Dr S A Ebrahimi

Historical overview

1950-1970 Most agents were discovered through

large scale screening of natural or synthetic chemicals on rapidly proliferating animal tumors e.g. murine leukemias

Most interacted with DNA synthesis Paclitaxel and etoposide are examples

Last thirty years

Novel molecular targets have been discovered

Some agents are specific for antigens present on some malignant cells e.g. herceptin

Some induce differentiation in malignant cells e.g. all-trans-retinoic acid

Current classes of antineoplastic agents

Alkylating agents Antimetabolites Natural Products Hormones and antagonists Miscellaneous agents

Current trends in chemotherapy

Development of molecularly targeted drugs Use of antibodies growing e.g. herceptin

Use of chemosensitivty testing on biopsied samples prior to drug administration

Use of drug combinations: Drugs should act via different mechanisms Each drug used at its highest tolerated dose Each drug must be administered as frequently as

possible Treatment cycle must be repeated many times to

ensure complete eradication of tumor

The cell cycle G1 Phase: Gap period between mitotic phase and

DNA synthesis phase Cells

increase in size produce RNA synthesize protein

The cell cycle S phase:

DNA synthesis phase and replication occurs

The cell cycle G2 phase:

Cells continue to produce protein and grow

The cell cycle M phase (mitotic phase):

Cell growth stops Cell division occurs

The cell cycle Each of the two daughter cells produced in in M

phase, can enter G1 phase.

The cell cycle Sometimes, daughter cells enter a non-

proliferative state called G0

In slow growing cancers, G0 period is very long

The cell cycle and anti-neoplastic agents Many of the most potent agents damage the

DNA and are therefore effective during the S phase

Some other agents block formation of mitotic spindle and are therefore effective during the M phase

Therefore currently, human neoplasms which are susceptible to chemotherapy are those with a large proportion of cells undergoing division

Also normal cells with high rate of division e.g. bone marrow and hair follicles, are also affected by these agents

Cancers with low percentage of dividing cells e.g. carcinoma of colon, show little susceptibility to these agents

The cell cycle Each transition is controlled

by: Cyclins: Kinase activating

proteins Cyclin-dependent kinases (CDK) Inhibitory proteins e.g. p16 and

retinoblastoma protein

Loss of Inhibitory proteins or enhanced activity of CDKs cause excessive proliferation

Research is currently focused on CDK’s as drug targets

The cell cycle At phase boundaries, check points exist for examining DNA

integrity G1-S boundary: If the protein p53 is expressed normally

cells are checked for DNA damage in G1 cells with damaged DNA undergo apoptosis

G2-M boundary: DNA integrity is checked again

Mutation in check point components can induce drug

resistance to cancer cells

Achieving optimal efficacy from chemotherapy Treatment of cancer is the application

of a complex mixture of Radiotherapy Surgery Chemotherapy

“standard” chemotherapy regimens have been devised for different cancers Efficacy not optimal in all patients Side-effects are usually great

Achieving optimal efficacy from chemotherapy

In order to limit side-effects and optimize efficiency Individual dose adjustments are made based on:

Body surface area but No solid data supports this use Recently

Pharmacokinetic monitoring has been shown to: Increase efficiency e.g. methotrexate in ALL treatment but

maintaining a targeted plasma level Decreasing toxicity e.g. thrombocytopenia induced by

carboplatin by dose adjustment based on renal clearance

Achieving optimal efficacy from chemotherapy Selecting the right treatment for a particular

patient Finding therapy responders

Checking existence of CD20 antigen prior to treatment with rituximab

Testing for HER2 receptor existence prior to treatment with trastuzumab antibody

Chemosensitivity testing

Checking drug metabolizing enzyme polymorphisms Risk of toxicity in patients with polymorphisms of

dihydropyrimidine dehydrogenase gene on 5-fluoruracil treatment

Breast cancer resistance gene profiling prior to treatment

Management of toxicity

Antineoplastic agents have variable kinetics and toxicity

Have to monitor for: Blood cell count Infections Delayed toxicities on the:

Heart Kidneys Lungs

Current classes of antineoplastic agents

Alkylating agents Antimetabolites Natural Products Hormones and antagonists Miscellaneous agents

Alkylating agents

Goodman and Gilman discovered cytotoxic effects of mustard gas on murine lymphomas

