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הוראה בנושאי טיפול תרופתי בסרטן. מנגנוני פעולה ועמידות של תרופות ציטוטוקסיות ו”תרופות מגינות” ערך: פרופ’ נ. חיים , מאי 2003, עודכן-יולי 0705 כתובת לשאלות והערות: [email protected] Drug Resistance. Various mechanisms…. Decreased drug accumulation….carrier…. MDR…..MRP….

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2975967

הוראה בנושאי טיפול תרופתי בסרטן

מנגנוני פעולה ועמידות של תרופות ציטוטוקסיות ו”תרופות מגינות”

ערך: פרופ’ נ. חיים , מאי 2003, עודכן-יולי 0705

כתובת לשאלות והערות:

[email protected]


Drug resistance

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”….


Drug resistance1

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


Drug resistance due to decreased drug accumulation

Drug resistance due to decreased drug accumulation

  • Decreased influx (e.g. defective drug carrier)

  • Enhanced drug efflux (MDR)


Impaired transport across cell membrane

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


Multiple drug resistance mdr pleiotropic resistance

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….


Multiple drug resistance mdr pleiotropic resistance contd

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


Mdr cytotoxic drugs associated with mdr contd

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


Multidrug resistance protein mrp

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.


Reduced intracellular activation

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.


Altered or increased amounts of intracellular target

Altered or increased amounts of intracellular target

  • Methotrexate & DHFR….

  • 5FU & TS….


Increased intracellular drug detoxification

Increased intracellular drug detoxification

Glutathione & glutathion S-transferase…..

See: alkylating agents….


Increased repair capacity

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….


General drug resistance perry mc the chemotherapy source book 3rd edition 2001 page 42

“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….


Alkylating agents similar drugs

Alkylating agents & similar drugs

  • Mechanism of action….

  • Cyclophosphamide-metabolic activation….

  • Hydrazine and triazine derivatives (=methylating agents)….

  • Nitrosoureas….

  • Platinum analogs….


Alkylating agents mechanism of action

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….


Alkylating agents mechanism of action contd

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.


Cyclophosphamide mechanism of metabolic activation

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).


Mafosfamide

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).


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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.


Temozolomide vs dacarbazine

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”)


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O-6-methyl(alkyl)guanine-DNA methyl (alkyl)transferase (MGMT or AGT) & sensitivity of GBM to temozolomide

  • MGMT is a DNA repair enzyme.

  • A state of methylated MGMT promoter (=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


Nitrosoureas

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


Platinum analogs

Platinum analogs

  • Cisplatin….

  • Carboplatin….

  • Oxaliplatin….

  • JM-216 (Satraplatin)=oral platinum analog….


Cisplatin analogues mechanism of action

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.


Alkylating agents similar drugs drug resistance

Alkylating agents & similar drugs: Drug resistance

  • GSH and…..

  • O**6-alkyltransferase activity and…..

  • Potential drugs to reverse….


Glutathione gsh resistance to alkylating agents

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 ).


O 6 alkyltransferase activity and resistance to hydrazine and triazine derivatives

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.


Potential drugs to reverse resistance to alkylating agents

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).


Chemoprotectors for alkylating agents other drugs

Chemoprotectors for Alkylating agents & other drugs

  • Mesna….

  • Amifostine….

  • Methylene blue & ifosfamide-induced encephalopathy….

  • Dexrazoxane….

  • Glutamine….


Mesna sodium 2 mercaptoethane sulfonate

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.


Amifostine military code name wr 2721

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.


Methylene blue in the experimental treatment prevention of ifosfamide induced encephalopathy

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….


Methylene blue in the experimental treatment prevention of ifosfamide induced encephalopathy contd

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…,


Dexrazoxane icrf 187 cardioxane mechanism of action

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…


Glutamine

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.


Antimetabolites

Antimetabolites

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


Antimetabolites1

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


Antimetabolites structure

Antimetabolites-structure

What is the natural analog of:

Methotrexate, 5FU,

Cytosine arabinoside (Ara C), Gemcitabine?…..


Antimetabolites structure1

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.


Methotrexate

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….


Key enzymes inhibited by mtx and its polyglutamates

“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.


