8. Pharmacogenomics of AD-Related Genes
The pharmacogenomics of AD is still in a very primitive stage. In over 100 clinical trials for dementia, APOE has been used as the only gene of reference for the pharmacogenomics of AD [6,7,8,99,100,101,190,191]. Several studies indicate that the presence of the APOE-4 allele differentially affects the quality and extent of drug responsiveness in AD patients treated with cholinergic enhancers (tacrine, donepezil, galantamine, rivastigmine), neuroprotective compounds (nootropics), endogenous nucleotides (CDP-choline), immunotrophins (anapsos), neurotrophic factors (cerebrolysin), rosiglitazone or combination therapies [6,7,8,99,100,101,102,190,191,192]; however, controversial results are frequently found due to methodological problems, study design, and patient recruitment in clinical trials. 
In long-term open clinical trials with a multifactorial treatment, APOE-4/4 carriers are the worst responders [6,7,8,99,100,101,102]. With a similar therapeutic protocol, PSEN1-1/1 homozygotes are the worst responders and PSEN1-2/2 carriers are the best responders [7]. Significant ACE-related therapeutic responses to multifactorial treatments have also been reported [7,102]. Among ACE-I/D variants, ACE-D/D patients were the worst responders (r = -0.58), and ACE-I/D carriers were the best responders (r = + 0.26), with ACE-I/I showing an intermediate positive response (r = + 0.01) [7,102]. ACE-related biochemical and hemodynamic phenotypes have been studied in patients with AD [8,11,16]. ACE-I/I patients tend to be younger than ACE-I/D or ACE-D/D patients at the time of diagnosis and also to show a more severe cognitive deterioration. Serum ApoE, total cholesterol, LDL-cholesterol, HDL-cholesterol, nitric oxide, histamine, and ACE levels are higher in ACE-I/I carriers than in patients with the other genotypes; in contrast, serum triglyceride and VLDL levels are notably lower in ACE-I/I patients compared to patients harboring the ACE-I/D or ACE-D/D genotypes, whereas Aβ levels do not show any clear difference among ACE-related genotypes. Cerebrovascular function tends to be worse in ACE-D/D, with lower brain blood flow velocities and higher pulsatility and resistance indices, than in ACE-I/D (intermediate cerebrovascular hemodynamics) or ACE-I/I (almost normal cerebrovascular function) [8,11,16,102]. The correlation between lipid levels and brain hemodynamics is very similar in this study to data observed in that of CYP2D6-related metabolizer profiles in which EM patients with moderate cholesterol and lipoprotein levels (as well as relatively high nitric oxide, histamine, ACE, and ApoE levels) tend to show a better cerebrovascular hemodynamic profile than AD patients with lower cholesterol and lipoprotein levels [102]. This apparently paradoxical correlation appears to indicate that major influences in cerebrovascular homeostasis and hemodynamic brain blood flow are cholesterol, lipoproteins, nitric oxide, ACE, and histamine, among many other factors, in AD, and that peripheral levels of Aβ are indifferent in this concern. On the other hand, it seems likely that low triglyceride levels may facilitate cerebrovascular function. It is also worth mentioning that ACE-I/I patients with the highest cholesterol levels are the worst in mental performance. Other interpretations of these data might suggest an association of poor cerebrovascular function with ACE-D/D and ACE-I/D, and an association of alterations in lipid metabolism with ACE-I/I [11,102]. 
