**2. GP and Tac**

antiproliferative agent + corticosteroid. Tacrolimus (Tac) and ciclosporin (CsA) are both CNI, and Tac has held most of the market in recent years. Mycophenolate (MMF) is the most widely used antiproliferative agent, which is a prodrug of mycophenolic acid (MPA) that has immunosuppressive activity [2]. There is also a monosodium salt of MPA that was developed to overcome the gastrointestinal side effects of MMF. Ahead of the "triple immunosuppressive therapy", many maintenance regimens also have induction therapy, which consists of antibodies to lymphocytes. The mammalian target of rapamycin (mTOR) is a more recent IS, which includes sirolimus (SRL) and everolimus (EVE). They were first envisaged as a primary IS in regimens without CNI [1, 3, 4], while now, most of the time they are used at low doses

To achieve the target blood concentration fast with less dose adjustment is clinically important, for the therapeutic window of these IS is narrow, which may lead to either acute rejection or over-immunosuppression [7–9]. There are also numerous drug-specific adverse effects, CNI has a closer relation to high blood pressure hypertension, nephrotoxicity, and new-onset diabetes after transplantation (NODAT) [10], steroids may also lead to NODAT [11], and the mTOR inhibitors are often associated with delayed wound healing and hyperlipidemia [8]. In the majority of cases, these adverse effects are associated with high drug concentrations in the

In consideration of inter-individual variation in PK of IS, monitoring the blood concentration has been essential. Therapeutic drug monitoring (TDM) has been widely used to ensure the blood concentration of CNI and SRL [13]. In daily clinical practice, the blood sample is taken

concentration). Although recent studies have suggested that, for measurement of CsA, 2 h

TDM is no doubt powerful and indispensable in the current management of IS, while many patients may still experience a significant delay in achieving target blood-concentrations, and drug dosage need to be adjusted repeatedly, which could significantly increase the risk of acute graft rejection [9, 16]. The narrow therapeutic window of the IS makes it impossible to use a higher initial dose for all patients, and here comes an unmet need for a strategy, which can lead to the fast achievement of IS target blood concentration in the period immediately following transplantation for all the patients. Fortunately, the investigation of GP combined with critical patient data and concomitant medications may help and thus potentially reduce the adverse events related to over- or under-exposure. In addition, studies may also possibly reveal the association between GP and IS PD, which may help clinicians to choose a better combination of IS for the specific group. A strategy based on GP is most likely to be effective when a single gene has a major influence on the absorption, disposition, elimination or tissue

This chapter discusses the published genetic associations with IS PK and PD, and their poten-

) correlates better with the area under the concentration-time curve (AUC)

[9]. This may also apply for Tac, but the practice has not been adopted widely

or trough

and measured at a single time-point immediately before the next dose (called C<sup>0</sup>

in CNI-sparing regimens [5, 6].

104 Genetic Diversity and Disease Susceptibility

after dosing (C<sup>2</sup>

compared to C<sup>0</sup>

[12, 14, 15].

blood, but this is by no means universal [12].

compartmentalization of a drug [12].

tial use in clinical practice to guide drug dosing in SOT.

Tac is a 23-membered macrolide lactone isolated from *Streptomyces tsukubaensis* in 1987 for the first time [17], and in 1994, the US Food and Drug Administration firstly approved Tac for liver transplantation. Due to its excellent efficacy, Tac has been extended as a first-line regimen for kidney, heart, lung, intestinal and bone marrow transplantation. Genetic factors including CYP3A5\*3, CYP3A4\*1B, CYP3A4\*22, ABCB1, and POR\*28 have been reported frequently for their influence on Tac dose requirement, which reveals the importance of GP of Tac.

#### **2.1. CYP3A5 and Tac**

Reportedly, polymorphisms in the CYP3A5 gene may explain 40–50% of the variability in Tac dose requirement [18, 19]. The hottest SNP studied in CYP3A5 is CYP3A5\*3, which is an A to G transition at position 6986 within intron 3 (rs776746) [20]. This mutation leads to alternative splicing, and truncation of the protein, which decreases the function of the CYP3A5 enzyme [21]. As a result, CYP3A5 expressers (CYP3A5\*1/\*1 or CYP3A5\*1/\*3 genotype) have significantly lower dose-adjusted C<sup>0</sup> compared to CYP3A5 non-expressers (CYP3A5\*3/\*3 genotype), and the requirement of Tac dose is CYP3A5\*1/\*1 > \*1/\*3 > \*3/\*3 [20]. A large number of retrospective studies have shown that kidney graft recipients who are CYP3A5 expressers require an approximately 2-fold higher Tac dose compared with non-expressers [22–24]. In addition, no matter in adult or pediatric heart recipients [25, 26], in lung transplantation recipients [27], as well as in liver transplantation recipients [28], the same relationship has also been observed.

