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
one 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
1. Altered Pteridine ring replacement at N8/5  Quinazolines
2. Lipid soluble lacking terminal glutamate  Trimetrexate,Piritrexim
3. Folate like with alteration in N10 bridge  Pralatrexate– increased
capacity for transport into tumor cells
4. TS inhibitor  Raltitrexed, Pemetrexed (Multi targeted antifolate)
5. Early steps in purine synthesis inhibitor  Lometrexol

10. Cellular pharmacology
Reduced Folate
Carrier
FolateReceptor
system
PHsensitive
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 – Folylpolyglutamyl 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 TSMRP-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 flora
• BA for doses of 50mg/m2 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
I. A rapid distribution phase
II. Renal clearance phase
III. 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