Polymorphisms affecting immunosuppressive drug response

Supervised by : Dr.Malik Zuhluf Presented by: Hayder Al Saraj Omar Al Rais

Introduction

THE IMMUNE RESPONSE• The immune system evolved to discriminate self from non self

antigens.• Multicellular organisms were faced with the problem of destroying

infectious invaders (microbes) or dysregulated self (tumors) while leaving normal cells intact.

• These organisms responded by developing a receptor-mediated sensing and effector mechanisms broadly described as natural and adaptive immunity

• natural, immunity is primitive, does not require priming, and is of relatively low affinity, but is broadly reactive

• Adaptive, or learned, immunity is antigen-specific, depends upon antigen exposure or priming, and can be of very high affinity

.• The two arms of immunity work closely together, with the

primitive immune system being most active early in an immune response and adaptive immunity becoming progressively dominant over time

• The major effectors of primitive immunity are complement, granulocytes, monocytes/macrophages, natural killer cells, mast cells, and basophils

• The major effectors of adaptive immunity are B and T lymphocytes. B lymphocytes make antibodies; T lymphocytes function as helper, cytolytic, and regulatory (suppressor) cells,These cells are important in the normal immune response to infection and tumors, but also mediate transplant rejection and autoimmunity

Immunosuppressive drugs • Immunosuppressive drugs are used to dampen the immune

response in organ transplantation and autoimmune disease. In transplantation, the major classes of immunosuppressive drugs used today are:

• Glucocorticoids.• calcineurin inhibitors. • antiproliferative/antimetabolic agents.• biologics (antibodies).These drugs have met with a high degree of clinical success in treating conditions such as acute immune rejection of organ transplants and severe autoimmune diseases. However, such therapies require lifelong use and nonspecifically suppress the entire immune system, exposing patients to considerably higher risks of infection and cancer. The calcineurin inhibitors and glucocorticoids, in particular, are nephrotoxic and diabetogenic, respectively, thus restricting their usefulness in a variety of clinical settings.

General Approach to Organ Transplantation Therapy

Organ transplant therapy is organized around five general principles:

• 1-careful patient preparation and selection of the best available ABO blood type-compatible HLA match for organ donation.

• 2-a multitiered approach to immunosuppressive drug therapy, similar to that in cancer chemotherapy, is employed. Several agents are used simultaneously, each of which is directed at a different molecular target within the allograft response Synergistic effects permit use of the various agents at relatively low doses, thereby limiting specific toxicities while maximizing the immunosuppressive effect

• 3-greater immunosuppression is required to gain early engraftment and/or to treat established rejection than to maintain long-term immunosuppression

• 4-careful investigation of each episode of transplant dysfunction is required, including evaluation for rejection, drug toxicity, and infection, keeping in mind that these various problems can and often do coexist

• 5-a drug should be reduced or withdrawn if its toxicity exceeds its benefit.

. Sites of Action of Selected Immunosuppressive Agents on T-Cell Activation

DRUG SITE OF ACTION

Glucocorticoids Glucocorticoid response elements in DNA (regulate gene transcription)

Muromonab-CD3 T-cell receptor complex (blocks antigen recognition)

Cyclosporine Calcineurin (inhibits phosphatase activity)

Tacrolimus Calcineurin (inhibits phosphatase activity)

Azathioprine Deoxyribonucleic acid (false nucleotide incorporation)

Mycophenolate Mofetil Inosine monophosphate dehydrogenase (inhibits activity)

Daclizumab, Basiliximab IL-2 receptor (block IL-2-mediated T-cell activation)

Sirolimus Protein kinase involved in cell-cycle progression (mTOR) (inhibits activity)

.

Induction Therapy• induction can be divided into 2 groups: the depleting agents

and the immune modulators. • The depleting agents consist of lymphocyte immune globulin,

antithymocyte globulin, and muromonab-CD3 mAb (the latter also produces immune modulation); their efficacy derives from their ability to deplete the recipient's CD3-positive cells at the time of transplantation and antigen presentation.

• The second group of biologic agents, the anti-IL-2R mAbs, do not deplete T lymphocytes, but rather block IL-2-mediated T-cell activation by binding to the a chain of IL-2R.

