Chromatographic Framework to Determine the Memantine Binding Mechanism on Human Serum Albumin Surface
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- Ibrahim, F., Guillaume, Y. & André, C. Chroma (2008) 68: 179. doi:10.1365/s10337-008-0675-6
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In this work, the interaction of memantine with human serum albumin (HSA) immobilized on porous silica particles was studied using a biochromatographic approach. The determination of the enthalpy change at different pH values suggested that the protonated group in the memantine–HSA complex exhibits a heat protonation with a magnitude around 65 kJ mol−1. This value agrees with the protonation of a guanidinium group, and confirmed that an arginine group may become protonated in the memantine–HSA complex formation. The thermodynamic data showed that memantine–HSA binding, for low temperature (<293 K), is dominated by a positive entropy change. This result suggests that dehydration at the binding interface and charge–charge interactions contribute to the memantine–HSA complex formation. Above 293 K, the thermodynamic data ΔH and ΔS became negative due to van der Waals interactions and hydrogen bonding which are engaged at the complex interface. The temperature dependence of the free energy of binding is weak because of the enthalpy–entropy compensation caused by a large heat capacity change, ΔCp = − 3.79 kJ mol−1 K−1 at pH = 7. These results were used to determine the potential binding site of this drug on HSA.
KeywordsColumn liquid chromatographyHuman serum albuminMemantine
N-Methyl-d-aspartate (NMDA) receptor antagonists have therapeutic potential in several central nervous system disorders, including neuroprotective treatment in chronic neurodegenerative diseases, and symptomatic treatment in other neurologic diseases .
Memantine, an NMDA antagonist, has been recently approved for the treatment of moderate to advanced Alzheimer’s disease (AD) . Memantine is a low-affinity voltage-dependent uncompetitive antagonist for glutamatergic NMDA receptors [3, 4]. By binding to the NMDA receptor with a higher affinity than Mg2+ ions, memantine is able to inhibit the prolonged influx of Ca2+ ions which form the basis of neuronal excitotoxicity . The low affinity and rapid off-rate kinetics of memantine at the level of the NMDA receptor-channel, however, preserves the physiological function of the receptor as it can still be activated by the relatively high concentrations of glutamate released following depolarization of the presynaptic neuron . Age-related changes in physiology and organ function alter drug pharmacokinetics. In addition, older persons take more medications in treating multiple disorders, increasing the risk of drug–drug and drug–disease interactions . Thus, the expanded pharmacokinetics studies, e.g. binding on plasmatic proteins, are important for drugs which are taken by aging patients as the drugs of Alzheimer’s disease. Memantine binds on plasmatic proteins to about 45%, it crosses the blood-brain barrier but its cerebrospinal fluid level is 20% to 50% lower than the serum level due to albumin binding in serum [8–10]. HSA is the most abundant protein in blood and can reversibly bind a large number of pharmacological substances. Few specific binding sites are present on HSA [11, 12]. The most important sites are benzodiazepine and warfarin binding sites. He et al.  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 corresponds to the pocket of subdomain IIIA, which has almost the same size as Site I, the interior of cavity is constituted of hydrophobic amino-acid residues and the cavity exterior presents two important amino-acids residues (Arg410 and Tyr411) [13, 14]. Two common methods that have traditionally been used in evaluating the binding of drugs to albumin include equilibrium dialysis and ultrafiltration [15–17]. These two methods suffer of several disadvantages, as the long periods of time which are required to establish an equilibrium during the dialysis process [15, 16]. Furthermore, it is necessary to correct for the alterations in free and bound analyte concentrations that occur during the dialysis procedure . Ultrafiltration requires less time to perform, but like dialysis it requires the use of a labeled drug and/or an additional analysis step for the actual measurement of the final free drug concentration. In addition, the effects of analyte adsorption to the ultrafiltration membrane must be considered [15, 16]. Other problems include difficulties with temperature changes during the separation and problems when working with highly bound drugs . Because of these limitations, there has been continuing research to find better, faster and more convenient approaches for the analysis of drug–protein binding. One such approach involves the use of affinity chromatography (AC) . AC is a liquid chromatography (LC)-based method in which the stationary phase consists of an immobilized biologically-related ligand. In the case of solute–albumin studies, this ligand consists of serum albumin which has been adsorbed or covalently linked to a support like silica. The association constants of many ligands have been determined by zonal elution  or frontal analysis . The thermodynamic process involved in the binding have also been studied [21–23]. One advantage of utilizing AC for solute–protein studies is the ability of this method to reuse the same ligand preparation for multiple experiments (small amount of protein is needed for a large number of studies), this helps to give good precision by minimizing run-to-run variations. Other advantages include the ease with which AC methods can be automated and the relatively short periods of time that are required in AC for most solute binding studies. The fact that the immobilized protein is continuously washed with an applied solvent is yet another advantage of AC [24, 25]. HSA was the model ligand used in a great number of studies. The main advantage of using HSA is the data available for its interaction with a wide range of organic and inorganic compounds .
In this study, AC was used to determine and quantify the forces driving the association between memantine and HSA by studying the energetic changes of this association as both a function of temperature and pH. Moreover, the number of protons linked to this memantine binding reaction of HSA was calculated. These results were used for estimating the binding site of memantine on HSA.
2.1 Reagents and Operating Conditions
The LC system consisted of a Shimadzu LC- 10ATvp pump (Champs sur Marne- France), a Rheodyne 7125 injection valve (Cotati, California, 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 I.D., 5 μm particle size) was used where HSA was covalently bound onto spherical 5 μm silica particles. The temperature was controlled with an Interchim oven TM701 (Monluçon, France).
3 Result and Discussion
3.1 Bulk Solvent pH Effects
3.2 Thermodynamic Analysis
Thermodynamic parameters ∆H (kJ mol−1) and ∆S (J mol−1 K−1) for the memantine binding to HSA at pH = 7 and for six temperatures
∆H kJ mol−1
∆S J mol−1 K−1
In this paper the memantine binding mechanism to human serum albumin was analyzed. This binding was accompanied with a proton uptake which can be attributed to an increase in the pKa of one or more groups of the memantine and/or HSA in the complex at the entire range of pH studied. The protonation heat showed that an arginine group of albumin binding site may become protonated in the complex. The binding was temperature dependent. In a low temperature domain (<293 K) it was entropically driven, indicating a contribution from hydrophobic effect due to the release of water molecules when memantine and HSA associated. Above 293 K, the thermodynamic data ∆H and ∆S became negative due to van der Waals interactions and hydrogen bonding which are engaged at the complex interface. By the use of known correlations between the heat capacity change and the burial of non-polar surface area, the surface area that is burried in the memantine–HSA complex was estimated. These results associated with the use of a zonal elution approach demonstrated that memantine seemed to be good candidate as ligand for the HSA Site II (indole-benzodiazepine site).