Other CYP3A5 SNPs include CYP3A5\*6 (rs10264272) and CYP3A5\*7 (rs41303343). CYP3A5\*6 encodes a G to A transition at position 14,690, causing a splice variant mRNA and deletion of exon 7, resulting in nonfunctional CYP3A5 protein [21, 29]. CYP3A5\*7 denotes a single base insertion at codon 346, causing a frame shift and resulting in a truncated mRNA and nonfunctional CYP3A5 [30].

As for the association between CYP3A5 and the early prediction of the risk of acute rejection, the results are quite inconsistent. Some studies involving Tac therapy did not find any significant association [23, 31–34], while some other studies reported that CYP3A5 expressers had a higher risk of experiencing biopsy-proven acute rejection (BPAR) [35, 36].

As for other kidney transplantation outcomes involving Tac therapy, such as chronic allograft nephropathy or delayed graft function (DGF). Some studies reported that CYP3A5 expressers had a higher risk of experiencing biopsy-proven Tac-related nephrotoxicity [37, 38] and reduced renal function during the first year after transplantation [36], and CYP3A5 nonexpressers were more likely to develop DGF [39] and early renal graft injury as assessed by the urine test [40].

#### **2.2. CYP3A4 and Tac**

As for CYP3A4 gene, two SNPs in relation to Tac PK have been investigated extensively: CYP3A4\*1B SNP (rs2740574) and CYP3A4\*22 SNP (rs35599367).

The CYP3A4\*1B SNP involves an A to G transition at position −392 in the promoter region of CYP3A4 and is associated with an increase of CYP3A4 activity [41]. It showed that the C0 /D ratio of Tac in patients with the \*1B mutation was reduced by 35% compared with that of wild-type homozygotes [42]. However, there is a linkage disequilibrium (LD) between CYP3A4\*1B and rs776746 of the CYP3A5 gene. It is possible that the effect of CYP3A4\*1B on Tac PK and PD is caused by rs776746, which has been shown in several published studies [43, 44]. Therefore, the exact effect of CYP3A4\*1B alone on Tac is still unclear.

results and suggested that 3435C>T and 2677G>T/A SNPs were associated with daily Tac dose requirements. In addition, the study of these three SNP haploids (1236C>T, 2677G>T/A and 3435C>T, which are in linkage disequilibrium) found that C-G-C (haplotype 1) and T-T/A-T (haplotype 2) accounted for 45.4 and 36.2% of the haplotypes, respectively; individuals with haplotype 1 required significantly higher daily doses of Tac than those with haplotype 2 [57]. Although there are many studies on the association of ABCB1 GP with Tac PK or PD, the results remain inconsistent. To further confirm the association, large-scale genotype-pheno-

Gene Polymorphisms of Immunosuppressants in Solid Organ Transplantation

http://dx.doi.org/10.5772/intechopen.75717

POR is essential for CYP-mediated drug oxidation as an electron donor [58]. POR\*28 (rs1057868; A503V) is a coding variant in POR gene, which is believed to be effective in increasing the activity of POR and thus leads to the increasing activities of CYP3A4 and CYP3A5 [59, 60].

/Tac dose in heart or kidney transplantation, no matter for the adult or the pediatric [61–64]. Although the strength of this association seems weak and has a limited clinical impact on Tac dose requirements (15–20%), POR\*28 may explain a part of Tac variability, and POR\*28 carri-

As for the association between POR\*28 allele and BPAR after Tac or CsA therapy, no significant evidence was found [63]. As for other graft clinical outcomes, the POR\*28 allele was not found to be associated with the higher risk of DGF after Tac therapy in patients with renal transplantation [63]. While it should be noted that one study in recipients with kidney transplantation demonstrated a higher risk of post-transplantation diabetes mellitus (PTDM) in

As a nuclear transcription, the human pregnane X receptor (PXR), which is encoded by NR1I2, regulates the expression of CYP3A and ABCB1. Polymorphisms of NR1I2 have been reported, but the results regarding their association with Tac dose requirement are conflicting

The expression and activity of CYP3A are also related to the nuclear receptor peroxisome proliferator-activated receptor alpha (PPAR-α). Two sequence variants in the PPAR-α gene (PPARA), PPARA c.209-1003G>A and c.208+3819A>G, can reduce the PPAR-α expression and contribute to the intra- and inter-individual variability of CYP3A [68]. At present, PPARA c.208+3819A>G appears to have the strongest influence on Tac PK, though it still needs

As for graft clinical outcome, one study in kidney transplant recipients demonstrated a higher

and Tac

107

it has also been reported frequently that POR\*28 carriers have lower adjusted Tac C<sup>0</sup>

type correlation trials are encouraged.

ers may experience faster Tac metabolism [61, 63, 65].

patients carrying the POR\*28 allele [60].

risk of PTDM who carry the rs4253728 SNP [59].

