**6. BACE-1 inhibitors in the treatment of Alzheimer's disease**

subsequently formed in the neurons. BACE-1 and the homologous BACE-2 are regulated differently and also control different processes. A disrupted intracellular calcium homeostasis may stimulate the genetic expression of BACE-1 *via* triggering the nuclear factor of activated T-cells of type 1 (NFAT1), which leads to over-production of Aβ. Expression of the BACE-1 can also be controlled by the level of Aβ1–42 (but not by the Aβ1–40) through some transcription factors. In addition, some plaques containing Aβ1–42 even increase the levels of BACE-1 in the adjacent neurons just before their death [10]. The homologous enzyme BACE-2 shares 64% of the sequence identity with BACE-1. The action of BACE-2 is in many aspects similar to the activity of α-secretase. BACE-2 triggers a cascade of cleavage of APP by the non-amyloido-

4 Alzheimer's Disease - The 21st Century Challenge

genic way. Its physiological function is associated with the organ pigmentation [11].

In order to clearly demonstrate the involvement of BACE-1 in the pathogenesis of AD, many prominent scientific groups worldwide dealt with developing a mouse model that had deactivated the gene for the production of BACE-1 (i.e., BACE-1 knockout (−/−) mice). At first, these strains of mice were viable, capable of reproduction, with the normal morphology of the body, without any obvious signs of damage of the tissues and normal blood picture [12]. This finding supported the idea that inhibition of BACE-1 can bring about the desired therapeutic effect without adverse effects. The results of this study also point to the fact that the related BACE-2 fails to offset the activity of BACE-1 in the formation of Aβ. It is interesting that hybridization of these BACE-1 knockout (−/−) mice with transgenic mice having the APP gene, which increasingly produce amyloid plaques, provided a generation, the newly born individuals of which did not exhibit the formation of Aβ, Aβ deposits or signs of memory impairment caused by production/accumulation of Aβ. As already mentioned, BACE-1 is located mostly in the presynaptic endings of neurons, where its physiological effects is assumed to occur. Over time, however, it was found that BACE-1 knockout (−/−) mice had impaired axonal conduction, experiencing hypomyelinization (i.e., disrupted formation of myelin, the substance that surrounds the axons and nerve fibers), memory disorders, disturbed neurochemical balance, pathological neurogenesis, astrogenesis, degeneration of neurons with increasing age, pathological changes in the retina and schizophrenic symptoms. All these discoveries observed in BACE-1 knockout (−/−) mice can serve as a model that reflects the potential adverse effects associated with the administration of BACE-1 inhibitors for normal animals or people [13].

The substrates subject to proteolysis by BACE-1 are in particular the membrane-bound proteins like APP. Many of these BACE-1 substrates undergo a process called ectodomain shedding (ectodomain is a part of a membrane protein which protrudes to the extracellular space), while at the same time, these substrates can be cleaved by proteases, called also disintegrins, and ADAM-related metaloproteases. The extent of cleavage of the substrate by ADAM related proteases or BACE-1 depends on the nature of the particular substrate. All the possible side effects caused by inhibition of BACE-1 thus may not be always exhibited, assuming that

The homology between BACE-1 and BACE-2 gave rise to arguments that BACE-1 inhibitors may simultaneously inhibit non-selectively also BACE-2. For this reason, transgenic BACE-2 knockout (−/−) mice were developed to clarify the physiological role of BACE-2 and to explore the benefits offered by inhibition of this enzyme. Similar to the BACE-1 knockout (−/−) mice, the

some substrates are hydrolyzed by another protease [14].

Currently, BACE-1 inhibitors have an exclusive position regarding the therapeutic options for introduction into clinical practice to treat AD [16]. Their mechanism of action is based on reducing the levels of Aβ in the brain. Although several of these inhibitors had already reached clinical testing, there are still important questions to answer, for instance, about their safety, the optimum degree of inhibition of BACE-1 needed to achieve the desired therapeutic effect without the presence of side effects, and the stage of the disease when these compounds are to be indicated in order to achieve the greatest assets [17].

