Cytotoxic Anticancer Drugs

Cytotoxic Anticancer Drugs

• Cytotoxic anticancer drugs' mechanisms of action, achievements, and limitations are covered in the notes.
• The notes review SHP3002 Molecular Pharmacology and SHB4015 Medical Pharmacology III.

Objectives

• Understand the origins of chemotherapy and its role in cancer treatment.
• Understand the different classes of cytotoxic drugs and their mechanisms of action.
• Understand the achievements and limitations of cytotoxic drug-based chemotherapy.

What is Chemotherapy?

• Cancer is treated in 4 main ways: surgery, radiotherapy, chemotherapy, and biologic therapy.
• Surgery and radiotherapy are most effective when the disease is localized.
• Once the disease has spread, the effectiveness of surgery and radiotherapy decreases.
• Chemotherapy is the administration of drugs to treat cancer.
• It's a systemic therapy capable of seeking and destroying cancerous cells throughout the body.

The Origin of Chemotherapy

• Paul Ehrlich (1854 – 1915) is considered the "father" of chemotherapy.
• Ehrlich's lab discovered arsphenamine, the first effective medicine for syphilis.
• Arsphenamine is toxic to the bacterium Treponema pallidum, which causes syphilis.
• Ehrlich screened many chemical compounds hoping for a drug with antimicrobial activity that wouldn't harm human patients, naming this approach "chemotherapy."
• He coined the term "magic bullet" to describe a compound that selectively targets a disease-causing organism.
• These principles are highly relevant in drug discovery today.

The Origin of Cancer Chemotherapy

• Chemotherapy began with sulphur mustard, a chemical warfare agent, which caused chemical burns and blisters.
• Sulphur mustard was later found to shrink tumors in mice.
• It caused suppression of hematopoiesis, significantly decreasing white blood cell counts.
• Nitrogen mustard, a derivative of sulphur mustard, became the first anti-cancer chemotherapy drug in the 1940s.

Nitrogen Mustards

• Sulphur mustards were too reactive, so mechlorethamine (mustine, nitrogen mustard) was developed
• Yale pharmacists Goodman and Gilman showed nitrogen mustard shrank tumors in mice.
• In 1946, they administered it to a lymphoma patient who saw his cancer shrink.
• Other scientists reported exposure to mustard gases caused myelosuppression

Cytotoxic Anti-Cancer Drugs

• Different chemotherapy agents have different mechanisms of action.
• They target different parts of the cell cycle.
• The cell cycle includes G1 phase (organelle duplication), S phase (DNA duplication), G2 phase (cell preparation for mitosis), and Mitotic phase.

The M Phase of the Cell Cycle

• Prophase: Centrosome duplicates and microtubules form.
• Metaphase: DNA aligns in the middle of the cell, and microtubules attach to centromeres.
• Anaphase: Chromosomes separate and migrate to opposite poles of the cell.
• Telophase: Constriction of cell membrane, new nuclear membrane forms, and cell prepares for cleavage.

Classes of Cytotoxic Anti-Cancer Drugs

• These drugs are classified as "cytotoxic anti-cancer drugs".
• Alkylating Agents
  • cyclophosphamide
  • ifosfamide
  • temozolomide
• DNA Linking Agents (platinum compounds)
  • carboplatin
  • cisplatin
  • oxaliplatin
• Anti-metabolites
  • capecitabine
  • 5-fluorouracil
  • methotrexate
  • gemcitabine
  • pemetrexed
• Anti-tumor Antibiotics
  • mitomycin
  • bleomycin
  • epirubicin
  • doxorubicin
• Plant Alkaloids and Microtubule Inhibitors
  • etoposide
  • paclitaxel
  • docetaxel
  • irinotecan
  • vincristine
  • vinorelbine
  • eribulin

Alkylating Agents

• An alkylating agent attaches an alkyl group (CnH2n+1) to DNA.
• It covalently binds to guanine, forming crosslinkages in DNA strands.
• Alkylating DNA arrests the cell cycle by making it difficult for DNA to unravel and replicate, normally arresting the G1 or S phase.
• Therefore, alkylating agents are anti-proliferative drugs.

