Topoisomerase Inhibitors

Question 1

What is the general function of topoisomerases? What 2 processes do they allow?

Answer 1

topoisomerases are nuclear enzymes that relax supercoiled ds DNA to allow DNA replication and RNA transcription

Question 2

What is the function of topoisomerase I? topoII?

Answer 2

topoisomerase I – > releases torsional strain via SINGLE strand nick

topoisomerase II – > releases torsional strain via DOUBLE strand nick

After nicks, swiveling of supercoiled DNA occurs at the nicks followed by re-ligation to relieve torsional strain

Question 3

Topo inhibitors bind and stabilize DNA/topo cleavable complex – > preventing what?

Answer 3

Topo inhibitors bind and stabilize DNA/topo cleavable complex – > preventing RE-LIGATION of DNA STRANDS

Irreversible damage results when advancing DNA replication fork encounters drug-stabilized cleavable complex – > lethal DS DNA breaks and cell death

Question 4

Name the 2 topoisomerase I inhibitors, what group are they in?

Answer 4

Topo I

Camtothecins (Topotecan & Irinotecan)

Question 5

Name the topo II inhibitors, what groups are they in?

Answer 5

Anthracyclines:

• Daunorubicin
• Doxorubicin
• Epirubicin (doxorubicin analog)
• Idarubicin (daunorubicin analog)

Anthracenediones:
- Mitoxantrone

Epipodophyllotoxins:
- Etoposide, Teniposide

Question 6

What are 2 types of resistance for the topoisomerase inhibitors (both I and II) ?

Answer 6

1. Topo II (NOT topo I) are substrates for p-glycoprotein and have MDR phenotype
2. For both topo I/II inhibitors, can see resistance w reduced topoisomerase levels or changes in the enzymes dt mutation

Question 7

Describe metabolism/elimination of topotecan

How administered?

Answer 7

Topotecan - NOT metabolized. Excreted unchanged by kidenys, so dose reduce if renal disease

PO or IV

Question 8

What is the active metabolite of irinotecan?

Answer 8

Irinotecan – > SN-38 (active)

Question 9

Irinotecan requires esterification by serum/tissue ________ to become activated to SN-38

Answer 9

Irinotecan requires esterification by serum/tissue ___carboxylesterases__ to become activated to SN-38

Question 10

Are topoisomerase I inhibitors cell phase specific?

Answer 10

NO but Cells in S-phase are very sensitive bc DNA replication requires topo I activity; topo associated ss breaks are converted to ds breaks

Question 11

What is most interesting side effect of irinotecan?

Answer 11

early onset diarrhea and other atropine-responsive cholinergic signs

Question 12

Name the doxorubicin analog and the daunorubicin analog

Answer 12

Epirubicin is the doxorubicin analog.

Idarubicin is the daunorubicin analog.

Question 13

Teniposide and etoposide are semisynthetic derivatives of podophyllotoxin, a _______ agent derived from the _______ plant

Answer 13

Teniposide and etoposide are semisynthetic derivatives of podophyllotoxin, a __antimitotic_ agent derived from the _mandrake__ plant

Question 14

DLT of etoposide at high doses: _____

What is another concern? ________

Answer 14

etoposide

1. mucositis at high dose
2. secondary leukemia

Question 15

Do etoposide and teniposide act against tubulin?

Answer 15

In contrast to the parent podophyllotoxin, the epipodophyllotoxins are INACTIVE against tubulin and are PURE inhibitors of DNA topoisomerase II

Question 16

Which is more potent, etoposide or teniposide?

Answer 16

teniposide is 10x more potent than etoposide

Question 17

Is etoposide or teniposide a radiosensitizer?

Which requires dose reduction in proportion to creatinine?

Answer 17

ETOPOSIDE for both

Question 18

Which topoisomerase II inhibitors also target free radicals?

Answer 18

Anthracyclines, Mitoxantrone

Question 19

Which topo II inhibitors also target DNA intercalation?

Answer 19

Anthracyclines, Mitoxantrone, Ellipticines

Question 20

Which topo II are considered PURE top II poisons?

Answer 20

epipodophyllotoxins (teniposide, etoposide)

**they are NON intercalators!

Question 21

What organs metabolize the epipodophyllotoxins?

