הכנה לבחינות התמחות באונקולוגיה

1. הכנה לבחינות התמחות באונקולוגיה טיפול תרופתי- מנגנוני פעולה ועמידות של תרופות ציטוטוקסיות ו”תרופות מגינות” ערך: פרופ’ נ. חיים , אוגוסט 2003 , עודכן- נובמבר 2003 כתובת לשאלות והערות: n_haim@rambam.health.gov.il

2. Drug Resistance • Various mechanisms…. • Decreased drug accumulation….carrier…. • MDR…..MRP…. • Reduced intracellular activation…. • Altered or increased amounts of intracellular target…. • Increased intracellular drug detoxification…. • Increased repair capacity…. • “General Drug resistance”….

3. Drug Resistance • Primary….. • Secondary…. Mechanisms of resistance • Impaired transport across cell membrane-including MDR • Reduced intracellular activation • Altered or increased amounts of intracellular target • Increased intracellular drug detoxification • Increased repair capacity

4. Drug resistance due to decreased drug accumulation • Decreased influx (e.g. defective drug carrier) • Enhanced drug efflux (MDR)

5. Impaired transport across cell membrane Some drugs require facilitated transport (in contrast to passive diffusion). These drugs bind to a carrier (= drug receptor protein on the cell membrane). examples: • transport systems for methotrexate: RFC (reduced folate carrier) • transport systems for gemcitabine: nucleoside transporter system

6. Multiple Drug Resistance (MDR) (pleiotropic resistance) • P-glycoproteinn (p170) is a cell surface glycoprotein of 170-KD size. • P-glycoprotein is encoded by mdr-1 gene and functions as a cellular pump for extruding toxic molecules. • Part of the P-gp is located in the interior aspect of the cell membrane, and bind in this area to the drug (by an ATP dependent process) Contd….

7. Multiple Drug Resistance (MDR) (pleiotropic resistance)(Contd.) • MDR is associated with overexpression of P-glycoprotein. • In-vitro MDR can be reversed by calcium channel blockers (e.g. verapamil) and cyclosporine Contd.

8. MDR (Contd)-Cytotoxic drugs associated with MDR Drugs associated with MDR tend to be large molecules that are derived from the natural environment: • Anthracyclines • Vinca alkaloids • Epipodophyllotoxins • Actinomycin D (Dactinomycin) • Taxanes

9. Multidrug-resistance protein (MRP) • mrp gene encodes a 190-kD protein that function in a similar action to that of p170 glycoprotein to mediate rapid efflux.

10. Reduced intracellular activation • 5 fluorouracil (analogue of uracil=base) and Ara C/Gemcitabine (analogues of deoxycytidine=base+sugar=nucleoside) must be converted into the appropriate nucleotide form (=nucleoside+phosphor =phosphorylated form) to be cytotoxic. • Impaired activity of the enzymes responsible for converting the drug to its phosphorylated form can be associated with resistance.

11. Altered or increased amounts of intracellular target • Methotrexate & DHFR…. • 5FU & TS….

12. Increased intracellular drug detoxification • Glutathione & glutathion S-transferase….. See: alkylating agents….

13. Increased repair capacity • Alkylation in the O-6 position of guanine can be removed by a specific methyl transferase (0-6 alkyl transferase). • Cells that have low methyltransferase repair capacity (Mer-) are resistant. See: alkylating agents….

14. “General Drug resistance”- (Perry MC. The chemotherapy source book, 3rd edition,2001, page 42) • All of the above mentioned drug-resistance mechanisms might be described as upsream resistance (interaction with cell membrane, intracellular target, repair processes). • There appear to be at least one further critical step in the drug action that occurs downstream from the drug-target interaction-the process of apoptosis………… …..p53-associated drug resistance….

15. Alkylating agents & similar drugs • Mechanism of action…. • Cyclophosphamide-metabolic activation…. • Hydrazine and triazine derivatives (=methylating agents)…. • Nitrosoureas…. • Platinum analogs….

