1. PHL 425 ANTIMETABOLITES Dr. Yieldez Bassiouni

2. Antimetabolites Antimetabolites are drugs which have similar structure to normal metabolites which are required for normal biochemical reactions. S-phase specific = CCS Antimetabolite are classified into: 1- Folic acid Antagonists: Methotrexate, Pemetrexed, Nolatrexed, Raltitrexed 2- Pyrimidine Antagonists: -5-FU -ORALLY BIOAVAILABLE 5′-FLUOROPYRIMIDINES (Capecitabine, Ftorafur ) - Cytidine Analogues - Ara-C - Gemcitabine - 5-Azacytidine 3- Purine Antagonists: -6-Mercaptopurine -Azathioprine Purine and pyrimidine antagonists are phosphorylated inside the body into nucleotid form in order to be cytotoxic

3. Indications - Leukemias - Non-Hodgkin's lymphoma - Treatment of non-neoplastic disorders including: - Rheumatoid arthritis, - Psoriasis - Inflammatory bowel disease - Ulcerative colitis - Immunosuppressant in transplantations

4. 1- Folic Acid Antagonists (Anti-folates) Antifolate compounds has been proven in the treatment of a variety of hematologic and non-hematologic malignancies. 1- Methotrexate 2- Pemetrexed 3- Nolatrexed 4- Raltitrexed

5. Methotrexate (MTX)

6. DFR

7. Methotrexate (MTX) Mechanism of action: Methotrexate inhibits DHFR enzyme, therefore, it inhibits the synthesis of DNA, RNA and proteins. DHFR catalyses the conversion of FH2 to the active FH4 which is needed for the de novo synthesis of the deoxynucleoside thymidine phosphate DTMP ( required for DNA synthesis

8. Metabolism: 1- Converted to MTX polyglutamates (cytotoxic metabolite) in normal and malignant tissues. 2- Converted to 7-OHMTX (Inactive metabolite) in liver by aldhyde oxidase. 3-Converted to diaminomethyl peteroic acid (DAMPA), Inactive metabolite, by bacterial carboxypeptidase enzyme in GIT Elimination: Primarily as interact drug in urine

9. High Dose MTX Therapy Rational: - MTX enter the cells through memberane folate binding protein (MFBP). - Some tumours lack or have reduced MFBP, therefore, the uptake of MTX in these tumours is not achieved after normal doses. - High dose MTX is required to increase the extracellular levels of MTX. Under these condition, MTX is transported by passive diffusion. - In high dose MTX therapy, the uptake of MTX into tumour cells is highly selective because the affinity of MFBP to MTX is higher (1 uM) than normal cells (90 uM). - Also, high dose MTX therapy improves its penetration into tumour sites such as testis, CNS and blood brain barrier.

10. High Dose MTX Therapy Precaution: - High dose MTX therapyis associated with severe nephrotoxicity due to precipitation of MTX and 7-OHMTX in renal tubules. - Dose adjustment in proportion to creatinine clearance. - Do not administer high-dose MTX to patients with abnormal renal function - Monitor plasma concentrations of MTX - Intensive hydration - Alkalinzation of urine to increase MTX solubility by Sodium Bicarbonate 3 gm/3 hours for 12 hours before therapy - Leucovorine Rescue: Lucovorin is tetrahydrofolate and competes with MTX for transport into mammalian cells and human targeted tissues as bone marrow and GIT. Leucovorin allowed the conventional doses of MTX to increased by 10-100 times. - Monitoring serum MTX level is an obligatory during high dose MTX therapy. - The dose of Leucovorin depends on serum MTX levels. - MTX drug levels should be measured every 24 hr and the dosage of leucovorin adjusted until the MTX level is <5 × 10 -8 mol/L.

11. Glucarpidase to combat toxic levels of methotrexate in patients In January 2012, glucarpidase (Voraxaze®) received approval from the US Food and Drug Administration for intravenous treatment of toxic plasma methotrexate concentrations due to impaired renal clearance. High-dose methotrexate has been particularly useful in the treatment of leukemias and lymphomas. However, even with aggressive hydration and urine alkalinization, such regimens can lead to acute renal dysfunction, as indicated by decreases in urine production and concomitant increases in blood urea nitrogen and serum creatinine levels. Because methotrexate is largely excreted by the kidneys, this can greatly potentiate tissue damage. Toxic levels of blood methotrexate can be rapidly and effectively decreased by intravenous administration of glucarpidase. Glucarpidase is a recombinant form of carboxypeptidase G2, a bacterial enzyme that rapidly cleaves methotrexate to form the amino acid glutamate and 2,4-diamino-N10-methylpteroic acid. Catabolites of methotrexate are much less toxic than the parent compound, and are primarily excreted by hepatic mechanisms

12. MTX Toxicity 1- Renal dysfunction 2- Hepatotoxicity. 3- Neurotoxicity 4- GIT mucositis 5- Bone marrow suppression 6- Pneumonitis (interstitial pulmonary infiltrate)

13. MTX Resistance 1- Decrease MTX transport into the cells. 2- Gene amplification of DHFR. 3- decrease binding affinity of DHFR to MTX.