Both APOE and ACE variants also affect behavior and the modification of behavioral changes (mood, anxiety) in dementia after non-psychotropic pharmacological treatment [6,8,11,100,103]. At baseline, all APOE variants show similar anxiety and depression rates, except the APOE-4/4 carriers who differed from the rest in significantly lower rates of anxiety and depression. Remarkable changes in anxiety were found among different APOE genotypes. Practically all APOE variants responded with a significant diminution of anxiogenic symptoms, except patients with the APOE-4/4 genotype who only showed a slight improvement. The best responders were APOE-2/4 ( r = -0.87) > APOE-2/3 (r = -0.77) > APOE-3/3 (r = -0.69) > APOE-3/4 carriers (r = -0.45). The potential influence of APOE variants on anxiety and cognition in AD does not show a clear parallelism, suggesting that other more complex mechanisms are involved in the onset of anxiety in dementia. Concerning depression, all APOE genotypes improved their depressive symptoms with treatment except those with the APOE-4/4 genotype, which worsen along the treatment period. The best responders were APOE-2/4 (r = -0.85) > APOE-2/3 (r = -0.77) > APOE-3/3 (r = -0.73) > APOE-3/4 (r = -0.16), and the worst responder was APOE-4/4 (r = + 0.31) [11,102]. Patients with each one of the 3 ACE-I/D indel variants were equally anxiogenic and depressive at baseline and all of them responded favorably to the multifactorial protocol by gradually reducing anxiety and depressive symptoms over the 12-month treatment period. The best responders were ACE-I/D (r = -0.89) > ACE-D/D (r = -0.68) > ACE-I/I (r = -0.08). Depressive symptoms were also similarly improved in all ACE-I/D variants. The best responders were ACE-I/D (r = -0.88) > ACE-D/D (r = -0.55) > ACE-I/I (r = -0.13). Comparatively, the worst responders among ACE-I/D variants were carriers of the ACE-I/I genotype which were also the poorest responders in anxiety and cognition [11,102,104].
The combination of APOE and ACE polymorphic variants in bigenic clusters yielded different anxiety and depression patterns at baseline and after one year of treatment. The most anxiogenic patients at baseline were those with the 23DD, 44ID, and 34II genotypes, and the least anxiogenic patients were those harboring the 23II, 44DD, and 23ID genotypes. The most depressive clusters at baseline were those harboring the 23DD, 33ID, and 33II genotypes, with a clear accumulation of APOE-3/3 carriers in these groups, and the least depressive clusters were those represented by carriers of the 23II, 44ID, and 23ID genotypes. All bigenic clusters showed a positive anxiolytic and anti-depressive response to the multifactorial treatment, except 44DD carriers who exhibited the worst response [11,102,104].
APOE influences liver function and CYP2D6-related enzyme activity probably via regulation of hepatic lipid metabolism. It has been observed that APOE may influence liver function and drug metabolism by modifying hepatic steatosis and transaminase activity. There is a clear correlation between APOE-related TG levels and GOT, GPT, and GGT activities in AD [11,102]. Both plasma TG levels and transaminase activity are significantly lower in AD patients harboring the APOE-4/4 genotype, probably indicating (i) that low TG levels protect against liver steatosis; and (ii) that the presence of the APOE-4 allele influences TG levels, liver steatosis, and transaminase activity. Consequently, it is very likely that APOE influences drug metabolism in the liver through different mechanisms, including interactions with enzymes such as transaminases and/or cytochrome P450-related enzymes encoded in genes of the CYP superfamily [11,101,104].
When APOE and CYP2D6 genotypes are integrated in bigenic clusters and the APOE + CYP2D6-related therapeutic response to a combination therapy is analyzed in AD patients, it becomes clear that the presence of the APOE-4/4 genotype is able to convert pure CYP2D6*1/*1 EMs into full PMs, indicating the existence of a powerful influence of the APOE-4 homozygous genotype on the drug metabolizing capacity of pure CYP2D6-EMs. In addition, a clear accumulation of APOE-4/4 genotypes is observed among CYP2D6 PMs and UMs [7].