Answer 21

Etoposide -hepatic metabolism and 35-40% renal excretion (unchanged)

Teniposide -probable hepatic metabolism

Question 22

Name 2 interesting toxicities of mitoxantrone

Answer 22

Cardiac dysfunction, especially after doxorubicin

BLUE tint to nails, sclerae, urine

Question 23

Name 2 mechanisms of resistance of mitoxantrone

Answer 23

1. Cross resistance w many other natural products (vincas, adria) - may be mediated by amplification of P170-glycoprotein (MDR1); others likely involved
2. Altered cellular pH in tumor cells

Question 24

Name 4 modes of cytotoxicity of mitoxantrone

Answer 24

Mitoxantrone:

1. Topo II inhibition
2. Avid binding to nucleic acids, inhibiting DNA/RNA synthesis (mode of binding includes intercalation bt opposing DNA strands, w preference for GC pairs)
3. One electron reduction (more than doxorubicin) but reduced potential for free radical generation
4. Readily induces apoptosis, like doxorubicin

Question 25

Describe the structure of mitoxantrone (anthracenedione)

Answer 25

*lacks the sugar and tetracyclic ring Is synthetic drug w 3 planar rings

Question 26

Mitoxantrone intercalates into DNA w a preference for which types of pairs? It also functions as a _________

Answer 26

1. GC pairs | 2. Topo II inhibitor

Question 27

What are the mechanisms of toxicity for the doxorubicin-related drugs: (4)

Answer 27

Cytotoxic Mechanisms for Doxorubicin-Related Drugs 1. Topo II inhibition - directly binds, preventing resealing of topo II-induced DNA cleavage 2. DNA intercalation - intercalates bt base pairs perpendicular to long axis of double helix -- > partial unwinding of helix 3. Free radical formation - Adria may undergo metabolism of its quinone ring to a SEMIQUINONE RADICAL that reacts w oxygen to yield a SUPEROXIDE RADICAL -- > oxidative dmg of cell membranes/DNA -- > death (=cardiotoxicity) 4. Affects cell membrane = bind cell membranes which can lead to death

Question 28

How does doxorubicin form a superoxide radical?

Answer 28

Doxorubicin can undergo metabolism of quinone ring, forming SEMIQUINONE RADICAL -- > which reacts w oxygen -- > superoxide radical. Leads to oxidative damage of cell membranes and DNA -- > death

Question 29

Anthracyclines: What are the products of one-electron reduction? Two-electron reduction?

Answer 29

1. One-electron reduction to semiquinone free radical -- > aerobic production of (1) superoxide anion, (2) hydrogen peroxide (3) hydroxyl radical 2. Two-electron reduction: (1) aglycone species which can be conjugated for export in bile

Question 30

1. What drug will increase doxorubicin cardiotoxicity? 2. What will bind doxorubicin, causing aggregation, and increase doxorubicin clearance? 3. What types of drugs will sensitize liver to anthracycline toxicity?

Answer 30

1. Paclitaxel - dt delayed adria clearance 2. Heparin 3. Drugs that decrease hepatic reduced glutathione pools (acetominophen, CCNU)

Question 31

What happens to Adria if.... 1. Intracellular acidosis 2. Acidification of extracellular fluid

Answer 31

1. Intracellular acidosis -- > enhanced drug accumulation bc un-ionized drug enters, gets protonated, then can't diffuse out of cell 2. Acidification of ECF -- > drug shift out of cell NOTE: Tumor masses 1cm + can have acidic ECF, so better to treat microscopic disease!

