Metabolism of ATS is an intricate pathway of different reactions that include glucuronidation [
7,
8,
38], lactonization [
39], and cytochrome P450-mediated oxidation [
40,
41]. A simplified scheme with the different metabolic pathways of ATS is depicted in . ATS is administered as the hydroxy acid form (calcium salt), and its active metabolites (
ortho-hydroxy atorvastatin (2OH-ATS) and
para-hydroxy atorvastatin (4OH-ATS)) are equipotent to the parent compound, being responsible for 70% of the HMG-CoA reductase inhibitory activity of ATS [
42]. The in vitro HMG-CoA reductase inhibitory activity (IC
50) values for ATS, 2OH-ATS, and 4OH-ATS are 3.71, 5.54, and 3.29 nM, respectively [
43]. Both metabolites, as the parent compound, are equilibrated with the corresponding lactone forms (ATS-L, 2OH-ATS-L, and 4OH-ATS-L) [
38,
39,
41]. It has been demonstrated that lactonization might occur non-enzymatically at pH < 6 [
44] or enzymatically, being the former pathway negligible at pH > 6. The formation of an acyl-glucuronide prior to lactonization is expected to be the major pathway for the enzymatic lactonization of ATS in humans, which is catalyzed by UDP-glucuronosyltransferases (UGTs) UGT1A1, UGT1A3, and UGT2B7. The isoenzyme UGT1A3 is the major contributor to this process with 200 times more activity than UGT2B7 [
7]. The mechanism proposed for the lactonization is the formation of an acyl-β-D-glucuronide conjugate of the ATS acid (parent), elimination of the glucuronic moiety, and final spontaneous cyclization to the corresponding lactone [
38]. ATS glucuronidation, and thus lactonization, follows non-linear kinetics with K
m values of 4 and 20 µM and V
max values of 2280 and 120 pmol/min/mg for UGT1A3 and UGT2B7, respectively [
7]. ATS lactonization is affected by polymorphisms in the
UGT1A locus and has been demonstrated both in vitro and in vivo in healthy volunteers [
8]. On the other hand, the hydrolysis of the lactone forms of ATS and its metabolites to the corresponding carboxylates takes place non-enzymatically at pH > 6 [
44] or can be catalyzed by plasmatic esterases or paraoxonases (PONs) [
38]. PONs are a family of esterase/lactonase enzymes whose encoding genes are located in tandem in the long arm of human chromosome 7 (7q 21–22) [
45], and PON1, PON2, and PON3 are highly involved in ATS metabolism. In addition, ATS increases the expression of PON2 [
46]. A 3.8-fold higher ATS-L hydrolysis rate through PON1 and PON3 has been demonstrated in vitro when compared to spontaneous hydrolysis in a pooled microsomal fraction [
47]. Additionally, results from incubation experiments in human liver microsomes (HLMs) show a median ATS formation rate through hydrolysis of the corresponding lactone of 309.70 pmol/min/mg protein [
47]. Hydrolysis of lactone forms has been demonstrated to occur in plasma [
48]. Therefore, this process must be considered when modelling ATS and its metabolites to better assess its pharmacokinetics.
Cytochrome P450-mediated oxidative metabolism has been described as a major pathway of biotransformation for statins in humans [
38], where CYP3A4 is the major enzyme involved in the formation of the two hydroxy-metabolites of ATS [
39,
41]. The CYP3A4-mediated oxidation is clearly polarized to the lactone forms, with K
m values of 3.9 and 1.4 µM and V
max values of 4235 and 14312 pmol/min/mg for the ortho- and para-hydroxylated metabolites, respectively [
39]. Differences in K
m and V
max values between the acid and lactone form of ATS result in an intrinsic clearance ratio lactone/acid equal to 73 [
40] and in specific metabolite clearance ratios for ortho-hydroxylation and para-hydroxylation of 20.2 and 83.1, respectively [
39]. Quantum mechanics/molecular mechanics (QM/MM) have revealed that the acid form of ATS must pay a desolvation penalty of 5 Kcal/mol to enter in the more hydrophobic active site of the enzyme [
39]. Moreover, the higher V
max value for the para-hydroxylation of ATS-L has been attributed to a shorter distance to the heme oxygen atom of CYP3A4 [
39]. Inhibition studies have demonstrated that ATS-L could be an inhibitor of the metabolism of the acid form [
39]. It could be concluded that ATS lactonization changes its affinity to CYP3A, affecting the preferred hydroxylation positions, and may be responsible for DDIs at this level.