1. GENOMICS AND PROTEOMICS BY RAJASHEKAR MANTHRI M.PHARMACY IISEMESTER DEPARTMENT OF PHARMACEUTICS VAAGDEVI COLLEGE OF PHARMACY

3. Genomics and Proteomics

4. Bioinformatics

5. PHARMACOGENOMICS

6. SNPs

7. Personalized medicine

8. Benefits of pharmacogenomics

9. HUMAN GENOME PROJECT

10. Birth and activity of HGP

11. Mapping of HG

12. Approaches for genome sequence

13. GENETIC POLYMORPHISMS INFLUENCING DRUG DISPOSITION

14. Metabolizing enzymes

15. Transporters

16. GENETIC POLYMORPHISMS IN DRUG TARGETS

17. β2 – receptors

21. Proteome plays a key role in intracellular signalling pathways of the immune system and intercellular metabolism as being the interface between the cell and the environment.

24. It broadly involves the computational tools and methods used to manage, analyze and manipulate volumes and volumes of biological data.

27. SWISS – PROT (www.expasy.ch/sport): it provides the description of the structure of a protein.

28. PIR (Protein Information Resource) is a database provided by the National Biomedical Research Foundation (NBRF) in USA

29. PROSITE: provides information on protein families and domains.

30. Nucleotide sequence database:

33. Finding of sites that can be cut by restriction enzymes.

34. Prediction of functional gene products.

35. Prediction of 3 – dimensional structure of proteins.

36. Designing of drugs for medical treatment.

38. Pharmacogenomic studies explain the inherited nature of these differences in drug disposition and effects.

39. It is the branch of pharmacology which deals with studying the influence of genetic variation on drug response in patients.

40. Study of pharmacogenomics helps to study how different drugs interact with multiple genes and biological molecules they encode.

43. Insertions

44. Deletions

45. Short Tandem Repeats (STRs)

49. SNPs represent as much as 90% of all human genetic variation. More than 1.4 million single nucleotide polymorphisms were identified in the initial sequencing of the human genome.

51. SNPs are believed to underlie susceptibility to such common diseases as cancer, diabetes, cardio vascular and inflammatory diseases and to contribute to the traits that make individuals unique.

53. Personalized medicineis the use of detailed information about a patient’s genotype or level of gene expression and a patient’s clinical data in order to select a medication, therapy or preventive measure that is particularly suited to that patient at the time of administration. Certain drugs work differently in different individuals. In some individuals they produce therapeutic effects and in some other individuals, they produce severe toxic effects at the same dose. Inter individual variability can be seen in drug absorption, distribution, elimination and excretion. This inter individual variation in drug efficacy and toxicity is related to inherited differences in the genes that encode drug metabolizing enzymes drug transporters or drug receptors.

56. The ADRs are the major cause on non - compliance and failure of treatment, particularly for chronic pathologies.

57. Many of these deaths can be avoided, if the physician has prior knowledge of patient’s genetic profile of drug metabolizing enzymes and receptors, which determine drug responses.

58. In the near future, the present prescription model of “one dose for all” (based on age, sex, gender, weight, drug interactions) will be replaced by “individualized prescription” (i.e. individualized / personalized therapy) – that is the right drug in right dose for right person.

60. Facilitate drug approvals: Pharmaceutical companies can discover new drugs by targeting genomes. Risk of adverse effects in clinical trials can be minimized by targeting only those who will respond to drug. Accuracy in dosing: By knowing a person’s genetic makeup, it becomes easier to determine, how the body processes the medicine. Thus instead of using conventional method of calculating dosage based on age and weight, dosage can be calculated based on person’s genetic profile. This reduces chances of overdose and under dose thereby optimizing drug therapy

61. The Human Genome Project Until the early 1970’s, DNA was the most difficult cellular molecule for biochemists to analyze. DNA is now the easiest molecule to analyze – we can now isolate a specific region of the genome, produce a virtually unlimited number of copies of it, and determine its nucleotide sequence overnight.

