Association mechanism of four acetylcholinesterase inhibitors (AChEIs) with human serum albumin: A biochromatographic approach

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Abstract

In this work, the interaction of a series of acetylcholinesterase inhibitors (AChEIs; donepezil, galanthamine, huperzine and neostigmine) with human serum albumin (HSA) immobilized on porous silica particles was studied using a biochromatographic approach. For all the tested AChEI molecules, linear retention plots were observed at all temperatures. An analysis of the thermodynamics (i.e. enthalpy (ΔH°), entropy ((S°*)) of the interaction of the AChEI molecules with the immobilized human serum albumin was also carried out. The (H° and (S°* values for donepezil, galanthamine and neostigmine, were negative due to van der Waals interactions and hydrogen bonding which govern this association with albumin. Whereas the positive values of (H° and (S°* of huperzine binding on HSA indicated a predominance of hydrophobic interactions. The association of AChEIs with HSA was increased linearly with pH. A comparative thermodynamic study with benzodiazepine molecules was also done to determine the potential binding site of these drugs on HSA.

Introduction

Alzheimer's disease (AD) is a progressive brain disorder that gradually destroys a person's memory and ability to learn, reason, make judgments, communicate and carry out daily activities. The greatly reduced presence of acetylcholine in the cerebral cortex is a significant factor in AD [1], [2]. The inhibition of acetylcholinesterase (AChE) activity may be one of the most realistic approaches to the symptomatic treatment of AD. AChE is responsible for degradation of the neurotransmitter acetylcholine (ACh) in the synaptic cleft of neuromuscular junctions and of neuronal contacts in the central nervous system [3], [4]. Many medicinal agents, as donepezil, huperzine or rivastigmine, used for treatment of Alzheimer's disease, belong to the important class of acetylcholinesterase inhibitors (AChEIs) [5].

Age-related changes in physiology and organ function alter drug pharmacokinetics and pharmacodynamics. In addition, older persons take more medications in treating multiple disorders, increasing the risk of drug–drug and drug–disease interactions [6]. Thus, the expanded pharmacokinetics studies are important for drugs which are taken by aging patients as the drugs of Alzheimer's disease.

HSA is the most abundant protein in blood and can reversibly bind a large number of pharmacological substances, such as AChEI molecules. Few specific binding sites are present on HSA [7], [8]. The most important sites are benzodiazepine and warfarin binding sites. He and Carter [8] have determined the three dimensional structure of HSA and have shown that these two binding sites are located in hydrophobic cavities in subdomains IIA and IIIA. Site I is formed as a pocket in subdomain IIA and involves the lone tryptophan of the protein (Trp214). The inside wall of the pocket is formed by hydrophobic side chains, whereas the entrance to the pocket is surrounded by positively charged residues. Site II correspond to the pocket of subdomain IIIA, which is almost the same size as site I, the interior of cavity is constituted of hydrophobic amino-acids residues and the cavity exterior presented two important amino-acids residues (Arg410 and Tyr411) [9], [10]. HSA was the model protein used in a great number of studies [11]. The main advantage of using HSA is the data available for its interaction with a wide range of organic and inorganic compounds [12]. Affinity chromatography with HSA immobilized on the support is specially suited to the study of drug–protein interactions. The association constants of many ligands have been determined by zonal elution [13] or frontal analysis [14]. The thermodynamic process involved in the binding has already been studied [15], [16], [17], [18], [19]. The aim of this work was to study the association mechanism of four AChEIs (donepezil, galanthamine, huperzine, neostigmine) with the HSA using a biochromatographic approach, and to determine their potential binding site by comparative thermodynamic approach between these drugs and a group of five benzodiazepines. Moreover, the pH effect of the bulk solvent on the AChEI–HSA association was determined.

Section snippets

Theory

Single and multi-component isotherms are now measured by dynamic methods. The most widespread of this is frontal analysis, but this technique is time consuming and requires large amounts of pure compounds [20]. Another popular method, elution by characteristic point (ECP), derives the isotherm from the profile of the diffuse front of the band obtained in response to a single injection of a highly concentrated sample [21]. This method is fast and needs only small amounts of sample, but it

Apparatus

The HPLC system consisted of a Shimadzu LC-10ATvp pump (Champs sur Marne-France), a Rheodyne 7125 injection valve (Cotati, CA, USA) fitted with a 20 μl sample loop, and a Shimadzu UV–visible detector. A chromtech HSA column (Interchim, Montluçon, France) (150 mm × 4 mm) was used in a controlled temperature oven TM701 (Interchim, Montluçon, France). The support was HSA immobilized onto spherical silica particles (diameter 5 μm; pore size 6 nm).

Solvents and samples

The four drugs AChEIs were depicted in (Fig. 1).

Langmuir distribution isotherms

So as to calculate the adsorption constants of the AChEI with HSA, the Langmuir distribution isotherms were calculated at pH 7.0 and 298 K. For each AChEI and for each AChEI concentration in the bulk solvent, the most concentrated AChEI sample was injected into the chromatographic system and its retention factor was determined (see Section 3.3). The variation coefficients of the k′ values were <0.4%, indicating a high reproducibility and a good stability for the chromatographic system. Using a

Conclusion

The mechanism of donepezil, galanthamine, huperzine, and neostigmine binding to human serum albumin (HSA) was analyzed. The results demonstrated that binding of donepezil, galanthamine and neostigmine with albumin was temperature-independent, and governed principally by hydrogen bonding and van der Waals forces. The binding of huperzine with albumin was also temperature-independent and characterized by predominance of hydrophobic interactions. The albumin affinity of the four AChEIs enhanced

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