Maintenance Immunotherapy• Therapy typically involves a calcineurin inhibitor,

glucocorticoids, and mycophenolate mofetil (a purine metabolism inhibitor)

Therapy for Established Rejection• Although low doses of prednisone, calcineurin inhibitors,

purine metabolism inhibitors, or sirolimus are effective in preventing acute cellular rejection, they are less effective in blocking activated T lymphocytes, and thus are not very effective against established, acute rejection or for the total prevention of chronic rejection

• Therefore, treatment of established rejection requires the use of agents directed against activated T cells. These include glucocorticoids in high doses (pulse therapy), polyclonal antilymphocyte antibodies, or muromonab-CD3 mAb.

Polymorphisms affecting immunosuppressive drug response

• The polymorphisms that hold the most potential for use in a drug selection algorithm are in genes CYP3A5, ABCB1, IMPDH1 and IMPDH2, TPMT, cytokines and growth factors.

ABCB1• P-glycoprotein 1 (permeability glycoprotein) also known as

multidrug resistance protein 1 (MDR1) or ATP-binding cassette sub-family B member 1 (ABCB1)

• ABCB1 is an ATP-dependent efflux pump with broad substrate specificity. It likely evolved as a defense mechanism against harmful substances.

• ABCB1 transports various substrates across the cell membrane including immunosuppressants (calcineurin inhibitors ).

Polymorphism in ABCB1 • The pharmacokinetic characteristics of calcineurin inhibitors

including bioavailability have shown large inter- and intra-individual variation.

• There are clear ethnic differences in dose requirements for those drugs. It has been noted that African American and non-white South American transplant recipients

require a higher dose of tacrolimus to achieve target blood concentrations than Caucasians or Asians. So is the case for CsA.

• Significant variability presents even within ethnic groups. The bioavailability of orally administered tacrolimus varies from 4% to 89% among individuals.

ABCB1 genotype vs. phenotype

• MDR1 variant homozygotes (3435CC )=wild type= higher expression of ABCB1

• MDR1 variant heterozygotes (3435CT )= intermediate expression of ABCB1

• MDR1 variant homozygotes (3435TT )=mutant type=lower expression of ABCB1

Cyclosporine:• studies showed that patients with wild genotype (3435CC) or

heterozygous mutant genotype (3435CT) did not require dose adjustment when compared their concentration / dose ratio , but those were homozygous for the mutation (3435 TT) required lower doses , because at the regular recommended dose they were exposed to much higher adverse effects.

• These differences in the immunosuppressant (especially cyclosporine )pharmacokinetics were noticed between day 1 to 3 after transplantation .

• with ongoing treatment and after the target plasma level was reached no further drug adjustment were required.

Tacrolimus• recipients with C/C genotype at C3435T would require a little

higher dose of tacrolimus compared to those with C/T and T/T genotypes

Graft failure• Studies found that the ABCB1 1236T, 2677T, and 3435T variant

alleles and the corresponding (1236T-2677T-3435T) variant haplotype in graft donors were associated with a higher risk of graft loss in the recipient.

Conflicting studies • conflicting results have been reported. Some studies found no

significant differences in CsA doses needed to maintain similar CsA trough concentrations in 124 stable Caucasian renal transplant recipients with different MDR1 C3435T genotypes

• . More over no association between 10 SNPs of the MDR1 gene and the tacrolimus concentration/dose ratio during the first postoperative days after liver transplantation.

• Others found no evidence supporting a role of MDR1 C3435T in tacrolimus dose or CsA dose-adjustment.

• These conflicting results might be explained by that the side-effects of immunosuppressive treatment frequently require the addition of concomitant medications (antihypertensive, antilipemic and uricosuric) that might interact with CsA ,tac,absorption, metabolism and excretion.

ABCB1 haplotype• There may be a linkage disequilibrium with SNPs in the MDR1

gene.

• SNPs in exon 21 and exon 12 have attracted more attention from researchers.

• the co-segregation of exon 26 3435T with the T allele of the nonsynonymous exon 21 G2677T SNP, and with the T allele of the synonymous exon 12 C1236T SNP. This disequilibrium led to a haplotype analysis of the MDR1 gene, to identify the links between the genomic variations represented by each haplotype on one hand and by altered MDR1 function on the other.

CYP3A5 genotype vs. phenotype

• CYP3A5*1= wild allele• CYP3A5*3= mutant allele

• CYP3A5*1/*1 genotype=functional expressers• CYP3A5*1/*3 genotype=intermediate expressers • CYP3A5*3/*3 genotype=non-functional expressers

SNPs affecting Cytochrome 3A enzymes :

• Drugs affected are calcinurein inhibitors (CNI) like cyclosporine(CsA), tacrolimus(Tac).