Reduced folates and thymidine synthesis

dUMP

CH2FH4

FH4

Thymidylate

Synthase

dTMP

FH2

DHFR

Reduced Folates and Thymidine synthesis


Methotrexate dhfr dependent mechanism of action

Methotrexate DHFR dependent mechanism of action

Methotrexate

Binding & inhibition

Dihydrofolate Reductase

FH4 (reduced folates)

FH2


Mechanism of resistance to mtx

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


Metabolites of mtx

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


Standard methods to neutralize mtx toxicity

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….


Experimental methods to neutralize mtx toxicity

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).


Anti folates other than methotrexte

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)….


Pemetrexed alimta mta multitargeted antifolate agent

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)


Pemetrexed alimta mta multitargeted antifolate agent1

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)


Folate status homocysteine and toxicity of mta

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)


Concomitant treatment to reduce toxicity of alimta

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.


Does vitamin supplementation impairs mta activity

Does vitamin supplementation impairs MTA activity?

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.


Trimetrexate

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


Tomudex raltitrexed a selective inhibitor of ts

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)


Cytarabine ara c gemcitabine

Cytarabine (Ara C) & Gemcitabine

  • Gemcitabine: mechanism of action….

  • Gemcitabine: mechanism of resistance….

  • Ara C: mechanism of action…..

  • Ara C: mechanism of resistance….


Gemcitabine mechanism of action

Gemcitabine-mechanism of action

  • Metabolizes intracellularly by nucleoside kinases into 2 active metabolites, dFCDP & dFCTP (gemcitabine diphosphate and gemcitabine triphosphate)(=nucleotides).

  • dFCDP inhibits ribonucleotide reductase, the enzyme responsible for catalyzing the synthesis of dCTP (inhibition of a key enzyme).

  • Gemcitabine triphosphate competes with dCTP for incorporation into DNA (incorporation of active nucleotide, derived from the antimetabolite).

  • DNA polymerase is unable to remove the incorporated gemcitabine triphosphate and repair the DNA strands.

  • In summary: Inhibition of DNA synthesis may result from both inhibition of deoxynucleotide pools (dCTP) and interference with DNA chain elongation (incorporation into DNA).


Mechanisms of resistance to gemcitabine

Mechanisms of resistance to Gemcitabine

  • Defect in nucleoside transporter system

  • Decreased activity of deoxycytidine kinase (converts gemcitabine to the monophosphate form)

  • Overexpression of ribonucleotide reductase (RR) (gemcitabine inhibits RR which results in depletion of intracellular deoxynucleotide triphosphates pool)


Cytosine arabinoside cytarabine ara c mechanism of action

Cytosine arabinoside=Cytarabine=Ara C-mechanism of action

  • Differ from the natural pyrimidine, cytidine, by the substitution of arabinose for the sugar moiety ribose.

  • Ara-C is phosphorylated to ara-CTP by deoxycytidine kinases.

  • Ara-CTP is incorporated into DNA and inhibits DNA synthesis.


Cytosine arabinoside cytarabine ara mechanisms of resistance

Cytosine arabinoside=Cytarabine=Ara -Mechanisms of resistance

  • impaired cellular uptake (nucleoside transport system)

  • decreased activation to Ara-CTP

  • Increased detoxification by cytidine deaminase (to uracil arabinoside)

  • increased intracellular deoxycytosine triphosphate (dCTP) pools.


Fludarabine

Fludarabine

  • Structure & mechanism of action….


Fludarabine1

Fludarabine

  • analogue of deoxyadenosine (=nucleotide), (=adenine+arabinose+monophsphate)

  • the mechanism of action is similar to that of Ara C:

    dephosphorylated in serum to fluoro-ara-A, which enter the cell through a carrier-mediated transport, and then phosphorylated to the triphosphate by deoxycytidine kinase…...


5fu other fluoropyrimidines

5FU & other fluoropyrimidines

  • 5FU: mechanism of action….

  • 5FU: catabolism….

  • 5FU: modulation….

  • 5FU: mechanisms of resistance….

  • 5FU pharmacogenetics and increased risk of toxicity….

  • FUdR vs. 5FU….

  • Capecitabine: mechanism of action….

  • UFT: mechaniism of action…..