From these studies we can conclude the following: (i) Most studies with acetylcholinesterase inhibitors indicate that the presence or absence of the APOE-4 allele influences the therapeutic outcome in patients with AD; (ii) Multifactorial treatments combining neuroprotectants, endogenous nucleotides, nootropic agents, vasoactive substances, cholinesterase inhibitors, and NMDA antagonists associated with metabolic supplementation on an individual basis adapted to the phenotype of the patient may be useful to improve cognition and to slow down disease progression in AD; (iii) The therapeutic response in AD seems to be genotype-specific under different pharmacogenomic conditions; (iv) In monogenic-related studies, patients harboring the APOE-4/4 genotype are the worst responders; (v) APP, PSEN1 and PSEN2 mutations influence the therapeutic response in AD; (vi) In trigenic-related studies (APOE + PSEN1 + PSEN2) the best responders are those patients carrying the 331222-, 341122-, 341222-, and 441112- genomic clusters; (vii) The worst responders in all genomic clusters are patients with the 441122+ genotype; (viii) The interaction of several AD-related genes seems to be determinant for drug efficacy and safety; (ix) APOE-CYP2D6 interactions might influence the therapeutic response in AD via changes in lipid metabolism and liver function; (x) APOE may also interact with PSEN1, ACE, A2M and other genes to regulate the effect of drugs on cognition and behavioral changes in dementia; (xi) The APOE-4/4 genotype seems to accelerate neurodegeneration anticipating the onset of the disease by 5-10 years; and, in general, APOE-4/4 carriers show a faster disease progression and a poorer therapeutic response to all available treatments than any other polymorphic variant; (xii) Pharmacogenomic studies using monogenic, bigenic, trigenic, tetragenic or polygenic clusters as a harmonization procedure to reduce genomic heterogeneity in clinical trials are very useful in order to widen the therapeutic scope of limited pharmacological resources [6,7,8,9,10,11,12,13,14,15,16,99,100,101,102,103,104].

8.1. APOE-Related Therapeutic Response to a Multifactorial Therapy in Alzheimer’s Disease
Patients with dementia (N=765, age: 69.44 ± 9.15 years, range: 50-96 years; 466 females, age: 69.18 ± 9.19 years, range: 50-96 years; and 299 males, age: 69.85 ± 9.09 years, range: 50-91 years; p < 0.01) received for three months a multifactorial therapy integrated by CDP-choline (500 mg/day, p.o.), Nicergoline (5 mg/day, p.o.), Sardilipin (E-SAR-94010)(LipoEsar®)(250 mg, t.i.d.), and Animon Complex® (2 capsules/day), a nutraceutical compound integrated by a purified extract of Chenopodium quinoa (250 mg), ferrous sulphate (38.1 mg equivalent to 14 mg of iron), folic acid (200 µg), and vitamin B12 (1 µg) per capsule (RGS: 26.06671/C). Patients with chronic deficiency of iron (<35 µg/mL), folic acid (<2.5 ng/mL) or vitamin B12 (<150 pg/mL) received an additional supplementation of iron (80 mg/day), folic acid (5 mg/day) and B complex vitamins (B1, 15 mg/day; B2, 15 mg/day; B6, 10 mg/day; B12, 10 µg/day; nicotinamide, 50 mg/day), respectively, to maintain stable levels of serum iron (50–150 µg/mL), folic acid (5–20 ng/mL) and vitamin B12 levels (500–1000 pg/mL) in order to avoid the negative influence of all these metabolic factors on cognition [102,103]. Patients with hypertension (>150/85 mmHg) received Enalapril (20 mg/day). The frequency of APOE genotypes was: APOE-2/3, 7.97%; APOE-2/4, 1.18%; APOE-3/3, 58.95%; APOE-3/4, 27.32%; and APOE-4/4, 4.58% (Figure 1). Blood pressure, psychometric assessment (Mini-Mental State Examination, MMSE; ADAS; Hamilton Rating Scale-Depression, HAM-D; Hamilton Rating Scale-Anxiety, HAM-A), and blood parameters (glucose, total cholesterol, HDL-cholesterol, LDL-cholesterol, triglyceride, iron, folate, vitamin B12, TSH, T4) were evaluated at baseline and after 3 months of treatment.
