Pharmacogenetics in solid organ transplantation - Future Medicine

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Pharmacogenetics in solid organ transplantation: genes involved in mechanism of action and pharmacokinetics of immunosuppressive drugs Allogenic solid organ transplantation has become the routine procedure in patients with end stage organ disease. Although the transplanted organ compensates deficient body functions, its allogenic nature requires institution of immune tolerance, nowadays provided by immunosuppressive drug administration. Both the safety and efficacy of immunosuppressive treatment depend on many factors, and maintaining levels of immunosuppressants within therapeutic range is the essential target for success in graft function preservation. It is obvious that drug and metabolite concentrations depend on efficiency of individual patient metabolism. Recently, many studies were undertaken to investigate the relationship between genetic factors, drug pharmacokinetics and therapy outcome, and interindividual variability apparently can be explained, at least in part, by genetically determined polymorphisms of xenobiotic-metabolizing enzymes, transport proteins and also in some cases, drug targets. This review presents the recent state of knowledge in the field of pharmacogenetics related to solid organ transplantation. KEYWORDS: azathioprine n cysclosporin A n genetic polymorphism n heart transplantation n kidney transplantation n liver transplantation n lung transplantation n mycophenolate mofetil n pharmacogenetics n tacrolimus

Allogenic solid organ transplantation has become a routine procedure in patients with end stage organ disease. Although the transplanted organ compensates deficient body functions, its allogenic nature requires the institution of immune tolerance, nowadays provided by the administration of immunosuppressive drugs. The final outcome of transplantation, that is, graft function maintenance, depends on many factors, for example, HLA matching, type and timing of surgical procedure, and immunosuppressive regimen administered, which also depends on the transplanted organ. Experimental data and clinical observations provide evidence that different organs vary in their immunogenicity, and thus immunosuppressive regimens are tailored for the organ graft. Currently approved immunosuppressive regimens for maintenance therapy include: calcineurin inhibitors (cyclosporin A [CsA] and tacrolimus [TAC]), mTOR inhibitors (sirolimus [SIR] and everolimus), anti proliferatives (azathioprine [AZA] and mycophenolic acid [MPA]) and biologic drugs (belatacept). The interpersonal differences in the immunosuppressive treatment efficacy and safety are in part determined by genetic factors. This review discusses the association between genetic factors coding for drug-metabolizing enzymes, drug transporters as well as drug targets, and immunosuppressive treatment outcome in solid organ transplant patients.

Kidney transplantation Immunosuppressive regimens in allogenic kidney transplant patients vary among transplantation centers, but most commonly it consists of a calcineurin inhibitor (most commonly TAC) and an adjuvant agent (most commonly mycophenolate mofetil [MMF]) with or without corticosteroids.

10.2217/PGS.13.89 © 2013 Future Medicine Ltd

Pharmacogenomics (2013) 14(9), 1099–1118

Mateusz Kurzawski1 & Marek Droździk*1 Department of Experimental & Clinical Pharmacology, Pomeranian Medical University, Powstancow Wlkp 72, 70–111 Szczecin, Poland *Author for correspondence: Tel.: +48 91 4661589 Fax: +48 91 4661600 [email protected] 1

Tacrolimus Pharmacokinetics

Numerous studies have evaluated the effects of genetic polymorphisms on TAC pharmaco kinetics (summarized in Table 1). TAC is primarily metabolized by the CYP3A subfamily, including CYP3A4 and the highly polymorphic CYP3A5 [1]. The available data point to CYP3A4/5 polymorphisms as determinants of the drug concentration. Owing to the presence of a common nonfunctional splicing defect, known as the CYP3A5*3 allele, which is determined by the rs776746 (G>A) single SNP, most Caucasians lack enzyme activity and are defined as CYP3A5 nonexpressors. Patients characterized by CYP3A5*3/*3 genotype require less TAC to reach target concentrations compared with CYP3A5*1 allele carriers. Another nonfunctional splicing defect variant, CYP3A5*6 (rs10264272, G>A) is an additional factor underlying lack of enzyme activity in non-Caucasian populations [2]. In the case of CYP3A4,

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Table 1. Relevant pharmacogenetic studies in kidney transplantation. Drug

Gene

Polymorphism

Patients (n)

Comments

Significant association

Ref.

TAC

CYP3A4 CYP3A5 ABCB1

CYP3A4*1B CYP3A5*1 and *3 ABCB1 3435C>T

64 C

PK study; no association with ABCB1 polymorphism

CYP3A4/5 polymorphism with PK

TAC

CYP3A5 ABCB1

CYP3A5*3 and *6 ABCB1 3435C>T 1236C>T and 2677G>T/A

50 C


CYP3A5*3 with PK

TAC

CYP3A4 CYP3A5

CYP3A4*1B CYP3A5*3

200 C

PK study; no association with 14 out of 16 studied drug-metabolizing enzymes

CYP3A4/5 polymorphism with PK

[6]

TAC

CYP3A4 CYP3A5

CYP3A4*22 CYP3A5*3

50 C

PK study

CYP3A4*22 and CYP3A5 with PK

[14]

TAC

CYP3A4 CYP3A5 ABCB1

CYP3A4*4, *5 and *18 CYP3A5*3 ABCB1 3435C>T 1236C>T and 2677G>T/A

210 Korean

PK study; no association with CYP3A4 and ABCB1 polymorphisms

CYP3A5*3 polymorphism with PK

[9]

TAC

CYP3A5 ABCB1

CYP3A5*3 ABCB1 3435C>T

72 JP


CYP3A5*3 polymorphism for once-daily formula PK

[10]

TAC

CYP3A5

CYP3A5*3

297 Mexican

PK study


[8]

TAC

CYP3A5

CYP3A5*3

22 pediatric PK study


[7]

TAC

CYP3A4 CYP3A5 CYP2C8 CYP2J2 ABCB1

CYP3A4*1B CYP3A5*3 CYP2C8*3 CYP2J2*7 ABCB1 3435C>T 1236C>T and 2677G>T/A

103 C

No association of CYP2C8, CYP2J2 and ABCB1 with PK; no association of CYP3A4, CYP3A5, CYP2C8 and CYP2J2 with PD

CYP3A4*1B and CYP3A5*3 polymorphisms with PK; ABCB1 haplotypes with PD

[13]

TAC

SCLO1B3

SCLO1B3 334T>G and 699G>A

38 C

PK study

SCLO1B3 334T>G and 699G>A with PK

[23]

TAC

226 genes

2031 SNPs

446 C

PK study


[25]

TAC

CYP3A5

CYP3A5*3

236

Prospective study: genotype-adjusted Faster achievement of the regimen vs standard regimen target C0 in CYP3A5adjusted regimen

[26]

TAC

CYP3A5 POR

CYP3A5*3 POR*28

298 C

PK study

POR*28T in CYP3A5*1/*1 with PK

[22]

TAC

CYP3A5

CYP3A5*3

32 C

PK study

CYP3A5*3 with PK

[11]

TAC

CYP3A4 CYP3A5 ABCB1

CYP3A4*1B CYP3A5*3 ABCB1 3435C>T and 2677G>T/A

304 C

PK study: no effects of the SNPs studied; PD study: drug-induced nephrotoxicity

CYP3A5*3 with PD

[20]

TAC

CYP3A5 ABCB1

CYP3A5*3 ABCB1 3435C>T and 2677G>T/A

136 C

PK study: no effects of ABCB1 SNPs; PD study: drug-induced nephrotoxicity – no effects of ABCB1 or CYP3A5 SNPs; acute rejection – no effects of ABCB1 SNPs

CYP3A5*3 polymorphism with PK CYP3A5*3 with acute rejection

[29]

[4]

[19]

AA: African–American; AUC: Area under the curve; AZA: Azathioprine; C: Caucasian; C0: Therapeutic trough concentration; CsA: Cyclosporin A; EVR: Everolimus; HC: Han Chinese; JP: Japanese; MMF: Mycophenolate mofetil; MPA: Mycophenolic acid; PD: Pharmacodynamics; PK: Pharmacokinetics; PRE: Prednisolone; SIR: Sirolimus; TAC: Tacrolimus; VNTR: Variable number tandem repeat.

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Table 1. Relevant pharmacogenetic studies in kidney transplantation (cont.). Drug

Gene

Polymorphism

Patients (n)

Comments


Ref.