16. Alkylating agents-mechanism of action • React with (alkylate) electron-rich atoms (electrophylic targets) to form covalent bonds. The most important reaction are with DNA bases, with formation of DNA adducts. • Electrophylic targets in DNA include: N-7 position of guanine (site of alkylation for intermediate intermediate/metabolite of nitrogen mustard/cyclophosphamide) or the O-6 position of cytosine (the site of alkylation for nitrosoureas). Contd….

17. Alkylating agents-mechanism of action (Contd.) • Monofunctional alkylating agents react with only one strand of DNA; • bifunctional alkylating agents react with two strands to produce a “cross-link” that covalently links the two strands of the DNA double helix.

18. Cyclophosphamide-mechanism of metabolic activation • Cyclophosphamide (a prodrug) is mmetabolized in the liver by cytochrome P-450 to active species. …….the active metabolite is phosphoramide mustard (bifunctional alkylating agent).

19. Hydrazine and triazene derivatives(DeVita…6th edition)or: “Methylating agents” (Perry MC. The chemotherapy source book, 3rd edition,2001, page 203) • Procarbazine (hydrazine derivative), Dacarbazine (DTIC), Temozolomide (Temodal)(triazene derivatives): • Like nitrsoureas decompose spontaneously or are metabolized to produce carbonium ion, which alkylates DNA. • These agents exert their activities mainly through the methylation of the O-6 position of guanylic acid in DNA.

20. Temozolomide vs. Dacarbazine • Both are prodrugs. DTIC requires liver microsomal metabolism for activation. Temozolomide does not require hepatic activation; this takes place by spontaneous degradation at physiological PH to form the cytotoxic methylating agent, MTIC. • A possible mechanism of resistance to these drugs is removal of the methyl groups from O-6-methylguanine by the enzyme O-6-alkylguanine-DNA-alkyltransferase (AGT)(See: “Drug Resistance / Increased repair capacity”)

21. Nitrosoureas Carmustine (BCNU), Lomustine (CCNU), Semustine (methyl-CCNU), Streptozotocin (Streptozocin): • Produce interstrand cross-links of DNA which occurs through generation of chloroethyldiazonium species. • Carbamoylation of proteins

22. Platinum analogs • Cisplatin…. • Carboplatin…. • Oxaliplatin….

23. Cisplatin & analogues-mechanism of action React preferentially at the N7 position of guanine and adenine residues to form a variety of monofunctional and bifunctional adducts.

24. Alkylating agents & similar drugs: Drug resistance • GSH and….. • O**6-alkyltransferase activity and….. • Potential drugs to reverse….

25. Glutathione (GSH) & Resistance to alkylating agents • Alkylating agents are potent electrophiles-reacting with electron reach molecules within the cell (especially with GSH-present in millimolar concentrations). • GSH is a tripeptide (glutamate, cysteine, glycine) with a free cysteine sulfhydril (and can serve as an alternative nucleopylic target). • Possible mechanisms of resistance (related to GSH): -elevated cellular GSH concentration, -increased activity of Glutathione S-transferase (a number of isoenzymes that catalyze the conjugation of GSH with electrophiles ).

26. O**6-alkyltransferase activity and resistance to hydrazine and triazine derivatives • Increased activity of O**6-AT can be associated with resistance to procarbazine, Dacarbazine (DTIC) or temozolomide.

27. Potential drugs to reverse resistance to alkylating agents • Buthionine sulfoximine (BSO): inhibitor of gamma-glutamylcysteine synthetase (necessary for GSH synthesis). • Inhibitors of O**6-alkylguanine:alkyltransferase (0**6AT): this enzyme enhances DNA repair by removing an alkyl group from O**6 position of guanine; (potentially reverse resistance to hydrazine & triazine derivatives).

28. Chemoprorectors for Alkylating agents & similar drugs • Mesna…. • Amifostine…. • Methylene blue & ifosfamide-induced encephalopathy….

29. Mesna (sodium-2- mercaptoethane sulfonate) A thiol (=with-SH group) compund (See:”Alkylating agents & similar drugs: Drug resistance” for the protective effect of thiols). • Functions as a regional detoxificant of the oxazaphosphorine metabolites. • Selective urinary tract protectant for oxazophosphorine-type alkylating agents through binding of the SH moiety to acrolein. • Undergoes dimerization in blood to inactive disulfide dimesna, reduced back to mesna in renal tubules and excreted in urine.

