of action can be described as neuroprotective. In addition, memantine is also being considered for the treatment of Parkinson disease, epilepsy, CNS trauma, amyotrophic lateral sclerosis, drug addiction and chronic pain [90-93]. Memantine is marketed under the commercial brand name Ebixa® since 2002.

Figure 9 Structure of memantine as a representative of NMDA antagonists in the treatment of AD.

6.2 Potential Therapeutic Approaches to AD

6.2.1 Vaccination and Immunization

Transgenic mice immunized with amyloid-beta (Aβ) bearing human transmembrane amyloid precursor protein (amyloid precursor protein, APP) exhibited reduced production of amyloid plaques in the brain, which belong to the pathological findings in the brain of AD patients. Furthermore, improved behavioral functions were also detected [94, 95]. This particular finding led to several vaccination clinical trials with AD patients. However, the administration of synthetic Aβ (AN1792) increased the incidence of meningo-encephalitis, which was discovered in six percent of patients and clinical trials were prematurely terminated [96]. Nevertheless, analysis of the clinical trials data has proven that administration of AN1792 produces antibodies against Aβ and produces significant improvements in cognitive functions [97]. Nevertheless, a magnetic resonance after immunization revealed a reduction of the brain volume [98]. Based on these findings, it was concluded that passive immunization should be safer and more effective then tested active form. Currently, several vaccines using selective Aβ monoclonal antibody as passive immunization tool are in the various stages of clinical development [99, 100].

6.2.2 Modulators of Secretases

Aβ is formed from APP by two enzymes known as β-secretase and γ-secretase. Connected research is mainly focused on the possibility to inhibit these secretases. Compound labeled KMI-429 (Fig. (10)) is one of the novel compounds that have experimentally proven inhibition ability towards β-secretase. In vivo experiments on transgenic mice have proven reduced formation of Aβ after its application [101]. However, one of the encountered problems is the molecular weight of these inhibitors, which does not allow a penetration of the blood-brain barrier. Furthermore, the application of KMI-429 to mice deficient in β -secretase produced problems with learning [102, 103]. For these reasons, the attention is paid to the development of small molecule inhibitors of β-secretase [104-106].

Figure 10 Modulators of secretases.

Decreased levels of Aβ in the brain, cerebrospinal fluid (CSF) and plasma were observed in rodents who received inhibitors of γ - secretase labeled Dapto, LY450139 dihydrate and BMS-299897 (Fig. (10)) [107-110]. The acquired results lead to the conclusion that Aβ may be associated with cognitive impairment in AD patients and also that the administration of an γ-secretase inhibitor can lead (particularly in the early stage of AD) to reversible changes during the formation of amyloidal plaques. The encouraging results from clinical trial of these inhibitors are on the other hand clouded by detrimental effects in the gastrointestinal compartment, thymus and spleen. This could be suggested by insufficient selectivity of tested compounds [111, 112].

Tarenflurbil (MPC-7869) belongs to a group of γ -secretase modulators, whose mechanism of action is to shift the production of longer Aβ42 towards short and less amyloidogenic peptides (e.g. Aβ37) without affecting any other physiological substrates (Fig. (10)) [113]. Results from tarenflurbil second phase clinical trials indicated possible benefit for patients with mild AD. Analysis of the trial data suggested that the best results were achieved in the group receiving the highest dose (800 mg twice a day). The third phase clinical trials reported adverse effects such as eosinophilia, mild anemia, hypertension or rash and tarenflurbil investigation concluded its withdrawal from other clinical trials [114, 115].

6.2.3 Amyloid-β Antagonists

Aβ forms fibrillar aggregates that cause destruction of neurons. Tramiprosate, which is the main representative of this class of compounds, inhibits the formation of fibrillar aggregates by binding to the soluble Aβ and thereby mimicking the effect of glycosaminoglycans (Fig. (11)). A reduction of soluble Aβ levels in the CSF reduces a chance of the possible formation of amyloid plaques in neurons. Open-label clinical study suggested that tramiprosate slows down deterioration of cognitive functions in early stage AD [116]. Tramiprosate is currently undergoing phase three clinical trials in North America and Europe, however its potential benefit in AD treatment remains unknown [117].