TAC

ABCB1

ABCB1 3435C>T

33 HC

PK study

ABCB1 with PK

[17]

TAC

ABCB1

ABCB1 3435C>T

92 Turkish

PK study

ABCB1 with PK

[18]

TAC

CYP3A5

CYP3A5*3

118 JP

PD study; interstitial fibrosis of the kidney

CYP3A5 with PD

[31]

TAC

CYP3A5 ABCB1

CYP3A5*3 ABCB1 3435C>T and 1236C>T

62 Korean

PK study; no association with ABCB1; CYP3A5 with PK and PD PD study: acute rejection

[30]

TAC

CYP3A4, CYP3A5, ABCB1

ABCB1 3435C>T and 2677G>T/A CYP3A4*1B and CYP3A5*3

252

Histological evaluation of renal Donor–recipient allograft biopsies obtained for 3 years homozygosity for the from transplantation ABCB1 3435C>T variant with increased susceptibility to chronic allograft damage

[32]

CsA

CYP3A4 CYP3A5

CYP3A4*22 CYP3A5*3

49 C

PK study

CYP3A4*22 and CYP3A5*3 with PK

[14]

CsA

CYP3A5 ABCB1

CYP3A5*3 and *6 ABCB1 3435C>T, 1236C>T and 2677G>T/A

50 C



[19]

SIR

CYP3A5 ABCB1

CYP3A5*3 and *6 ABCB1 3435C>T

50 HC



[33]

SIR

ABCB1 CYP3A4 CYP3A5 IL10 TNF

ABCB1 1236C>T, 2677G>T/A and 3435C>T, CYP3A4*1B CYP3A5*3 and *6 IL10 -1082G>A TNF -308G>A

86 C

PK study; no association with ABCB1 1236C>T and 2677G>T/A or with CYP3A4, CYP3A5 and TNF polymorphisms

ABCB1 3435C>T and IL10 -1082G>A with PK

[34]

SIR

ABCB1 CYP3A5

ABCB1 1236C>T, 2677G>T/A, 3435C>T, CYP3A5*3

85 C

PK study; no association

[35]

EVR

ABCB1, CYP3A5, CYP2C8, PXR

ABCB1 -129C>T, 1236C>T, 2677G>T/A and 3435C>T CYP3A5*3 and *6 CYP2C8*3 NR1I2 7635A>G

53 C


[36]

MMF

UGT1A9

UGT1A9 -275A>T and -2152C>T

100 C

5‑year clinical follow-up study of MPA exposure: significantly higher proportion of MPA AUC0–12 h measurements in the low range in variant carriers

-275A>T and/or -2152C>T with PK

[40]

MMF

UGT1A8 UGT1A9 UGT2B7 ABCC2

Several SNPs, including UGT1A9 -275T>A and -2152C>T

338 C

PK study; pharmacodynamic effects: acute rejection; no association of UGT1A8, UGT2B7 and ABCC2 polymorphisms and PK and PD

UGT1A9 with PK and PD

[41]

MMF

UGT1A9

UGT1A9 -275T>A and -2152C>T

133 C

PK study; PD effects: gastrointestinal side effects

UGT1A9 with PK and PD

[42]

MMF

UGT1A8 UGT1A9

UGT1A8*2 and *3 UGT1A9*3 -275T>A and -2152C>T

106 C

PK study

UGT1A8 and UGT1A9 with PK

[43]



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Table 1. Relevant pharmacogenetic studies in kidney transplantation (cont.). Drug

Gene

Polymorphism

Patients (n)

Comments


Ref.

MMF

UGTs SLCOs ABCB1 ABCC2 ABCG2

Multiple SNPs

80 JP

PK study, AUC measured on day 28

SLCO1B1 and SLCO1B3 with AUC

MMF

UGT1A9 UGT2B7 ABCC2

Multiple SNPs

40 C

PK study

UGT1A9 with PK

MMF

ABCC2

ABCC2 24C>T and 3972C>T

66 C

PK study

ABCC2 24C>T with PK

[47]

MMF

IMPDH1

Multiple SNPs

191 C

PD study

106G>A and 25G>A with acute rejection

[52]

MMF

IMPDH1

106G>A and 125G>A

82 JP

PD study: subclinical acute rejection, no associations

MMF

10 genes

21 SNPs

237 C

PD study

ABCB1 SNPs and IMPDH2 3757T>C with acute rejection

[54]

AZA

TPMT

TPMT*2, *3A and *3C

36 (22 C + 14 AA)

Prospective study, 30 days

TPMT carrier status with blood indices

[63]

AZA

TPMT

TPMT*2, *3A and *3C

112 C

1-year observation

TPMT carrier status with leukopenia and AZA dose

[64]

AZA

TPMT ITPA

TPMT*2, *3A and *3C ITPA 94C>A IVS2 +21A>C

157 C

1-year observation

TPMT carrier status with blood indices and AZA dose

[65]

AZA

TPMT

TPMT*2, *3A and *3C

150 HC

TPMT genotype and activity measured

TPMT carrier status with enzyme activity and adverse effects

[66]

AZA

TPMT

TPMT*2, *3A and *3C

139 Thai

6-month observation, TPMT genotype and activity measured

TPMT carrier status with enzyme activity and leukopenia

[67]

AZA

TPMT

TPMT*2, *3A and *3C, and VNTR

172 (95% C)

1-year observation

TPMT carrier status with leukopenia and AZA dose

[68]

AZA

TPMT

TPMT*2, *3A and *3C

122 HC

1-year observation

TPMT carrier status with leukopenia and blood indices

[69]

AZA

ITPA

ITPA 94C>A IVS2 +21A>C

155 HC

ITPA genotype and activity measured ITPA genotype with enzyme activity, 94AA variant homozygote with adverse effects

AZA

XDH AOX1 MOCOS

12 SNPs

156 C

1-year observation

MOCOS rs594445 with lower AZA dose

[73]

PRE

CYP3A5 ABCB1 NR1I2

CYP3A5 (A6986G, CYP3A5*3), ABCB1 3435C>T, 1236C>T and 2677G>T/A NR1I2 A7635A>G

95 JP

PK study; no association of CYP3A5 and ABCB1 polymorphisms with PK

NR1I2 with PK

[74]

[45]

[53]

[72]


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a key drug-metabolizing enzyme in humans, there are no common polymorphisms associated with complete lack of activity. However, an intronic variant designated as the CYP3A4*22 allele (rs35599367, C>T in intron 6) has been described in relation to altered response to statin drugs [3]. Recently published reports also suggest a potential association between the CYP3A4*22 allele and TAC concentrations, with higher drug levels in *22 variant carriers. Hesselink et al. reported that TAC doseadjusted trough levels (C 0) were higher in CYP3A5*3/*3 patients than in *1/*3 and *1/*1 patients [4]. Haufroid et al. provided data that the C 0 of TAC were threefold higher in CYP3A5*3/*3 patients than in CYP3A5*1/*3 patients. The difference was even more striking when considering CYP3A5*1/*1 patients showing C 0 of 5.8-fold lower than CYP3A5*3/*3 patients [5]. Multiple regression analyses showed that the CYP3A5*1/*3 polymorphism explained up to 45% of the variability in TAC dose requirement. Similar findings were reported by Tavira et al., who pointed out that from 16 genes of drug metabolizing enzymes (CYP1A1, CYP1A2, CYP2B6, CYP2C19, CYP2C8, CYP2C9, CY P2D6, CY P3A4, CY P3A5, GSTM1, GSTM3, GSTP1, GSTT1, NAT2, TPMT and ABCB1), CYP3A5 genotype (*3 allele, defined by SNP rs776746) was the strongest predictor of TAC dose requirements [6]. Patients who were CYP3A5*3/*3 carriers received significantly higher TAC doses at 1 week, 6 months and 1 year from therapy initiation. At 1 week, 41% of the CYP3A5 nonexpressors achieved target blood concentrations compared with 26% of the CYP3A5 expressors. Significant association between TAC pharmacok inetics and CYP3A5 polymorphism is also observed in pediatric populations. Zhao et al. revealed that weightnormalized oral clearance was lower in patients with CYP3A5*3/*3 compared with patients with CYP3A5*1/*3 (32.2 ± 10.1 vs 53.5 ± 20.2 l/h; p = 0.01) [7]. Likewise, reports from populations other than Caucasian indicate a significant association between CYP3A5 genotype and TAC pharmacok inetics. Data from a Mexican population provided by García-Roca et al. show that CYP3A5 phenotype had a significant influence on TAC pharmacokinetics, since wild-type CYP3A5 carriers required higher doses in comparison with CYP3A5 variant carriers to produce similar trough concentrations of the drug [8]. Subjects characterized by CYP3A5*1/*1 genotype had a median dose future science group

