**3.1. Calcineurin inhibitors**

**Immunosuppressant Pharmacology Adverse effects Enzymes** 

Nephrotoxicity, hepatotoxicity, Hyperlipidaemia, hypertension, Tremor, hyperkalaemia, hypomagnaesemia,

Hypertrichosis, gingiva hyperplasia

Nephrotoxicity, hypertension, diabetes, cholestasis, diarrhea, Tremor, hyperkalaemia, hypomagnaesemia

Thrombocytopenia, anaemia, leukopenia, lymphocele, pneumonitis

Thrombocytopenia, anaemia, leukopenia, lymphocele, pneumonia Hyperlipidaemia, hypertonia, wound-healing complications

Bone marrow suppression, leukopenia, anaemia, thrombocytopenia, myeloid

Cholestasis, hepatotoxicity

Adrenal cortex suppression Hypercholesterolemia, diabetes, hypertension, osteoporosis, osteonecrosis, cataracta, skin atrophy

muscle weakness, Anaemia, leucopenia

Vomiting, diarrhea, abdominal pain

Stomatitis aphtosa, wound-healing

Hyperlipidaemia,

complications

dysplasia,

T-cell dependent immune

T-cell dependent immune

Calcineurin inhibition, Inhibition of cytokine

Calcineurin inhibition, Inhibition of cytokine

response:

412 Organ Donation and Transplantation - Current Status and Future Challenges

production

response:

production

proliferation

proliferation

metabolism

Selective inhibition of inosine monophosphate dehydrogenase,

Inhibition of B- and T-cell

proliferation

**Table 1.** Immunosuppressants with low molecular weight.

Inhibition of T-cell migration and production of T-cell lymphokines

*Calcineurin inhibitors*:

*mTOR inhibitors*:

*Purine analogues*:

*Inosine monophosphate dehydrogenase inhibitors*: **Mycofenolate mofetil Mycofenolate**

*Corticosteroids:* **Prednisone**

**Methyl-prednisolone**

**Ciclosporin** Selective inhibition of

**Tacrolimus** Selective inhibition of

**Sirolimus** Inhibition of B- and T-cell

**Everolimus** Inhibition of B- and T-cell

**Azatioprin** Inhibition of purine

**responsible for the metabolism**

CYP3A4/5

CYP3A4/5

CYP3A4/5

CYP3A4/5

Thiopurine S-methyltransferase, Xantine oxidase

UDP-glucuronyl transferase, CYP3A4/5

CYP3A4/5

For solid organ transplant recipients, the mainstay of the immunosuppressive regimens is calcineurin inhibitor (CNI) therapy with ciclosporin or tacrolimus which selectively blocks several signaling processes, resulting in the inhibition of T-cell activation and proliferation (**Figure 1**) [29, 30]. These drugs effectively treat allograft rejection; however, they display large interindividual variability in their pharmacokinetics, requiring monitoring of blood concentrations for optimal safety and therapeutic efficacy.

Ciclosporin A is an 11-amino acid cyclopeptide that blocks the production of IL-2 by inhibition of calcineurin and, as a consequence, the activation of T-cells (**Figure 1**) [31]. Ciclosporin undergoes extensive metabolism by CYP3A enzymes, producing more than 30 metabolites. The major metabolic pathways are *N*-demethylation to 4-*N*-demethyl ciclosporin, hydroxylation at several positions (1-, 6-, 9-monohydroxy and 1,9- or 6,9-dihydroxy-metabolites), and oxidation to carboxylic acid [32]. Some of the metabolites (e.g., 1,9-dihydroxy-ciclosporin,

**Figure 1.** Molecular action of calcineurin inhibitors and corticosteriods. AP-1 activator protein 1, CAR constitutive androstane receptor, CSA ciclosporin, FKBP tarcolimus binding protein, GR glucocoticoid receptor, IL-2 interleukin 2, JNK c-Jun N-terminal kinase, MAP3K mitogen-activated protein 3 kinase, MPK-1 mitogen-activated protein kinase 1, NFAT nuclear factor of activated T-cells, PXR pregnane X receptor, Tacr tacrolimus.