Mechanism of Action of Alkylating Agents

• SN2 reactions are the chemical basis for their mechanism of action.
• Nucleophilic substitution involves a nucleophile attacking an electrophile.
• A nucleophile is attracted to positively charged nuclei and is generally negatively charged.
• Electrophiles are attracted to electrons and have a positive charge.
• The nucleophile attacks the carbon atom attached to the leaving group, displacing it.

Nitrogen Mustard

• The amine nitrogen causes displacement of Cl to form an aziridine ring.
• The N7 position of guanine on DNA is a good nucleophile (relative negative charge) and this attacks the aziridine ring leading to alkylation.
• A second reaction crosslinks 2 guanines on opposite strands: an inter-strand DNA crosslink.

Nitrogen Mustard Compound Development

• Sulphur mustards too toxic, so scientists developed chlormethine.
• Aliphatic mustards (chlormethine) have sufficient therapeutic index to be used in humans.
• Aromatic mustards (chlorambucil) are less electrophilic, react more slowly with DNA, and can be administered orally.
• Estramustine targets oestrogen-dependent tumors.
• Cyclophosphamide is an attempt to release the mustard agent through enzymatic degradation.
• Melphalan enhances cellular uptake via the phenylalanine-transport mechanism.

Temozolomide

• Temozolomide was developed by scientists at Aston University without targeting specific proteins; it was "chemistry led."
• The lab was interested in tetrazines, which led to imidazotetrazine ring structure and eventually temozolomide.
• Clinical trials for Temozolomide were sponsored by CRUK.
• Temozolomide treats certain brain tumors such as glioblastoma, astrocytoma, neuroendocrine tumors, and adrenal gland cancer.

Temozolomide

• A prodrug requiring chemical activation.
• Stable under acidic conditions.
• At physiological pH, hydrolytic cleavage of the tetrazinone ring releases an unstable monomethyl triazine.
• Undergoes further cleavage to generate the methyldiazonium ion.
• Results in DNA methylation of guanine.

Platinum Compounds

• Cisplatin is one of the most active anti-cancer drugs and is platinum-based.
• Used to treat sarcomas, carcinomas (eg, lung, ovarian cancer), lymphomas, and germ cell tumours (testicular cancer).
• The class now includes carboplatin and oxaliplatin.
• Serious toxic ADRs from DNA damage, but can also interact with proteins.

Discovery and Development of Cisplatin

• A biophysicist at Michigan investigated electrical currents' role in cell division.
• Electrical current was passed through ammonium chloride buffer with E.coli.
• "Inert" platinum electrodes supplied the current.
• E.coli cells elongated due to inhibition of cell division.
• The effect was due to hydrolysis products from the platinum electrodes, not electricity.
• Discovery of Cisplatin a revolutionised treatment, particularly of testicular cancer.

Mechanism of Action of Cisplatin

• Cisplatin acts like an alkylating agent.
• High chloride levels in plasma keep the drug intact after administration.
• In the cell, chloride levels fall and are replaced by water (aquation).
• This forms a reactive, positively charged species that can't leave.
• Reacts with the N7 position of guanine, forming a mono adduct.
• The second chloro group undergoes a similar reaction creating an intra-strand crosslink.
• This causes apoptosis or DNA repair.

Consequences of DNA Alkylation

• Types of damage that can occur:
  • Inter-strand crosslink (crosslink between two strands of DNA)
  • Intra-strand crosslink (crosslink on the same strand of DNA)
  • Mono-alkylation (no crosslinking)
• Consequences:
  • Inhibits cell replication
  • Blocks transcription
  • Induces apoptosis

Anti-Metabolites

• Anti-metabolites interfere with normal cell metabolism of nucleic acids.
• Broadly speaking, they interrupt DNA/RNA metabolism/production.
• This arrests the cell cycle during the S phase.

Anti-Metabolites Example

• Thymine is disrupted from thymidine, making it difficult to replicate DNA.

Synthesis of Thymidine

• Thymidine is synthesised when deoxyuridine monophosphate (dUMP) is converted to deoxythymidine monophosphate (dTMP).
• Thymidylate synthase uses methylene tetrahydrofolate as the methyl group donor in the process of synthesising dTMP.
• In the process of thymidine synthesis, the methyl THF is converted to dihydrofolate (DHF).
• Conversion of DHF back to active THF requires dihydrofolate reductase.