**2.4. POR and Tac**

**2.5. PXR and Tac**

[18, 66, 67].

confirmation.

**2.6. PPAR-α and Tac**

C0

The CYP3A4\*22 SNP (rs35599367) contains a transition of C to T in intron 6 and is associated with reduced CYP3A4 mRNA expression and CYP3A4 enzyme activity in vitro [45]. In clinic observation of kidney transplantation, the CYP3A4\*22 required less Tac dose to achieve the target exposure. What's more, it was not influenced by the CYP3A5 genotype [46]. However, it should be noted that the frequency of CYP3A4\*22 is relatively low. About 5% of the Caucasian population, 3% in the American population, and not found in Asians or Africans [47].

Other CYP3A4 SNPs such as CYP3A4\*18 (rs28371759) may also have an impact on Tac PK. This SNP is located in intron 10, with a transition of T to C at position 878. This mutation may increase the activity of the CYP3A4 enzyme and thereby increase the Tac clearance rate and plasma drug concentration [48].

There is also a new and rare CYP3A4 variant, which is now designated as CYP3A4\*26. This variant is an 802C>T transition and results in a premature stop codon at position 268 in exon 9 (R268\*) [49]. The truncated CYP3A4 protein is non-functional.

When combining CYP3A4 and CYP3A5 genotypes, Elens et al. [50] were able to predict Tac dose requirements better compared with the CYP3A4 or CYP3A5 genotype alone. Based on these observations, it has been proposed to prescribe different Tac doses for ultrarapid (CYP3A5 expressers and CYP3A4 \*1/\*1), intermediate (CYP3A5 non-expressers and CYP3A4\*1/\*1) and poor (CYP3A5 non-expressers and CYP3A4\*22 carriers) CYP3A metabolizers, respectively [51].

## **2.3. ABCB1 gene and Tac**

P-gp, also known as ABCB1 or MDR1 is a glycoprotein encoded by the human ABCB1 gene, which serves as drug transporter of Tac, and plays an important role in Tac PK. Recently, P-gp has been found to contain more than 50 SNPs. Among them, the ABCB1 3435C>T (rs1045642), 1236C>T (rs1128503) and 2677G>T/A (rs2032582; Ala893Ser/Thr) SNPs have drawn the most attention after intensive investigation [52–54].

The ABCB1 3435C>T (rs1045642) might be the hottest locus among all the ABCB1 gene SNPs. Reportedly, the frequency of this mutation in orientals is 37–49% [55]. The variation of rs1045642 locus might reduce the expression and function of P-gp in the duodenum, and thus potentially affect the bioavailability of Tac [56].

As for ABCB1 2677G>T/A SNP, wild-type patients required 40% higher Tac dose compared with homozygous carriers of 2677G>T/A SNP (P ≤ 0.05), while the concentration/dose ratio was 36% lower in the wild-type patients (P ≤ 0.02). The haplotype analysis further confirmed the results and suggested that 3435C>T and 2677G>T/A SNPs were associated with daily Tac dose requirements. In addition, the study of these three SNP haploids (1236C>T, 2677G>T/A and 3435C>T, which are in linkage disequilibrium) found that C-G-C (haplotype 1) and T-T/A-T (haplotype 2) accounted for 45.4 and 36.2% of the haplotypes, respectively; individuals with haplotype 1 required significantly higher daily doses of Tac than those with haplotype 2 [57].

Although there are many studies on the association of ABCB1 GP with Tac PK or PD, the results remain inconsistent. To further confirm the association, large-scale genotype-phenotype correlation trials are encouraged.