30. Amifostine (military code name: WR-2721) • A thiol compound. • A prodrug . • Dephosphorylated at the tissue level to its active metabolite, the free thiol WR-1065, by membrane-bound alkaline phosphatase. • Mechanism of protection: free radical scavenger and by hydrogen donation to repair damaged target molecules. • In tumor tissue: lower concentrations of alkaline phosphatase and lower PH are associated with lower rate of prodrug activation by alk. Phosph.

31. Methylene blue in the treatment & prevention of ifosfamide-induced encephalopathy Kupfer A et al. Lancet 343: 763-64, 1994 Aeschlimann C et al. Drug Metabol Dispos 26: 883-90, 1998 Pelgrims J et al. Brit J Cancer 82: 291-4, 2000 Peter C et al. Eur J Clin Pharmacol 56: 247-50, 2000 • Anecdotal reports…. • Methylene blue can be given by IV or oral administration. • the mechanism is not clear…. Contd….

32. Methylene blue in the treatment & prevention of ifosfamide-induced encephalopathy (Contd.) • After the systematic study of a patient receiving overdose of ifosfamide revealed a glutaric aciduria-the hallmark of a type II defect in mitochondrial electron transfer-methylene blue was introduced in the treatment of ifosfamide encephalopathy (Kupfer A et al. Lancet 343: 763-64, 1994). • Further investigations showed a relationship between glutaric aciduria and chloroethylamine but not with any other metabolites of ifosfamide. This led to the conclusion that chloroethylamine may be the principal neurotoxic metabolite of ifosfamide…..Methylene blue counteracts some of these metabolic pathways…,

33. Antimetabolites • Structure, mechanism (principles) & phase of action….

34. Antimetabolites Structure: • Structural similarity to naturally occurring compounds Mechanisms of action (principles): • 1. Competitive inhibition of a “key enzyme” • 2. Incorporation of the antimetabolite (or an active nucleotide derived from the antimetabolite) into DNA or RNA Phase during which active: • S phase

35. Antimetabolites-structure What is the natural analog of: Methotrexate, 5FU, Cytosine arabinoside (Ara C), Gemcitabine?…..

36. Antimetabolites-structure • Methotrexateis an analog of folic acid. • 5FU is an analog of uracil (=base). • Gemcitabine & Ara-C : pyrimidine analogs of deoxycytidine. deoxycytidine is a nucleoside, I.e. base (cytosine) + sugar (deoxyribose). • Ara-C: The sugar (arabinose) differ from deoxyribose in one hydroxyl group. • Gemcitabine: The sugar (arabinose) differ from deoxyribose in two atoms of fluorine.

37. Methotrexate • “Key enzymes” inhibited…. • Mechanisms of resistance to…. • Metabolites and their activity…. • Standard & experimental methods to neutralize toxicity… • Trimetrexate: similarity & difference from MTX…. • Tomudex (Raltitrexed): similarity & difference from MTX….

38. “Key enzymes” inhibited by MTX and its polyglutamates • DHFR- …depletion of intracellular FH4, necessary (as one carbone carrier) for synthesis of purines (through GAR and AICAR transformylases) and thymidylate (through TS), with inhibition of DNA and RNA synthesis. • TS, GAR, and AICAR-…inhibited mainly by polyglutamates.

39. Mechanism of resistance to MTX • Defect in RFC (reduced folate carrier) or FR (folate receptor -binding protein). • Decreased polyglutamation (by folylpoly-gamma-glutamate synthetase) • Alteration in the target-DHFR: -increased expression -mutated DHFR with decreased affinity to MTX

40. Metabolites of MTX • 7-OH-MTX-undergoes polyglutamation by folylpolyglutamyl. Active metabolite • DAMPA (2,4, diamino-N10-methyl pteroic acid) a product of bacterial degradation of MTX in the gut lumen. Non-active

41. Standard methods to neutralize MTX toxicity • 5-formyltetrahydrofolate (leucovorin): This compound is a reduced folate... undergoes intracellular polyglutamation, competes with MTX and MTX polyglutamates on inhibition of TS and de novo purine synthesis and on cellular transport. (the commercially available form is a mixture of d and l -forms. Only the l-isomer is active). • Vigorous hydration & urine alkalization….