62. Aims of the project: To identify the approximate 100,000 genes in the human DNA. Determine the sequences of the 3 billion bases that make up human DNA. Store this information in databases. Develop tools for data analysis. Address the ethical, legal, and social issues that arise from genome research.

64. The primary objective of HGP was to determine the nucleotide sequence of the entire human nuclear genome.

65. First director of HGP was James Watson (who elucidated DNA structure).

66. In 1997, united states established the national human genome research institute (NHGRI).

68. Total expenditure of $ 3 billion and a time period of 10 to 15 years for the completion of HGP was expected and it was completed in June 2000.

69. A second human genome project was setup by a private company —Celera genomics of Maryland USA in 1998. This team was led by Craig Venter. ANNOUNCEMENT OF THE DRAFT SEQUENCE OF HUMAN GENOME: Working drafts of human genome sequence was announced in the year of 2000 on June 26th by the leaders of the two HGPs, Francis Collins and Venter.

70. Mapping of the Human Genome:- The most important objective of human genome project was to construct a series of maps for each chromosomes. Cytogenetic map: This is a map of the chromosome in which the active genes respond to a chemical dye and display themselves as bands on the chromosomes. Gene linkage map: A chromosome map in which the active genes are identified by locating closely associated marker genes. The most commonly used DNA markers are restriction fragment length polymorphism (RFLP), variable tandem repeats(VNTRs) and short tandem repeats (STRs). VNTRs are also called as minisatellites while STRs are microsatellites.

71. Restriction fragment map: This consists of the random DNA fragments that have been sequenced. Physical map: This is the ultimate map of the chromosome with highest resolution base sequence. Cytogenetic map Gene linkage map Gene Gene Restriction fragment map Restriction fragments Physical map Base sequence

74. Human genome is composed of 3.2 billion base pairs.

75. Approximately 1.1 to 1.5% of the genome codes for proteins.

76. Genes and DNA sequences associated with many diseases such as breast cancer, muscle diseases, deafness and blindness have been identified.

77. Between the humans the DNA differs only by 0.2% or one in 500 bases.

79. If a typist types at the rate of 60 words per minute for 8 hours a day, he would take around 50 years to type human genome.

81. Earlier detection of predisposition to disease

82. Gene therapy and control systems for drugs

84. Exonerate wrongly accused persons

85. Identify crime victims

89. Aldehyde dehydrogenase

94. Antipsychotics,

95. Selective serotonin reuptake inhibitors,

96. Anti hypertensive agents e.g. debrisoquine,

102. Metabolism of CYP2D6 Drug Substrates in Relation to Genotypes Nortriptyline was one of the first clinically important drug metabolized by CYP2D6. Nortriptyline was given as a single oral dose to 21 healthy subjects with different genotypes. The plasma concentrations of the parent drug were extremely low in one subject with 13 CYP2D6 genes. The plasma concentrations of the formed metabolite 10-hydroxynortriptyline show the opposite pattern, i.e., highest concentrations in the subject with 13 genes and lowest in the PMs. This study clearly shows the impact of the CYP2D6*4 allele as well as the duplication/amplification of the CYP2D6*2 gene on the metabolism of nortriptyline.

104. CYP4502C sub family In humans the CYP2C subfamily of CYP450 catalyzes roughly 20% of the CYP mediated metabolism of drugs . The first polymorphisms discovered in the CYP2C subfamily was a well described deficiency in the ability to metabolize the anticonvulsant drug mephenytoin. The inherited variability in the metabolism of mephenytoin was due to genetic variations in the gene coding for CYP2C19. CYP2C9 is the major CYP2C subfamily member in the liver and is primarily responsible for the oxidative metabolism of drugs including warfarin, phenytoin, glipizide, and losartan. Variants of CYP2C9 are CYP2C9*1, *2, *3, *4, *5 and *6. Patients with CYP2C9 genetic variants *2 and *3 have higher risk of acute bleeding complications than patients with a *1 variant upon administration of warfarin.