Figure 11 Amyloid-β antagonists.

The next compound that belongs among amyloid-β antagonists is colostrinin, which is a mixture of proline rich polypeptides derived from colostrum. It showed a reasonable improvement in cognitive function in patients with mild AD (note: for moderate AD test results were inconclusive, when compared with placebo). Nevertheless, colostrinin did not demonstrate up-to-date any long term benefits [118-120].

Particularly interesting drug in this group is scyllo-inositol (AZD-103, Fig. (11)). It stabilizes un-clustered, non-toxic Aβ complexes and reduces the harmful impact of Aβ clusters on neurons. Furthermore, in terms of long-term use it is even able to recover some memory functions [121, 122].

6.2.4 Statins in the Treatment of AD

The statin group is primarily used for the treatment of hyperlipidemia acting through the inhibition of 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG CoA reductase). However, some studies have also showed decrease of Aβ levels after statin therapy in vivo [123, 124]. The effect of statins may be particularly seen, when they are regularly used by AD patients before 80 years of age [125]. Moreover, several epidemiological studies suggest statins reduce the AD risk [126-130]. However, large cohort and randomized, placebo-controlled studies on the prevention of coronary heart disease do not confirm that statins have positive effect on cognitive functions in patients with AD [131-133]. Based on these findings, it can be concluded that statins in AD may slow the neurodegenerative processes, but cannot help those with already manifested AD [134]. Two basic structural types of statins are shown in Fig. (12).

Figure 12 Simvastatin (first-generation statin), rosuvastatin (third generation statin).

6.2.5 Peroxisome Proliferator-Activated Receptor Agonists

Abnormalities in the structure of insulin and insulin resistance may contribute to the neuropathology and clinical symptoms associated with AD [135]. Thiazolidinedione derivative rosiglitazone (note: in 2010 withdrawn from the market because of possible adverse cardiovascular effects, Fig. (13)) increases peripheral insulin sensitivity by agonistic effect on peroxisome proliferator-activated receptor (PPAR-γ). A study conducted on 30 selected individuals diagnosed with AD suggested a possible benefit in the rosiglitazone group particularly concerning cognitive symptoms, when compared to the placebo group [136]. In addition, individuals without the ApoE ε4 gene had considerably better cognitive functions than patients carrying this gene. Another agonist acting on PPAR-γ tested in clinical trials is pioglitazone (Fig. (13)) [137]. Furthermore, it has been observed that the actual administration of intranasal insulin improves memory function [138]. The close correlation between the misbalance of insulin and cerebral glucose metabolism in the brain of AD diagnosed patients indicate recent studies with metformin. Data shows that metformin reduces insulin resistance and could be beneficial in the treatment of AD [139].

Figure 13 Peroxisome proliferator-activated receptor agonists.

6.2.6 Metal Chelators

Aβ interacts with biogenic elements such as zinc, copper and iron, and together form a cytotoxic aggregates [140-142]. Clioquinol was the first drug in this group, which has been tested against the formation of Aβ aggregates (Fig. (14)). Within the 36-week trial, it demonstrated Aβ decrease in the brain by 49%. However, two serious side effects appeared during this period (syncopal episodes and cardiac arrhythmia) [143, 144]. Other chelators contemplated for use in AD connection contain compounds labeled XH1, DP-109 and (-)-epigallocatechin-3-gallate. All consistently demonstrated significant reduction of Aβ caused brain damage (Fig. (14)) [145-148].

Figure 14 Metal chelators.