Aβ is produced by neurons in the brain, partly also by astrocytes and other glial cells, which are involved in the formation of this protein in particular during the stress conditions accompanying the AD development. For the production of Aβ, the activity of both enzymes, BACE-1 and γ-secretase, is necessary [10]. The biochemical processes involving the activity of these enzymes are often referred in the literature as the amyloid pathway. Importantly, modulation or inhibition of these enzymes can reduce the formation of Aβ in the brain of patients with AD. On the other hand, activation of the non-amyloidogenic pathway by supporting the α-secretase activity may also reduce the formation of Aβ and currently it is alternatively considered as a promising approach for therapy of AD. An important role for the accumulation of Aβ is also played by the genetic aspects of AD. Nowadays, more than 200 autosomal-dominant mutations in APP and presenilin (PS) have been identified which contribute to the occurrence of familial forms of AD [18]. Without any exception, all these mutations increase the production of all Aβ isoforms, in particular the toxic Aβ containing 42 amino acids (Aβ1–42). An example might be seen in Swedish mutation of APP in the amino acids Lys670 and Met671, that is, the places where BACE-1 enzyme cleaves APP. This mutation results in higher proteolytic efficacy of BACE-1, which promotes an increased rate of the C99 fragment formation and thereby the total production of Aβ [19]. The *APOE-ε4* allele represents a major genetic risk factor for the development of AD with the late onset and it is also associated with an increased production and accumulation of Aβ. Similarly, mutation of ADAM10 metaloprotein, which is endowed with physiologically similar activity to that of α-secretase in neurons, causes the late onset of the AD by suppressing this enzyme activity, while the amyloidogenic cleavage of APP by BACE-1 prevails [20]. Recently, at least five different genes whose mutation contributes significantly to the increased formation of Aβ have been identified. Based on all these mutations and their effects, we can conclude that Aβ is responsible for the pathogenesis leading to the breakout and development of AD. Accordingly, some mutations in APP can represent a protecting mean to suppress the progression of AD. For example, a mutation in the Ala673 region (so-called Ala673Thr mutation) causes a lower affinity of APP to BACE-1, bringing the production of Aβ reduced by up to 40% [21].

Extensive research is dedicated to the development of small molecule inhibitors of BACE-1, capable to act centrally. The first experimental inhibitors were derived from short fragments of APP, being therefore peptide derivatives. These differed from APP by modified amino acid sequence and increased metabolic resistance against cleaving by BACE-1. In *in vitro* conditions these bulky peptide derivatives showed high affinity for BACE-1, especially due to the fact that the active site of BACE-1 is so large that it is able to cleave very large substrates. The disadvantage of these derivatives is that they do not possess true drug-like properties, exerting low oral availability and short half-life in the plasma. Such drug candidates are quickly metabolized and have low permeability through the blood-brain barrier. For these reasons, the researchers have focused on the development of small BACE-1 inhibitors that have high affinity for this enzyme, but are small enough to penetrate through the blood-brain barrier and, at the same time, to exhibit suitable pharmacokinetic properties. In addition, these compounds must be lipophilic enough to permeate through the cytoplasmic and endosome membrane to block the active site of BACE-1 located inside of the lumen. A large number of these compounds, however, reached only a limited concentration in the brain because in most cases, they reached high efflux mediated by P-glycoprotein (P-glycoprotein is an ATP-dependent pump, which removes xenobiotics and protects the brain from the effects of these compounds) [22].

The latest generations of BACE-1 inhibitors are characterized by a good capacity to permeate through the blood-brain barrier, by a suitable pharmacokinetic profile, and the ability to induce reduction of the cerebral levels of Aβ. The result of the research is a panel of several inhibitors of BACE-1, which have entered various stages of clinical testing [23].