42. Experimental methods to neutralize MTX toxicity Experimental methods: • Exogenous thymidine: • IV Carboxypeptidase -G2: bacterial enzyme which hydrolyses MTX • hemoperfusion over a charcoal column • oral administration of activated charcoal or cholestyramine (anion-exchange resin) to increase enterohepatic drug loss (biliary clearance).

43. Anti-folates other than methotrexte • Pemetrexed (Alimta): “Key enzymes” inhibited…. Transport across cell membrane & polyglutamation…. Folate status, homocysteine, and toxicity ofMTA…. Concomitant treatment to reduce toxicity…. • Trimetexate…. • Raltitrexed (Tomudex)….

44. Pemetrexed (Alimta)=MTA (multitargeted antifolate agent) • A novel folate based anticancer drug which inhibits the following key enzymes (involved in purine and pyrimidine synthesis): -thymidylate synthase (TS), -dihydrofolate reductase (DHFR) -and glycinamide ribonucleotide transformylase (GARFT) (purine synthesis). Adjei AA. Ann Oncol 11: 1335-41, 2000 (review)

45. Pemetrexed (Alimta)=MTA =multitargeted antifolate agent • Enters the cell primarily through RFC. • Undergoes extensive intracellular polyglutamation by folylpoly-gamma-glutamate synthetase (which leads to persistently elevated intracellular concentrations). Adjei AA. Ann Oncol 11: 1335-41, 2000 (review)

46. Folate status, homocysteine, and toxicity of MTA Severe toxicities to MTA were linked to high blood levels of homocysteine and methylmalonic acid at study entry, suggesting that such toxicity and possibly some deaths may be related to reduced folic acid and B12 pools. Niyikiza C et al. Mol Cancer Ther 1: 545-52, 2002 Plasma homocysteine is a much more sensitive measure of the functional folate status (induced by MTA) than red blood cell or serum folate. Adjei AA. Ann Oncol 11: 1335-41, 2000 (review)

47. Concomitant treatment to reduce toxicity of Alimta • Folic acid 350-600 micrograms (or equivalent) PO, daily, beginning approximately 1 to 2 weeks before the first dose of Alimta and continue until treatment discontinuation. • Vitamine B12 injection IM, 1 to 2 weeks prior to the first dose, and should be repeated approximately every 9 weeks. • Dexamethasone (for prevention of severe cutaneous toxicity): 4 mg (or its equivalent) PO, twice a day on the day before, the day of, and the day after Alimta.

48. Does vitamin supplementation impairs MTA activity? • Adjei AA. Ann Oncol 11: 1335-41, 2000 (review): “while folic acid clearly ameliorates the toxicity of MTA, the effect of concomitant folate supplementation on the clinical activity of MTA remains unsettled”. • Scagliotti GV et al. J Clin Oncol 21: 1556-61, 2003: MTA was given as a single agent in patients with pleural mesothelioma. Patients supplemented with folic acid and vitamin B12 tolerated treatment better (less toxicity and more cycles of treatment) and had better survival. Vogelzang NJ et al. J Clin Oncol 21: 2636-44, 2003 MTA + cisplatin in malignant pleural mesothelioma….Addition of folic acid and vitamin B12 significantly reduced toxicity without adverely affecting survival time.

49. Trimetrexate Similarity to MTX: • inhibits DHFR Difference from MTX: • more lipid soluble • does not undergo polyglutamation • does not require RFC for cellular transport • does serve as a substrate for P-glycoprotein (I.e., affected by expression of the classic MDR phenotype). • clearance-mainly hepatic

50. Tomudex (Raltitrexed)-a selective inhibitor of TS Similarity to MTX: • requires RFC • undergoes polyglutamation • clearance-mainly renal Difference from MTX: • main target is TS (and not DHFR)