**1. Introduction**

Brain is exposed to a variety of neuromodulating agents given as therapy. The consequential action of these agents should be investigated to have the knowledge of their side influence. The primary degenerative disease of brain is dementia subsequently causing Alzheimer's disease. It is instigated in late life with cumulative properties like diminishing of memory, cognition, linguistic ability, and judgment. It is an advanced brain disorder relating to a person's inability to learn, reason, and carry out daily activities [1, 2]. The significant neurotransmitter acetylcholine is associated with normal functioning of the brain, and if the level of acetylcholine goes down in the cerebral cortex, this promulgates to Alzheimer's disease, the debilitating brain condition [3, 4].

Alzheimer's disease (AD) patients have lesser level of acetylcholine with the progressive abnormalities in cholinergic neurons. One line of attack to

reduce the impact of these abnormalities is to obstruct the relevant enzyme AChE (acetylcholinesterase) which acts as a foremost agent in the breakdown of acetylcholine (ACh) [2]. Acetylcholine hydrolysis into choline and acetate is prevented by AChE inhibitors in the synaptic clefts and ensuing in activating cholinergic transmission [3]. Donepezil (**Figure 1**) is a piperidine-class *(piperidine is a widely used building block and chemical reagent in the synthesis of organic compounds, including pharmaceuticals; piperidine is a widely used secondary amine)* AChE inhibitor, sensibly designed especially for Alzheimer's disease [5, 6]. It is used to improve cognitive function in patients of AD and shows no sign of hepatotoxicity [7–9]. The trade name of this drug is Aricept and it functions as an acetylcholinesterase inhibitor [10]. It has 100% of an oral bioavailability and easily passes the blood–brain barrier. Shows the half life of 70 hrs. The primary action of donepezil is by plummeting the breakdown of acetylcholine thus amassing the concentration of acetylcholine in the brain retaining back to its normal function [11].

The major neuropathological hallmarks of AD are senile plaque, neurofibrillary tangles, and neuronal loss. Both cathepsins and cystatins (cystatin A and B) are found in close association with senile plague, cerebrovascular amyloid deposits, and neurofibrillary tangles in Alzheimer's disease, Parkinson's, and patients suffering from senile dementia supporting the fact that they are amyloid constituents. Recent researches have shown that cathepsins are one of the most important proteases involved in the processing of neuropeptides in the central nervous system (CNS) of brain (**Figure 2**) [12], while cystatin C is also existing in high concentration and their concentration relevant in brain diseases. Cysteine proteinases in normal persons help in -peptide clearing. The disturbance between the accumulative action of cathepsins and cystatins may lead to aggregation of potentially amyloidogenic fragment collection aggravates to form amyloid fibrils in nerve cells of AD patients causing cystatin concentration to shrink (**Figure 3**). Generated by imbalance of cathepsins and cystatin, these potentially amyloidogenic fragments at liberty into the extracellular space cause the aggravation of disease [12].

The powerful regulatory system is constituted by cystatins for endogenous cysteine proteinases (cathepsins) which are often permeable from the lysosomes of dying or diseased cells [13]. Cystatins are the natural inhibitors of cysteine proteases with wide occurrence in tissues and cells which belong to a super family of proteins [14]. Cystatin super family has been divided into three families on the basis of homology, inhibition of target enzymes, and presence or absence of disulfide bonds.

Family 1, also called as stefins, includes members of low-molecular-weight proteins (11 kDa) which lack disulfide bonds and carbohydrate contents. This family includes cystatins A and B and stefins C and D.

**63**

cystatin C.

**Figure 3.**

*Graphical abstract-2.*

**Figure 2.**

plasma [15].

*Putative Involvement of Thiol Protease Inhibitor in the Function of Alzheimer Drug*

*Graphical abstract – Showing the function of cystatin in brain, how it is combating the several diseases.*

The figure shows the detailed interaction of cystatin-cathepsin imbalance and the role of acetylcholine esterase inhibitor as function in restoration of Alzheimer's diseases. They are found both in the body fluids and cells. Common illustration is

Kininogens or family 3 cystatins are large precursor molecules of the vasoactive kinins. They are glycoproteins of single chain that perform the multiple biological functions such as kinin delivery, induction of endogenous blood coagulation cascade, and acute phase response intercession. They are inhabitants of blood

*DOI: http://dx.doi.org/10.5772/intechopen.83578*

**Figure 1.** *Structure of donepezil [17].*

*Putative Involvement of Thiol Protease Inhibitor in the Function of Alzheimer Drug DOI: http://dx.doi.org/10.5772/intechopen.83578*

#### **Figure 2.**

*Redirecting Alzheimer Strategy - Tracing Memory Loss to Self Pathology*

normal function [11].

disease [12].

bonds.