Subsequently tested a number of agents clinically

Five major groups are used today: Nitrogen mustards Ethyleneimines Alkyl sulfonates Nitrosoureas Triazenes

Alkylating agents They all produce a carbonium

intermediate Carbonium can attack a nucleophile

such as Phosphate Amino Sulfhydryl Hydroxyl Carboxyl Imidazole

Alkylating agents

Nitrogen number 7 (N7) on guanine is particularly sensitive to this alkylation

Guanine is probably most important biological target

Other atoms in DNA susceptible to attack are: N1 and N3 of adenine N3 of cytosine O6 of guanine

The result is cross linking of DNA chains to each other or to proteins

Pharmacological actions

Inhibition of DNA synthesis Inhibition of cell division Rapidly dividing cells are affected

more Delayed damage also seen in tissues

with low mitotic indices: liver kidney mature lymphocytes

Monofunctional versus bifunctional agents Bifunctional agents

create interstrand cross-links This stops DNA replication with little chance

of DNA repair Monofunctional agents

alkylate the chains, but no cross-linkages are formed

DNA repair processes may be able to overcome the damage

Mutations may occur because of alkylation-repair sequence Causing drug resistance Carcinogenesis is normal tissue

Mechanisms of resistance in alkylating agents

Decreased drug transport Increased intracellular nucleophile

concentrations e.g. increased glutathione

Increased activity of DNA repair mechanism

Increased rate of metabolism of active drugs

Toxicity of alkylating agents

Dose-dependent bone marrow suppression Acute myelosuppression

Peak in 6-10 days after initiation of therapy Recovery in 14-21 days after cessation of therapy

Mucosal toxicity Oral mucosal ulceration Intestinal denudation

Neurotoxicity Nausea and vomitting Some agents induce seizures, cerebellar ataxia

Therapeutic uses of Nitrogen mustards

Mechlorethamine As part of MOPP regimen

(mechlorethamine, vincrsitine (oncovin), procarbzaine and prednisone) for the treatment of Hodgkin’s disease (cancer of the lymph tissue as in lymph nodes, spleen etc)

Topically for the treatment of cutaneous T-cell lymphoma

Therapeutic uses of Nitrogen mustards Cyclophosphamide

Breast cancer Lymphomas Chronic lymphocytic leukemia Non-Hodgkin’s lymphomas Ovarian cancer Solid tumors in children Burkitt’s lymphoma, associated with Epstien-

Barr virus, complete remission reported Also as an immunosuppressant

Therapeutic uses of Nitrogen mustards Ifosfamide

Germ cell testicular cancer Sarcomas

Melphalan Multiple myeloma

Chlorambucil Chronic lymphocytic leukemia (CLL)

Other alkylating agents

Altretamine Persistent or recurrent ovarian cancer

when cisplatin or other agents have failed

Busulfan Chronic myeloid leukemia

Carmustine It passes the blood-brain barrier Used in treatment of Malignant gliomas

Other alkylating agents

Dacarbazine Hodgkin’s lymphoma Less effective for treatment of

melanoma’s and adult sarcomas

Platinum coordination complexes

Platinum complexes were found to have antiproliferative activity in the 1960’s

Cis-diaminedichloro-platinum (II) was the most potent

It inhibits DNA synthesis by formation of inter and intra strand cross-linkages.

N7 of guanine appears to be most susceptible to the attack

Other alkylating agents

Platinum coordination complexes (Cisplatin) Have broad antineoplastic activity With etoposide, vinblastine, bleomycin or

ifosfamide, cis-platin cures 90% of cases of testicular cancer

In carcinoma of ovaries, with paclitaxel, induces complete response in most cases

Used for the treatment of: Carcinomas of the lung Cancers of neck, head, bladder, endometrium

and cervix Rectal and anal carcinomas Enhances effects of irradiation in some cancers

e.g. esophegal and lung

Antimetabolites

Folic acid analogs: Historically important

1st agents to produce temporary remission in leukemia

1st agents to produce cure of a solid tumor (choriocarcinoma of the uterus)

Structures of folic acid analogs

Folic acid: Mode of action

Deoxyuridine monophosphate is converted to thymidine monophosphate using tetrahydorofolate and producing dihydrofolate