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requirement of 0.16 mg/kg/day, CYP3A5*1/*3 patients were administered a median TAC dose of 0.13 mg/kg/day and CYP3A5*3/*3 carriers had a median dose of 0.07 mg/kg/day. Similarly, CYP3A5 status was shown to affect TAC pharmacok inetics in a Korean population, leading to significantly higher TAC C0 at months 1, 3, 6 and 12 after transplantation in patients with the CYP3A5*3 alleles [9]. The effects of the CYP3A5 polymorphism was also evaluated in the case of a new TAC formula given once daily by Niioka et al. [10]. The authors defined that median dose-adjusted area under the curve (AUC)0–24 h was markedly smaller for CYP3A5 expressors than nonexpressors for both once- and twice-daily administered TAC formulations in a Japanese population. The C0 was also significantly smaller for CYP3A5 expressors than nonexpressors for each formulation. Moreover, the dose-adjusted AUC0–24 h was approximately 25% lower for once-daily TAC formula in CYP3A5 expressors (CYP3A*1 allele carriers), but not individuals with CYP3A*3/*3 genotype than for twice-daily TAC formula. Similar observations were reported in Causasians by Glowacki et al. [11]. The authors observed a significant decrease in the dose-adjusted AUC24 h for both CYP3A5 nonexpressor and expressor patients after a switch from twice-daily to oncedaily TAC formula. However, in the nonexpressor group, mean blood trough concentration C0 was comparable for both formulations, while it decreased significantly in the expressor group after the switch. The rs2740574 (G) allele that encodes a variant form of CYP3A4 (known as CYP3A4*1B) was identified in the gene promoter, which could potentially inf luence transcriptional activity, increasing gene expression compared with wild-type CYP3A4*1 [12]. This variant has been extensively investigated in CYP3A4 pharmacogenetics studies for the last 20 years, providing contradictory results. Hesselink et al. stated that CYP3A4*1B carriers required more TAC to reach target trough concentrations compared with CYP3A4*1 homozygotes [4]. Likewise, Tavira et al. documented that the CYP3A4 polymorphism was related to TAC bioavailability in kidney transplant recipients [6]. CYP3A4*1 homozygotes required lower TAC dose in comparison with CYP3A4*1B carriers. The most significant effect of the CYP3A4 genotype on TAC dose requirements was observed in patients who were CYP3A5 nonexpressors. An association of CYP3A4*1B as well as an effect of combined CYP3A4*1B and CYP3A*3 genotype www.futuremedicine.com

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was also addressed by Gervasini et al. [13]. The authors reported that carriers of the CYP3A4*1B variant allele displayed TAC predose concentrations that were on average 59% lower than those of subjects with the CYP3A4*1/*1 genotype. However, the mean TAC dose-adjusted C 0 concentrations within 1 year of the study period were 145.59, 86.89 and 58.21 ng/ml per mg/kg/day for the 3A4*1–3A5*3, 3A4*1–3A5*1 and 3A4*1B–3A5*1 haplotypes, respectively, and were significantly different from 1 month to 1 year after kidney transplantation. Contrary to those Caucasian studies, Cho et al. did not document any significant association between CYP3A4*4, CYP3A4*5 and CYP3A4*18 polymorphisms and TAC pharmacokinetics in a Korean population [9]. The recently identified CYP3A4*22 polymorphic locus (rs35599367, C>T in intron 6) has been demonstrated to influence TAC pharmaco kinetics independently of CYP3A5 genotype. Elens et al. reported that the first dose-adjusted concentrations of TAC were 2.0-fold higher in T-variant allele carriers compared with CC homozygotes [14]. Moreover, it was found that combined CYP3A4/CYP3A5 genotypes affected the concentration more strikingly, that is, CYP3A poor metabolizer status was characterized by 1.6and 4.1-fold higher TAC dose-adjusted concentrations than the intermediate and extensive metabolizer groups, respectively. Multiple linear regression analysis revealed that, taken together, both CYP3A4 intron 6 and CYP3A5*3 SNPs explained more than 60% of the variability observed in dose-adjusted TAC dose. It is known that the calcineurin inhibitors TAC and CsA are transported by P-gp, encoded by the ABCB1 gene (formerly designated as MDR1) (Figure 1) [15]. Therefore, all the factors influencing P-gp activity may result in altered bioavailability of both drugs, including common SNPs present in the ABCB1 gene. The most extensively studied ABCB1 variant is the rs1045642 (3435C>T) variant. This silent polymorphism was linked to decreased P-gp activity, probably due to altered protein structure in the presence of a rare codon encoded by the 3435T allele [16]. Contradictory data are available for associations of ABCB1 polymorphisms and TAC pharmacokinetics. The study by Li et al. in a rather limited number of Chinese kidney transplant recipients demonstrated an impact of 3435C>T polymorphism on TAC concentration:dose ratio, with 3435CC patients displaying significantly lower TAC levels per dose than 3435CT/TT patients [17]. Likewise, Akbas et al. reported from a Turkish population 1104


that TAC daily doses were significantly lower in subjects with the 3435TT genotype at months 1 and 6 after kidney transplantation [18]. At 6 and 12 months following surgery, ABCB1 3435CC carriers showed significantly lower TAC C 0 compared with those of 3435TT and CT genotypes. Conversely, no evidence was found supporting a role for the ABCB1 polymorphism in TAC dose requirements in Caucasian populations [4,6,13,19,20]. Similar data were reported from Korean [9] and Japanese [10] populations. A total of 13 papers concerning 1327 individuals were included in the meta-analysis. The overall results showed that the 3435C>T SNP could influence pharmacokinetic parameters in different posttransplant times. Subjects with the CC genotype had a lower concentration:dose ratio and needed higher TAC doses than individuals with CT or TT genotypes [21]. Only limited information from single studies on the influence of other genetic factors on TAC pharmacokinetics is available. However, some significant associations were reported in the case of genes indirectly involved in TAC metabolism or transport. de Jonge et al. evaluated the effects of a recently defined functional polymorphism in POR*28 (rs1057868C>T) that has been associated with an increased in vivo CYP3A activity [22]. The authors found that in kidney transplant recipients characterized by the CYP3A5*1 allele who were administered a standard loading dose of TAC, POR*28 allele-inheriting subjects had lower TAC C0 concentrations in the first days following transplantation, and reached target C0 levels significantly later as compared with patients without the POR*28 allele (rs1057868CC homozygous). The POR*28 allele carriers had significantly higher TAC dose requirements throughout the first year. In CYP3A5 none xpressors (CYP3A5*3/*3) the POR*28 SNP did not affect TAC pharmacok inetics. In another study, no association between analyzed polymorphic loci and pharmacok inetic parameters of TAC were found for CYP2C8 and CYP2J2 [13]. An association between SLCO1B3 and TAC pharmacokinetics was shown by Boivin et al. [23]. The authors reported significant correlations between two linked coding nonsynomymous polymorphisms of the SLCO1B3 gene (334T>G, rs4149117 as well as 699G>A, rs7311358) and mean dose-adjusted TAC trough blood concentrations during the first week following kidney transplantation when target dose (10–12 ng/ml) was obtained. Patients carrying the homozygous mutant haplotype had 14.3-fold higher risk (95% CI: 1.43–100; p = 0.02) of having blood future science group


Tacrolimus, cyclosporin A

Mycophenolate mofetil

ABCB1 (intestinal uptake)

IMPDH1/2 (target)

CYP3A4/5 (intestinal metabolism)

UGT1A7/8/9 (hepatic metabolism) UGT2B7 (hepatic metabolism)

CYP3A4/5 (hepatic metabolism)

Review

Azathioprine

TPMT (metabolism/inactivation) ITPA (hepatic metabolism)

ABCC2 (hepatic excretion)

Figure 1. Main factors involved in interindividual variation of pharmacokinetics and pharmacodynamics of the immunosuppressive agents, potentially influenced by genetic polymorphism.