Question 32

Doxorubicin can directly inhibit which enzyme, which can produce alterations in vascular tone, w/in the heart (and tumors)

Answer 32

doxo can directly inhibit NO synthase

Question 33

One-electron reduction (adria) -- > 1. leads to formation of __A____ in presence of ___B____ , An electron is then donated to form superoxide anion, which yields ___C____. 2. Both A and __D__ cleave H2O2 to produce the ___E____ --- > this initiates apoptosis or necrosis secondary to.... - Oxidative damage to cell memb -- > can incite Fas/FasL induced apoptosis - Mitochondrial membrane injury -- > leads to release of cytochrome c - DNA base oxidation - Altered calcium sequestration - Energy loss

Answer 33

One-electron reduction (adria) -- > 1. leads to formation of _SEMIQUINONE RADICAL_ in presence of _OXYGEN_ , An electron is then donated to form superoxide anion, which yields _HYDROGEN PEROXIDE_. 2. Both SEMIQUINONE RADICAL and _IRON_ cleave H2O2 to produce the _HYDROXYL RADICAL__ --- > this initiates apoptosis or necrosis secondary to.... - Oxidative damage to cell memb -- > can incite Fas/FasL induced apoptosis - Mitochondrial membrane injury -- > leads to release of cytochrome c - DNA base oxidation - Altered calcium sequestration - Energy loss

Question 34

These 3 molecules act as intracellular protectants to reduce superoxide, H2O2, and lipid hydorperoxides, to WATER w/o formation of hydroxyl/peroxyl radicals (ADRIA)

Answer 34

1. Superoxide dismutase 2. Catalase 3. Glutathione peroxidase Glutathione also reacts w radicals and helps glutathione peroxidase

Question 35

Cardiotoxicity of doxorubicin results from these 4 things:

Answer 35

1. Low levels of cardiac CATALASE 2. High levels of mitochondria/myoglobin that enhance drug activation 3. Cardiac glutathione peroxidases are very sensitive to free radical attack -- > free radicals destroy this enzyme while also stimulating cardiac H2O2 formation 4. Doxorubicinol = alcohol metabolite of doxorubicin that is produced by TWO-electron reduction of C-13 side chain carbonyl group -- > causes toxicity

Question 36

Doxorubicin can directly inhibit which enzyme, which can produce alterations in vascular tone, w/in the heart (and tumors)

Answer 36

doxo can directly inhibit NO synthase

Question 37

One-electron reduction (adria) -- > 1. leads to formation of __A____ in presence of ___B____ , An electron is then donated to form superoxide anion, which yields ___C____. 2. Both A and __D__ cleave H2O2 to produce the ___E____ --- > this initiates apoptosis or necrosis secondary to.... - Oxidative damage to cell memb -- > can incite Fas/FasL induced apoptosis - Mitochondrial membrane injury -- > leads to release of cytochrome c - DNA base oxidation - Altered calcium sequestration - Energy loss

Answer 37

One-electron reduction (adria) -- > 1. leads to formation of _SEMIQUINONE RADICAL_ in presence of _OXYGEN_ , An electron is then donated to form superoxide anion, which yields _HYDROGEN PEROXIDE_. 2. Both SEMIQUINONE RADICAL and _IRON_ cleave H2O2 to produce the _HYDROXYL RADICAL__ --- > this initiates apoptosis or necrosis secondary to.... - Oxidative damage to cell memb -- > can incite Fas/FasL induced apoptosis - Mitochondrial membrane injury -- > leads to release of cytochrome c - DNA base oxidation - Altered calcium sequestration - Energy loss

Question 38

These 3 molecules act as intracellular protectants to reduce superoxide, H2O2, and lipid hydorperoxides, to WATER w/o formation of hydroxyl/peroxyl radicals (ADRIA)

Answer 38

1. Superoxide dismutase 2. Catalase 3. Glutathione peroxidase Glutathione also reacts w radicals and helps glutathione peroxidase

Question 39

Cardiotoxicity of doxorubicin results from these 4 things:

Answer 39

1. Low levels of cardiac CATALASE 2. High levels of mitochondria/myoglobin that enhance drug activation 3. Cardiac glutathione peroxidases are very sensitive to free radical attack -- > free radicals destroy this enzyme while also stimulating cardiac H2O2 formation 4. Doxorubicinol = alcohol metabolite of doxorubicin that is produced by TWO-electron reduction of C-13 side chain carbonyl group -- > causes toxicity

Question 40

Doxorubicin increases intracellular iron by: 1. ____ 2. _____

Answer 40

1. Releasing it from ferritin and microsomes | 2. Increasing iron uptake into cells by transferrin-mediated process (enhancing intracellular iron availability)

Question 41

The hydroquinone structure of doxo/dauno binds what form of iron? This leads to what 3 effects:

Answer 41

FERRIC IRON (doxo is v potent chelator of other metal ions too) 1. Oxidative destruction of MEMBRANES 2. Oxidation of SULFHYDRYL groups 3. Binding to DNA (differently than intercalation mechanism)

Question 42

Dexrazoxane is an iron chelator that prevents doxorubicin-induced lipid peroxidation and cardiac toxicity. It is a PRODRUG that undergoes _____ to become active. This is markedly enhanced after uptake into cardiac myocytes, with rapid conversion to active drug.