#### **2.4. POR and Tac**

The CYP3A4\*1B SNP involves an A to G transition at position −392 in the promoter region of CYP3A4 and is associated with an increase of CYP3A4 activity [41]. It showed that the

/D ratio of Tac in patients with the \*1B mutation was reduced by 35% compared with that of wild-type homozygotes [42]. However, there is a linkage disequilibrium (LD) between CYP3A4\*1B and rs776746 of the CYP3A5 gene. It is possible that the effect of CYP3A4\*1B on Tac PK and PD is caused by rs776746, which has been shown in several published studies [43,

The CYP3A4\*22 SNP (rs35599367) contains a transition of C to T in intron 6 and is associated with reduced CYP3A4 mRNA expression and CYP3A4 enzyme activity in vitro [45]. In clinic observation of kidney transplantation, the CYP3A4\*22 required less Tac dose to achieve the target exposure. What's more, it was not influenced by the CYP3A5 genotype [46]. However, it should be noted that the frequency of CYP3A4\*22 is relatively low. About 5% of the Caucasian

Other CYP3A4 SNPs such as CYP3A4\*18 (rs28371759) may also have an impact on Tac PK. This SNP is located in intron 10, with a transition of T to C at position 878. This mutation may increase the activity of the CYP3A4 enzyme and thereby increase the Tac clearance rate

There is also a new and rare CYP3A4 variant, which is now designated as CYP3A4\*26. This variant is an 802C>T transition and results in a premature stop codon at position 268 in exon

When combining CYP3A4 and CYP3A5 genotypes, Elens et al. [50] were able to predict Tac dose requirements better compared with the CYP3A4 or CYP3A5 genotype alone. Based on these observations, it has been proposed to prescribe different Tac doses for ultrarapid (CYP3A5 expressers and CYP3A4 \*1/\*1), intermediate (CYP3A5 non-expressers and CYP3A4\*1/\*1) and poor (CYP3A5 non-expressers and CYP3A4\*22 carriers) CYP3A metabo-

P-gp, also known as ABCB1 or MDR1 is a glycoprotein encoded by the human ABCB1 gene, which serves as drug transporter of Tac, and plays an important role in Tac PK. Recently, P-gp has been found to contain more than 50 SNPs. Among them, the ABCB1 3435C>T (rs1045642), 1236C>T (rs1128503) and 2677G>T/A (rs2032582; Ala893Ser/Thr) SNPs have drawn the most

The ABCB1 3435C>T (rs1045642) might be the hottest locus among all the ABCB1 gene SNPs. Reportedly, the frequency of this mutation in orientals is 37–49% [55]. The variation of rs1045642 locus might reduce the expression and function of P-gp in the duodenum, and thus

As for ABCB1 2677G>T/A SNP, wild-type patients required 40% higher Tac dose compared with homozygous carriers of 2677G>T/A SNP (P ≤ 0.05), while the concentration/dose ratio was 36% lower in the wild-type patients (P ≤ 0.02). The haplotype analysis further confirmed the

population, 3% in the American population, and not found in Asians or Africans [47].

44]. Therefore, the exact effect of CYP3A4\*1B alone on Tac is still unclear.

9 (R268\*) [49]. The truncated CYP3A4 protein is non-functional.

and plasma drug concentration [48].

106 Genetic Diversity and Disease Susceptibility

lizers, respectively [51].

**2.3. ABCB1 gene and Tac**

attention after intensive investigation [52–54].

potentially affect the bioavailability of Tac [56].

C0

POR is essential for CYP-mediated drug oxidation as an electron donor [58]. POR\*28 (rs1057868; A503V) is a coding variant in POR gene, which is believed to be effective in increasing the activity of POR and thus leads to the increasing activities of CYP3A4 and CYP3A5 [59, 60].

it has also been reported frequently that POR\*28 carriers have lower adjusted Tac C<sup>0</sup> and Tac C0 /Tac dose in heart or kidney transplantation, no matter for the adult or the pediatric [61–64]. Although the strength of this association seems weak and has a limited clinical impact on Tac dose requirements (15–20%), POR\*28 may explain a part of Tac variability, and POR\*28 carriers may experience faster Tac metabolism [61, 63, 65].

As for the association between POR\*28 allele and BPAR after Tac or CsA therapy, no significant evidence was found [63]. As for other graft clinical outcomes, the POR\*28 allele was not found to be associated with the higher risk of DGF after Tac therapy in patients with renal transplantation [63]. While it should be noted that one study in recipients with kidney transplantation demonstrated a higher risk of post-transplantation diabetes mellitus (PTDM) in patients carrying the POR\*28 allele [60].

#### **2.5. PXR and Tac**

As a nuclear transcription, the human pregnane X receptor (PXR), which is encoded by NR1I2, regulates the expression of CYP3A and ABCB1. Polymorphisms of NR1I2 have been reported, but the results regarding their association with Tac dose requirement are conflicting [18, 66, 67].