5fu mechanism of action

5FU-Mechanism of action

  • 5FU undergoes intracellular activation to the following active nucleotides:

  • -fluorodeoxyuridine monophosphate (FdUMP)

  • (TS inhibition= inhibition of DNA synthesis)

  • -5-fluorouridine triphosphate (FUTP) (incorporation into RNA= RNA damage)

  • -5-fluordeoxyouridine triphosphate (FdUTP) (incorporation into DNA= DNA damage)

  • Currently, it is thought that bolus 5FU acts mainly via the RNA pathway, whereas prolonged or infusional 5FU acts mainly via damage to DNA (Sobrero AF et al. J Clin Oncol 15: 368-81, 1997).


5 fu catabolism

5 FU- Catabolism

  • The rate-limiting enzyme in 5FU catabolism is dihydropyrimidine dehydrogenase (DPD), which converts 5FU to dihydrofluorouracil (DHFU). More than 80% of administered 5FU is catabolized primarily in the liver, where DPD is abundantly expressed.

  • Because the majority of 5FU is metabolized, minor changes in catabolism can have profound effect on cytotoxicity.


Association between dpd deficiency due to mutation in the dpd gene and severe 5fu toxicity

Association between DPD deficiency due to mutation in the DPD gene and severe 5FU toxicity

A point mutation in DPD gene- Exon-14-skipping mutation- is associated with severe 5FU toxicity, especially in homozygous cases(Raida M et al. Clinical Cancer Research 7: 2832-9, 2001).

(the incidence of this mutation is approximately 1%, and , therefore, could be responsible for only small percentage of cases with severe 5FU toxicity).


Modulation of 5fu

Modulation of 5FU

  • Leucovorin: reduced folates are necessary for optimal binding of FdUMP to TS ( a ternary complex from the association of FdUMP, TS, and reduced folate).

  • Methotrexate: inhibition of purine synthesis by MTX increases the levels of phosphoribosyl pyrophosphate (PRPP), which is the cofactor required to for conversion of 5FU to FUMP….increased incorporation to RNA.

    contd


Modulation of 5fu contd

Modulation of 5FU (contd)

  • Inhibitors of DPD: 1. Uracil (UFT), 2. Eniluracil, 3. Avoidance of DPD-mediated degradation in the liver (capecitabine)


5fu mechanisms of resistance

5FU-mechanisms of resistance

  • Alterations in the target enzyme-TS

  • decreased intracellular activation

  • loss of p53 (tumor suppressor gene)


5fu pharmacogenetics and increased risk of toxicity

5FU pharmacogenetics and increased risk of toxicity

DPD deficiency is associated with increased toxicity….


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Thymidylate Synthase (TS) and Methylenetetrahydrofolate reductase (MTHFR) gene polymorphism in normal tissues as predictores of fluorouracil sensitivity

  • The analysis of gene polymorphism was performed on normal intestinal tissues and lymphocytes.

  • The response rate was significantly higher in pts with TS 3R/3R or MTHFR677 TT gene polymorphism compared with the other groups.

  • (MTHFR catalyzes an irreversible conversion of 5-10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. The former is essential to DNA synthesis by acting as a cofactor in the conversion of deoyuridine monophosphate to deoxythymidine monophosphate by TS. It also stabilizes the binding of FUDMP to TS, resulting in prolonged inhibition of the enzyme.

  • Lower activity of MTHFR will result in an accumulation of 5-10-methylenetetrahydrofolate, prolonged inhibition of TS and increased sensitivity to 5FU.

  • (Jakobsen A et al. J Clin Oncol 23: 1365-9, 2005).


Fudr vs 5fu

FUdR vs. 5FU

  • 5FU=analogue of pyrimidine base; FUdR=analogue of nucleoside (differs from 5FU only in the addition of the deoxyribose sugar moiety).Can be metabolized to FdUMP….

  • FUdR-more rapid intracellular degradation; therefore-not useful for systemic administration; is useful for intra-hepatic arterial administration.

    (FUdR has higher total body clearance and higher first-pass).


Capecitabine xeloda

Capecitabine (Xeloda)

  • An oral fluoropyrimidine (a prodrug of 5FU) that must undergo a 3-step enzymatic conversion to 5FU, with the final conversion catalyzed by thymidine phosphorylase (TP).TP activity is increased in tumor tissue compared to normal tissues.