1c9-dihydroxy-ciclosporin, 1-carboxy-ciclosporin) are toxic contributing to the nephrotoxic and hepatotoxic properties of the parent compound [33, 34]. Consequently, high CYP3A activity increases the rate of ciclosporin metabolism and decreases the immunosuppressive effect, which requires dose modification [35]. However, high CYP3A activity also increases the toxic metabolite formation and the risk of nephrotoxicity and hepatotoxicity. Therefore, immunosuppressive strategy must consider the blood concentrations of both ciclosporin and the toxic metabolites, especially if they are accompanied with symptoms indicating nephrotoxicity or hepatotoxicity.

The structural similarities can explain some common metabolic pathways of mTOR inhibitors and tacrolimus, such as *O*-dealkylation and hydroxylation at several positions [42]. Sirolimus is primarily metabolized by CYP3A enzymes and by CYP2C8 at lower extent, producing hydroxylated and *O*-demethylated metabolites (e.g., 12-hydroxy-, 16-*O*-demethyl-, 39-*O*-demethyl-, 27–39-*O*-didemethyl- and dihydroxy-sirolimus as major metabolites) [46, 47]. The metabolism of sirolimus leads to inactivation, despite the fact that some metabolites display some pharmacological activity less than one tenth of the parent drug. Everolimus is also metabolized by CYP3A and CYP2C8 enzymes; however, the elimination rate of everolimus is more rapid than sirolimus (with 30 h *vs.* 62 h elimination half-lives, respectively). Everolimus is *O*-demethylated and hydroxylated at several positions (forming both mono- and dihydroxy-metabolites); furthermore, a ring-opened metabolite is also formed from everolimus [46]. Everolimus-induced adverse effects are associated with the exposure rather to the parent compound than to its metabolites.

Metabolic Drug Interactions with Immunosuppressants http://dx.doi.org/10.5772/intechopen.74524 415

One of the oldest agents with immunosuppressive activity introduced for kidney transplant recipients was the purine analogue 6-mercaptopurine, which acts by inhibiting purine nucleotide synthesis and, as a consequence, cell proliferation. The prodrug of 6-mercaptopurine, azathioprine with more favorable side-effect profile was later introduced to prevent rejection. Azathioprine is converted to 6-mercaptopurine by nonenzymatic cleavage of the thioether in enterocytes and hepatocytes or in erythrocytes. The major active metabolites, 6-thioguanine nucleotides, are formed *via* 6-thioinosine monophosphate in natural purine synthetic pathways. Inhibition of cell proliferation is mediated by incorporation of the thiopurine nucleotide analogues into DNA (and RNA), causing DNA damage [48]. 6-Mercaptopurine, independently from either direct administration or production from azathioprine, undergoes metabolic inactivation by xanthine oxidase and thiopurine S-methyl transferase and is excreted in the urine, leaving less parent compound available to form thiopurine nucleotides [49]. Due to genetic polymorphism, the thiopurine S-methyl transferase activity is highly variable in patients; namely, those subjects who carry one or two nonfunctional thiopurine S-methyl transferase alleles are unable to tolerate normal doses of azathioprine and can experience serious myelosuppression [50]. Therefore, genotyping assay is recommended before starting azathioprine therapy to identify high-risk

patients, and dosage reduction or alternative therapy is recommended for these patients.

Mycophenolic acid is a selective inhibitor of inosine monophosphate dehydrogenase, which is responsible for *de novo* biosynthesis of guanosine monophosphate, one of the building blocks of DNA. Depletion of the guanosine pool in the cell arrests the lymphotic cell proliferation and suppresses the subsequent immune response triggered by allogenic transplanted organ [51]. In several rapidly dividing cells (e.g. enterocytes), an alternative salvage pathway exists for purine synthesis in addition to *de novo* synthetic pathway; however, lymphocytes seem to be dependent on the *de novo* pathway. Consequently, mycophenolic acid is able to selectively block proliferation of T- and B-cells. Mycophenolic acid is available as enteric-coated mycophenolate sodium and as mycophenolate mofetil ester prodrug that is extensively hydrolyzed

**3.4. Inosine monophosphate dehydrogenase inhibitors**

to the active metabolite mycophenolic acid by carboxylesterases.