6.2.7 M1 Muscarinic Receptor Agonists

Presynaptic M1 muscarinic receptors influence the AD at several levels of its pathogenesis [149]. Based on results from in vitro studies, it has been concluded that the muscarinic receptor subtype 1 (M1) plays a vital role in the AD pathogenesis. Furthermore, the M1 muscarinic agonist (AF267B) supported these findings in the in vitro and in vivo tests, where it was found to mediate cleavage of APP by α-secretase through a non-amyloidogenic pathway. Thus, the soluble APPsα with neuroprotective character is produced instead of the toxic Aβ (Fig. (15)). Moreover, the M1 agonist also reduced Aβ levels in the CSF and the incidence of inflammation and cognitive disorders [150-154]. Other studies have demonstrated that AF267B also prevents hyperphosphorylation of the τ-protein and thus retains its normal physiological function [155]. Talsaclidine is another novel compound introduced into clinical testing (Fig. (15)). Like AF267B, talsaclidine also reduced levels of CSF Aβ [156]. Similarly, xanomeline is capable to reduce Aβ levels, positively affect τ-protein and also potentiate the effect of AChE inhibitors, thereby improving the cognitive symptoms of AD patients(Fig. (15)) [157-159]. While the complex of mechanism of action does not give much space to further define it in more detail, it is obvious that the activation of postsynaptic and pre-synaptic M1 receptors influences the pathogenesis of AD at several stages [159]. However, it is an interesting approach that might prove to be a great benefit for patients diagnosed with AD [160].

Figure 15 M1 muscarinic receptor agonists.

6.2.8 RAGE Receptor Modulation

RAGE (Receptor for Advanced Glycation Endproducts) is a transmembrane receptor that belongs to the immunoglobulin family. It was firstly described in 1992 by Neeperem et al. [161]. Its name is derived from the ability to bind glycation end products derived from glycoproteins. RAGE in the presence of Aβ and in contact with endothelial cells of the pallets can induce migration of monocytes through endothelial cells of the brain. The monocyte diapedesis plays an important role in the inflammatory processes associated with AD [162]. The ligands for this receptor are currently being produced, which could suppress the Aβ accumulation and thereby contribute to the improvement of the AD prognosis [163, 164].

6.2.9 Peripherally Acting Scavenger of AΒ

The reduction of Aβ levels is still considered as one of the therapeutic targets in the AD treatment. While active immunization can directly reduce Aβ levels in the brain, peripherally acting scavengers like gelsolin (actin-binding protein) have a high affinity for peripheral Aβ, which also contribute to the overall AD prognosis [165]. An interesting group of compounds are also dihydropyridine antihypertensives. In AD relation and besides alleviating Aβ production, they also reduce Aβ caused brain damage, while they facilitate the clearance of this pathological protein at same time [166].

6.2.10 Glycogen Synthase Kinase-3 (GSK-3)

Glycogen synthase kinase-3 (GSK-3) is a cellular serine/threonine protein kinase. This enzyme regulates countless number of cellular processes and its dysregulation is crucial to the pathogenesis of diverse diseases such as AD, Parkinson disease, diabetes mellitus type 2, bipolar disorder and cancer [167].

There are two isoforms of this enzyme: GSK-3α and GSK-3β, both are expressed ubiquitously in the brain with an increased incidence in hippocampus, cerebral cortex and in the Purkinje neurons in the cerebellum. These two isoforms have similar catalytic domains and almost the same substrate specificity [168].

Activity of GSK-3 depends on phosphorylation of specific sites. However, deactivation of this enzyme has bigger importance than its activation, because the enzyme is active all the time in varying degrees as it undergoes the process of autophosphorylation. GSK-3 is the enzyme which plays an important role in both accumulation of extracellular deposits, so called senile plaques (the main component is Aβ) and formation of intracellular neurofibrillary tangles (the major element is τ-protein) [169, 170].

GSK-3β is important in physiological and also pathological phosphorylation of τ-protein. Under the physiological conditions, phoshorylation of τ -protein determines its affinity for microtubule binding, whereas during the pathogenesis comes to the hyperphosphorylation, which leads to the dissociation of τ -protein from the microtubules and its subsequent aggregation. In vivo tests showed that overexpression of GSK-3β results in τ-neurodegeneration, while inhibition of this kinase decreases τ-toxicity [167].