  • Absorbed as an intact molecule through the intestinal tract, thus avoiding the intra-luminal release of 5FU.


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UFT

  • Tegafur (=Ftorafur, a prodrug of 5FU)+Uracil in a 1:4 molar ratio

  • Tegafur is metabolized to 5FU especially through hepatic microsomal cytochrome P450.

  • Uracil inhibits DPD…

  • Relatively higher concentrations of 5FU in tumor tissue….


Tpoisomerase i topoisomerase ii interactive agents

Tpoisomerase I & Topoisomerase II interactive agents

  • Topo I ….-Drugs & mechanism of action….resistance….irinotecan pharmacogenetics and increased risk of toxicity….

  • Topo II ….-Drugs & mechanism of action….


Topoisomerase i interactive agents

Topoisomerase I interactive agents

  • Camptothecins: Irinotecan (CPT-11) & Topotecan

  • Topo I “inhibition”, with production of single- strand DNA breaks:

  • Topo I breaks DNA to allow strand to uncoil.

  • The drug stops topo I from repairing the strand in one spot.

  • Completion of DNA replication is prevented.

    Note: the term “Topo I inhibition”, although commonly used, is inaccurate (since the DNA breakage by the enzyme is enhanced). “Topoisomerase I interactive agents” or “poisoning topo I” are more accurate terms.


Mechanisms for irinotecan resistance

Mechanisms for irinotecan resistance

  • Low carboxylesterase (=the enzyme that catalyzes the conversion of irinotecan to SN-38) expression

  • Overexpression of UGT (=the enzyme that inactivates SN-38)

    Xu Y and Villalona-Calero A. Ann Oncol 13: 1841-51, 2002


Irinotecan pharmacogenetics risk of severe neutropenia

Irinotecan pharmacogenetics & risk of severe neutropenia

  • SN-38 is conjucated into SN-38 glucuronide by uridine diphosphate glucuronosyltransferase 1A1 (UGT-IAI), the same isoenzyme responsible for glucuronidation of bilirubin. (UGT1A1 genetic variation has been extensively investigated in relation to hyperbilirubinemic syndromes).

  • UGT1A1 variants in pts treated with irinotecan 350 mg/m2 every 3 weeks were genotyped.

  • UGT1A1 genotype (UGT1A1*28 allele) and total bilirubin levels were strongly associated with severe neutropenia, and could be used to identify pts predisposed to severe toxicity of irinotecan.

    -Innocenti F et al. J Clin Oncol 22: 1382-8, 2004

    -McLeod H and Watters JW. J Clin Oncol 22: 1356-9,

    2004(editorial)


Topoisomerase ii as a target for drugs

Topoisomerase II as a target for drugs

  • Anthracyclines & Epipodopylotoxins

  • Topo II is a molecular target for epipodophyllotoxins and anthracyclines. These drugs form a covalent topo II-DNA complexes that prevent the enzyme from completing the religation portion of the process (and induce protein-linked breaks in the DNA).

    (Topo II inhibition with stabilization of the DNA-Topo II complex and production of DNA double-strad breaks).


Tubulin binding agents

Tubulin binding agents

  • Vinca alkaloids-mechanism of action….

  • Vinca alkaloids-mechanism of resistance….

  • Taxanes-mechanism of action….

  • Taxanes-mechanism of resistance….

  • Epothilones….


Vinca alkaloids

Vinca Alkaloids

Mechanism of action:

binding to specific site on tubulin with prevention of polymerization, inhibition of microtubule assembly and mitotic spindle formation (leading to metaphase arrest)


Vinca alkaloids1

Vinca Alkaloids

Mechanisms of resistance:

  • MDR phenomenon (P-gp overexpression)

  • mutations in alpha and beta tubulin


Mechanism of action of taxanes

Mechanism of action of taxanes

  • Bind to polymerized tubulin (beta subunit)(microtubules).

  • .Paclitaxel and Docetaxel bind to the same sites (different from vinca alkaloides).

  • Binding is reversible and stabilize the microtubules against depolymerization (induce tubular polymerization), thereby disrupting normal microtubule dynamics (halts mitosis)(and lead to arrest at G2/M phase).

    contd.


Mechanism of action of taxanes contd

Mechanism of action of taxanes contd.

