Mechanism of Action of Commonly Used Anti-cancer Agents

Question 1

mustards were first used to treat what in the 1940s

Answer 1

leukemia and lymphoma

Question 2

examples of nitrogen mustards

Answer 2

chlormethamine, mechlorethamine, mustine

very reactive and excessively toxic

Question 3

nucleophilicity is controlled by what

Answer 3

steric, electronic and hydrogen bonding effects

Question 4

alkylating agents are potent

Answer 4

electrophiles

Question 5

intrastrand definition

Answer 5

linking between two bases on the same strand

Question 6

interstrand definition

Answer 6

cross-linking of two separate strands

Question 7

alkylating agents react with many nucleophiles other than DNA bases…

Answer 7

Thiols especially reactive, amines also reactive

Cysteine and lysine residues in proteins. Protein adducts occasionally trigger an allergic response

Glutathione in cells can react with alkylating agents and ”quench” their activity.=

Question 8

toxicity to cancer cells results from _____________ and _____________. DNA-protein cross-links also inhibit DNA function

Answer 8

DNA alkylation; DNA cross-linking

Question 9

cells are more susceptible in late G1 and S phases of cell cycle but alkylating agents are considered ____________

Answer 9

non cell cycle specific

Question 10

mechlorethamine (mustargen, mustine, chlormethine) facts

Answer 10

First used clinically in the 1940’s – An important prototype that resulted in the development of field of chemotherapy. Not used commonly anymore.

Extremely reactive compound – half-life in plasma ~ 1 min

• Rapidly alkylates all nucleophiles – modifies DNA, RNA and protein
• Antitumor activity correlates with DNA cross-links

Side effects for all alkylators

• Myelosuppression
• Nausea and vomiting
• Carcinogenic and teratogenic

Question 11

Two strategies to reduce reactivity and increase selectivity of nitrogen mustards

Answer 11

1. decrease nucleophilicity of nitrogen by adding aryl groups
2. pro-drug strategy

Question 12

examples of compounds that decrease nucleophilicity of nitrogen by adding aryl groups

Answer 12

chlorambucil

bendamustine

melphalan

Question 13

examples of compounds that utilize the pro-drug strategy

Answer 13

cyclophosphamide

Question 14

for chemically stable prodrugs…

Answer 14

…requires hydroxylation by hepatic Cytochrome P450

Question 15

cyclophosphamide facts

Answer 15

most useful and commonly used alkylating agent

SE modest compared to most alkylating agents:

• mild bone marrow toxicity: sparing to marrow stem cells (and platelets) because of high ADH levels in these cell types
• hemorrhagic cystitis: acrolein is toxic to bladder mucosa

also in this class: ifosfamide has longer half life than cyclophosphamide, but also has increased CNS toxicity

Question 16

what is mesna

Answer 16

Cyclophosphamide is toxic to the bladder
-Acrolein accumulates in urine and damages bladder mucosa

Mesna containing a charged (anionic) sulfonate group so it does not penetrate cells

• Anion transporters in proximal tubule excrete via kidney
• Mesna accumulates in the urine

The free thiol on Mesna reacts with and inactivates acrolein metabolites in urine

Administered with cyclophosphamide to block hemorrhagic cystitis

Question 17

what are nitrosoureas

Answer 17

Chemically unstable - non-enzymatic conversion to diazonium ion

Diazonium ion-highly electrophilic and has extremely short half-life
-Conversion to diazonium ion must take place inside cell

examples: bis-chlorethylnitrosourea (BCNU) = carmustine

also in this class = lomustine

Question 18

nitrosoureas: highly lipophilic alkylating agents which means they…

Answer 18

…readily cross the blood-brain barrier

used to treat glioblastoma multiforme and other brain tumors. structurally different DNA adducts so different DNA repair mechanisms

toxicities typical of other alkylating agents: significant bone marrow toxicity: myelosuppression is delayed (3-4 weeks) and prolonged. requires longer interval between doses than other agents. suggests that toxicity is greater to earlier progenitor cells

Question 19

alkyl sulfonates facts

Answer 19

Given in high doses with cyclophosphamide before a bone marrow transplant – designed to eradicate all hematopoetic cells

Pulmonary fibrosis - “busulfan lung”

example compound: busulfan

Question 20

Mitomycin C (Mutamycin)

Answer 20

Aziridine-containing natural product

Similarities to nitrogen mustard compounds. Functions as an alkylating agent, although toxicity pattern differs slightly from other alkylating agents

Myelosuppression is dose-limiting

Question 21

mitomycin C can form ___________ adducts

Answer 21

bifunctional

Question 22

What alkylator reacts via the more reactive (and positively charged) aziridinium?