#### **2.6. PPAR-α and Tac**

The expression and activity of CYP3A are also related to the nuclear receptor peroxisome proliferator-activated receptor alpha (PPAR-α). Two sequence variants in the PPAR-α gene (PPARA), PPARA c.209-1003G>A and c.208+3819A>G, can reduce the PPAR-α expression and contribute to the intra- and inter-individual variability of CYP3A [68]. At present, PPARA c.208+3819A>G appears to have the strongest influence on Tac PK, though it still needs confirmation.

As for graft clinical outcome, one study in kidney transplant recipients demonstrated a higher risk of PTDM who carry the rs4253728 SNP [59].

#### **2.7. Other SNPs and Tac**

The multidrug resistance-associated protein 2 (MRP2), which is encoded by the ABCC2, may also be associated with Tac metabolism [69].

significant associations between SRL exposure and the CYP3A5\*3 genotype [96–99]. The difference between the two kinds of results may be caused by the co-treated use of CNI. The effect of CYP3A5\*3 genotype on SRL may only be notable in the patients taking no CNI [100].

The CYP3A4\*22 allele was found to be associated with a moderately lower SRL hepatic metabolism in vitro, which, is inconsistent with another study [101]. A 113 stable recipients post renal transplantation switched from a CNI to SRL and found no significant association

As for CYP3A4\*1B allele and SRL PK, a study including 149 recipients with renal transplanta-

of 69 patients taking no CNI [96, 97], as the same case in CYP3A5\*3. However, another study

As for ABCB1 gene, two studies showed no significant association between the ABCB1

On the other hand, some reported that ABCB1 haplotype combination has a significant influ-

lower in Chinese renal transplantation recipients carrying ABCB1 CGC/CGC as than those carrying the CGC/TTT or TTT/TTT combinations (no effect of ABCB1 individual SNPs was found).

As for the POR\*28 allele, the PPARA rs4253728 SNP, and CYP2C8\*3, no significant association

Reported data about the influence of P450 or ABCB1 gene variants on the PK/PD of SRL are inconsistent [96, 97, 101, 105, 106]. CYP3A5\*3 genotyping might be potentially useful in kidney transplant recipients with no CNI because of the possible competition for CYP3A5 metabolism [100]. As was stated in [47], at the present time, data are insufficient to recom-

EVE is the hydroxyethyl derivative of SRL, with a similar mechanism of action but much more predictable PK. Clinical trials using EVE followed, first in combination with CNI then in CNI-

Again, the reported data about the association between SNP polymorphisms (including CYP3A5\*3, CYP3A4\*22, ABCB1 c.3435C>T, CYP2C8 and PXR) and the PK/PD of EVE are

/dose [96, 98]. In another study, no association was found

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109

/dose and any of the ABCB1 exon 12, exon 21, and exon 26 SNPs, nor with

/dose in the subgroup

/dose was approximately 30%

tion confirmed a significant association between this allele and SRL C<sup>0</sup>

ence on SRL PK [44]. According to the report, the mean SRL C<sup>0</sup>

was found between these SNPs and SRL PK [60, 68, 102–104].

mend any genotype test for this immunosuppressant.

**4.2. CYP3A4 and SRL**

between this allele and SRL PK.

c.3435C>T SNP and the SRL C<sup>0</sup>

reported differently [97].

**4.3. ABCB1 and SRL**

between SRL C<sup>0</sup>

their haplotype [94].

**4.4. Other SNPs and SRL**

**5. GP and EVE**

sparing regimens [100].

The CYP2C8 enzyme, which is highly expressed in the liver, can also be found in extrahepatic tissues like kidney [70]. Reported by Suarez-Kurtz et al. [71], the CYP2C8\*3 was associated with higher Tac C<sup>0</sup> /D, but only in CYP3A5 non-expressers. Furthermore, CYP2C8\*3 and CYP2J2 -76G>T SNPs were reported to influence the renal function of the patients and the occurrence of adverse events during treatment with Tac and mycophenolate sodium [72].

Genetic polymorphisms in IL-18 (e.g., rs5744247) and IL-10 (e.g., −819 C/T and −592 C/A) can also affect Tac dose requirements [50, 62]. However, the exact mechanism by which they affect Tac dose requirements is unknown [73, 74].

Recently, it was also reported that IL-3 rs181781 and CTLA4 rs4553808 genetic polymorphisms probably influence the Tac dose requirements in Chinese kidney transplant recipients [75].