Systolic (p < 0.0002) and diastolic blood pressure (p < 0.001), cognitive function (as assessed by MMSE, 20.51 ± 6.51 vs. 21.45 ± 6.95, p < 0.0000000001; ADAS-Cog, 22.94 ± 13.87 vs. 21.23 ± 12.84, p < 0.0001; ADAS-Non-Cog, 5.26 ± 4.18 vs. 4.15 ± 3.63, p < 0.0000000001; ADAS-Total, 27.12 ± 16.93 vs. 24.28 ± 15.06, p < 0.00009), and mood (HAM-A, 11.35 ± 5.44 vs. 9.79 ± 4.33, p < 0.0000000001; HAM-D, 10.14 ± 5.23 vs. 8.59 ± 4.30, p < 0.0000000001) improved after treatment. Glucose levels did not change. Total cholesterol levels (224.78 ± 45.53 vs. 203.64 ± 39.69 mg/dL, p < 0.0000000001), HDL-cholesterol levels (54.11 ± 14.54 vs. 52.54 ± 14.86 mg/dL, p < 0.0001), and LDL-cholesterol levels (148.15 ± 39.13 vs. 128.89 ± 34.83 mg/dL, p < 0.0000000001) were significantly reduced, whereas triglyceride levels increased (111.99 ± 67.14 vs. 120.69 ± 67.14 mg/dL, p < 0.0006) after 3 months of combined treatment. Folate (7.07 ± 3.61 vs. 18.14 ± 4.23 ng/mL, p < 0.000000001) and vitamin B12 levels (459.65 ± 205.80 vs. 689.78 ± 338.82 pg/mL, p < 0.000000001) also increased, and both TSH and T4 levels remained unchanged after treatment. The response rate in terms of cognitive improvement was as follows: 59.74% responders (RRs), 24.44% non-responders (NRs), and 15.82% stable responders (SRs)(no change in MMSE score after three months of treatment). The response rate in cholesterol levels was very similar: 57.78% RRs, 28.50% NRs, and 13.72% SRs. 
In this study, the basal MMSE score differed in APOE-2/3 carriers with respect to APOE-2/4 (p < 0.02), APOE-3/4 (p < 0.004), and APOE-4/4 carriers (p < 0.0009); in APOE-3/3 vs. APOE-3/4 (p < 0.0005), and APOE-3/3 vs. APOE-4/4 (p < 0.002). The best responders were APOE-3/3 (p < 0.0000000001) > APOE-3/4 (p < 0.00001) > APOE-4/4 carriers (p < 0.05). Patients harboring the APOE-2/3 and APOE-2/4 genotypes did not show any significant improvement. The response rate by genotype was the following: APOE-2/3: 44.26% RRs, 36.07% NRs, 19.67% SRs; APOE-2/4: 55.56% RRs, 44.44% NRs, 0.0% SRs; APOE-3/3: 63.42% RRs, 21.06% NRs, 15.52% SRs; APOE-3/4: 56.94% RRs, 27.75% NRs, 15.31% SRs; APOE-4/4: 51.43% RRs, 28.57% NRs, 20.00% SRs. 
Systolic blood pressure (SBP) was significantly reduced in patients with the APOE-3/3 (p < 0.00007) and APOE-3/4 genotypes (p < 0.01), and diastolic blood pressure exhibited a similar pattern (APOE-3/3, p < 0.005; APOE-3/4, p < 0.01), with no changes in either SBP or DBP in APOE-2/3, APOE-2/4 and APOE-4/4 carriers.
Figure 1  APOE-Related total cholesterol levels response to a multifactorial therapy in patients with dementia.    Glucose levels tended to decrease in APOE-4 allele carriers, but only patients with the APOE-3/4 genotype showed a significant reduction in glucose levels (p < 0.02). In contrast, APOE-2/3 carriers showed a tendency to increased glucose levels.

8.2. APOE-related blood lipid response to Sardilipin (E-SAR-94010)
Basal cholesterol levels were significantly different in patients with the APOE-2/3 genotype vs. APOE-3/3 (p < 0.007), vs. APOE-3/4 (p < 0.001), vs. APOE-4/4 (p < 0.00002); APOE-2/4 vs. APOE-4/4 (p < 0.01); APOE-3/3 vs. APOE-4/4 (p < 0.005); and APOE-3/4 vs. APOE-4/4 (p < 0.01). 
The highest cholesterol levels were seen in APOE-4/4 > APOE-3/4 > APOE-3/3. All patients showed a clear reduction in cholesterol levels after treatment with Sardilipin. This was particularly significant in APOE-3/3 (p < 0.0000000001) > APOE-3/4 (p < 0.00000008) > APOE-4/4 (p < 0.002) > APOE-2/3 (p < 0.02) > APOE-2/4 carriers (p: 0.26) (Figure 1). The response rate by genotype was as follows: APOE-2/3: 63.93% RRs, 29.51% NRs, 6.56% SRs; APOE-2/4: 44.44% RRs, 22.22% NRs, 33.34% SRs; APOE-3/3: 54.32% RRs, 28.16% NRs, 17.52% SRs; APOE-3/4: 53.59% RRs, 31.58% NRs, 14.83% SRs; APOE-4/4: 65.71% RRs, 20.00% NRs, 14.29% SRs. 