Furthermore, the relationship between Aβ production and the GSK-3 activity was found out, when the enhanced activity of GSK-3 was induced by increased Aβ production. It was also discovered that APP and presenelin 1 are substrates for GSK-3α and exactly this kinase is considered to be a regulator of the Aβ production by the mechanism of interaction between APP and γ-secretase during the process of APP cleavage [171].

By the above-mentioned reasons, the attention of a huge amount of scientific groups is concentrated exactly on the most potent inhibitors of GSK-3. The inhibitors could be divided into 2 major groups – direct and indirect inhibitors, while the direct ones interact with the enzyme directly and the indirect increase N-terminal phosphorylation of GSK-3 (whereby deactivate it) [172].

Direct inhibitors are further subdivided into smaller groups: lithium, small molecule inhibitors and peptide or protein inhibitors. Lithium was the first one and at the same time the only one drug, which was clinically used for the treatment of bipolar disorders. This monovalent cation inhibits both GSK isoforms. The mechanism of action consists in the direct competitive binding to the ATP-dependent magnesium-sensitive catalytic site of the enzyme and also indirectly in the modulation of post-translational modifications of GSK-3. Lithium reduces τ-phosphorylation and Aβ production from APP as well. Lithium established positive influence on the prevention of β-amyloid toxicity, facilitation of neurogenesis and also on rescue of β-amyloid induced cognitive impairments [172].

The small molecule inhibitors could interact by the mechanism of ATP or non-ATP competitive inhibition. Currently, the bigger importance is attached to the non-ATP competitive inhibitors, because of their better ADME properties due to the absence of endogenous ATP competition. They bind outside the ATP pocket, what could show better kinase selectivity and last but not least reason is that they should have lower IC50 values. An important group of non-ATP competitive inhibitors are thiadiazolidindions. There are two natural representatives of this group – manzamine, which binds to the allosteric site of the GSK-3β and palinurin, whose mechanism of action has not been yet recognized (Fig. (16)) [173, 174]. Among the synthetic analogues, tideglusib is the most promising that is in the IIb phase of AD clinical trials (Fig. (16)). The observation of relationship between the structure and the effect discovered that presence of 1,3-dicarbonyl moiety, where the atom of nitrogen is between two carbonyl groups, had the biggest influence on the GSK-3β inhibition [172].

Figure 16 Structures of GSK-3 inhibitors.

The peptide and protein inhibitors are the last subgroup of direct inhibitors, which were essentially developed for the therapy of early depressive behavior induced by mild traumatic brain injury. The representative of indirect inhibitors of GSK-3 is valproic acid. Nowadays, it is prescribed mostly for epileptic patients, but it also undergoes phase II trials with the AD indication [175].

7 Conclusions

AD therapy is based on the so-called cholinergic theory, which assumes that cholinesterase activity plays an important role in the AD pathogenesis. Up-to-date, there are available almost exclusively only AChE inhibitors and the NMDA receptor antagonist memantine on the market. Here are discussed additional ten different possible therapeutic approaches in the AD treatment. An accurate determination of the AD etiology is still to be determined and thoroughly revised. Despite the worldwide effort, AD is a growing problem of mankind, not only in terms of health, but also in terms of sociological and economic parameters.

Acknowledgements

This study was supported by the specific research (SV/FVZ201201, SV/FVZ2011/04), by the Grant Agency of the Czech Republic (No. P303/11/1907 and P303/12/0611), by Post-doctoral project (No. CZ.1.07/2.3.00/30.0044), by Long Term Development plan – 1011, by MH CZ - DRO (University Hospital Hradec Kralove, No. 00179906), by project MSM0021620849 given by Ministry of Education, Youth and Sports.

Conflict of Interest

The authors confirm that this chapter contents have no conflict of interest.

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