Answer 22

nitrogen mustards

Question 23

Monoalkylating agents definition

Answer 23

alkylates DNA but does NOT cross-link DNA

still inhibits DNA replication by blocking activity of replication enzymes on DNA

example compound: temozolomide, decarbazine (DTIC)

Question 24

temozolomide (TMZ)

Answer 24

Inhibits DNA, RNA and protein synthesis

Can be combined with other alkylating (cross-linking) agents

Toxicities typical of other alkylating agents but generally less severe

• Bone marrow toxicity is milder
• Nausea and vomiting is severe
• Temozolomide is 100% bioavailable (Readily crosses blood-brain barrier – used for brain cancers)

Question 25

temozolomide fact to remember

Answer 25

readily crosses blood-brain barrier. Used for brain cancers

Question 26

decarbazine

Answer 26

Also converted to MTIC intermediate, but requires activation via N-demethylation catalyzed by hepatic P-450 enzyme
-N-demethylated intermediate is unstable and has a short half-life

Dacarbazine is poorly absorbed (IV administration)

Question 27

TMZ resistance

Answer 27

promoter methylation predicts whether glioblastoma patients respond to TMZ

Question 28

Temozolomides alkylate DNA via a diazonium intermediate. What crosslinker also alkylates via a diazonium?

Answer 28

carmustine

Question 29

platinum drugs

Answer 29

Originally described as a therapeutic when a soluble platinum complex generated at platinum electrodes inhibited cell division in E. coli

Covalent crosslinkers, but not alkylating agents (no alkyl groups)

Cisplatin is the original prototype Square planar complex with leaving groups having cis geometry (not trans!)

Question 30

platinum drugs continued

Answer 30

Cisplatin undergoes reversible hydrolysis in aqueous solution

• Equilibrium favors Cisplatin in plasma (where there is a high chloride concentration)
• Equilibrium favors aquo form inside cell (low chloride concentration)
• Aquo form is highly reactive and a potent electrophile

Aquo form reacts rapidly with other nucleophiles, especially thiols

Question 31

platinum crosslink geometry

Answer 31

Aquo form reacts primarily at Guanine N-7 and Adenine N-7 in DNA

Because of bond lengths and angles, cross-links are often INTRASTRAND (but can be interstrand)

Intrastrand cis-Pt cross-link imposes severe geometrical constraints on DNA

• Introduces sharp bend in cross-linked strand
• Lesion not readily repaired by standard DNA repair enzymes

Question 32

Cisplatin

Answer 32

Highly effective agent for many solid tumors

Drug requires non-enzymatic conversion to the active aquo form

Aquo form produces primarily intrastrand cross-links

Cross-links generally form more slowly than for other alkylating agents

Some tumor cells more sensitive in G1 than in S phase

Question 33

Cisplatin SE profile

Answer 33

Dose-limiting nephrotoxicity (proximal tubule)

Severe nausea and vomiting (centrally mediated)

Minimal bone marrow toxicity (so good in combination with other drugs)

Peripheral neuropathy – related to cumulative dose

Ototoxicity (hearing loss)

Question 34

Drug Resistance - General Mechanisms

Answer 34

Multiple cellular mechanisms are involved but the major mechanisms are specific for alkylating agents and platinum drugs

Increased expression of DNA repair enzymes

• Excision repair – excise alkylated base(s) and replace
• Alkyl group removal – Guanine O-alkyl transferase

Increased intracellular concentration of non-protein thiols, especially glutathione

• Glutathione is a cysteine-containing tripeptide (-Glu-Cys-Gly)
• Free thiols have extremely high reactivity toward electrophilic intermediates
• Thiols intercept the reactive intermediates of alkylating agents
• Free thiol levels > 10-fold higher in alkylating agent-resistant cells

Increased expression of cellular glutathione S-transferase (GST)

• GST’s are a family of Phase II metabolic enzymes – P1-1 is key isozyme
• GST catalyzes the reaction of glutathione with alkylating agents (parent drugs as well as reactive intermediates)