Bcl-2 inactivation by taxanes:

Taxanes phosphorylates the anti-apoptotic gene, Bcl-2, leading to its inactivation and eventual cell death by apoptosis.

Haldar S et al. Cancer Res 56: 1253-5, 1996


Mechanisms of resistance to taxanes

Mechanisms of resistance to taxanes

  • P-gp overexpression ;MDR (mediated by the 170-kD p gp pump efflux, encoded by the mdr1 gene)

  • Mutations of tubulin

  • p53 mutations (taxanes induce apoptosis, induced by p53)

  • overexpression of Bcl-2


Epothilones

Epothilones

Goodin S et al. J Clin Oncol 22: 2015-25, 2004 (review)

  • A new class of microtubule targeting drugs.

  • Bind to and stabilize microtubules in a manner that is similar but not identical to that of paclitaxel.

  • May be effective in paclitaxel resistant tumors.


2975967

  • Intercalation….

  • Doxorubicin-mechanisms of cytotoxicity & cardiotoxicity…

  • Pegylated liposomal doxorubicin (Doxil)

  • Mitoxantrone….

  • Actinomycion D-mechanism of action….

  • Bleomycin….

  • Mitomycin C….

  • Mitotane….

  • Ecteinascidin (ET-743)


Dna intercalation

DNA intercalation

DNA intercalators insert a planar moiety between two adjacent base pairs in the duplex DNA (causing single-stranded and double-stranded DNA breaks).


Doxorubicin

Doxorubicin

Mechanism of cytotoxicity:

1. Topo II inhibition (the most important mechanism)

2. DNA intercalation of aglycone between base pairs, with inhibition of nucleic acid synthesis

3. Generation of hydroxyl radicals (relevant mainly for cardiac toxicity)

Contd


Doxorubicin contd

Doxorubicin (Contd)

Generation of hydroxyl radicals:

Anthracyclines undergo chemical reduction through enzimatically catalyzed or iron catalyzed pathways to yield reactive free radical intermediates (hydrogen peroxide and hydroxyl radical) that can cause oxidative damage to cell membrane proteins and DNA.

(in addition, iron-drug complexes are formed, that bind to DNA).


Pegylated liposomal doxorubicin doxil

Pegylated liposomal doxorubicin (Doxil)

  • Doxil is doxorubicin encapsulated in liposomes with surface-bound methoxypolyethylene glycol. This process is known as pegylation and protects liposomes from destruction by the mononuclear phagocyte system, which increases blood circulation time.

  • Same mechanism of action as doxorubicin

  • different P/K profile (lower peak plasma concentration) and different spectrum of toxicity & activity).


Mitoxantrone

Mitoxantrone

  • Belongs to the anthracenedione class of antitumor compounds.

  • It lacks the sugar moiety of the anthracycline drugs’ but retains the planar polycyclic aromatic ring that permits its intercalation to DNA.

  • Mechanism of action: See Doxorubicin….previous slides:1.& 2.- same as for doxorubicin; 3.-much less prominent due to impaired ability to form free radicals (therefore, less cardiotoxic).

  • Is associated with MDR.


Dactinomycin actinomycin d mechanism of action

Dactinomycin (Actinomycin D)-mechanism of action

Binds to DNA by DNA intercalation: blocks the ability to serve as a template for RNA (and DNA) synthesis.


Bleomycin mechanism of action

Bleomycin-mechanism of action

  • DNA binding (intercalation), with production of single-and double-strand breaks.

  • (generation of oxygen-free radicals)

  • BLM is inactivated by BLM hydrolase; pulmonary toxicity as a result of low enzyme concentration and high oxygen tension.


Mitomycin c mechanism of action

Mitomycin C-mechanism of action

Alkylation of DNA with formation of interstrand crosslinks; generation of oxygen free radicals.


Mitotane o p ddd

Mitotane (o,p,`DDD)

  • Used only in adrenocortical carcinoma.

  • Blocks adrenal steroid 11-beta-hydroxylation, decreases cortisol production, and leads to cortical atrophy.


Ecteinascidin et 743

Ecteinascidin (ET-743)

Ecteinascidin (ET-743) is a novel tetrahydroisoquinoline compound isolated from the marine ascidiaqn Ecteinascidia turbinata. It is a unique DNA-intercalating agent with covalent binding to the DNA minor groove. It blocks cell cycle progression in G2/M phase through a p-53-independent apoptotic process and inhibits the transcriptional activation of inducible genes.

