7. CYP2D6-Related Therapeutic Response to a Multifactorial Treatment in Dementia
Few prospective clinical trials have been performed to elucidate the influence of CYP2D6 variants on the therapeutic outcome in AD in response to cholinesterase inhibitors or other anti-dementia drugs. We have performed the first prospective study in AD patients who received a combination therapy with (i) an endogenous nucleotide and choline donor, CDP-choline (500 mg/day); (ii) a nootropic substance, piracetam (1600 mg/day); (iii) a vasoactive compound, 1,6 dimethyl 8β-(5-bromonicotinoyl-oxymethyl)-10α-methoxyergoline (nicergoline) (5 mg/day); and (iv) a cholinesterase inhibitor, donepezil (5 mg/day), for one year. With this multifactorial therapeutic intervention, EMs improved their cognitive function (MMSE score) from 21.58 ± 9.02 at baseline to 23.78 ± 5.81 after 1 year of treatment. IMs also improved from 21.40 ± 6.28 to 22.50 ± 5.07 (r = + 0.96), whereas PMs and UMs deteriorate from 20.74 ± 6.72 to 18.07 ± 5.52 (r = -0.97), and from 22.65 ± 6.76 to 21.28 ± 7.75 (r = -0.92), respectively. According to these results, PMs and UMs were the worst responders, showing a progressive cognitive decline with no therapeutic effect, and EMs and IMs were the best responders, with a clear improvement in cognition after one year of treatment. Among EMs, AD patients harboring the *1/*10 genotype responded better than patients with the *1/*1 genotype. The best responders among IMs were the *1/*3, *1/*6 and *1/*5 genotypes, whereas the *1/*4, *10/*10, and *4/*10 genotypes were poor responders. Among PMs and UMs, the poorest responders were carriers of the  *4/*4 and  *1xN/*1 genotypes, respectively [7,8,11,25,99,100,101,102]. In a recent study, Pilotto et al. [186] have confirmed the influence of CYP2D6 variants (rs1080985) on the efficacy of donepezil in AD. 
From all these data we can conclude the following: (i) The most frequent CYP2D6 variants in the Southern European population (Iberian peninsula) are the *1/*1 (57.84%), *1/*4 (22.78%), *1xN/*1 (6.10%), *4/*4 (2.56%), and *1/*3 (2.01%) genotypes, accounting for more than 80% of the population; (ii) the frequency of EMs, IMs, PMs, and UMs is about 59.51%, 29,78%, 4.46%, and 6.23%, respectively, in the general population, and 57.76, 31.05%, 5.27%, and 5.90%, respectively in AD cases; (iii) EMs are more prevalent in GP (59.51%) than in AD (57.76%); IMs are more frequent in AD (31.05%) than in GP (29.78%); the frequency of PMs is slightly higher in AD (5.27%%) than in GP (4.46%); and UMs are more frequent in GP (6.23%) than in AD (5.90%); (iv) there are differences between females and males in the distribution and frequency of CYP2D6 genotypes which might be of relevance in therapeutic terms and risk of ADRs; (v) there is an accumulation of AD-related genes of risk in PMs and UMs; (vi) PMs and UMs tend to show higher transaminase activities than EMs and IMs; (vii) EMs and IMs are the best responders, and PMs and UMs are the worst responders to a combination therapy with cholinesterase inhibitors, neuroprotectants, and vasoactive substances; and (viii) the pharmacogenetic response in AD appears to be dependent upon the networking activity of genes involved in drug metabolism and genes involved in AD pathogenesis [6,7,8,11,25,99,100,101,102].

7.1. CYP Clustering in Alzheimer’s Disease
Since over half of the available drugs are metabolized via different CYP enzymes and other metabolic pathways, it is convenient to understand the networking activity of CYP genes and the genomic profiles of these genes in particular groups of risk. In the case of dementia, 73.71% of AD patients are CYP2C19-EMs, 25.12% IMs, and 1.16% PMs. The distribution and frequency of CYP2C9 genotypes is as follows: *1/*1-EM 60.87%, *1/*2-IM 23.98%, *1/*3-IM 10.17%, *2/*2-PM 2.54%, *2/*3-PM 2.16%, and *3/*3-PM 0.25%, globally representing 60.87% CYP2C9-EMs, 34.16% IMs, and 4.97% PMs [7]. This is especially important because the CYP2C9-Ile359Leu (CYP2C9*3 allele) and CYP2C9-Arg144Cys (CYP2C9*2 allele) variants are associated with warfarin sensitivity. Clustering together CYP2C9 and VKORC1 variants, we can estimate that approximately 30% of the elderly population is sensitive to warfarin anticoagulants. 