TAC concentrations above the median level and, thus, being classified as poor OATP1B3 transporters, than carriers of one or two copies of the wild-type haplotype. OATP1B3, encoded by SLCO1B3, is highly expressed in hepatocytes, serving as an uptake transporter of many xenobiotics. However, the results of the study should be treated with caution, as there is no direct evidence that OATP1B3 is involved in TAC transportation, and a recent study has shown that contrary to CsA, TAC is not a potent inhibitor of OATP1B3 [24]. A good summary of this chapter is a recent study by Birdwell et al., who genotyped 2031 polymorphisms in drug-metabolizing enzyme and transporter genes in kidney transplant recipients, and associated the data with TAC blood concentration:dose ratios. The study replicated the significant association of TAC blood concentration:dose ratios with CYP3A5*3 allele status (rs776746), and identified associations with nine variants in linkage disequilibrium with rs776746, including eight CYP3A4 variants [25]. The vast majority of the studies on TAC pharmacogenetics is based on retrospective evaluation of transplant patients’ data, and one of the main issues that should be addressed in the future is confirmation of the findings in prospective cohorts. An exceptional prospective study by Thervet et al. demonstrated an advantage of a CYP3A5-adjusted regimen (0.30 mg/kg/day for CYP3A5 expressors and 0.15 mg/kg/day for nonexpressors) over the standard TAC regimen in renal transplant recipients (0.20 mg/kg/day) [26]. The authors observed that patients receiving CYP3A5-adjusted TAC dose were more likely to achieve targeted C 0 therapeutic concentrations on day 3 from therapy onset (after six oral doses, 43.2% vs 29.1% in standard regimen group; p = 0.03), required fewer dose modifications (p = 0.004) and achieved C0 in significantly reduced time (p = 0.001). At the same time, the future science group

clinical end points were similar in both groups. However, the lack of clinical relevance might be owing to a low-risk population being included in the study and a relatively short observation time (3 months), as was concluded by the authors. Recently, an algorithm was described by Passey et al., allowing for prediction of TAC apparent clearance by taking into account all the variables shown to be relevant, including CYP3A5 genotype of a patient [27]. It was also suggested that the novel CYP3A4*22 allele should be included in the algorithm as a potential predictive factor [28]. Pharmacodynamics

There are some data available on the effects of gene polymorphisms affecting TAC pharmacokinetics and their impact on pharmacodynamic measures. Kuypers et al. demonstrated that carriers of the CYP3A5*1 allele were at significantly higher risk of chronic irreversible drug-induced nephrotoxicity, which is associated with higher drug exposure, than CYP3A5*3 allele subjects [20]. No effects of the CYP3A4 and ABCB1 polymorphisms were demonstrated. Quteineh et al. reported significantly higher number of acute rejection episodes in CYP3A5*1 homoz ygotes compared with carriers of the CYP3A5*1/*3 and CYP3A5*3/*3 genotypes (38 vs 10 and 9%) [29]. However, CYP3A5 polymorphisms were not associated with TAC-related nephrotoxicity. This study also evidenced significantly higher TAC dose requirements in CYP3A5*1/*1 genotype carriers. ABCB1 3435C>T and 2677G>T/A polymorphisms were not related to transplantation outcome. Similar results were provided by Min et al. [30]. The authors observed that CYP3A5 expressors were characterized by significantly higher incidence of early T-cell-mediated rejection of at least Banff grade 1 in severity, including clinical rejection within 10 days and subclinical rejection in biopsies at postoperative day 10. The severity of the rejection according www.futuremedicine.com

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to Banff 07 classification was associated with CYP3A5 genotypes. It was also found that CYP3A5 expressors displayed significantly lower estimated glomerular filtration rates until 1 month and lower estimated glomerular filtration rates by 12 months. Genetic polymorphisms of the ABCB1 gene exerted no significant effects. A quantitative analysis of interstitial fibrosis in biopsy sections in living kidney recipients at 1 month and 1 year after kidney transplantation was reported by Miura et al., who demonstrated that CYP3A5 nonexpressor status significantly correlated with development of kidney interstitial fibrosis, raising the risk more than twofold, as evaluated in a multivariate analysis [31]. No CYP3A4/CYP3A5, CYP2C8 and CYP2J2 SNPs or haplotypes have shown a significant effect on TAC-induced toxicity [13]. By contrast, both the ABCB1 2677GG and 1236CC wild-type genotypes increased by threefold, in a significant manner, the risk for neurotoxic events [29]. The study by Gervasini et al. also demonstrated that the T–G–C haplotype (derived from 1236C>T, 2677G>A/T and 3435C>T genotypes) significantly increased by 4.7-fold the risk for TAC-induced nephrotoxicity in comparison with the wild-type C–G–C haplotype. In addition, the mutant T–T–T haplotype was observed to moderately reduce the risk for neurotoxicity (OR: 0.34; 95% CI: 0.1–1.0; p = 0.05) [13]. Conversely, Naesens et al. reported that combined donor–recipient ABCB1 3435TT genotype (leading to lower P-gp expression) was significantly associated with increased susceptibility to chronic allograft damage independent of graft quality at implantation [32]. That was in concordance with P-gp expression investigated in that study, as absence of P-gp expression at the apical membrane of tubular epithelial cells was also a significant determinant of chronic allograft damage. The authors suggested that chronic histological damage of renal allograft were probably mediated by local renal accumulation of TAC rather than systemic TAC exposure. Cyclosporin A Pharmacokinetics

Contrary to TAC, the available data on pharmacogenetic effects on CsA medication in kidney transplant recipients are still debatable as the reports are contradictory. Haufroid et al. [19] revealed lower C0 in CYP3A5 expressors than in nonexpressors for daily dose and the last dose of CsA administered. CsA dose requirement was not statistically different between expressor and nonexpressor patients. However, the CYP3A5*3 1106


allele was the most significant independent variable for CsA trough blood levels, together with the postgraft time, contributing to 18% of the total variance. Other studies confirmed lack of pharmacogenetic association between CsA pharmacokinetic measures not only for CYP3A5 but also for ABCB1 as well as CYP3A4 [4,19]. However, Elens et al. reported that the first dose-adjusted concentrations of CsA were 1.6fold higher in CYP3A4*22 allele carriers compared with homozygous CC subjects [14]. Moreover, it was found that combined CYP3A4/ CYP3A5 genotypes affected the concentration more strikingly; that is, CYP3A poor metabolizer status was characterized by 1.5- and 2.2fold higher CsA dose-adjusted concentrations than the intermediate and extensive metabolizers, respectively. Multiple linear regression analysis revealed that, taken together, both CYP3A4*22 and CYP3A5*3 SNPs explained more than 20% of the variability observed in dose-adjusted CsA dose. Sirolimus Pharmacokinetics

The association between SIR trough concentration and dose requirements in Chinese stable renal transplant recipients and the ABCB1 3435C>T SNP and CYP3A5*3 allele was reported by Miao et al. [33]. It was found that the drug concentration:dose ratios in CYP3A5*3/*3 patients were significantly higher than in CYP3A5*1 allele carriers, indicating higher SIR demands in the latter subjects. No significant influence of the ABCB1 SNPs on SIR trough concentration and dose requirements were revealed. The aforementioned observations were confirmed by Sam et al., who evaluated the ABCB1 1236C>T, 2677G>T/A and 3435C>T, CYP3A4*1B, CYP3A5*3, CYP3A5*6, IL-10 -1082G>A and TNF -308G>A polymorphisms with SIR dose-adjusted, weightnormalized trough concentrations at 7 days, and at 1, 3, 6 and 12 months following kidney transplantation [34]. From the studied polymorphisms, the ABCB1 3435C>T and IL-10 -1082G>A were significantly associated with logtransformed dose-adjusted, weight-normalized trough concentrations of the drug. Mean SIR dose-adjusted, weight-normalized trough concentrations were 48% higher in patients with the ABCB1 3435CT/TT genotype than those with the 3435CC genotype, and was 24% higher in IL-10 -1082GG compared with -1082AG/AA. However, Mourad et al. found no association between SIR-adjusted trough concentrations future science group


and dose requirements in patients stratified by CYP3A5 (*3 allele) and ABCB1 (1236C>T, 2677G>T/A and 3435C>T) polymorphisms [35]. Everolimus Pharmacokinetics

Moes et al. found no clinically relevant effects of ABCB1, CYP3A5, CYP2C8 and PXR poly morphisms on everolimus pharmacokinetics in kidney transplant patients switched from a triple therapy of CsA, MMF and prednisolone (PRE) to a calcineurin inhibitor-free dual therapy of everolimus and PRE [36]. Mycophenolic acid Pharmacokinetics

Pharmacogenetic issues may also be important as a cause of intersubject variability in dosage requirement for MPA, as summarized by Tett et al. [37]. Both mycophenolate formulations, MMF and enteric-coated mycophenolate sodium, are prodrugs producing the same active compound – MPA. MPA is primarily metabolized by glucuronidation, mainly catalyzed by the UDP-glucuronosyltransferases: UGT1A7, UGT1A8, UGT1A9 and UGT2B7 [38,39]. Among these genes, UGT1A9 promoter polymorphisms were reported as significantly affecting MMF pharmacokinetic parameters, and this has been confirmed in independent studies [40–42]. A significantly higher proportion of MPA AUC 0–12 h measurements in recipients carrying the UGT1A9 -275A>T and/or -2152C>T SNP were in the low MPA exposure range (23.7, 16.6 and 12.6%, ranges 60 mg/l × h-1, respectively; p = 0.02) in a prospective, clinical follow-up study by Kuypers et al. [40]. MPA exposure and biopsy-proven acute rejections during a 1‑year follow-up along with UGT1A8, UGT1A9, UGT2B7 and ABCC2 polymorphisms in kidney transplant patients medicated with TAC or CsA and MMF were evaluated by van Schaik et al. [41]. It was revealed that TAC-treated patients who were UGT1A9 -275T>A (rs6714486) and/or -2152C>T (rs17868320) variant allele carriers displayed a significantly (20%) lower MPA AUC0–12 h. Conversely, in carriers of the rare UGT1A9*3 lack-of-function allele, MPA AUC0–12 h was significantly higher compared to noncarriers (49% higher in patients cotreated with TAC and 54% in patients cotreated with CsA). CsA-treated UGT1A8*2/*2 (518GG) patients had an 18% higher MPA AUC 0–12 h in comparison with noncarriers. Similarly, Sánchez-Fructuoso et al. reported that kidney future science group