La Cesne A et al. J Clin Oncol 23: 576-84, 2005


Recombinant hematopoietic growth factors

Recombinant hematopoietic growth factors

  • CSFs & human G-CSF….

  • Filgrastim vs. Lenograstim….

  • Mechanism of action of GCSF….

  • PEG-modification of filgrastim….

  • Antiemetics-mechanism of action….

  • Octreotide-mechanism of action in chemotherapy induced diarrhea….

  • Erythropoietic proteins….


Colony stimulating factors csfs

Colony Stimulating Factors (CSFs)

CSFs are a series of cytokines (or growth factors) that act on various stages of blood cell proliferation and differentiation.

  • The human G-CSFgeneis located on chromosome 17 q11-21 and encodes a mature polypeptide of 175amino acids with a molecular weight of 18,800 Daltons.

  • The human G-CSF is produced in-vivo in only very small quantities.


G csf biologic activities

G-CSF-Biologic activities

  • Stimulates the production of neutrophil precursors.

  • Stimulates neutrophil maturation.

  • Activates phagocytic action of mature neutrophiles.

    Overall, G-CSF regulates the formation and development of neutrophils within the bone marrow and their release into the peripheral circulation. It also increase the expression of chemotactic receptors on mature neutrophils, thereby enhancing chemotaxis and phagocytosis.


Recombinant granulocyte colony stimulating factors g csf

Recombinant Granulocyte Colony Stimulating Factors (G-CSF)

  • Filgrastim (Neupogen):

    non-glycosylated methionyl G-CSF; produced in culture by bacteria; has an additional amino acid and is not glycosylated (addition of a saccharide residue following the assembly of amino acids).

  • Lenograstim (Granocyte):

    glycosylated G-CSF; produced in culture by mammalian cells; identical to the natural molecule.


Peg modification of filgrastim

PEG-modification of filgrastim

  • Filgrastim is cleared by renal and neutrophil-mediated mechanisms. (filgrastim is a low molecular weight protein, and is freely filtered by the kidney, leading to rapid clearance).

  • Filgrastim has a plasma half-life of 3-4 hrs (and requires daily administration).

  • Proteins can be modified to significantly increase their half-life by the addition of polyethylene glycol (PEG). The addition of the 20 kDa polyethylene glycol moiety virtually eliminate renal clearance.

    contd


Peg modification of filgrastim contd

PEG-modification of filgrastim-contd

  • PEG-modification of filgrastim results in a new molecule called pegfilgrastim (neulasta) with increased plasma half-life (46-62 hrs).

  • A single fixed dose of fegfilgrastim administered once per cycle of chemotherapy was comparable to multiple daily injections of filgrastim.

    Green MD et al. Ann Oncol 14: 29-35, 2003

    Crawford J. Ann Oncol 14: 6-7, 2003 (editorial)


Erythropoietic proteins

Erythropoietic proteins

Recombinant form of erythropoietin-a glycoprotein hormone produced by the liver and kidney, and is the hematologic growth factor that regulates the proliferation, maturation, and differentiation of RBCs:

  • Epoietin alpha (Eprex)

  • Epoietin Beta (Recormon)

  • Darbepoietin alpha (Aranesp)


Drugs to prevent chemotherapy induced nausea vomiting

Drugs to prevent chemotherapy-induced nausea & vomiting


Nausea and vomiting mechanism of action of antiemetic drugs

Nausea and Vomiting-mechanism of action of antiemetic drugs

  • serotonin & dopamine: neurotransmitters that stimulate receptors in the CTZ or GIT

  • 5-HT3 receptors (Type3 5-Hydroxytryptamine receptors) antagonists (=serotonin receptor antagonists):

    ondancetron (zofran), granisetron (setron, kytril)

  • dopamine receptor antagonists:

    metoclopramide (pramin), sulpiride (modal)

  • corticosteroids:

    dexamethasone

    Contd.


Palonosteron

Palonosteron

  • Palonosteron is 5-HT3 antagonist which has the highest affinity for 5-HT3 receptor and a terminal half-life of elimination of at least 4 –times longer than any either drug of this type.

