The role of Chromatography in Alzheimer’s Disease Drug Discovery.

Alzheimer’s disease (AD), the most common form of dementia in adults, is a neurodegenerative disorder whose physiopathological events include progressive cognitive impairment and memory loss, associated with a deficit in cholinergic neurotransmission (Davies and Maloney 1976). Histological hallmarks that characterize this disorder comprise plaques of β-amyloid (Aβ) peptide, neurofibrillary tangles (NFT), a dramatic loss of synapses and neurons, and a decreased level of choline acetyltransferase that correlates with a decline in mental status scores (Bartus et al., 1982). This disease is still a serious burden for the society, since no effective pharmacological treatment has yet been found. The only symptomatic drugs being available on the market can just partially restore the cholinergic deficit in the first stages of the disease. Therefore, the research is strongly focused on the discovery of new effective drugs which can halt and reverse the disease. Owing to the multi-factorial nature of AD, one of the most promising drug discovery approaches for AD treatment is addressed to compounds with a multitarget biological profile, the so-called multitarget-directed ligands (MTDLs) (Cavalli et al., 2008). MTDLs developed so far include derivatives that can simultaneously restore brain acetylcholine (ACh) levels, decrease oxidative stress, inhibit Aβ aggregation and formation, decrease tau protein hyperphosphorylation and protect neuronal cells against toxic insults (Prati et al., 2015). The research efforts in drug discovery field are based on the knowledge of the molecular aspects of the disease and on the development of new techniques necessary to investigate the biological systems at molecular level. The selection of new leads to enter clinical trials is therefore a challenging task and involve various essential steps, the first being the selection of molecules able to bind to the AD validated target(s), and then the study of the effects of hitting the target at molecular, cellular, whole animal and human level. In the case of Alzheimer’s disease (AD), acetylcholinesterase (AChE) has been the first target for the development of new drugs since the discovery of the cholinergic deficit in the central nervous system. However, basic research showed that cognitive impairment could be due not only to a cholinergic deficit but also to a cascade of biochemical events leading to the accumulation in the brain of proteins such as ß-amyloid (A) and hyper-phosphorylated tau protein. Important targets are amyloid fibrillogenesis, beta-secretase amyloid precursor protein cleaving enzyme (BACE1), one of the enzymes which cleave APP (amyloid precursor protein) and GSK3, a tau protein phosphorylating kinase. On the other hand, other non cholinergic role of AChE in the AD has been discovered: some evidences suggest that AChE peripheral binding site may play a key role in the development of senile plaques, accelerating A deposition. Once the disease targets have been selected, the determination of the activity of the new compounds must be carried out quickly and in a way that allows the verification of the design hypothesis. Drug activity is in fact mediated by different types of interactions with specific biological targets and the esteem of these interactions may elucidate the mechanism of action. To this aim, in a first instance, high throughput screening methods (HTS) of a large number of compounds for the selection of few lead compounds are required. Secondly, specific methods, which elucidate the selected compound mechanism of action in vitro, have to be employed, before the ultimate and most advanced tools, transgenic animal models of the disease, can be used to study the effects of single compounds on the disease phenotype in vivo, followed by clinical trials on real patients. Towards all these aims, separation science is a demand and a resource for solving the urging analytical problems associated with all the steps in the long pathways which go from the selection of active compounds to the complete development of new drugs. Here we review the purposely designed chromatographic methodologies which have contributed in defining the most important steps towards the discovery of new drugs for AD. To start with, an important contribution is given by in vitro assessment of the activity of chemical libraries on isolated target by the affinity chromatography on HPLC immobilized-enzyme column (or immobilized enzyme reactors, IMERs). A further step is the in vivo verification of activity of the lead selected compound by monitoring the appropriate biomarkers in biological fluids in animals, which are any isolated and characterized molecules capable of probing the activity/toxicity of the lead compound. Then, the verification of adsorption, distribution, metabolism and excretion (ADME) profile in humans is essential to prove that the potential new AD drugs reaches the central nervous system (CNS), hitting the AD targets