14. Drug interactions: - Toxicity to normal tissues is rescued by leucovorin calcium (ANTIDOT) - L-Asparaginase blocks toxicity and antitumor activity. - Pretreatment with MTX increases 5-fluorouracil and cytosine arabinoside nucleotide formation Toxicity: - Myelosuppression - Mucositis, - Gastrointestinal epithelial denudation - Renal tubular obstruction and injury - Hepatotoxicity - Pneumonitis - Neurotoxicity

15. Interaction between MTX and 5-FU 1- MTX preceding 5-FU: SYNERGISTIC CYTOTOXICITY Interaction between MTX and 5-FU 2- 5-FU preceding MTX : ANTAGONISTIC CYTOTOXICITY

16. Interaction between MTX and L-Asparaginase L-Asparaginase conversts L-Asparagine (required for acute leukemia cells) into aspartic acid and amonia. L-Asparaginase pretreatment blocks the antitumour effect of MTX due to: 1- Inhibition of protein synthesis and prevention of cell entry into S-phase of cell cycle 2- Inhibition of MTX polyglutamation with the consequent decrease in MTX retention in tumour cells. 3- Inhibition of DNA synthesis lasts 10 days, a period during which cells are refractory to MTX. Therefore, Cycles of MTX-L-Asparaginase are repeated at 14 days interval. This combination is effective against Acute Leukemia of Childhood.

17. Regimen for Combined MTX and L-Asparaginase in Treatment of Refractory Acute Leukemia DrugsDays 013142728 L-ASPARAGINASE + + + (500 IU/kg) MTX + + (100 mg/m2)

18. 2- Pemetrexed: Thymidylate synthetase (TS) represents a logical target for pemetrexed. Pemetrexed is transported into cells via the reduced-folate carrier (memberane folate binding protein (MFBP). and possibly by a unique transporter identified in mesothelioma cell lines. It is metabolized to the polyglutamated forms, which are potent inhibitors of thymidylate synthetase (TS) enzyme.

19. 3- Nolatrexed Nolatrexed is a non-classic inhibitor of TS specifically designed to avoid potential resistance mechanisms that can limit the activity of the classic antifolate antimetabolites. Nolatrexed is a lipophilic molecule. Nolatrexed is a non–glutamate-containing molecule. Nolatrexed does not require facilitated transport for uptake. Nolatrexed does not undergo, nor require, intracellular polyglutamylation for activity. Nolatrexed exhibited S-phase cell-cycle arrest.

20. 4- Raltitrexed Raltitrexed is a water-soluble TS inhibitor that appears to have an acceptable toxicity profile, convenient dosing schedule, and antitumor activity in colorectal, breast, pancreatic, and a variety of other solid cancers. This drug is a second-generation agent designed to overcome the major toxicity associated with its predecessor.

21. 2- Pyrimidine antagonists -5-Fluorouracil -ORALLY BIOAVAILABLE 5′-FLUOROPYRIMIDINES - Capecitabine - Ftorafur - Cytidine Analogues - Ara-C - Gemcitabine - 5-Azacytidine

22. 5-Fluorouracil 5-FU resembles the Pyrimidine base URACIL (Component of RNA) and Thymidine base (Component of DNA )

23. Mechanism of action: 1- Incorporation of FUTP (false nucleotide) instead of UTP into RNA interferes with RNA synthesis and function. 2- Inhibition of thymidylate synthase by (FdUMP) leads to depletion of TMP and TTP. 3- Incorporation of FdUTP into DNA may affect DNA stability. Intracellular activation of 5-fluorouracil (5-FU). dUTP, deoxyuridine triphosphate; FdUDP, fluorodeoxyuridine diphosphate; FdUMP, fluorodeoxyuridylate; FdUrd, 5-fluoro-2′-deoxyuridine; FdUTP, fluorodeoxyuridine triphosphate; FUDP, fluorouridine diphosphate; FUMP, fluorouridine monophosphate; FUrd, 5-fluorouridine; FUTP, fluorouridine triphosphate; PPRP, phosphoribosyl phosphate.