The enzyme dihydrofolate reductase, converts dihydrofolate to tetrahydrofolate

The cycle can repeat for the production of the next TMP molecule

Folic acid analogs: Mode of action

Main mechanism of action is to inhibit dihydrofolate reductase

Dihydrofolate is not reduced to tetrahydrofolate

Tetrahydrofolate reserves become depleted

Production TMP is inhibited Production of DNA strands

becomes inhibited Cell division is blocked

Methotrexate Critical in the treatment of acute

lymphoblastic leukemia (ALL) in children Of little value in adult leukemias except

leukemic meningitis With dactinomycin, can produce cure in 75%

of advanced cases of choriocarcinoma and 90% of early diagnosed cases

Beneficial effects are seen in combination therapies in Burkitt’s lymphomas

Is a component of drug regimens in treatment of carcinomas of the: Breast, head, neck, ovary and bladder

DNA nucleotides

Purines: Two fused rings Adenosine Guanine

Pyrimidines: Single 6 member rings Thymine Cytosine Uracil

The bases are converted to deoxynucleoside triphosphates (dNTP)

dNTP is the substrate for DNA polymerase

Pyrimidine analogs 5-fluorouracil

It is transformed to 5-fluoro-2-deoxyuridine-5-phosphate (FdUMP)

FdUMP covalently inhibits the enzyme Thymidylate synthetase reposnsible for synthesis of TMP

Thus DNA synthesis becomes inhibited It also is incorporated into RNA Thus interferes with RNA function

Used with some success, for treatment of carcinomas of the colon, upper digestive tract and breast

Cytarabine (cytosine-arabinoside)

Enters the cell via active transport mechanism Becomes incorporated into DNA during synthesis Inhibits Base stacking and normal DNA

conformation Interferes with DNA replication Effective in

Acute Myelocytic Leukemia

Newer Agents: Azacitidine Gemcitabine

Purine analogs

6-Mercaptopurine Converted to 6-thioinosine-5-

monophosphate (T-IMP) T-IMP accumulation:

Inhibits formation of purine bases To a small extent is incorporated into DNA

Used for the treatment of acute leukemia

Natural products

Vinca alkaloids Obtained from a plant indigenous to

Madagaskar Three clinically important agents have

been isolated Vincristine Vinblastine Vinorelbine

Vinca alkaloids: mode of action

These are cell-cycle-specific agents They bind beta-tubulin stiochiometrically Alkaloid bound tubulin can not

polymerize with alpha-tubulin to form microtubules

Mitotic spindle can not form Chromosomes can not organize

themselves at the mitotic plate Mitosis does not proceed Cells undergo apoptosis

Vinblastin Curative in combination with bleomycin

and cisplatin for treatment of testicular cancer

A component of curative therapy for Hodgkin’s disease

Acitve in: Kaposi’s sarcoma due to infection by human

herpes virus 8 (HHV8) Neuroblastoma Carcinoma of the breast Choriocarcinoma

Other vinca alkaloids

Vincristine With glucocorticoids as treatment of

choice in childhood leukemia As part of MOPP regimen for treatment

of adult lymphomas Vinorelbine

Non small cell carcinoma of the lung with cisplatin

Carcinoma of the breast

Taxanes Paclitaxel was 1st isolated from the bark of Yew

tree It binds beta-tubulin at a site different from

vincristine It promotes microtubule formation

Taxanes: mode of action

Binds beta-tubulin Antagonizes break down of

microtubules The cell becomes locked in the mitotic

phase Cell death follows

Taxanes: Uses

Paclitaxel Metastatic cancers of:

ovaries Breast Lung head and neck

Docetaxel Hormone-refractory prostate cancer

Camptothecin

Initially isolated from a Chinese tree in 1966 It was found to be too toxic in vivo for clinical

application

Clinically useful analogs were developed in the 1980’s

Camptothecin analogs: mode of action Topoisomerases are enzymes that reduce

torsional stress in a selected region of DNA This is achieved by untangling the DNA strand This allows the two DNA strands to separate Separation is necessary before