HDL-cholesterol levels significantly decreased in APOE-3/3 (p < 0.001) > APOE-3/4 (p < 0.05), with no significant changes in patients with other genotypes. In contrast, LDL-cholesterol levels showed identical changes to those observed in total cholesterol, with similar differences among genotypes at baseline and almost identical decreased levels after treatment (APOE-3/3, p > 0.0000000001; > APOE-3/4, p < 0.00001; > APOE-2/3, p < 0.0004; > APOE-4/4, p < 0.001; > APOE-2/4, p:0.31).
Paradoxically, triglyceride levels tended to increase in all APOE genotypes (APOE-3/3, p < 0.01; > APOE-4/4, p < 0.03; > APOE-2/3, p:0.12; > APOE-3/4, p:0.17), except in APOE-2/4 carriers, who showed a tendency to decrease. Basal triglyceride levels were significantly lower in APOE-4/4 carriers than in APOE-2/3 (p < 0.03) and APOE-3/4 carriers (p < 0.04).
Sardilipin (E-SAR-94010, LipoEsar®, LipoSea®) is a natural product extracted from the marine species Sardina pilchardus, by means of non-denaturing biotechnological procedures [193]. The main chemical compounds of LipoEsar® are lipoproteins (60–80%) whose micelle structure probably mimics that of physiological lipoproteins involved in lipid metabolism. In preclinical studies, sardilipin has shown to be effective in (i) reducing blood cholesterol (CHO), triglyceride (TG), uric acid (UA), and glucose (Glu) levels, as well as liver alanine aminotransferase (ALT), and aspartate aminotransferase (AST) activity; (ii) enhancing immunological function by regulating both lymphocyte and microglia activity; (iii) inducing antioxidant effects mediated by superoxide dismutase activity; and (iv) improving cognitive function [16,193,194]. 
According to these results, it appears that the therapeutic response of patients with dyslipidemia to sardilipin is APOE-related. The best responders were patients with APOE-3/3 > APOE-3/4 > APOE-4/4. Patients with the other APOE genotypes (2/2,  2/3, 2/4) did not show any hypolipemic response to this novel compound [16,194]. In patients with dementia, the effects of sardilipin were very similar to those observed in patients with chronic dyslipidemia, suggesting that the lipid-lowering properties of sardilipin are APOE-dependent (Figure 1).
Clinical studies have revealed that sardilipin reduces blood total cholesterol (T-CHO) (20–30%), Glu (5–10%), UA (10–15%), TG (30–50%), ALT and AST, after 1–3 months of treatment at a daily dose of 250-500 mg (t.i.d). The effect on T-CHO is the result of decreasing LDL-CHO levels and increasing HDL-CHO levels in parallel with an improvement in hepatic protection reflected by reduction in ALT, AST, and GGT activity, as the result of reducing liver steatosis. Both LDL and HDL levels are modulated by dietary, behavioral and genetic factors [16]. Most of these therapeutic effects on the regulation of lipid metabolism tend to show an age-dependent pattern and are also associated with specific genomic profiles in the population. In addition, sardilipin diminishes the size of xanthelasma plaques by 30–60% after 6–9 months of treatment, and specifically protects against the hepatotoxicity induced by statins. Similar effects can be observed on atheromatous plaques on the aortic wall of patients with familial and sporadic dyslipidemia/hyperlipidemia. The daily administration of 1000–1500 mg/day of E-SAR p.o. for three months tends to reduce the average size of atherosclerotic plaques on the aortic wall by 10%. This effect is more significant in patients harboring the APOE-3/3 than in APOE-3/4 carriers in whom the size of the plaque is approximately 30–40% larger than in APOE-3/3 carriers [16,195].