MK-8931 was applied in three different doses (12, 40 or 60 mg) and the effect was compared with the placebo over a period of 7 days. The markers of Aβ1–40, Aβ1–42 and sAPP-β were also monitored. As in the previous phase, decrease in the levels of Aβ, depending on the dose of the drug (for the Aβ1–40 57% (12 mg), 79% (40 mg), 84% (60 mg)) was observed and, in addition, without the presence of the more serious side effects. The results of this phase of clinical trials are especially important because the pharmacokinetic and pharmacodynamic properties of this BACE-1 inhibitor are not affected by the quantity of Aβ present in the brain of patients with AD. At the end of 2012, MK-8931 advanced to the clinical phase (II/III) with patients suffering from mild to moderate dementia of AD type. This substance was administered in dosages of 12, 40 and 60 mg and controlled with placebo in the total sample of 200 patients. According to the initial promising results, extension of the third phase of clinical trials by another 1960 patients with AD is expected. Further evaluation of MK-8931 is simultaneously monitored within the III phase of clinical testing on 1500 patients with AD. The

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A non-peptidic BACE-1 inhibitor LY2811376 (**Figure 2**), which was analyzed in a study with oral administration, demonstrated satisfactory pharmacokinetic and pharmacodynamic properties in animal models, which promoted the compound to the first phase of clinical trials. These clinical studies, however, were soon discontinued due to adverse reactions, in particular in the area of inflammation of the retina and the occurrence of stroke. Although all other studies

results of both studies are to be expected in 2017–2018 [24].

**Figure 2.** Chemical structures of BACE-1 inhibitors in clinical testing.

**6.2. LY2886721**

#### **6.1. MK-8931**

In 2012, the results of the first phase of clinical trials with inhibitor MK-8931 were presented (**Figure 2**), which had been performed in 88 healthy individuals aged between 18 and 45 years. The safety, tolerability, pharmacokinetic and pharmacodynamic parameters after single or repeated administration were experimentally determined. MK-8931 was generally well tolerated, and no severe side effects were observed. The main goal in this first phase was to determine whether MK-8931 was capable of penetration into the brain to inhibit the activity of BACE-1. Biomarkers monitoring the levels of Aβ1–40, Aβ1–42 and soluble fragment of APP (sAPP-β), which is formed by BACE-1, were intensively studied. MK-8931 significantly decreased the concentrations of cerebrospinal Aβ, depending on the dose administered, and even in repeated oral administration a reduction of Aβ in the CSF of up to about 90% has been observed. The plasma half-life of MK-8931 after a single administration was around 20 h, which assumes the dosing schedule within the range of a single daily dose. This was followed by a clinical study 1b, where the safety, tolerability, pharmacokinetics and pharmacodynamics in 32 patients with mild to moderate dementia of the AD type were determined. Amyloid Beta Hypothesis: Attention to β- and γ-Secretase Modulators http://dx.doi.org/10.5772/intechopen.75629 7

**Figure 2.** Chemical structures of BACE-1 inhibitors in clinical testing.

MK-8931 was applied in three different doses (12, 40 or 60 mg) and the effect was compared with the placebo over a period of 7 days. The markers of Aβ1–40, Aβ1–42 and sAPP-β were also monitored. As in the previous phase, decrease in the levels of Aβ, depending on the dose of the drug (for the Aβ1–40 57% (12 mg), 79% (40 mg), 84% (60 mg)) was observed and, in addition, without the presence of the more serious side effects. The results of this phase of clinical trials are especially important because the pharmacokinetic and pharmacodynamic properties of this BACE-1 inhibitor are not affected by the quantity of Aβ present in the brain of patients with AD. At the end of 2012, MK-8931 advanced to the clinical phase (II/III) with patients suffering from mild to moderate dementia of AD type. This substance was administered in dosages of 12, 40 and 60 mg and controlled with placebo in the total sample of 200 patients. According to the initial promising results, extension of the third phase of clinical trials by another 1960 patients with AD is expected. Further evaluation of MK-8931 is simultaneously monitored within the III phase of clinical testing on 1500 patients with AD. The results of both studies are to be expected in 2017–2018 [24].