24. 5-flurouracil (5-FU)

25. Metabolism: Converted enzymatically to active nucleotide forms intracellularly. Dihydropyrimidine dehydrogenase (DPD) catalyzes the initial, rate-limiting step in 5- fluorouracil (5-FU) catabolism. Pharmacokinetics: Primary half-life is 8–14 minutes after IV bolus. Nonlinear pharmacokinetics from saturable catabolism: Total-body clearance decreases with increasing doses; clearance is faster with infusional schedules. Volume of distribution slightly exceeds extracellular fluid space. Elimination: Approximately 90% is eliminated by metabolism. <3% and < 10% unchanged drug excreted by kidneys with infusional and bolus 5-FU. Reduction of 5- FU to dihydrofluorouracil by DPD is rate-limiting. Thereafter: dihydrofluorouracil → fluoroureidopropionic acid → fluoro-β-alanine. 5-FU and its catabolites undergo biliary excretion. Toxicity: Gastrointestinal epithelial ulceration Myelosuppression Dermatologic Ocular Neurotoxicity (cognitive dysfunction and cerebellar ataxia) Cardiac (coronary spasm) Biliary sclerosis

26. Precautions: - Nonlinear pharmacokinetics: difficulty in predicting plasma concentrations and toxicity at high doses. (nonlinear elimination kinetics, the drug clearance decreases with increasing drug concentration) - Patients with deficiency of Dihydropyrimidine dehydrogenase (DPD) may have life-threatening or fatal toxicity if treated with 5-FU. - Duration of DPD inhibition with eniluracil (a potent inactivator of DPD) may be prolonged (8-week washout period recommended). - Older, female, and poor-performance–status patients have greater risk of toxicity. - Closely monitor prothrombin time in patients receiving concurrent warfarin.

27. Clinical Predictive Value of Dihydropyrimidine Dehydrogenase (DPD) in 5-Fluorouracil (5-FU) Clinical Toxicity DPD deficiency is a pharmacogenetic syndrome in which molecular defects in the DPD gene result in a complete (profound) or partial loss of DPD enzyme activity. This results in markedly altered 5-FU pharmacokinetics and in turn severe toxicity following administration of standard doses of 5- FU. The cause for this toxicity appears to be decreased drug clearance, resulting in markedly prolonged exposure to 5-FU. Clinical Monitoring of DPD in cancer patients before 5-FU therapy is essential to avoid severe 5-FU toxicity. Monitoring of DPD in peripheral Blood Mononuclear Cells. Genetic Deficiency of DPD in patients with colorectal carcinoma is associated with severe 5-FU toxicity.

28. Eniluracil Combined With 5-Fluorouracil: The uracil analog eniluracil is a potent mechanism-based inactivator of DPD. Administration of eniluracil results in complete inhibition of DPD throughout the body. When given with eniluracil, renal excretion of 5-FU becomes the predominant route of elimination. Oral administration of 5-FU with eniluracil renders 5-FU completely bioavailable. Although eniluracil appears to be nontoxic when given alone, it shifts the 5-FU dose toxicity-response curves to lower doses.

29. ORALLY BIOAVAILABLE 5′-FLUOROPYRIMIDINES Capecitabine

30. Capecitabine Capecitabine [N (4-pentoxycarbonyl-5′-deoxy-5-fluorocytidine, Xeloda] is the first oral 5-FU prodrug to be approved in the United States, on the basis of its activity in patients with metastatic breast cancer whose disease is refractory to two earlier regimens. This agent is absorbed intact as the parent drug through the GI mucosa. It then undergoes three-step enzymatic conversion to 5-FU. By carboxylesterase (CES), cytosolic carboxylesterase and Cytidine deaminase, [In the liver, 5′-deoxy-5-fluorocytidine (5′-dFCyd) formation is catalyzed by carboxylesterase (CES), which is mainly expressed in microsomes, but a cytosolic carboxylesterase, CES1A1, also contributes to formation of 5′- dFCyd. Cytidine deaminase, a widely distributed enzyme, produces 5′-dFUrd, and dThd phosphorylase then generates 5-FU. Clinical studies have documented rapid GI absorption of the parent drug]

31. Ftorafur : Ftorafur is a prodrug and is slowly metabolized to 5- FU by two major metabolic pathways One pathway is mediated by microsomal cytochrome P-450 oxidation at the 5′-carbon of the tetrahydrofuran moiety, resulting in the formation of a labile intermediate (5′-hydroxyftorafur) that spontaneously cleaves to produce succinaldehyde and 5-FU. The second pathway occurs in the cytosol, and is thought to be mediated by thymidine phosphorylase.