Anti-Metabolites Disrupt Synthesis of Thymidine

• 5-Fluorouracil (5-FU) inhibits thymidylate synthase.
• Methotrexate (MTX) inhibits dihydrofolate reductase.
• Mercaptopurine (6MCP) and thioguanine (6TG) inhibit production of purines.

Development of Methotrexate

• Folic acid was discovered to worsened leukaemia in the 1900s.
• A diet deficient in folic acid could, conversely, produce improvement.
• In 1947, aminopterin induced remission in children with acute lymphoblastic leukemia.
• By 1950, methotrexate (amethopterin) was being proposed as a treatment for leukemia.
• In 1956, Methotrexate was better than aminopterin.

Development of Methotrexate

• Folic acid is key to DNA metabolism.
• Folic acid is needed for the synthesis of thymidine and purines.
• Folic acid stimulates cell proliferation in acute lymphoblastic leukaemia (ALL) cells.
• Methotrexate is a folate analogue and inhibitor.
• It prevents folate-requiring enzymes such as dihydrofolate reductase.
• This is rational drug design

Mechanism of Action of Methotrexate

• Methotrexate is structurally similar to DHF.
• DHF is a substrate for dihydrofolate reductase (DHFR), converting DHF to tetrahydrofolate (THF).
• Due to its structural similarity, MTX binds strongly to DHFR and prevents THF synthesis.
• Methotrexate prevents the biosynthesis of thymidine, blocking cell proliferation in S phase.

5-Fluorouracil (5-FU)

• 5-FU was designed after learning that tumors incorporated greater amounts of uracil than normal cells.
• The theory was that incorporating fluorine at the 5 position of uracil would interfere with its metabolism.
• 5-FU requires enzymatic conversions to active metabolites, including:
  • Fluorodeoxyuridine monophosphate (FdUMP)
  • Fluorodeoxyuridine triphosphate (FdUTP)
  • Fluorouridine triphosphate (FUTP)

Mechanism of Action of 5-FU

• FdUMP inhibits thymidylate synthase.
• It is a structural analogue of dUMP, the main substrate for this reaction.
• The inclusion of a fluorine atom means dUMP cannot be methylated by thymidylate synthase.
• dTMP synthesis is prevented, arresting DNA replication in the S phase of the cell cycle.

Fluorouracil (5-FU) Summary

• 5-FU has three main metabolites:
  • FdUMP inhibits thymidylate synthase, leading to dUTP accumulation and dTTP depletion, with deleterious consequences for DNA synthesis/repair.
  • FdUTP incorporates into DNA, leading to DNA damage and cell death.
  • FUTP mimics UTP, replacing it in RNA synthesis and interfering with normal RNA processing/function.

Anti-Tumour Antibiotics

• Topoisomerase I and II enzymes fix tangled supercoils during DNA replication.
• Topoisomerase I makes a break in 1 strand, while Topoisomerase II makes a break in both strands.
• Various types of antitumour antibiotics include:
  • Anthracyclines (doxorubicin, daunorubicin, epirubicin)
  • Dactinomycin (actinomycin D)
  • Bleomycin
  • Mitomycin C
• Mechanisms of action include DNA intercalation, inhibition of topoisomerases, DNA alkylation, and free radical damage of cellular and membrane macromolecules.

Bleomycin

• A glycopeptide antibiotic produced by Streptomyces verticillus fungus.
• Discovered in 1962 by Umezawa; observed anticancer activity.
• FDA approved Bleomycin in 1973 for Hodgkin's lymphoma.

Mechanism of Action of Bleomycin

• Contains a DNA-binding region (bithiazole domain) and an iron-binding domain.
• Binds to DNA and Fe2+, then redox cycles between Fe2+ and Fe3+ in the presence of oxygen causing reactive oxygen species (ROS) formation.
• This results in DNA strand breaks:

Mitomycin C

• Discovered in the 1950s by Japanese scientists who isolated it from Streptomyces caespitosus cultures.
• It is an alkylating agent.
• Mitomycin's crosslinking inhibits DNA, RNA and protein synthesis activity for cancers: breast, colorectal, lung, and bladder.

Mechanism of Action of Mitomycin C

• Mitomycin C is a prodrug; the parent compound is inactive.
• The first step is a reduction catalyzed by oxidoreductases.
• The quinone group reduced, reactions lead to DNA crosslinking.
• A "bio-reductive" drug, it targets hypoxic cells.

