Cytotoxic Chemotherapy (2)

describe the MOA of anthracyclines (antitumour antibiotics)

• DNA intercalation- drug physically inserts between base pairs and causes distortion of the DNA helix and inhibition of RNA transcription and DNA synthesis

• Poisoning of topoisomerase II- leads to stabilisation of the topoisomerase II-DNA cleavage complex and consequently the accumulation of dsDNA breaks

  • Topo-II is the enzyme responsible for untangling the DNA during replication

• Redox cycling of the quinone group which causes generation of ROS

describe the MOA of doxorubicin (DOX), how it is administered and when it is used

MOA of DOX- it intercalates preferentially in GC-rich regions of DNA

Administered intravenously

Used for breast cancer, lymphomas, sarcomas, leukaemia and solid tumours

though cell cycle non-specific which cycle phase are cells more sensitive to DOX and why

cytotoxicity of DOX is enhanced at the S/G2 phase

• S phase- cells have increased sensitivity in this phase due to active DNA replication and topoisomerase II-mediated strand cleavage

• G2 phase- it is dependent on topo II for decatenation of sister chromatids before mitosis

describe the side effects of DOX and what it means to be a vesicant chemotherapeutic

Side effects- emetogenic, alopecia, harmless urine discolouration for 1-2 days post-dose (this can be alarming to patients if they are not warned)

vesicant chemotherapeutic

• if the drug leaks out of the vein during infusion it causes severe tissue damage that may create a need for skin grafting

• Once the drug leaks it out it enters healthy cells, binds to their DNA and kills the cells which causes them to undergo lysis

• Lysis causes the release of DOX into the environment and it is then taken up more normal cells

• DOX is cleared slowly by the local microenvironment so it continues to have prolonged toxicity and the injury evolves over time leading to severe tissue

name the major organ toxicities of DOX and state which is the primary dose limiting toxicity

• Cardiotoxicity- this is the primary dose-limiting toxicity

• Myelosuppression

• Mucositis (inflammation and ulceration of GI mucosa)

• Hepatotoxicity (though less prominent)

name and describe the 3 types of cardiotoxicity DOX causes

acute (rare)

• ECG changes, transient arrhythmias, rarely myocarditis

chronic (dose limiting)

• Progressive dilated cardiomyopathy resulting from cumulative cardiomyocyte injury

• OS, MD, TOP2-beta inhibition causes reduced left ventricular ejection fraction

• progress to heart failure due to myofibrillar deterioration and intracellular calcium dysregulation

• huge risk seen at total doses ≥450 mg/m²

late/delayed

• manifest years/decades after treatment, particularly in childhood cancer survivors

name the mechanisms of DOX cardiotoxicity

oxidative stress

disrupts mitochondrial function

topoisomerase II

ferroptosis

calcium dysregulation and cardiomyocyte injury

Mechanisms of cardiotoxicity- describe the process of DOX-mediated oxidative stress

• DOX is enzymatically reduced into an unstable semiquinone radical

• This radical reacts with oxygen and that triggers a redox cycle that amplifies ROS production

• ROS induces lipid peroxidation, protein oxidation and DNA damage

• Cardiomyocytes are particularly susceptible due to their low antioxidant capacity (e.g. low levels of catalase)

Mechanisms of cardiotoxicity- describe how DOX disrupts mitochondrial function

• DOX impairs mitochondrial function via ROS-dependent and independent mechanisms

  • ROS-dependent: mitochondrial lipid peroxidation, impaired ATP production, oxidative damage to respiratory chain complexes

  • ROS-independent (inhibition of TOP2-beta): altered transcription of genes regulating mitochondrial biogenesis and oxidative phosphorylation which leads to impaired mitochondrial respiratory chain function

• Mitochondrial dysfunction promotes the opening of mPTP which leads to a loss of mitochondrial membrane potential and activation of cell death pathways

• Interaction of DOX with cardiolipin in the inner mitochondrial membrane promotes mitochondrial  retention of DOX and facilitates ROS generation, contributing to a self-amplifying cycle of redox cycling, energy failure and cell injury in the cardiomyocytes

Mechanisms of cardiotoxicity- describe the effect of DOX on topoisomerase II

• ATP-dependent enzyme that regulates the DNA topology- supercoiling, decatenation and chromosome segregation