#### **6.2. LY2886721**

these mutations and their effects, we can conclude that Aβ is responsible for the pathogenesis leading to the breakout and development of AD. Accordingly, some mutations in APP can represent a protecting mean to suppress the progression of AD. For example, a mutation in the Ala673 region (so-called Ala673Thr mutation) causes a lower affinity of APP to BACE-1,

Extensive research is dedicated to the development of small molecule inhibitors of BACE-1, capable to act centrally. The first experimental inhibitors were derived from short fragments of APP, being therefore peptide derivatives. These differed from APP by modified amino acid sequence and increased metabolic resistance against cleaving by BACE-1. In *in vitro* conditions these bulky peptide derivatives showed high affinity for BACE-1, especially due to the fact that the active site of BACE-1 is so large that it is able to cleave very large substrates. The disadvantage of these derivatives is that they do not possess true drug-like properties, exerting low oral availability and short half-life in the plasma. Such drug candidates are quickly metabolized and have low permeability through the blood-brain barrier. For these reasons, the researchers have focused on the development of small BACE-1 inhibitors that have high affinity for this enzyme, but are small enough to penetrate through the blood-brain barrier and, at the same time, to exhibit suitable pharmacokinetic properties. In addition, these compounds must be lipophilic enough to permeate through the cytoplasmic and endosome membrane to block the active site of BACE-1 located inside of the lumen. A large number of these compounds, however, reached only a limited concentration in the brain because in most cases, they reached high efflux mediated by P-glycoprotein (P-glycoprotein is an ATP-dependent pump, which

removes xenobiotics and protects the brain from the effects of these compounds) [22].

inhibitors of BACE-1, which have entered various stages of clinical testing [23].

**6.1. MK-8931**

The latest generations of BACE-1 inhibitors are characterized by a good capacity to permeate through the blood-brain barrier, by a suitable pharmacokinetic profile, and the ability to induce reduction of the cerebral levels of Aβ. The result of the research is a panel of several

In 2012, the results of the first phase of clinical trials with inhibitor MK-8931 were presented (**Figure 2**), which had been performed in 88 healthy individuals aged between 18 and 45 years. The safety, tolerability, pharmacokinetic and pharmacodynamic parameters after single or repeated administration were experimentally determined. MK-8931 was generally well tolerated, and no severe side effects were observed. The main goal in this first phase was to determine whether MK-8931 was capable of penetration into the brain to inhibit the activity of BACE-1. Biomarkers monitoring the levels of Aβ1–40, Aβ1–42 and soluble fragment of APP (sAPP-β), which is formed by BACE-1, were intensively studied. MK-8931 significantly decreased the concentrations of cerebrospinal Aβ, depending on the dose administered, and even in repeated oral administration a reduction of Aβ in the CSF of up to about 90% has been observed. The plasma half-life of MK-8931 after a single administration was around 20 h, which assumes the dosing schedule within the range of a single daily dose. This was followed by a clinical study 1b, where the safety, tolerability, pharmacokinetics and pharmacodynamics in 32 patients with mild to moderate dementia of the AD type were determined.

bringing the production of Aβ reduced by up to 40% [21].

6 Alzheimer's Disease - The 21st Century Challenge

A non-peptidic BACE-1 inhibitor LY2811376 (**Figure 2**), which was analyzed in a study with oral administration, demonstrated satisfactory pharmacokinetic and pharmacodynamic properties in animal models, which promoted the compound to the first phase of clinical trials. These clinical studies, however, were soon discontinued due to adverse reactions, in particular in the area of inflammation of the retina and the occurrence of stroke. Although all other studies with the substance faded away, at present, LY2811376 has become a lead structure, which could be administered orally and reach its biological target behind the blood-brain barrier.