Review

recipient carriers of UGT1A9 -275A>T and/or -2152T>C were characterized by a significantly smaller AUC of MPA [42]. However, Johnson et al. suggested that the pharmacogenetic effect of UGT polymorphisms on mycophenolate could be modified by coadministered calcineurin inhibitors [43]. The authors found that MPA C0 were 60% higher in subjects heterozygous or homozygous for UGT1A8*2 than in UGT1A8 wild-type, but the effect was dependent on concomitant calcineurin inhibitor administration. The UGT1A8*2 effect was only seen in patients medicated with TAC, whereas MPA C0 were lower in carriers of the UGT1A9 -275T>A/-2152 C>T polymorphism in subjects administered CsA. There was no effect of the UGT1A9 -275T>A/-2152C>T polymorphism in the TAC medicated patients. Glucuronidated metabolites of MPA are excreted from hepatocytes into bile via MRP2, encoded by the ABCC2 gene [44]. Although human ABCC2 displays the functional -24C>T polymorphism (rs717620), most of the studies failed to find an association with MPA treatment course [40,45,46]. Lloberas et al. provided data suggesting an association between steadystate MPA concentrations and ABCC2 polymorphisms in kidney transplant patients medicated with MMF, steroids, low-dose CsA and standard-dose CsA, TAC and SIR. The study revealed significantly reduced exposure to MPA in ABCC2 24T variant allele carriers, with the association most prominent in patients medicated with TAC or SIR along with macrolides. However, the number of evaluated subjects was rather limited, especially in the case of patients treated according to different protocols [47]. Pharmacodynamics

van Schaik et al. reported that carriers of UGT1A9 -275T>A and/or -2152C>T polymorphisms were at significantly higher risk of acute rejection in fixed-dose MMF-treated patients receiving TAC [41]. UGT2B7 is the major enzyme responsible for the formation of acyl mycophenolate glucuronide (AcMPAG), an MPA metabolite that is thought to contribute to the development of MPA adverse effects such as diarrhea and leukopenia [48]. AcMPAG is also a substrate for MRP2 protein, a product of the ABCC2 gene. Hence, the ABCC2 polymorphism was also evaluated in relation to MPA adverse effects, revealing some weak associations with diarrhea [49] in one of the studies, that was not confirmed by other investigators [39,50,51]. Alternatively, Woillard et al. associated increased risk of diarrhea with TAC or SIR vs CsA www.futuremedicine.com

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treatment, as well as with noncarrier status of the UGT1A8*2 allele (rs1042597, 518CC genotype; HR: 1.876; p = 0.0192) [50]. Patients who carried either the UGT1A9 -275T>A or the -2152C>T polymorphism (or both) experienced more admissions owing to gastrointestinal side effects, as observed by Sánchez-Fructuoso et al. [42]. MPA is a selective inhibitor of IMPDH, with two isoenzymes, encoded by two separate genes in humans, IMPDH1 and IMPDH2. Two SNPs within the IMPDH1 gene, 106G>A and 125G>A SNPs (rs2278293 and rs2278294) were significantly associated with the incidence of biopsy-proven acute rejection in the first year post-transplantation in a study of 191 patients [52]. IMPDH1 polymorphism association with acute rejection episodes in kidney transplant recipients medicated with MMF was also studied by Kagaya et al. [53]. The authors revealed no significant differences in the incidence of subclinical acute rejection between IMPDH1 rs2278293 or rs2278294 polymorphisms. However, in the high MPA night-time exposure range (AUC>60 µg × h/ml and C0 ≥1.9 µg/ml), there was a significantly lower incidence of subclinical acute rejection in IMPDH1 rs2278293AA patients that was not influenced by IMPDH1 rs2278294 genotype. The presence of at least one IMPDH2 3757C allele (3757T>C, rs11706052) tripled the odds of developing biopsy-proven acute rejection in a study by Grinyo et al. (OR: 3.39; 95% CI: 1.42–8.09; p = 0.006) [54]. It is in concordance with the observation that the IMPDH activity was significantly higher for patients with at least one IMPDH2 3757C variant compared with wild-type homozygotes (336 vs 227 µmol/s/mol adenosine monophosphate; p = 0.04) [55]. Azathioprine Pharmacodynamics

AZA is a thiopurine drug, an inhibitor of purine synthesis, and has been used as an immuno suppressant since the 1960s. Recently, AZA was replaced in kidney transplantation by MMF, a more selective inhibitor of purine synthesis, based on data that revealed a significant reduction in acute rejection rates with use of MMF compared with AZA or placebo along with CsA and steroids [56]. However, it is still used in some centers, as well as in management of acute lymphoblastic leukemia and autoimmunologic disorders such as Crohn’s disease. The first genetic factor influencing thiopurine metabolism – the TPMT polymorphism – was identified at the enzyme activity level over 30 years ago 1108


[57].

The TPMT gene polymorphism is a model example in pharmacogenetics: deficiency of this cytosolic enzyme, which is responsible for converting AZA metabolites to inactive compounds, is associated with severe hematopoietic toxicity after administration of standard doses of thiopurine drugs, usually developing several weeks after therapy initiation. The TPMT-deficient AZA-treated renal transplant recipients (up to 0.3% in Caucasian populations), inheriting two variant alleles accumulate high concentrations of toxic thioguanine nucleotides in tissues [58,59]. Individuals heterozygous for one of the impaired alleles (most frequently TPMT*2, *3A or *3C) show intermediate enzyme activity and lower accumulation of thioguanine metabolites, although these patients are still at greater risk of AZA toxicity compared with TPMT wildtype homozygotes [60]. Although TPMT polymorphism was most extensively studied in acute lymphoblastic leukemia and Crohn’s disease, leading to recommendations for dose adjustment based on the genotype status [61,62], some also report data from AZA-treated kidney transplant recipients [63–69]. Fabre et al. reported that 58% of heterozygous subjects required AZA dose reduction because of leukopenia during the first year of immunosuppressive therapy, compared with 30% of homozygous wild-type patients (p = 0.04) [68]. In another study the frequency of leukopenia episodes registered in the same period was significantly higher in heterozygous patients (53.8%) compared with those with TPMT wildtype genotype (23.5%). AZA dose was significantly reduced in 69.3% of variant allele carriers and 40.8% of homozygous wild-type patients [64]. Kurzawski et al. demonstrated lower AZA dose, white blood cell count and platelet count at different time points in the course of treatment in carriers of variant TPMT alleles. Frequency of leukopenia episodes was also significantly elevated in that group of patients (41.2 vs 18.0% among wild-type homozygotes during the first year of treatment) [65]. In a prospective study of 36 patients, heterozygotes for a variant TPMT allele demonstrated a significant decrease in red blood cell count, hematocrit and hemoglobin over an initial 30‑day period of AZA treatment, confirmed in multivariate regression analysis [63]. Significance of TPMT genotype was also confirmed in a study by Song et al., who reported hematological toxicity in 50% of heterozygous patients compared with only 3.5% of wild-type patients and a significant decrease of red blood cell, hematocrit and hemoglobin levels during the first year of treatment after transplantation future science group


[69].