Concerning CYP3A4/5 polymorphisms, 82.75% of AD cases are EMs (CYP3A5*3/*3), 15.88% are IMs (CYP3A5*1/*3), and 1.37% are UMs (CYP3A5*1/*1) [7]. The human CYP3A subfamily plays a dominant role in the metabolic elimination of more drugs than any other biotransformation enzyme. CYP3A enzyme is localized in the liver and small intestine and thus contributes to first-pass and systemic metabolism. CYP3A expression varies as much as 40-fold in liver and small intestine donor tissues. Unlike other human P450s (CYP2D6, CYP2C19) there is no evidence of a ‘null’ allele for CYP3A4. Over 50 SNPs have been identified in the CYP3A4 gene. The most common variant, CYP3A4*1B, is an A-392G transition in the 5'-flanking region with an allele frequency ranging from 0% (Chinese and Japanese) to 45% (African-Americans). CYP3A5 is polymorphically expressed in adults with readily detectable expression in about 10-20% in Caucasians, 33% in Japanese and 55% in African-Americans. The primary causal mutation for its polymorphic expression (CYP3A5*3) confers low CYP3A5 protein expression as a result of improper mRNA splicing and reduced translation of a functional protein. The CYP3A5*3 allele frequency varies from approximately 50% in African-Americans to 90% in Caucasians. Functionally, microsomes from a CYP3A5*3/*3 liver contain very low CYP3A5 protein and display on average reduced catalytic activity towards midazolam. Additional intronic or exonic mutations (CYP3A5*5, *6, and *7) may alter splicing and result in premature stop codons or exon deletion. As CYP3A5 is the primary extrahepatic CYP3A isoform, its polymorphic expression may be implicated in disease risk and the metabolism of endogenous steroids or xenobiotics [187]. 
The construction of a genetic map integrating the most prevalent CYP2D6 + CYP2C19 + CYP2C9 polymorphic variants in a trigenic cluster yields 82 different haplotype-like profiles. The most frequent trigenic genotypes in the AD population are *1*1-*1*1-*1*1 (25.70%), *1*1-*1*2-*1*2 (10.66%), *1*1-*1*1-*1*1 (10.45%), *1*4-*1*1-*1*1 (8.09%), *1*4-*1*2-*1*1 (4.91%), *1*4-*1*1-*1*2 (4.65%), and *1*1-*1*3-*1*3 (4.33%). These 82 trigenic genotypes represent 36 different pharmacogenetic phenotypes. According to these trigenic clusters, only 26.51% of the patients show a pure 3EM phenotype, 15.29% are 2EM1IM, 2.04% are pure 3IM, 0% are pure 3PM, and 0% are 1UM2PM (the worst possible phenotype) [7].
Taking into consideration the data available, it might be inferred that at least 10-15% of the AD population may exhibit an abnormal metabolism of cholinesterase inhibitors and/or other drugs which undergo oxidation via CYP2D6-related enzymes. Approximately 50% of this population cluster would show an ultrarapid metabolism, requiring higher doses of cholinesterase inhibitors in order to reach a therapeutic threshold, whereas the other 50% of the cluster would exhibit a poor metabolism, displaying potential adverse events at low doses. If we take into account that approximately 60–70% of therapeutic outcomes depend upon pharmacogenomic criteria (e.g., pathogenic mechanisms associated with AD-related genes), it can be postulated that pharmacogenetic and pharmacogenomic factors are responsible for 75–85% of the therapeutic response (efficacy) in AD patients treated with conventional drugs [6,7,8,99,100,101]. Of particular interest are the potential interactions of cholinesterase inhibitors with other drugs of current use in patients with AD, such as antidepressants, neuroleptics, antiarrhythmics, analgesics, and antiemetics which are metabolized by the cytochrome P450 CYP2D6 enzyme. Although most studies predict the safety of donepezil and galantamine, as the two principal cholinesterase inhibitors metabolized by CYP2D6-related enzymes, few pharmacogenetic studies have been performed so far on an individual basis to personalize the treatment, and most studies reporting safety issues are the result of pooling together pharmacological and clinical information obtained with routine procedures. In certain cases, genetic polymorphism in the expression of CYP2D6 is not expected to affect the pharmacodynamics of some cholinesterase inhibitors because major metabolic pathways are glucuronidation, O-demethylation, N-demethylation, N-oxidation, and epimerization. However, excretion rates are substantially different in EMs and PMs. For instance, in EMs, urinary metabolites resulting from O-demethylation of galantamine represent 33.2% of the dose compared with 5.2% in PMs, which show correspondingly higher urinary excretion of unchanged galantamine and its N-oxide [188]. Therefore, there are still many unanswered questions regarding the metabolism of cholinesterase inhibitors and their interaction with other drugs (potentially leading to ADRs) which require pharmacogenetic elucidation. It is also worth mentioning that dose titration (a common practice in AD patients treated with cholinesterase inhibitors, e.g., tacrine, donepezil) is an unwise strategy, since approximately 30-60% of drug failure or lack of therapeutic efficacy (and/or ADR manifestation) is not a matter of drug dosage but a problem of poor metabolizing capacity in PMs. Additionally, inappropriate drug use is one of the risk factors for adverse drug reactions (ADRs) in the elderly. The prevalence of use of potentially inappropriate medications in patients older than 65 years of age admitted to a general medical or geriatric ward ranges from 16% to 20% [189], and these numbers may double in ambulatory patients. Overall, the most prevalent inappropriate drugs currently prescribed to the elderly are amiodarone, long-acting benzodiazepines and anticholinergic antispasmodics; however, the list of drugs with potential risk also include antidepressants, antihistaminics, NSAIDs, amphetamines, laxatives, clonidine, indomethacin, and several neuroleptics [189], most of which are processed via CYP2D6 and CYP3A5 enzymes. Therefore, pre-treatment CYP screening might be of great help in order to rationalize and optimize therapeutics in the elderly, by avoiding medications of risk in PMs and UMs.