Over the last few years, it has turned out that four main factors are responsible for the enzymatic activity of γ-secretase complex: presenilin, anterior pharynx-defective, presenilin enhancer 2

Amyloid Beta Hypothesis: Attention to β- and γ-Secretase Modulators

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9

In recent years, a series of potential inhibitors of γ-secretase has been designed and synthesized. Unfortunately, most of them are not specific to cleaving APP with γ-secretase, and, like in the case of BACE-1, they prevent processing of other γ-secretase substrates that do not have any or at least no obvious role in the pathogenesis of AD. For these reasons, the inhibition of γ-secretase has been associated with serious side effects, which adumbrated the end for most

Historically, the first inhibitor of γ-secretase that underwent clinical studies was BMS-299897 (**Figure 3**) compound prepared by Bristol-Myers Squibb. In 2001, clinical trials of this molecule

and nicastrin, which are described further in this chapter [27].

drug candidates in clinical testing.

**Figure 3.** Inhibitors of γ-secretase in various stages of clinical testing.

**8. Inhibitors of γ-secretase in the clinical development**

The molecule marked with LY2886721 (**Figure 2**) represents the next evolutionary generation of orally acting BACE-1 inhibitors, which has entered into the second phase of clinical trials. Compared to its predecessor LY2811376, the novel drug LY2886721 did not exhibit any side effects in the area of the retina and any stroke. During the first phase of clinical trials on 47 healthy volunteers, no adverse effects were observed in 14 days (different dosing schemes repeated administration of 5, 15 and 35 or 70 mg single administration). The biological half-life fluctuated around 12 h, allowing the dosing once per day, when the drug holds the necessary biological effect even after substantial elimination from the body. Treatment with LY2886721 resulted in the reduction of the plasma and cerebrospinal levels of Aβ1–40 by up to 74% (i.e., after the highest dose of 70 mg). Similar decreasing changes were detected in the cerebrospinal levels of Aβ1–42 and sAPP-β, while the blood level of sAPP-*α* was increased, which is logically explainable by relative excess of α-secretase in comparison with BACE-1.

The second phase of clinical trials with LY2886721 was carried out in 130 patients with moderate to severe AD dementia type. This testing, however, was terminated because of liver abnormalities, but, presumably, this is not associated with inhibition of BACE-1 [25].

#### **6.3. E2609**

E2609 (**Figure 2**) is an orally available, nonpeptidic spirocyclic inhibitor of BACE-1, which induced a significant decline of brain levels of Aβ in preclinical studies. Based on this success, E2609 entered the first phase of clinical testing in which 73 volunteers, administered uniformly with increasing dose from 5 to 800 mg of the drug, and 50 volunteers, administered with different doses in the range of 25–400 mg, participated. The plasma half-life of E2609 is around 12–16 h, which again allows one-day dosing schedule. At the maximal single dose (400 mg), decrease of the cerebrospinal Aβ levels by up to 85% has been observed. The concentration of sAPP-β has been similarly reduced, while sAPP-*α* has been increased. Currently, the drug is in the third phase of clinical determinations [26].

## **7. γ-Secretase**

γ-Secretase is a member of aspartic protease family that cleaves glycoproteins of type I including APP. Unlike β-secretase, γ-secretase has a regulated intramembrane proteolytic activity (RIP), thus, it breaks down domains inside of the cytoplasmic membrane. It is known that it breaks down multiple substrates, and to this day more than 50 such substrates, including APP, have been identified. Among these substrates are Notch, Jagged and Nectin-1α. The signal transmission by RIP is implemented so that the released intracellular domain is moved into the nucleus, as it is in the case of Notch, which regulates specific gene expression. Notch is therefore cleaved to Notch intracellular domain, NICD, which causes in the nucleus the mentioned regulation. In relation to AD, this signal pathway is interesting from the perspective of development and function of the nervous tissue.

Over the last few years, it has turned out that four main factors are responsible for the enzymatic activity of γ-secretase complex: presenilin, anterior pharynx-defective, presenilin enhancer 2 and nicastrin, which are described further in this chapter [27].
