הוראה ×‘× ×•×©××™ טיפול תרופתי בסרטן

1. הוראה בנושאי טיפול תרופתי בסרטן מנגנוני פעולה ועמידות של תרופות ציטוטוקסיות ו”תרופות מגינות” ערך: פרופ’ נ. חיים , מאי 2003, עודכן: מרץ 2007 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-glycoprotein (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-Cytotoxic drugs associated with MDR (Contd) 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 considered more sensitive. 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 upstream 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 • Potent electrophiles and react with (alkylate) electron-rich atoms to form covalent bonds . The most important reaction are with DNA bases, with formation of DNA adducts. • Electron reach 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), which requires oxidation by hepatic cytochrome P450 enzymes. It is metabolized in the liver by cytochrome P-450 to active species-4-hydroxycyclophosphamide, which is unstable and undergoes spontaneous decomposition to biologically active alkylating species such as phosphoramide mustard (bifunctional alkylating agent).

19. Mafosfamide • A chemically stable 4-thioethane sulfonic acid salt of 4-hydroxycyclophosphamide, a preactivated cyclophosphamide derivative that does not require hepatic activation. • Used for IT administration in phase I trial (Blaney SM et al. J Clin Oncol 23: 1555-63, 2005).

20. Hydrazine and triazene derivatives(DeVita…7th 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.

21. 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* (=active metabolite). *[(methyl-triazeno)-imidazole-caboxamide] • 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”)

22. O-6-methyl(alkyl)guanine-DNA methyl (alkyl)transferase (MGMT or AGT) & sensitivity of GBM to temozolomide (and alkylator-based chemotherapy) • MGMT is a DNA repair enzyme. • High endogenous MGMT activity is associated with resistance to temozolomide. • A state of methylated MGMT promoter (silencing of the MGMT by a promoter methylation) (=non-functional enzyme) in the biopsy specimen was associated with better outcome than non-methylated MGMT in temozolomide treated patients. Hegi ME et al. Clin Cancer Res 15: 1871-4, 2004 • Overexpression of MGMT in childhood malignant gliomas is strongly associated with an adverse outcome in children treated with alkylator-based chemotherapy. Pollack IF et al. J Clin Oncol 24: 3431-7, 2006

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

24. Platinum analogs • Cisplatin…. • Carboplatin…. • Oxaliplatin…. • JM-216 (Satraplatin)=oral platinum analog….

25. 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.

26. Alkylating agents & similar drugs: Drug resistance • GSH and….. • O**6-alkyltransferase activity and….. • Expression of ERCC1 protein in operative specimens of NSCLC and sensitivity to adjuvant cisplatin…. • Potential drugs to reverse….

27. 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 ).

28. O**6-alkyl-guanine-alkyltransferase activity(AGAT)* and resistance to hydrazine and triazine derivatives Increased activity of AGAT can be associated with resistance to procarbazine, Dacarbazine (DTIC) or temozolomide. *AGAT= MGMT (O6-methylguanine-DNA methyltransferase)

29. Expression of ERCC1 protein in operative specimens of NSCLC and sensitivity to adjuvant cisplatin Olaussen KA et al. N Engl J Med 355: 983-991, 2006 (IALT investigators) • DNA repair mechanisms are important in the resistance to cisplatin. • The excision repair cross-complementation group 1 (ERCC1) enzyme recognizes and removes cisplatin-induced DNA adducts and, therefore, is likely to play a role in resistance to cisplatin. • Pts with completely resected NSCLC and ERCC1-negative tumors (as assessed by immunohystochemistry) appear to benefit from adjuvant cisplatin-based chemotherapy, whereas pts with ERCC1 positive tumors do not.

30. 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).

31. Chemoprotectors for Alkylating agents & other drugs • Mesna…. • Amifostine…. • Methylene blue & ifosfamide-induced encephalopathy…. • Dexrazoxane…. • Glutamine….

32. 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 (RS-SR), reduced back to mesna (RSH) in renal tubules and excreted in urine.

33. 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.

34. Methylene blue in the (experimental) 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….

35. Methylene blue in the (experimental) 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…,

36. Dexrazoxane (ICRF-187) (cardioxane)-mechanism of action A derivative of EDTA that acts as an intracellular chelating agent. The mechanism: chelation of intracellular iron, which may decrease doxorubicin-induced free radical generation. See: Doxorubicin…

37. Glutamine Savarese DMF et al. Prevention of chemotherapy and radiation toxicity with glutamine. Cancer Treatment Reviews 29: 501-13, 2003 (Review) • Malignancy produces a state of physiologic stress that is characterized by relative deficiency of glutamine (which is needed for glutathione (GSH) synthesis). • Glutamine deficiency may impact on normal tissue tolerance to antitumor treatment. • The available evidence suggests that dietary glutamine supplementation may reduce the incidence and/or severity of chemotherapy-associated mucositis, irinotecan-associated diarrhea, paclitaxel-induced neuropathy, hepatic veno-occlusive disease in the setting of high-dose chemotherapy, and cardiotoxicity of anthracyclines.

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

39. 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

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

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

42. 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….

43. “Key enzymes” inhibited by MTX and its polyglutamates • DHFR- …depletion of intracellular FH4, necessary (as one carbon 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.

44. Methotrexate DHFR dependent mechanism of action Methotrexate Binding & inhibition Dihydrofolate Reductase FH4 (reduced folates) FH2

45. dUMP CH2FH4 FH4 Thymidylate Synthase dTMP FH2 DHFR Reduced Folates and Thymidine synthesis

46. 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 See: Serra M et al. Analysis of DHFR and RFC gene status in relation to MTX resistance in osteosarcoma cells. Ann Oncol 15: 151-60, 2004

47. 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

48. Standard methods to neutralize MTX toxicity • 5-formyltetrahydrofolate (leucovorin): This compound is a reduced folate... undergoes intracellular polyglutamation, competes with MTX and with 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….

49. Experimental methods to neutralize MTX toxicity Experimental methods: • Exogenous thymidine: • IV Carboxypeptidase -G2: bacterial enzyme which hydrolyses MTX (Widemann BC et al. J Clin Oncol 15: 2125-34, 1997) • hemoperfusion over a charcoal column • oral administration of activated charcoal or cholestyramine (anion-exchange resin) to increase enterohepatic drug loss (biliary clearance).

50. 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)….