• 1)Cyclosporine

• The cytochrome P450 (CYP) 3A subfamily is responsible for the metabolism of approximately 50% of drugs and endogenous substances, such as the calcineurin inhibitor. The CYP3A subfamily consists of various isozymes, including CYP3A4, CYP3A5, CYP3A7 and CYP3A43

• Intestinal P-gp levels and hepatic CYP3A4 and CYP3A5 activities are determinants for up to 75% of the CsA variation after oral administration,

• CYP3A4 also demonstrates interindividual variation in a metabolic capacity However, functional SNPs are few in the CYP3A4 gene and most studies found no association with pharmacokinetics of CsA, and controversial associations with Tac.

• the CYP3A5*3 polymorphism was found to cause alternative splicing and protein truncation, resulting in the absence of a functional CYP3A5 gene in hepatic and intestinal tissue.

• The CYP3A5*1 allele is necessary for the production of a fully catalytic CYP3A5 protein and also influences the ratio of the expression level of CYP3A4 and CYP3A5 proteins.

• CYP3A5 may constitute up to 50% of total CYP3A protein in the liver and small intestine, which carry at least one CYP3A5*1 allele.

• (Saeki et al. ) reported that CYP3A5*3 an SNP 6896 A > G (A6896G), is a predominantly defective allele and total CYP3A activity is correlated with CYP3A5 genotype.

• The findings show that the median cyclosporine dose-adjusted C0 was significantly lower in subjects carrying the CYP3A5*1/*1 genotype than in patients with the CYP3A5*3/*3 genotype.

• thus, patients with the CYP3A5*1/*1 genotype could require a higher dose of cyclosporine to achieve target blood concentrations than those with the CYP3A5*3/*3 genotype immediately after renal transplantation.

2) Tacrolimus:• The active barrier to the absorption of tacrolimus in the gut is

composed of the metabolic enzymes CYP P450 3A4 and 3A5 and the drug efflux pump P-gp.

• Individuals are either functional CYP3A5 expressers or nonexpressers based on a SNP in the 5´ end of the gene.

• Individuals with at least one wild-type *1 allele are expressers and individuals homozygous for the *3 mutation are nonexpressers,

• CYP3A5 expressers take longer to achieve target blood tacrolimus concentrations after transplantation and experience episodes of acute rejection earlier than nonexpressers

• It is well established that the CYP3A5 A6986G (*3) SNP influences the pharmacokinetics of Tac in renal recipients,

• Almost all studies have reported that recipients with the CYP3A5*3/*3 genotype (nonexpressers) exhibited higher dose-adjusted Tac exposure (C0/dose, C2/dose or AUC/dose), and a lower dose requirement compared with the CYP3A5*1/*1 or *1/*3 carriers (expressers)

• In addition, the CYP3A5*3/*3 homozygotes required a lower dose of CsA to reach target levels compared with the CYP3A5*1/*1 or *1/*3 genotypes

• CYP3A5*1/*1 or CYP3A5*1/*3 genotype group required on average a 80% higher tacrolimus dose and showed a greater tendency toward CNI toxicity after transplantation than the CYP3A5*3/*3 genotype group

SNPs affecting IMPDH enzymes• Mycophenolic acid (MPA)• IMPDH catalyses the rate- limiting step in de novo guanosine

monophosphate synthesis. Because lymphocytes rely on de novo synthesis to provide guanine nucleotides while most other cells use the salvage pathway, inhibition of IMPDH results in immunosuppression

• Two different IMPDH isoforms are encoded by IMPDH1 and IMPDH2( IMPDH2 expression is inducible and, thus, it is more highly expressed in many tissues than the constitutively expressed IMPDH1)

• Genetic polymorphisms in both IMPDH1 and IMPDH2 have been shown to influence outcome in transplantation patients treated with MPA, suggesting that response to MPA might be influenced, in part, by inheritance.

• Examples include:

• 1) a nonsynonymous single nucleotide polymorphism (SNP) in IMPDH2 that was shown to reduce IMPDH activity ,

• 2) an IMPDH2 intron SNP that increased IMPDH activity in MPA-treated transplantation patients

• 3) an IMPDH2 intron SNP that reduced the antiproliferative effects of MPA on lymphocytes

• 4) two IMPDH1 intron SNPs associated with biopsy-proven acute transplant rejection ,

• Gene resequencing, performed with 288 DNA samples from three ethnic groups, resulted in the identification of 73 SNPs in IMPDH1 (59 novel) and 25 SNPs in IMPDH2 (24 novel). We then pursued the potential functional implications of nonsynonymous SNPs as well as SNPs that might alter transcription.