Answer 42

hydrolysis.

Question 43

Two electron reduction leads to formation of ____. These are far less toxic than parent drug. Two electron reduction mainly leads to ______ of anthracyclines.

Answer 43

Two electron reduction leads to formation of _deoxyaglycone metabolites_. These are far less toxic than parent drug. Two electron reduction mainly leads to __inactivation_ of anthracyclines.

Question 44

Cell membrane damage from anthracyclines leads to activation of which pathway? (a phospholipid in the cell membrane) This leads to formation of _____ (a bioactive lipid) --- > activates PKC and leads to ______ membrane permeability --- > apoptosis

Answer 44

Cell membrane damage from anthracyclines leads to activation of which pathway? SPHINGOMYELIN PATHWAY This leads to formation of CERAMIDE (a bioactive lipid) --- > activates PKC and leads to _MITOCHONDRIAL_ membrane permeability --- > apoptosis

Question 45

Cell membrane damage from anthracyclines leads to alterations in which pathway? ______ In the heart, it decreases ____ phosphorylation (critical for cell survival); this is blocked by pretreatment with dexraz

Answer 45

Cell membrane damage from anthracyclines leads to alterations in which pathway? PI3K/AKT PATHWAY In the heart, it decreases AKT/ERK phosphorylation (critical for cell survival); this is blocked by pretreatment with dexraz

Question 46

What is the major pharmacokinetic risk for adria-induced cardiotoxicity? How do you lower the risk?

Answer 46

PEAK DRUG CONCENTRATION is major risk. Prolonged infusion will significantly reduce risk.

Question 47

Are the topo II drugs given IV or PO?

Answer 47

All given IV except Idarubicin, which is given PO

Question 48

Anthracyclines induce apoptosis by: 1. stimulating interaction between ______ & ______ . 2. This leads to _______ generation (secondary to cell membrane damage) -- > increased mitochondrial membrane permeability and activation of proapoptotic _________ and ___________ protein phosphatases. (note: altered mitochondrial membr permeability leads to cytochrome c release which can cause apoptosis AND necrosis) 3. Causing ______ damage (of cell membrane, mitochondrial membrane, DNA) 4. Leading to elevated ______.

Answer 48

Anthracyclines induce apoptosis by: 1. stimulating interaction between _FAS_ & _FASL_ . 2. This leads to _CERAMIDE_ generation (secondary to cell membrane damage) -- > increased mitochondrial membrane permeability and activation of proapoptotic _CASPASES__ and __SERINE-THREONINE__ protein phosphatases. (note: altered mitochondrial membr permeability leads to cytochrome c release which can cause apoptosis AND necrosis) 3. Causing __FREE RADICAL_ damage (of cell membrane, mitochondrial membrane, DNA) 4. Leading to elevated __p53_.

Question 49

Doxorubicin is metabolized in liver to _____

Answer 49

doxorubicinol (which retains some activity) and other metabolites...all excreted in bile

Question 50

5 modes of resistance for anthracyclines

Answer 50

1. P53 mutations (diminish apoptosis, increase doxorubicin resistance) 2. Enhanced drug efflux (P170 glycoprotein-mediated anthracycline efflux) - also, vincas, actino D, epipodophyllotoxins 3. Altered topo II activity 4. Altered free radical biochemistry, sensitivity to apoptosis (via overexpression of bcl-2, p53 mutations...) 5. Coadministration of heparin (increased doxorubicin clearance)

Question 51

P170-glycoprotein-mediated anthracycline efflux reversed with which 4 drugs?

Answer 51

verapamil cyclosporine A calmodulin inhibitors tamoxifen