Vannaprasaht et al. observed significantly higher risk for AZA-induced myelosuppression in patients with heterozygous TPMT*1/*3C genotype compared with TPMT wild-type individuals [67]. Another genetic factor evaluated in thiopurine therapy was the ITPA polymorphism. ITPA catalyzes the pyrophosphohydrolysis of inosine triphosphate, and its deficiency in patients treated with AZA may lead to the accumulation of thio inosine metabolites with the potential for adverse metabolic effects [70]. ITPA activity is genetically determined by the presence of two common polymorphisms: 94C>A missense (rs1127354, Pro32Thr), coding for nonfunctional enzyme, and intronic IVS2+21A>C (rs7270101), associated with reduced ITPA activity. The ITPA polymorphism was primarily investigated in Crohn’s disease patients treated with AZA, giving contradictory results [71]. The study in renal transplant recipients demonstrated that ITPA genotype did not influence AZA therapy, including AZA dose, hematological parameters and leukopenia risk [65]. However, that study did not include ITPA-deficient homozygotes for the nonfunctional 94A allele. A study by Xiong et al. revealed that patients homozygous for the ITPA 94C>A variant allele were at significantly higher risk for developing AZA-related gastrointestinal toxicity and flu-like symptoms [72]. Among other polymorphic enzymes involved in the metabolism of thiopurines, a rs594445 SNP (His703Asn) in the MOCOS gene was associated with AZA dosage in renal transplant recipients: patients inheriting the rs594445 minor allele required significantly lower doses of AZA for efficient treatment compared with wild-type heteroz ygotes starting from the third month following the transplantation [73]. Glucocorticoids Pharmacokinetics

Associations between polymorphisms of CYP3A5 (6986A>G, CYP3A5*3), ABCB1 (1236C>T, 2677G>T/A and 3435C>T) and NR1I2 (PXR, rs6785049, 7635A>G) and the pharmacokinetics of PRE in renal transplant recipients medicated with PRE, TAC and MMF were studied by Miura et al. [74]. It was observed that the mean PRE AUC0–24 h and Cmax values in carriers of NR1I2 7635G allele were significantly lower than in patients carrying the 7635AA NR1I2 genotype. No significant differences in PRE pharmacokinetics in patients stratified for CYP3A5 and ABCB1 genotypes were found.


Review

Liver transplantation The most frequent immunosuppressant combinations in maintenance therapy after liver transplantation include TAC, MMF and corticosteroids; TAC and MMF; or TAC and corticosteroids [75]. TAC is replaced by CsA and MMF by AZA only in a minority of cases in some centers. It is also known that the liver is less likely to be rejected in comparison with other organs. Operational clinical tolerance, that is, stable normal graft function in the absence of maintenance immunosuppression, may be developed in approximately 20% of all liver transplant recipients, with an even higher rate reported by some centers [76]. The main reason for the differences between pharmacogenetic studies performed in liver transplantation compared with other patient groups is the fact that efficacy of liver drug metabolism is mainly determined by the genot ype of the donor, but also overlaps with the recipient’s genetic variation influencing intestinal transport and metabolism, which makes pharmacogenetic investigations more complex and interpretation of the results potentially challenging. Tacrolimus Pharmacokinetics

In liver transplant recipients, the same potential genetic determinants of TAC pharmaco kinetics were studied as in kidney transplantation: CYP3A4/5 and ABCB1 polymorphic loci (summarized in Table 2). A significant role of the CYP3A5*3 allele (SNP rs776746) as a determinant of TAC pharmacokinetics in populations other than Caucasian, that is, Chinese, was provided by Shi et al. [77]. The authors observed significantly higher daily doses of TAC in patients with CYP3A5*1/*1 (AA) genotype than in those with CYP3A5*3/*3 (GG) genotype (3.0 [2.0–4.0] versus 2.0 [1.5–2.5] mg/day, only recipients’ genotypes were evaluated). The TAC concentration:dose ratio of recipients with CYP3A5*1/*1 genotype was the lowest compared with carriers of CYP3A5*3/*3 or CYP3A5*1/*3 genotypes. In the same study no evidence of the role of ABCB1 3435C>T and 1236C>T poly morphisms in pharmacokinetics of TAC was revealed, and the CYP3A4*22 allele was not found in the studied population [77]. However, data from a Han Chinese population reported by Yu et al. indicate that the ABCB1 SNP at 3435C>T as well as a haplotype are predictive for TAC pharmacokinetics [78]. The authors found that plasma concentration of TAC divided by the daily dose per bodyweight ratios of liver www.futuremedicine.com

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Table 2. Relevant pharmacogenetic studies in liver, heart and lung transplantation. Drug

Gene

Polymorphism

Patients (n)

Comments


Ref.

TAC

ABCB1

ABCB1 3435C>T, 1236C>T and 2677G>T/A

62 HC

PK study; only recipient’s genotype

ABCB1 haplotype and 3435C>T with PK

[78]

TAC

CYP3A5 ABCB1

CYP3A5*3, CYP3A4*22 ABCB1 3435C>T and 1236C>T

216 HC

PK study; pharmacodynamic effects: early renal dysfunction; only recipient’s genotype; no association of ABCB1 polymorphism with PK and PD

CYP3A5*3 with PK and PD

[77]

TAC

CYP3A5

CYP3A5*3

204 JP

PK study; donors and recipients genotyped

CYP3A5*3 (of donor and recipient) with PK

[79]

TAC

CYP3A5

CYP3A5*3

60 JP

PK study; PD study: renal dysfunction

CYP3A5*3 with PK and PD

[80]

TAC

CYP3A5

CYP3A5*3

52 JP

Pharmacokinetic study; donors and recipients genotyped

CYP3A5*3 (of donor and recipient) with PK

[81]

TAC

CYP3A5 ABCB1

CYP3A5*3 ABCB1 3435C>T and 2677G>T

32 C

PK study

CYP3A5*3 with PK

[82]

2677G>T,A and 3435C>T

120 C

PD study: renal dysfunction measured by serum creatinine

ABCB1 2677TT with lower frequency of renal dysfunction

[85]

3435TT with lower concentration/dosage

[84]

Liver

CsA and ABCB1 TAC CsA

ABCB1

3435C>T

44 C

1-month observation

AZA

TPMT ITPA MTHFR

TPMT*2, *3A and *3C ITPA 94C>A IVS2 21A>C, MTHFR 677C>T and 1298A>C

65 C

Various observation time from transplantation; adverse effects evaluated; no association

TAC

CYP3A5

CYP3A5*3

15 C

PK study

CYP3A5*3 with PK

[88]

TAC

CYP3A5

CYP3A5*3 CYP3A4*1B ABCB1 3435C>T

65 C

PK study

CYP3A5*3 with PK

[89]

CsA

ABCB1

ABCB1 3435C>T, 2677G>T and 1236C>T

337 C

PK study; PD study: acute rejection and renal impairment

ABCB1 haplotype with PK

[90]

CsA

CYP3A4 CYP3A5 ABCB1

CYP3A4*1/*1B CYP3A5*1/*3 ABCB1 3435C>T

28 C


CsA

ABCB1 CYP3A4

ABCB1 3435C>T, 2677G>T, 1236C>T; CYP3A4 3 SNPs

14 Asian

PK study

EVR

CYP3A5

CYP3A5*1/*3

30 C


MMF

UGT1A1 UGT1A7 UGT1A8 UGT1A9 UGT2B7 ABCC2

Coding sequences and promoter sequencing

32 C

PK study; PD effects analyzed together with lung transplant patients (n = 36)

[86]

Heart

[92]

ABCB1 haplotype with PK

[88]

UGT2B7*2 with PK, other SNPs (ABCC2, UGT1A7 and UGT2B7) with PD

AZA: Azathioprine; C: Caucasian; CsA: Cyclosporin A; EVR: Everolimus; HC: Han Chinese; JP: Japanese; MMF: Mycophenolate mofetil; PD: Pharmacodynamics; PK: Pharmacokinetics; TAC: Tacrolimus.

1110


[91]


[93]


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Table 2. Relevant pharmacogenetic studies in liver, heart and lung transplantation (cont.). Drug

Gene

Polymorphism

Patients (n)

Comments


Ref.

Heart (cont.) MMF

IMPDH1 IMPHD2 ABCC2

IMPDH1 (5 SNPs) IMPDH2 3757T>C ABCC2 -24C>T

59 pediatric PD study: evaluation of MMF adverse events

ABCC2 -24T, IMPDH1 125A and rs2228075A alleles with gastrointestinal intolerance; IMPDH2 3757C with neutropenia

[94]

MMF

IMPDH1

5 SNPS

59 pediatric PD study: evaluation of MMF adverse events

IMPDH1 haplotype with gastrointestinal intolerance

[95]

AZA

TPMT

TPMT*2, *3A and *3C

30 C

PD study: evaluation of blood Variant allele carrier status indices and adverse effects with blood indices and during the first month of therapy agranulocytosis

[96]

TAC

CYP3A5 ABCB1

CYP3A5*1/*3 ABCB1 3435C>T and 2677G>T

125 C

PK study

CYP3A5*3 with PK

[97]

TAC

ABCB1

ABCB1 3435C>T, 2677G>T and 1236C>T

91 C

PK study

ABCB1 haplotypes with PK

[98]

TAC

ABCB1 CYP3A5

ABCB1 3435C>T and 2677G>T CYP3A5*3 and *6

125 C

PD study: treatment efficacy (acute rejection rate) studied

ABCB1 3435C>T with acute persistent rejection

[99]

MMF

UGT1A1 UGT1A7 UGT1A8 UGT1A9 UGT2B7 ABCC2

Coding sequences and promoter sequencing

36 C

PK study; pharmacodynamics UGT2B7*2 with PK, other effects analyzed together with SNPs (ABCC2, UGT1A7 heart transplant patients (n = 32) and UGT2B7) with PD