32. Cytidine Analogues - CYTOSINE ARABINOSIDE (Ara-C) - Gemcitabine - 5-Azacytidine

33. CYTOSINE ARABINOSIDE (Ara-C) Cytarabine Ara-C is one of the most effective agents in the treatment of acute myeloid leukemia (AML) and is incorporated into all standard induction regimens for this disease, generally in combination with an anthracycline (daunorubicin hydrochloride or idarubicin hydrochloride). Ara-C is also a component of consolidation and maintenance regimens in AML after remission is attained. Ara-C is also active against other hematologic malignancies, including non- Hodgkin's lymphoma, ALL, and CML but has little activity as a single agent against solid tumors. This limited spectrum of activity has been attributed to the lack of metabolic activation of this agent in solid tumors and its selective action against rapidly dividing cells.

34. Mechanism of action: I nhibits DNA polymerase α, is incorporated into DNA, and terminates DNA chain elongation. Metabolism: Activated to triphosphate (Ara CTP)in tumor cells. Degraded to inactive ara-U by deamination Elimination: Deamination in liver, plasma, and peripheral tissues—100% Drug interactions: Methotrexate sodium increases ara-CTP formation Ara-C blocks DNA repair, enhances activity of alkylating agents Fludarabine phosphate increases ara-CTP formation Toxicity: Myelosuppression Gastrointestinal epithelial ulceration Intrahepatic cholestasis, pancreatitis Cerebellar and cerebral dysfunction (high dose) Conjunctivitis (high dose) Noncardiogenic pulmonary edema

35. Resistance to Ara-C 1- Increased dCTP pools, presumably through increased cytidine-5′-triphosphate (CTP) synthetase activity or deficiency of dCMP deaminase. 2- deletion of deoxycytidine (CdR) Kinase. 3- Increased cytidine deaminase activity which is reversed by the cytidine deaminase inhibitor tetrahydrouridine. 4- inhibition of nucleoside transporters can prevent the influx of gemcitabine through the cell membrane 5- Resistance to ARA-c has not been associated with increased P-glycoprotein expression

36. GEMCITABINE -Gemcitabine (2,2-difluorodeoxycytidine, dFdC) is the most important cytidine analog to enter clinical trials since ara-C. [- deoxycytidine kinase phosphorylates gemcitabine intracellularly to produce difluorodeoxycytidine monophosphate (dFdCMP), from which point it is converted to its diphosphate and triphosphate difluorodeoxycytidine (dFdCDP, dFdCTP).]

37. Mechanisms of Resistance TO GEMCITABINE 1- overexpression of ribonucleotide reductase. 2- Induction of cytidine deaminase 3- high concentrations of heat-shock protein 4- inhibition of nucleoside transporters can prevent the influx of gemcitabine through the cell membrane 5- Resistance to gemcitabine has not been associated with increased P-glycoprotein expression

38. Azacytidine Mechanism of action: Incorporated into DNA and RNA; prevents DNA methylation Metabolism: Activated to a triphosphate, Degraded to inactive, unstable 5-azauridine by cytidine deaminase Pharmacokinetics and elimination : Plasma half-lives not known, but the drug is chemically unstable and is rapidly deaminated. Drug interactions: Tetrahydrouridine (is a potent competitive inhibitor of cytidine deaminase) inhibits deamination, increases toxicity Toxicity: Myelosuppression Nausea, vomiting after bolus dose Hepatocellular dysfunction Muscle tenderness, weakness Lethargy, confusion, coma

39. 3- Purine Antagonists 6−Mercaptopurine, (6−MP): - It is immunosuppressive cytotoxic drug. It is widely used in transplantations to control rejection reactions. - It is acts as a purine analogue and once enter the cell, it is converted to 6-MP- ribosephophate and can be incorporated into RNA and DNA resulting in non functioning RNA & DNA & finally inducing cell cycle arrest and apoptosis. - It also inhibits purine ring biosynthesis Adverse reactions Diarrhea, nausea, vomiting, loss of appetite, Allergic reaction include rash, itching, swelling, dizziness, trouble breathing. Mercaptopurine cause myelosuppression. Those taking mercaptopurine should get permission from a doctor in order to receive immunizations and vaccinations. Azathioprine: It is one of the main immunosuppressive cytotoxic substance. It is widely used in transplantations to control rejection reactions. It is nonenzymatically cleaved to 6 - MP that acts as a purine analogue and inhibits DNA synthesis