Methotrexate

Methotrexate pharmacology

1. Methotrexate

2. Introduction Antifolate drug DHFR inhibitor Anti cancer Immunosuppression

3. Basics Nucleus  DNA , RNA Nucleotides  precursor of Nucleic acids Storage and transfer of genetic information Nucleotides  1.Nitrogenous base ( purine or pyrimidine) 2.Pentose sugar ( ribose /deoxyribose) 3.Phosphate groups esterified to sugar

4. Purines  Adenine , Guanine Pyramidines  Thymine , Cytosine , Uracil Adenine Guanine Thymine Cytosine Uracil

5. How are they made ? Dietary purines and pyrimidines are neither converted to nucleotides nor incorporated into nucleic acids They are directly catabolized De novo synthesis All cells especially liver ; cytoplasm multistep process In synthesis – C2 and C8 of Purines is donated by o ne carbon groups (AICAR transformylase & GAR transformylase)

6. Pyramidines – Thymine synthesis needs one carbon groups (Thymidylate synthesis) One carbon groups are contributed by amino acids and carried by THFA which is produced from folic acid By enzyme – Dihydro folate reductase

7. Purine Pyrimidine DNA GAR transformylase (C8) AICAR transformylase(C2) Thymidylate synthase (dUMP  dTMP) THFA THFA FA FA DHFR DHFR Methotrexate Polyglutamate forms Folyl polyglutamate synthetase

8. Methotrexate Competitive inhibitor of DHFR Pteridine ring P-amino benzoic acid Glutamyl residues 2,4 diamino

9. 2,4 diamino  pyrimidine analogues  pyrimethamine & Trimethoprim Other modified compounds Altered Pteridine ring replacement at N8/5  Quinazolines Lipid soluble lacking terminal glutamate  Trimetrexate ,Piritrexim Folate like with alteration in N10 bridge  Pralatrexate– increased capacity for transport into tumor cells TS inhibitor  Raltitrexed, Pemetrexed (Multi targeted antifolate) Early steps in purine synthesis inhibitor  Lometrexol

10. Cellular pharmacology Reduced Folate Carrier Folate Receptor system PH sensitive transporter THFA , Methotrexate , Leucovorin (5 –formyl tetrahydro folate ) Pemetrexed , Pralatrexate, Raltitrexed Folic acid MTX PGs Raltitrexed Pemetrexed Acidic , poorly perfused areas & CNS. Pemetrexed

11. Intra cellular transformation Natural folates  glutamated forms – Folylpoly g lutamyl synthetase Adds up to 8 Glutamyl groups in peptide linkage Helps in intracellular folate accumulation as monoglutamates are freely transportable into and out of the cells Prolongs intracellular half life under conditions of reduced folate availability MTX – Polyglutamates are slightly more potent inhibitors of DHFR and more potent antagonists of TS , AICAR transformylase , GAR transformylase compared to MTX monoglutamate

12. Pralatrexate is 10 fold more avidly polyglutamated than MTX GGH gamma Glutamyl hydrolase is a peptidase which removes the terminal Glutamyl residues and returns MTX PGs to their monoglutamate forms Cellular activity of FPGS is directly proportional to cell growth  modest PG formation in normal tissues compared to malignant and inversely to level of intracellular folates  Leucovorin reduces polyglutamation Relatively selective rescue of the normal tissue by Leucovorin Deprivation of essential amino acids leads to inhibition of polyglutamation

13. Ability of antifolate analogs to polyglutamate influence cytotoxic property Aminopterin is a better substrate to FPGS , so more cytotoxic than MTX A fluorinated MTX analogue PT430 is a weak substrate for FPGS and has little cytotoxic activity Defective polyglutamation  MTX resistance Hyperdiploid status in ALL is a good prognostic feature  higher synthesis of cytotoxic MTX

14. Cell death Cessation of DNA synthesis More DNA strand breaks High dUMP  misincorporation into DNA  again strand breaks by enzyme Uracil DNA glycosylase Cell cycle arrest and apoptosis

15. Mechanism of resistance Altered DHFR /less affinity to MTX Reduced Polyglutamates Increased DHFR MRP Increased TS MRP-1 MRP-2 MRP-3 BCRP

16. DHFR gene amplification  usually after prolonged selective pressure of drug exposure Highly MTX resistant cell may be generated by gene amplification within a single cell cycle Early S phase cell exposed transiently to DNA synthesis blockers reduplication of multiple genes including DHFR

17. Lipid soluble non glutamated antifolates – Trimetrexate , Piritrexim do not require active cellular transport ,demonstrates effect against transport resistant mutants but lack polyglutamation and have minimal antitumor activity Pralatrexate – cutaneous T cell lymphomas – 10 fold affinity to RFC than MTX Newer antifolate inhibitors use other transports Low folate  up regulation of FR in normal tissue  severe myelosuprression

18. Pk and determinants of cytotoxicity S phase specific Drug concentration and duration of exposure are important Duration of exposure is more important  more cells are allowed to enter the vulnerable DNA synthetic phase of the cell cycle Leucovorin  is effective when given within 24 – 36 hours after MTX It can reverse cytotoxic effect of both host and malignant cells  so minimum dose of Leucovorin needed to rescue host cell should be used

19. Absorption Variable and incomplete at higher doses (15mg) Degradation by intestinal flor a BA for doses of 50mg/m 2 or more is enhanced by subdividing the dose rather than single dose Larger routes – better systemic Drug induced epithelial damage , motility changes and alters intestinal flora

20. Distribution Vd approximates total body water 60% PPB Weak organic acids can displace MTX  sulfonamides , salicylates, Tetracyclines, Chloramphenicol , Phenytoin Drug disappears from blood in a triphasic fashion A rapid distribution phase Renal clearance phase Terminal phase  prolonged in renal failure

21. At normal dose only 3% reach CNS High dose MTX >1.5g/m2 is cytotoxic in CNS

22. Elimination 90% excreted unchanged in urine within 48 hours by glomerular secretion and active tubular secretion Minimal metabolism only  7-OH MTX  potentially nephrotoxic Drugs which reduce RBF (NSAID) , nephrotoxic (cisplatin ) , weak organic acids (Aspirin , Piperacillin ) increase toxicity

23. Therapeutic uses Psoriasis Refractory RA ALL in children Meningeal carcinomatosis Intraventricular Omayya reservoir Choriocarcinoma ,GTD CA Breast , Head and Neck , Ovary , Bladder Osteosarcoma , CNS lymphoma MTX + Misoprostol  for First trimester MTP

24. Toxicity M/t Inj.Leucovorin mg/Kg im S.MTX levels more than 1microM  Leucovorin 100 mg /m2 till value is less than 50nanoM If oliguric  intermittent hemodialysis MTX cleaving enzyme  Glucarpidase (recombinant carboxypeptidase G2) is an orphan drug

25. Thank you