Mitotic Spindle Poisons

• Cell replication requires formation of spindle complexes during mitosis.
• Inhibiting spindles, a suitable anticancer strategy.
• Early anaphase in Hela cells, spindle microtubules (shown in red) and the kinetochore (where spindles attach to the chromosome) are shown in green.

Mitotic Spindle Poisons.

• Microtubules have functions in cell movement, cell shape, intracellular transport and separation of chromosomes.
• Microtubules are dynamic structures, being assembled and disassembled throughout the cell cycle.
• Since assembling during mitosis and disassembling during remaining phases.
• It makes logical sense to target microtubule assembly and disassembly to kill rapidly dividing tumour cells.
• This can be done by preventing polymerisation or depolymerisation:

Vinca Alkaloids

• They prevent microtubule formation
• Vincristine was extracted from the periwinkle plant.
• Vincristine binds to tubulin dimers and disrupts their assembly into microtubules.
• This prevents chromosomes separating during mitosis.
• It causes cells to arrest Mitosis in metaphase - cell phase specific drug.

Microtubule Stabilisers – Paclitaxel

• First isolated from bark of Pacific Yew tree: (Taxus brevifolia)
• Synthesised by endophytic fungi.
• Paclitaxel accelerates microtubule assembly, stabilising established microtubules, it inhibits microtubule depolymerisation.
• This inhibits dynamic microtubule network essential for cells to function.
• Blocks mitosis and is a phase specific - treats breast cancer non-small cell lung cancer and Kaposi’s Sarcoma.

Classification According to Effects

• Cytotoxic chemotherapy targets DNA replication at various cancer stages.
• Cell cycle describes phases cells undergoing replication.
• Drugs react differently dependent on phases:
  • Some kill during any part of cell cycle. (cycle nonspecific)
  • Others only phases where cycle active. (cycle specific).

Achievements: Chemo Cures.

• Testicular cancer, deadliest amongst young males, (20-39 yrs).
• Before cisplatin, this was death sentence
• Now treatment, involves Chemotherapy.
• Bleomyrin, etopside or carbo, normally used
• Very effective, >90% cure rates.

Limitations of Chemotherapy

• Chemotherapy works BUT only against a few comparatively rare cancer types.
• Common cancers as lung, brain, colorectal and prostate do not respond well to chemotherapy.
• Other tumours, ie: pancreatic cancer, have very poor prognosis.
• Failure factors:
  • Toxicity
  • Drug Resistance
  • Induction of secondary cancers.

Toxicity

• Classical anticancer drugs disrupt cell division, targeting key replication processes. (DNA synthesis, spindle formation etc).
• Cannot distinguish cancer from normal ones.
• All drugs have separate spectrums relating to toxicology, but normally they include bone marrow suppression, hair loss, or gastrointestinal problems.
• Highly toxic.
• All classical agents limit the effective killing factors due to patients’ tolerance, if high enough to effective kill, its fatal.

Myelosupression

• nearly all chemo related drugs case a cell drop (blood cell count).
  • Anemia - decease of red blood.
  • Neutropenia - decrease of main defence
  • Thrombocytopenia - blood clots
  • Pancytopenia - decrease red/white plus platelets.
• chemotheraphy is a dividing toxicity.
  • its a threatening toxicty
  • risk of fatigue, infection, bleeding.
  • dose limits toxicity

Gastrointestinal Toxicity

• 40% of patients get therapy.
• Main drugs that cause mucositis: - anti metabolites. - Vinca Alkaloids.
• Caused by direct effects/indirect effects killing/attacking cells.

Hair

• Dermatological effects.
• Damages the skin/hair.
• Common side effect (psychologically painful!)
• Target: dividing hair follicles
• Cold caps can be used to vasoconstrict.

Summary

• Chemo is the use of drugs to treat cancers systemically.
• it can ‘seek and destroy’ cells
• conventional cells are cytotixc
• Interferes with DNA by interfering with synthesis and replication.
• Can classify on mechanisms or effect
• Used in combines
• Had major successes
• MAJOR PROBLEMS due to toxicity
• Resistance.
• New drugs that selectively target must be created.