105. Thiopurine methyl transferases: Thiopurine methyl transferase (TPMT) catalyzes the S- methylation of the thiopurines like azathiopurine, mercaptopurine and thioguanine. These agents commonly used in the treatment of leukemia, rheumatic diseases, inflammatory bowel diseases etc. TPMT activity is highly variable and polymorphic, such that approximately 90% of individuals have high enzyme activity, 10% have intermediate activity and 0.3% have low or no detectable activity. At least 8 TPMT variant alleles have been identified, among 8, three alleles (TPMT*2, TPMT*3A, TPMT*3C) accounting for about 95% of patients with intermediate or low enzyme activity.

106. The mutant allele TPMT*2 is due to single nucleotide transversion (G238C) in the coding sequence, leading to an amino acid substitution at codon 18 (ala>pro). TPMT*3A contains two nucleotide transition mutations (G460A &A719G), leading to amino acid substitutions at codon 154 (Ala>Thr) and codon 240(Tyr>Cys). TPMT*3C contains only the A719G transition mutation. Phenotypic deficiency in TPMT activity is rare but the mutant alleles differs among various ethnic populations. phenotypecommon mutant allele Caucasians TPMT*3A Africans TPMT*3c Asians TPMT*3C

109. 6-Mercaptopurine is metabolized by several pathways one of which is catalyzed by TPMT and leads to inactive methyl-thiopurine metabolites. Other pathways catalyzed by several other enzymes leads to the active thioguanine nucleotides (6-TGN).

110. The resulting 6-TGNs act as purine antagonists through their incorporation into the DNA molecule and subsequent prevention of DNA replication. The reduction in DNA replication suppresses various immunological functions in lymphocytes, T cells, and plasma cells.

111. TPMT-deficient patients are at very high risk of developing severe hematopoietic toxicity. At high concentrations, 6-TGNs may cause toxicity and bone marrow suppression (result of low TPMT activity due to TPMT polymorphism).

113. DRUG TRANSPORTERS Transport proteins have an important role in regulating the absorption, distribution, and excretion of many drugs. The most widely studied class of membrane transporters belong to the ABC (ATP – binding cassette) family involved in the drug disposition are P – Glycoprotein MRP – 6(multidrug resistant proteins) OCT (organic cation transporter) OAT (organic anion transporter) P-gp is encoded by the human ABCB1 gene (also called MDR1). MDR1 gene is located on chromosome 7 and consists of 28 exons.

116. Pgp also plays a role in the excretion of xenobiotics into urine, bile and the intestinal lumen.

117. At blood brain barrier, Pgp in the choroid plexus limits the accumulation of many drugs in the brain, including digoxin, vinblastin, dexamethasone, cyclosporine, domperidone and loperamide.

122. Individuals with the CC genotype had an approximately two fold higher P-gp expression in small intestine in comparison to subjects with the TT genotype. Heterozygous subjects had an intermediate P-gp expression

125. Data on the influence of MDR1 single nucleotide polymorphisms (SNPs) on disposition of P-gp substrates

127. The major objective of drug target pharmacogenomics research is to identify the inherited basis for interindividual variability in drug response and toxicity, particularly when the variability is not explained by differences in pharmacokinetics.

130. These receptors are widely distributed and play an important role in regulating cardiac, vascular, pulmonary and metabolic functions.

131. In heart activation of β2 receptors results in an increased rate and force of cardiac muscle, whereas stimulation in the lungs acts to relax air way smooth muscle.

132. The β2 adrenoreceptor is coded by the gene called ADRB2.

135. ApoE2 contains two cysteines

136. ApoE4 contains two argininesThese differences have profound effects on both the physical stability and biological function of ApoE.

137. For example, while both apoE3 and apoE4 bind to the LDL receptor with high affinity, apoE2 exhibits defective LDL receptor binding. In addition the presence of the apoE4 allele is associated with elevated plasma cholesterol levels and an increased risk for both coronary artery and Alzheimer’s disease. Tacrine (acetyl cholinesterase inhibitor) used in the treatment of Alzheimer’s disease. Individuals without the apoE4 genotype showed improvement in total response compared to patients with the apoE4 genotype.

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