• It creates transient dsDNA breaks to relieve torsional stress and this is essential for DNA replication and transcription

• 2 isoforms: alpha (dividing cells, antitumour effects), beta (non-dividing, cardiotoxicity)

• DOX poisons Topo-II in tumour cells (mainly alpha) and cardiomyocytes (beta) this leads to persistent dsDNA breaks, increased p53 signalling and apoptosis

• DOX also causes cytotoxicity through Topo-II independent mechanisms

  • forms covalent DNA adducts after formaldehyde-mediated activation

Mechanisms of cardiotoxicity- describe how DOX causes ferroptosis

• this is an iron-dependent, non-apoptotic cell death process

• DOX disrupts iron regulation via iron regulatory proteins IRP1/2, leading to increase in labile iron pool

• Excess iron drives Fenton chemistry (Fe²⁺ + H₂O₂ = hydroxyl radical)

• also enhances DOX redox cycling which increases ROS generation and leads to lipid peroxidation

• DOX also causes loss of glutathione peroxidase 4 (GPX4) activity due to glutathione depletion or oxidative inactivation which prevents GPX4-mediated detoxification

Mechanisms of cardiotoxicity- describe how DOX causes calcium dysregulation and cardiomyocyte injury

• DOX disrupts SR Ca2+ storage and homeostasis

• DOX binds and blocks SERCA2a and RyR2 which reduces Ca2+ reuptake into SR and increases abnormal Ca"+ leak respectively

• RyR Ca2+ leak is partly driven by ROS-mediated oxidative modification of RyR2

• As a results, there is reduced Ca2+ storage in SR and increased cytosolic Ca2+

• Mitochondria take up the excess Ca2+ leading to mPTP opening, CytC release and apoptosis

• Cytosolic Ca+ overload also activates calpain-mediated proteolysis and other Ca2+ dependent pro-apoptotic pathways

describe the MOA of mitotic inhibitors (M-phase specific) and name 2 examples

• These drugs disrupt the mitotic spindle dynamics, thereby preventing proper chromosome segregation

• leads to activation of the spindle assembly checkpoint which consequently leads to metaphase arrest and apoptosis

• 2 subgroups:

  • Vinca alkaloids e.g. Vincristine 

  • Taxanes e.g. docetaxel

describe the MOA of vinca alkaloids

• MOA- bind to vinca domain on the beta subunit of tubulin, which is the building blood of MT and inhibits its polymerisation and therefore the formation of functional mitotic spindles

describe the MOA of taxanes

MOA- enter cells via passive diffusion (primary route) or membrane transporters (depending on cell type) and bind to beta tubulin. It stabilises the microtubules, prevents depolymerisation, thereby disrupting spindle function

describe the toxicity profile for taxanes and how some can be managed

• narrow therapeutic window

• dose limiting toxicity is the myelosuppression especially neutropenia

• peripheral neuropathy

  • stocking glove pattern- numbness and tingling in hands and feet; moves distally to proximally

  • due to disrupted axonal MT transport

• fluid retention

  • hallmark of docetaxel not vincristine

  • requires dexamethasone premedication

• hypersensitivity

  • managed with corticosteroids and antihistamines

• nail changes and alopecia

• hepatoxicity due to metabolism by CYP3A4/5

MOA of cytotoxicity of antimetabolites and name 2 examples

• S-phase specific agents that result in DNA synthesis failure

• examples- methotrexate and 5-fluorouracil

describe the MOA of methotrexate and the toxicity profile

• MOA- inhibit dihydrofolate reductase (DHFR) which prevents generation of tetrahydrofolate. This depletes folate pools required for synthesis of thymidylate (dTMP) and purines and therefore impairs DNA replication 

TOXICITY PROFILE

• Myelosuppression is common and dose-limiting

• GI toxicity- mucositis (dose limiting), stomatitis

• Hepatotoxicity especially with chronic use

• Nephrotoxicity because it crystallises in renal tubules

  • Clinicians alkalinise the urine using sodium bicarbonate to avoid this

• Cardiac effects are rare

describe the metabolism of 5-fluorouracil and how this relates to its MOA

• This can be converted to multiple active metabolites:

  • FUTP- this incorporates into the RNA and disrupts RNA processing and function

  • FdUMP- this inhibits thymidylate synthase and blocks conversion of dUMP to dTMP which leads to dTMP depletion, thymidine deficiency and 'thymineless death'

describe the toxicity profile of 5-fluorouracil

• Myelosuppression is common

• GI toxicity- mucositis (dose limiting)

• Neurotoxicity

• Hand-foot syndrome- patient experiences redness, soreness and peeling in palm and soles of their food

• Important to check for DPD deficiency as 5% of population have partial or complete deficiency of this enzyme → 5-FU accumulation

• Can cause acute coronary vasospasm

describe the MOA of leucovorin (folinic acid) in the presence of MTX or 5-FU

MTX

• rescue cells from MTX effect by bypassing DHFR block and providing reduced folate to healthy cells.

• This restores nucleotide synthesis in normal cells

• needs to be gap between MTX and leucovorin administration

• because MTX needs to damage tumour cells then leucovorin is given to rescue the normal cells

• Cancer cells have reduced capacity to take up leucovorin and retain MTX longer than normal cells

5-FU

• inhibits thymidylate synthase to stop DNA synthesis 

• does this increasing 5,10-methylene-THF which is the specific form required to stabilise binding of FdUMP to thymidylate synthase

• leads to strong TS inhibition and increases drug efficiency

describe the peripheral pathway of chemotherapy induced nausea and vomiting

peripheral (within 24hrs)

• This is primarily driven by 5HT

• It damages enterochromaffin cells in the small intestine, release large amounts of 5HT

• 5HT binds to 5HT3R on the vagal afferent nerves; these receptors are the primary drivers of acute emesis

• Signals are then transmitted to the vomiting centre

• Clinical implication- 5HT3R antagonists such as ondansetron are effect in preventing acute CINV

describe the central pathway of chemotherapy induced nausea and vomiting

central

• Dopamine acts primarily on D2R area postrema in the CTZ located in the medulla outside the BBB

• This means that the CTZ is directly exposed to drugs and toxins

• This sends signals to the NTS and activates the vomiting central

describe the delayed phase (>24hrs) pathway of chemotherapy induced nausea and vomiting

• Driven by SP which is the primary driver of delayed emesis

• SP acts on NK1R in the brainstem

• Clinical implications- NK1R antagonists e.g. aprepitant are used to target this pathway

define the emetogenic risk

The emetogenic risk is the likelihood that a medication will cause nausea and vomiting (emesis) without preventative treatment

using specific examples of drugs with different levels of emetogenic risk, state the drug used for antiemetic prophylaxis

Antiemetic prophylaxis depends on the emetogenic risk of the chemotherapy regimen

corticosteroids e.g. dex enhance antiemetic efficiency

describe mucositis as a chemotherapy induced side effect

• Mucositis is inflammation and ulceration of the oral and GI mucosa caused by damage to rapidly dividing mucosal epithelial cells, including intestinal crypt cells

• MOA- ROS generation and DNA damage leads to epithelial apoptosis which activates NFkB signalling, induces proinflammatory cytokine release (TNF-alpha, IL1-beta, IL6) which causes mucosal breakdown and ulceration

• clinically seen as stomatitis- pain, dysphagia, reduced oral intake

describe the chemotherapy agents associated with mucositis and how it be managed

• associated with antimetabolites, some alkylating agents and anthracyclines

management

• oral hygiene, analgesics such as cryotherapy where ice chips are given during 5-FU infusion, and palifermin which is a keratinocyte growth factor given to high risk patients

  • The ice chips cause vasoconstriction and so slow down how fast the drug reaches the GI are

discuss the clinical implications of known cell cycle specificity of tumour cells

• Tumours contain cells in different cell cycle phases at any given time

• Resistance- repeated exposure kills sensitive cancer cells but allows resistant ones to survive and these may repair DNA damage or change how they divide making the drug less effective over time

• Combination regimens are now designed to: target tumour cells across multiple cycle phases by combining phase specific and non-specific agents, exploit different cytotoxicity mechanisms, reduce likelihood of resistance and use non-overlapping toxicity profiles (e.g. avoid 2 nephrotoxic agents)