reduce the impact of these abnormalities is to obstruct the relevant enzyme AChE (acetylcholinesterase) which acts as a foremost agent in the breakdown of acetylcholine (ACh) [2]. Acetylcholine hydrolysis into choline and acetate is prevented by AChE inhibitors in the synaptic clefts and ensuing in activating cholinergic transmission [3]. Donepezil (**Figure 1**) is a piperidine-class *(piperidine is a widely used building block and chemical reagent in the synthesis of organic compounds, including pharmaceuticals; piperidine is a widely used secondary amine)* AChE inhibitor, sensibly designed especially for Alzheimer's disease [5, 6]. It is used to improve cognitive function in patients of AD and shows no sign of hepatotoxicity [7–9]. The trade name of this drug is Aricept and it functions as an acetylcholinesterase inhibitor [10]. It has 100% of an oral bioavailability and easily passes the blood–brain barrier. Shows the half life of 70 hrs. The primary action of donepezil is by plummeting the breakdown of acetylcholine thus amassing the concentration of acetylcholine in the brain retaining back to its

The major neuropathological hallmarks of AD are senile plaque, neurofibrillary tangles, and neuronal loss. Both cathepsins and cystatins (cystatin A and B) are found in close association with senile plague, cerebrovascular amyloid deposits, and neurofibrillary tangles in Alzheimer's disease, Parkinson's, and patients suffering from senile dementia supporting the fact that they are amyloid constituents. Recent researches have shown that cathepsins are one of the most important proteases involved in the processing of neuropeptides in the central nervous system (CNS) of brain (**Figure 2**) [12], while cystatin C is also existing in high concentration and their concentration relevant in brain diseases. Cysteine proteinases in normal persons help in -peptide clearing. The disturbance between the accumulative action of cathepsins and cystatins may lead to aggregation of potentially amyloidogenic fragment collection aggravates to form amyloid fibrils in nerve cells of AD patients causing cystatin concentration to shrink (**Figure 3**). Generated by imbalance of cathepsins and cystatin, these potentially amyloidogenic fragments at liberty into the extracellular space cause the aggravation of

The powerful regulatory system is constituted by cystatins for endogenous cysteine proteinases (cathepsins) which are often permeable from the lysosomes of dying or diseased cells [13]. Cystatins are the natural inhibitors of cysteine proteases with wide occurrence in tissues and cells which belong to a super family of proteins [14]. Cystatin super family has been divided into three families on the basis of homology, inhibition of target enzymes, and presence or absence of disulfide

Family 1, also called as stefins, includes members of low-molecular-weight proteins (11 kDa) which lack disulfide bonds and carbohydrate contents. This family

includes cystatins A and B and stefins C and D.

**62**

**Figure 1.**

*Structure of donepezil [17].*

*Graphical abstract – Showing the function of cystatin in brain, how it is combating the several diseases.*

The figure shows the detailed interaction of cystatin-cathepsin imbalance and the role of acetylcholine esterase inhibitor as function in restoration of Alzheimer's diseases. They are found both in the body fluids and cells. Common illustration is cystatin C.

Kininogens or family 3 cystatins are large precursor molecules of the vasoactive kinins. They are glycoproteins of single chain that perform the multiple biological functions such as kinin delivery, induction of endogenous blood coagulation cascade, and acute phase response intercession. They are inhabitants of blood plasma [15].

Cystatins tightly bind and impede the activity of cathepsins; if the activity of cathepsins is not regulated, it instigates chronic diseases [13]. Senile plaque, cerebrovascular amyloid deposits, and neurofibrillary tangles in Alzheimer's disease are all the ramifications of imbalance between proteinases and their endogenous inhibitor cystatins [1].

Normal functioning of the brain is balanced by maintaining the level of acetylcholine and acetylcholinesterase inhibitor [16]. A previous report showed that donepezil binds along with HSA modifying it conformationally by effecting its free concentration in plasma [17].

The supplementation of donepezil was explored to find out any effect on cystatin (major regulator of thiol proteases: cathepsins B, H, and L, etc.) in the mammalian system. If the activity of these proteases is not controlled, it will lead to protease and antiprotease imbalance and repercussion to several diseases [18]. Therefore, it was thought worthwhile to investigate the donepezil cystatin binding and its role in the proper accomplishment of drug delivery and if it lead to kind of side effects as well as to gain knowledge about any conformational change in cystatin effecting its activity?

The study shows that the imbalance of protease-antiprotease purportedly leads the way to Alzheimer's disease, while the presence of drug donepezil unfolds cystatin which becomes unfit to bind cathepsins leading to a number of diseases as a considerable side effect of the drug. As cystatins play significant role in several diseases like, cancer and cardiovascular diseases [19]. Therefore, the usage of donepezil in such patients requires additional attention.

## **2. Materials**

Papain (99% purity) was obtained from Sigma Chemical Company (St. Louis, USA). Donepezil (an Alzheimer drug) was purchased from Ranbaxy (India). The solutions were prepared in 50 mM phosphate buffer of pH 7.4. Salts were purchased from Merck (India). The protein concentration was determined spectrophotometrically. All other reagents were of analytical grade, and double distilled water was used throughout.