  • Single dose has protracted effect and greater efficacy in controlling delayed emesis.

    Sharma R et al. Lancet Oncol 6: 93-102, 2005 (review)

    contd.


Nausea and vomiting mechanism of action of antiemetic drugs contd

Nausea and Vomiting-mechanism of action of antiemetic drugs (Contd)

Aprepitant (Emend)-NK1 receptor antagonist:

  • Substance P (SP) belongs to the neurokinin (NK) family of neuropeptides and exerts its biological effect via interaction with the NK-1 receptor. SP is released by chemotherapy and acts on NK1 receptors.

  • Aprepitant is NK1 antagonist and has both central and peripheral action (5-HT3 receptors antagonists has mainly peripheral action).

    Contd


Nausea and vomiting mechanism of action of antiemetic drugs contd1

Nausea and Vomiting-mechanism of action of antiemetic drugs (Contd)

Aprepitant protects against both acute and delayed cisplatin-induced nausea & vomiting. Compared to standard therapy, (i.e. 5-HT3 antagonists+dexamethasone) it provides better protection(Hesketh PJ et al. J Clin Oncol 21: 4112-9,2003) sustained over multiple cycles (de Wit R et al. J Clin Oncol 21: 4105-11, 2003)

See also: Kris MG. J Clin Oncol 21: 4077-9, 2003 (Editorial).


Drugs used to treat chemotherapy induced diarrhea

Drugs used to treat chemotherapy induced diarrhea

  • Loperamide….

  • Octreotide in chemotherapy -induced diarrhea….


Loperamide imodium

Loperamide (imodium)

  • Opioids have significant constipating effect. They increase colonic phasic segmenting activity through inhibition of presynaptic cholinergic nerves in the submucosal and myenteric plexuses and lead to increased colonic transit time and fecal water absorption.

  • Loperamide is an opioid agonist that does not cross the blood brain barrier and has no analgesic properties or potential for addiction

    [Katzung BG– Basic & Clinical Pharmacology, 9th edition, (Lange and McGraw-Hill, publishers), chapter 63)pp 511-2].


Somatostatin analogs

Somatostatin analogs

Oberg K et al. Consensus report of the use of somatostatin analogs for the management of neuroendocrine tumors of the gastroenteropancreatic system. Ann Oncol 15: 966-73, 2004

  • Naive somatostatin is of limited clinical value due to very short half-life (< 3 min), which requires IV administration, and due to the post-infusion rebound hypersecretion of hormones.

  • Octreotide is a synthetic somatostatin analog. Elimination half-life after SC administration is 2 hrs and rebound secretion of hormones does not occur.

  • Somatostatin and its analogs exert their effects through interaction with somatostatin receptor subtypes 1-5. Contd….


Octreotide in chemotherapy induced diarrhea cid

Octreotide in chemotherapy -induced diarrhea (CID)

  • Octreotide is a synthetic octapeptide, that have been shown to be effective in the control of diarrhea associated with a number of conditions, including CID.

  • Acts directly on epithelial cells to reduce the secretion of a number of pancreatic and gastrointestinal hormones, which include serotonin, VIP, gastrin, insulin, glucagon, GH, secretin, motilin, and pancreatic polypeptide.

    (Wadler S et al. Recommended guidelines for the treatment of chemotherapy-induced diarrhea. J Clin Oncol 16: 3169-3178, 1998) & updated guidelines (Benson Al B et al. J Clin Oncol 22: 2918-26, 2004).


Rasburicase recombinant urate oxidase for the prevention of tumor lysis syndrome

Rasburicase (recombinant urate oxidase) for the prevention of tumor lysis syndrome


Rasburicase recombinant urate oxidase for the prevention of tumor lysis syndrome1

Rasburicase (recombinant urate oxidase) for the prevention of tumor lysis syndrome

  • Urate oxidase is an endogenous enzyme found in most mammals but not in humans. It catalyzes the oxidation of uric acid to allantoin (a metabolite 5-10 times soluble in urine than uric acid.

  • Rasburicase is a recombinant form of urate oxidase that has been found efficacious in preventing tumor lysis syndrome in patients with lymphoma.

    Coiffier B et al. J Clin Oncol 21: 4402-6, 2003


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