**3.3. Antimetabolite purine analogues**

Immunosuppressive properties of tacrolimus are similar to ciclosporin; however, for the same pharmacological effect, significantly lower blood concentration of tacrolimus is required than that of ciclosporin. Tacrolimus, the 23-membered macrocyclic lactone, is converted by demethylation, hydroxylation, and ring rearrangement to at least 15 metabolites, and only a minor proportion of tacrolimus dose is eliminated as unchanged parent drug [36]. Metabolism of tacrolimus leads to the inactivation of the molecule, except for the major 13-*O*-demethyl and the minor 31-*O*-demethyl metabolites. The 13-*O*-demethyl-tacrolimus possesses some immunosuppressive effect; however, it is about one tenth as active as tacrolimus, whereas the 31-*O*-demethyl metabolite displays an immunosuppressive activity comparable to tacrolimus [37, 38]. On the other hand, high blood concentration of 15-*O*-demethyl-tacrolimus metabolite has been reported to be associated with nephrotoxicity and myelotoxicity and with higher incidence of infections [39]. Similarly to ciclosporin, tacrolimus is metabolized by CYP3A enzymes, anticipating great interindividual and intraindividual differences in pharmacokinetics of tacrolimus: (1) CYP3A activity of enterocytes contributes to the first-pass metabolism of tacrolimus; (2) substantial interindividual differences in hepatic CYP3A activity result in great variability in the rate of tacrolimus metabolism, which requires continuous drug monitoring and dose modification primarily in the early postoperative period; (3) concomitant treatment with CYP3A inhibitors is the potential source of metabolic drug interactions; (4) genetic polymorphisms of CYP3A5 also contribute to the high interindividual variability. Since the relative contribution of CYP3A5 to tacrolimus biotransformation is significantly higher than that of CYP3A4 [40], the recipients carrying wild type *CYP3A5\*1* allele or transplanted with liver grafts carrying *CYP3A5\*1* are able to metabolize tacrolimus more rapidly than CYP3A5 nonexpressers [35, 41].

#### **3.2. mTOR (mammalian target of rapamycin) inhibitors**

The mTOR inhibitors prevent cell proliferation by blocking cell cycle progression from the G1-phase to the S-phase. The immunosuppressive activity is mediated *via* blocking mTOR protein kinases, resulting in inhibition of growth factor–mediated T-cell proliferation in response to IL-2 trigger [42]. Sirolimus is a 31-membered macrolide, whereas everolimus is a sirolimus derivative having a 2-hydroxyethyl chain substitution at position 40. Although the chemical structures of sirolimus and everolimus are similar to that of tacrolimus, the mechanism of action of mTOR inhibitors is distinct from calcineurin inhibitors, which allows the application of combination regimens. Additionally, the main advantages of mTOR inhibitors are their nonnephrotoxic properties; therefore, mTOR inhibitors in combination with reduced dose calcineurin inhibitors can augment the calcineurin inhibitor–induced nephrotoxicity [43–45].

The structural similarities can explain some common metabolic pathways of mTOR inhibitors and tacrolimus, such as *O*-dealkylation and hydroxylation at several positions [42]. Sirolimus is primarily metabolized by CYP3A enzymes and by CYP2C8 at lower extent, producing hydroxylated and *O*-demethylated metabolites (e.g., 12-hydroxy-, 16-*O*-demethyl-, 39-*O*-demethyl-, 27–39-*O*-didemethyl- and dihydroxy-sirolimus as major metabolites) [46, 47]. The metabolism of sirolimus leads to inactivation, despite the fact that some metabolites display some pharmacological activity less than one tenth of the parent drug. Everolimus is also metabolized by CYP3A and CYP2C8 enzymes; however, the elimination rate of everolimus is more rapid than sirolimus (with 30 h *vs.* 62 h elimination half-lives, respectively). Everolimus is *O*-demethylated and hydroxylated at several positions (forming both mono- and dihydroxy-metabolites); furthermore, a ring-opened metabolite is also formed from everolimus [46]. Everolimus-induced adverse effects are associated with the exposure rather to the parent compound than to its metabolites.