• Four nonsynonymous SNPs were identified in IMPDH1 and one in IMPDH2,

• To date, there are no data on the use of these genotypes in guiding mycophenolate dosing.

SNPS affecting thiopurine-s-methyl transferase (TPMT) enzyme

• Thiopurines, including 6-thioguanine, azathioprine, and 6-mercaptopurine, are currently indicated for treatment of ALL, as well as immunosuppressants for autoimmune disorders and after organ transplantation.

• These drugs are metabolyzed by TPMT, a cytosolic enzyme

that methylates the aromatic ring of these drugs

• Polymorphisms at the TPMT coding gene determine enzyme activity and consequently, development of thiopurine-related toxicity

• clinical studies have shown that patients with exceptionally low TPMT activity (approximately 1 in 300 individuals) are at high risk for developing severe and potentially fatal toxicity

• It has been reported that the three most frequent variant alleles are TPMT*2, -*3A, and -*3C, and which are responsible for[95% of cases with low enzyme activity ,

• These polymorphisms are associated with a higher risk of myelosuppression, particularly in homozygous patients, who have very low TPMT-enzyme activity and who develop severe and potentially fatal toxicity

• Frequency of TPMT polymorphisms is related with ethnicity.

• 1) TPMT*3A is the most common genotype in Caucasian, and is characterized by a G to A transition at position 460 (G460A), with a substitution of alanine by tyrosine at amino acid 154 (A154Y) and a transition of A to G at nucleotide 719(A719G), resulting in a change of tyrosine into cysteine at position 240 (Y240C).

• 2)Isolated substitution at exon 7 (position 460), which defines the *3B polymorphism, has been reported with a lower frequency (approximately 2%) in European population

• 3)substitution of G by C at position 238 (G238C) changes the amino acid alanine into proline (Ala80Pro) and defines the *2 allele.

• 4)In contrast, the *3C polymorphism defined by the isolated transition of A to G at nucleotide 719 (A719G) and resulting in a change of tyrosine into cysteine at position 240 has been documented, predominantly in Asia, Africa, and Latin America within a range of 2–8%

• The determination of TPMT gene polymorphisms have described three types of methylator populations:

• 1)TPMT *1 (wt functional), define the normal–high methylator population,(normal/high methylators), (TPMT*1/*1)

• 2) heterozygous functional polymorphisms are associated with an increased risk of thiopurines toxicity, (intermediate methylators)

• TPMT*1/*2, or • TPMT*1/*3A, or • TPMT*1/*3C or • TPMT*1/*4• 3)individuals with homozygous non- functional polymorphisms are

in risk of fatal toxicity.(deficient methylators), have 2 allele variants of (*2, *3A, *3B, *3C, or 4)

• The Clinical Pharmacogenomics Implementation Consortium (CPIC) guidelines:

• Heterozygous TPMT genotype (intermediate activity): In patients who possess a single TPMT functional (*1) and nonfunctional allele (*2, *3A, *3B, *3C, or *4), the initial dose of AZA or 6-MP should be reduced by 30- 70%,

• Homozygous TPMT genotype (variant mutant, low, or deficient activity): Reduce the initial dose of AZA, 6-MP or TG by 10-fold and extend the dosing frequency from daily to three times weekly, or select an alternative drug,

• the US Food and Drug Administration (FDA) and prescribing information for AZA and 6-MP recommend either TPMT genotyping or phenotyping prior to initiating therapy to help identify patients who are at an increased risk of developing toxicity

Cytokines and growth factors • Those that got most of the attention in studies were 1 Interleukin-10.2 Interferon-gamma.3 Transforming Growth Factor-beta.4 Tumor Necrosis Factor-alpha Gene .

.• Most of the studies showed polymorphisms in the genes that

regulate IL-10, IFN-γ, TGF-β, and TNF-α cytokines do not play a major role in renal allograft survival, and other potential factors in this regard should be considered.

• Keeping in mind That IFNG +874 A/T genotype showed a significantly higher frequency among kidney recipients of the rejection group than the control groups.

The end