[93]

Lung

AZA: Azathioprine; C: Caucasian; CsA: Cyclosporin A; EVR: Everolimus; HC: Han Chinese; JP: Japanese; MMF: Mycophenolate mofetil; PD: Pharmacodynamics; PK: Pharmacokinetics; TAC: Tacrolimus.

transplant recipients varied significantly among different haplotype groups of the ABCB1 gene composed of 1236C>T (rs1128503), 2677G>A/T (rs2032582) and 3435C>T (rs1045642). Patients carrying the T–T haplotype, formed by 1236C>T and 2677G>A/T, and an additional T/T homo zygote at either position, required significantly higher dose of TAC. As for 3435C>T SNPs, patients with CC genotype were characterized by a significantly lower ratio of plasma drug concentration divided by daily dose of TAC per bodyweight. An interplay between recipient intestinal CYP3A5 polymorphism and a donor hepatic CYP3A5 polymorphism was investigated by Uesugi et al. in a Japanese population [79]. It was found that the TAC concentration:dose ratio was lower in recipients characterized by CYP3A5*1/*1 and *1/*3 genotype compared with CYP3A5 nonexpressors. Further analysis revealed that CYP3A5 expressors in both the graft liver and the native intestine produced the lowest concentration:dose ratio of the drug for future science group

35 days following living-donor liver transplantation. Patients with the intestinal CYP3A5*1 genotype tended to require a higher dose of TAC compared with subjects carrying the same hepatic CYP3A5 genotype. These findings were partially replicated by Fukudo et al., who found that carriers of the CYP3A5*1 allele in the native intestine, but not in the graft liver, showed a significantly higher recovery of oral clearance with time than patients with the intestinal CYP3A5*3/*3 genotype. The authors also reported that initial oral clearance immediately after liver transplantation was significantly affected by the intestinal ABCB1 mRNA level [80]. Muraki et al. reported that CYP3A5 expressors in both the liver and intestine showed significantly lower TAC concentration:dose ratios compared with CYP3A5*3/*3 nonexpressors in both organs [81]. Data from Caucasian populations are in keeping with the aforementioned observations in Asian patients. Provenzani et al. reported data from Italian liver recipients [82]. Significantly www.futuremedicine.com

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higher TAC dose requirements were found in liver recipients with transplanted livers from CYP3A5 expressors, compared with those homozygous for the *3 allele at 3 and 6 months after transplantation. Recipients possessing at least one copy of the CYP3A5*1 allele tended to be administered increased TAC doses. The ABCB1 2677G>T and 26 3435C>T polymorphisms did not affect TAC medication. In another study of patients who underwent primary liver transplantation and were observed for 3 months, the presence of the CYP3A5*1 allele seemed to be related to greater TAC requirements [83]. However, no proper statistical evaluation was performed. Pharmacodynamics

Shi et al. reported that Chinese patients medicated with TAC who were homozygous for the CYP3A5*3 allele were at significantly increased risk of early renal glomerular injury compared with TAC recipients carrying the CYP3A5*1 allele (CYP3A5 expressors), as evaluated by urine transferrin, without significant differences in urine microalbumin, urine immunoglobulin G or a1-microglobulin [77]. Similar observations were reported by Fukudo et al. The authors revealed that the cumulative incidence of renal dysfunction within 1 year after transplantation was significantly associated with the recipient’s but not donor’s CYP3A5 genotype, with higher risk in CYP3A5*3/*3 subjects in comparison with CYP3A5 expressors [80].

Azathioprine Pharmacodynamics

Data on pharmacogenetics of AZA in liver transplantation is very limited. In the only study, Breen et al. evaluated TPMT, ITPA and MTHFR genotypes in relation to adverse effects of AZA in liver graft recipients treated with AZA, TAC and PRE at various times (up to >10 years) from the transplantation [86]. No significant association between any of the studied genotypes and drug adverse effects was recorded. However, two patients who suffered nodular regenerative hyperplasia were both heterozygous for the TPMT*3A mutation. That observation might be coincidental, but another case of nodular regenerative hyperplasia in a patient heterozygous for both TPMT*3C and ITPA 94C>A polymorphism was independently reported [87].

Heart transplantation Tacrolimus Pharmacokinetics

In a preliminary report including Swiss liver transplant recipients, ABCB1 3435C>T was associated with CsA dose requirement 1 month following liver transplantation [84]. During the first 3 days following liver transplantation (when each patient received a similar CsA weight-adjusted dose), CsA concentrations were significantly higher in 3435TT patients, and 1 month after surgery (when dose was adjusted to maintain CsA concentration within therapeutic range) CsA weight-adjusted dose in 3435TT patients was approximately 50% lower than in 3435CC subjects.

Studies in patients following heart transplantation on TAC maintenance therapy reported by Kniepeiss et al. evaluated an association between CYP3A5 polymorphism, TAC dosing and its blood concentrations [88]. Average TAC dose was found to be significantly higher in patients who were expressors of CYP3A5 compared with CYP3A5*3/*3 nonexpressors, whereas TAC concentrations were similar in both study groups. Likewise, DiazMolina et al. [89], who reported higher weightadjusted doses of TAC in CYP3A5 expressors in comparison with CYP3A5*3/*3 homo zygotes in the 12th month following transplantation (0.014 vs 0.08 mg/kg/day), and significantly lower concentration:dose ratio (84 vs 206 ng/ml/mg/kg/day). No associations between CYP3A4 and ABCB1 polymorphisms and TAC dosing were found.

Pharmacodynamics

Cyclosporin

Hebert et al. studied two SNPs in the ABCB1 gene (2677G>T,A and 3435C>T) among liver transplant recipients of non-Hispanic Caucasian origin, describing reduced frequency of renal dysfunction (measured by serum creatinine level) in patients with an ABCB1 2677TT genotype, as

Pharmacokinetics

Cyclosporin A Pharmacokinetics

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compared with those with a 2677GG genotype, as well as compared with all the other genotypes. The majority of patients were administered CsA, while the remainder received TAC, which is also a P-gp substrate [85].


Significant dose- and weight-adjusted CsA trough concentration associations with ABCB1 haplotypes in heart transplant patients were defined by Taegtmeyer et al. [90], with 1236CC/2677GG/3435CC subjects future science group


having significantly higher concentrations than 1236TT/2677TT/3435TT individuals at years 1 and 5 following transplantation. The above observations were supported by Chowbay et al. in patients of Asian origin [91]. The authors demonstrated in stable heart transplant patients that CsA AUC0–4 h, AUC0–12 h and Cmax were the highest in patients homozygous for ABCB1 T–T–T haplotype in comparison with C–G–C haplotype carriers. Any associations of CYP3A4*1B and *3, CYP3A5*3 and *6, and ABCB1 3435C>T polymorphisms and pharmacokinetic parameters in kidney and heart transplant patients were observed by Hesselink et al. [92]. Pharmacodynamics

There was no difference in the incidence of acute rejection, steroid weaning or renal impairment between ABCB1 3435C>T, 2677G>T and 1236C>T genotypes or haplotypes in heart transplant patients within a 5‑year observational period after transplantation [90]. Everolimus Pharmacokinetics

Kniepeiss et al. did not reveal any association between everolimus doses and blood levels of the drug and CYP3A5 polymorphism in heart transplant patients [88].

of subjects, the authors observed some positive associations (ABCC2 -24T, IMPDH1 125A and rs2228075A alleles with MMF gastrointestinal intolerance; IMPDH2 3757C allele with more frequent neutropenia requiring dose holding). The effect of IMPDH1 polymorphism was further investigated in their subsequent paper, in which haplot ypes derived from five IMPDH1 SNPs were analyzed in relation to gastrointestinal intolerance in the same group of patients [95]. The authors identified a common haplotype, containing alleles previously associated with gastrointestinal intolerance (125A and rs2228075A), as being of predictive value comparable to individual SNP analysis. However, those observations would surely require further confirmation of their clinical significance in a larger, multicenter patient cohort. Azathioprine Pharmacodynamics

In a study of 30 consecutive heart transplant recipients, a more prominent drop in leukocyte count in TPMT variant allele carriers was observed compared with wild-type patients evaluated at the third week following transplantation [96]. Presence of TPMT variants was also associated with a greater likelihood of agranulocytosis.

Mycophenolate mofetil

Lung transplantation Tacrolimus

Pharmacokinetics

Pharmacokinetics

Ting et al. sequenced full coding sequences of the UGT1A1, UGT1A7, UGT1A8, UGT1A9, UGT2B7 and ABCC2 genes, as well as the 3´-UTR of UGT1 in thoracic transplant recipients (36 lung and 32 heart) at steady state during oral MMF [93]. In both groups MPA pharmacok inetics (AcMPAG AUC 0–12 h and the AcMPAG:MPA ratio) were significantly influenced by the presence of the UGT2B7*2 allele (rs7439366). Contrary to some studies in renal transplant recipients, no role was observed for either the UGT1A9 or the ABCC2 polymorphism.

A significant difference in TAC dose:concentration ratio was found between lung transplant patients characterized by CYP3A5 expressor status versus nonexpressor subjects within the first year following transplantation. The ABCB1 2677G>T and 3435C>T polymorphisms had only minimal effects on the dose:concentration ratio within the first 3 months following transplantation [97]. The impact of ABCB1 haplotypes derived from three polymorphic loci (1236C>T, 2677G>T and 3435C>T) on TAC dose:concentration ratio within the first year after transplantation was also evaluated by Wang et al. [98]. The study was limited to lung transplant patients who were CYP3A5 nonexpressors. TAC concentration:dose ratio in the C–G–C/C–G–C patients was significantly lower than in C–G–C/T–T–T and T–T–T/T–T–T carriers throughout the first posttransplant year.

Pharmacodynamics

The UGT2B7*2 allele and concomitant treatment with CsA increased rejection risk in all thoracic (heart and lung) transplant recipients, as evaluated by means of multivariate analysis [93]. Ohmann et al. investigated several SNPs within the IMPDH1, IMPDH2 and ABCC2 genes in relation to MMF adverse events in 59 pediatric heart recipients [94]. Despite the small number future science group

Review

Pharmacodynamics

Zheng et al. described significant associations between ABCB1 3435C>T genotype and acute www.futuremedicine.com

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Table 3. Genetic polymorphisms most extensively investigated in relation to immunosuppressive therapy in solid organ transplantation. Gene

Polymorphism

Consequences

Drugs affected

CYP3A4

rs35599367, C>T (*22 allele) SNP in intron 6, recently associated with lower mRNA expression and hepatic enzyme activity

TAC, CsA and SIR

CYP3A4

rs2740574, A>G (*1B allele) Promoter variant associated with higher gene expression compared with wild-type

TAC, CsA and SIR

CYP3A5

rs776746, G>A (*3 allele)

Splicing defect (complete lack of activity)

TAC, CsA and SIR

ABCB1

rs1045642, C>T (3435C>T)

Silent polymorphism linked to decreased P-gp activity, probably due to altered protein structure in the presence of a rare codon encoded by the 3435T allele

TAC, CsA and SIR

UGT1A9

rs6714486 (-275T>A) and rs17868320 (2152C>T)

Promoter polymorphisms correlated with higher hepatic expression of UGT1A9 and increased in vitro glucuronidation activity

MMF

IMPDH1

rs2278293 (106G>A) and rs2278294 (125G>A)

Intronic SNPs, clinically associated with reduced risk of biopsy-proven acute rejection, mechanism not explained

MMF

ABCC2

rs717620 (-24C>T)

Lower expression measured at protein level

MMF

TPMT

rs1800460 (460G>A), rs1142345 (719A>G) and rs1800462 (238G>C)

Missense SNPs, altering amino acid sequence (A154T, Y240C and A80P), associated with lack of activity of encoded protein, most frequent TPMT variant alleles are *3A (460A+719G), *3C (719G) and *2 (238C)

AZA

AZA: Azathioprine; CsA: Cyclosporin A; MMF: Mycophenolate mofetil; SIR: Sirolimus; TAC: Tacrolimus.

persistent rejection, as 72% of patients with the C allele had acute persistent rejection in comparison with 52% for TT patients [99]. MMF Pharmacokinetics

The aforementioned study by Ting et al. [93] revealed an association between MPA pharmacokinetics (AcMPAG AUC 0–12 h and the AcMPAG:MPA ratio) and the presence of the UGT2B7 *2 allele (rs7439366), both in lung and heart transplant patients.

Conclusion & future perspective A vast majority of the abovementioned studies were performed in renal transplant recipients. As a result, conclusions related to pharmaco genetics and immunosuppressive treatment are the most informative in that group of patients. This is mainly because kidney transplantation is becoming routine procedure, applied in many centers all over the world. However, the same drugs are administered to patients after other organ transplantations, and observations from kidney graft recipients can usually be extrapolated and interpreted in a wider context. Variations of many genes involved in the metabolism and transport of immunosuppressive drugs used in transplantation have been investigated to date (Table 3). For some polymorphisms, including CYP3A5 in TAC administration, UGT1A9 in patients medicated with 1114


MMF or TPMT in the case of regimens including AZA, effects on the pharmacokinetics and pharmacodynamics of the respective drugs are well documented, and should be considered as important variables influencing the treatment. A recently described polymorphism within the CYP3A4 gene (the CYP3A4*22 allele), encoding for one of the most important enzymes in xenobiotic metabolism, should also be treated as a functional variant, determining (in part) individual metabolic efficiency in relation to CYP3A4-metabolized drugs. Additional studies are required to finally confirm or exclude the impact of other genetic variants that have been studied, and have resulted in contradictory conclusions, possibly owing to differences in study design, ethnicity, drug dosage or other variables yet to be defined. Among them, future studies on ABCB1 in calcineurin inhibitor transport, ABCC2 in MPA metabolite (AcMPAG) transport and IMPDH1/2 as an MPA target should be observed with attention. Apart from clinical conclusions, all the pharmacogenetic studies in transplantology performed to date have provided much data that helped to define the contribution of respective enzymes and transporters to the metabolism and pharmacokinetics of immunosuppressive drugs. However, possibilities for the clinical application of genetic testing are rather limited, as the monitoring of drug level is much more informative in most cases, providing current future science group


status information and also including nongenetic environmental variables. Acute toxicity of AZA in TPMT-deficient patients is unquestionable, but complete lack of enzyme activity is relatively rare and AZA use is becoming more restricted nowadays; it is no longer the first-choice medication in transplant recipients. Conversely, it seems reasonable to utilize genetic tests in individual subjects, especially in the case of adverse effects or treatment inefficacy, to obtain a more complete picture of a patient. In that circumstance, pharmacogenomic tools could be useful for the individualization of immunosuppressive regimens,

Review

leading to dose modification or the replacement of a toxic or inefficient drug with a more suitable alternative. Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.

Executive summary Kidney transplantation As kidney transplantation has become a routine procedure, pharmacogenetic studies have become numerous, performed in relatively large patients’ groups, and are therefore more conclusive. The impact of several polymorphisms is well-documented: CYP3A4/CYP3A5 on tacrolimus pharmacokinetics, UGT1A9 in the case of mycophenolate mofetil (mycophenolic acid [MPA]) treatment and TPMT in patients medicated with azathioprine. Significant influence on pharmacokinetic parameters does not necessarily translate into therapy outcome in all cases. Several other gene variants were investigated (i.e., ABCB1 and SLCO1B3 in calcineurin inhibitors transport, other UGTs in MPA metabolism, ABCC2 in MPA metabolite [AcMPAG] transport, and IMPDH1/2 as an MPA target), but results are inconsistent or need to be verified in further studies. Liver, heart & lung transplantation Most of pharmacogenetic studies in liver/heart/lung transplant recipients were performed in relatively small groups of patients, often lacking sufficient power for evaluation of therapeutic end points in relation to genetic polymorphism. Immunosuppressive treatment of patients after liver/heart/lung transplantation consists of the same drugs that are administered to renal transplant recipients; hence, most conclusive observations from kidney graft recipients can usually be generalized and interpreted in a wider context. Associations between genetic variants of CYP3A4/CYP3A5, ABCB1, ABCC2 and UGTs were among the most extensively evaluated. A specific issue in liver transplantation is the fact that transplanted liver drug metabolism overlaps with the recipient’s genetic constitution influencing intestinal transport and metabolism, which makes pharmacogenetic investigations more complex, and interpretation of the results potentially more challenging. Conclusion & future perspective In the case of some polymorphisms, namely CYP3A5 in tacrolimus administration, UGT1A9 in patients medicated with mycophenolate mofetil or TPMT in regimens including azathioprine, the role of genetic factors in pharmacokinetics and pharmacodynamics of the respective drugs is well documented, and should be considered as an important variable influencing therapeutic approach. Other genetic factors that seem to be of relevance to pharmacotherapy include the recently described polymorphism within the CYP3A4 gene (CYP3A4*22 allele), and variation of IMPDH1/2, the MPA target. Clinical application of genetic testing is limited, as therapeutic drug monitoring is much more informative in most cases. It seems reasonable to utilize genetic tests as an additional diagnostic tool in individual subjects, especially in the case of adverse effects or treatment inefficacy, to obtain a more complete picture of a patient and introduce necessary treatment modifications.

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