Replication Repair Transcription

Topoisomerase are a family of enzymes with two subtypes: I and II

These agents are inhibitors of Topoisomerase I

Camptothecin analogs: mode of action Tompoisomerase I binds covalently to double

stranded DNA through a reversible trans-esterfication reaction

This reaction attaches tyrosine on the enzyme to phosphate on the DNA

This causes a break in the DNA strand to which the enzyme has attached

The other end of the DNA strand is free to rotate, unraveling the DNA

The enzyme then reattaches the two broken ends of DNA

Camptothecin analogs: mode of action Camptothecins binds the topoisomerase I-

DNA complex Stabilises the normally reversible ester bond. The agents do not affect the rate of formation

of topoisomerase-DNA complex Religation becomes inhibited Single strand-breaks become accumulated This makes normal DNA replication

impossible Cells ultimately undergo apoptosis

Camptothecin analogs: mode of action These agents are S-phase specific Trials have shown that low dose,

long term usage is more effective that high dose short term use

There is some cytotoxicity in cells which are not synthesizing DNA

This suggests a mixed effect

Clinical use Topotecan

Ovarian cancer Small cell lung cancer CML

Irinotecan With fluoropyrimidines in advanced

colorectal cancer in patients which have not been treated before

Other possible uses include: Small cell and non-small cell long cancer Cervical, gastric, ovarian cancers and brain

tumors

Antibiotics Dactinomycin (Actinomycin D)

The palanar sing structure appears to intercalate between adjacent guanine-cytosine base pairs

The amino acid chains align themselves along the minor groove

A stable dactinomycin-DNA complex is formed The complex inhibits DNA and RNA

polymerases Also, agent appears to induce nicks in the DNA

structure

Dactinomycin

Is cytotoxic to rapidly dividing cells Is used clinically for

Rhabdomyosarcoma in children Kaposi’s sarcoma Soft tissue sarcoma’s With methotrexate, has been used in

advanced choriocarcinoma

Anthracyclin antibiotics

A tetracycline ring structure attached to a sugar, daunosamine

A number of mechanisms suggested for their antitumor activity

Anthracyclin antibiotics: Mode of action These agents form a complex with

DNA-bound topoisomerase II enzyme This, inhibits religation of nicked DNA

strand Normal DNA replication and repair

become impossible Cells under apoptosis

Also, the agents generate free radicals Important in their cardiotoxicity

Anthracyclin antibiotics: Uses

Daunorubicin AML AIDS-related kaposi’s sarcoma

Doxorubicin Kaposi’s sarcoma Malignant lymphomas Carcinoma of breast Small cell carcinoma of the lung

Epipodophyllotoxins

Extracted from Mandrake tree, endogenous to north America

They form a complex with DNA bound topoisomerase II

This leads to cell death

Epipodophyllotoxins

Etoposide Testitular cancer Small cell carcinoma of the lung Non-Hodgkin’s lymphomas Kaposi’s sarcoma May cause acute nonlymphocytic leukimia

Teniposide Glioblastomas Neuroblastomas Brain metastases from small cell lung carcinoma

Bleomycin Appears to induce single and double

stranded breaks in the DNA This is through oxidative damage to the

deoxyribose of thymidylate Requires Fe and oxygen for its actions It causes accumulation of cells in G2 phase Used for treatment of

Germ cell tumors of testis and ovaries Malignant pleural effusions As part of ABVD therapy in Hodgkin’s disease

L-Asparginase

An enzyme which converts aspargine to aspartic acid

This decreases free serum aspargine As cells in some lymphoid

malignancies can not synthesize this amino acid, their proliferation becomes inhibited

Used in combination for treatment of acute lymphoblastic leukemia (ALL)

Differentiating agents

Tretinoin A retinoid Under physiological conditions

Retinoic acid receptor-alpha (RAR-alpha) dimerizes with RAR-X receptor to form a complex with all-trans-retinoid acid (ATRA)

This complex induces cell differentiation i.e. stops malignant proliferation of cells

In some malignancies, the level of ATRA is too small to form the tripartite complex in adequate amounts

Tretinoin is given to compensate low ATRA levels Very effective for Acute promyelocytic leukemia

Tyrosine kinase inhibitors

Human genome contains code for 550 different protein kinases

These can be divided into 3 groups Tyrosine Kinases

Receptor tyrosine kinases (have extracellular ligand binding site)

Simple Enzymatic (in cytoplasm or nuclear compartment)

Serine/threonine kinases Nonselective kinases (serin,threonine

and tyrosine)

Tyrosine kinase inhibitors

The enzyme acts as on-off switch for many protein functions

Some mutations cause the enzyme to remain locked in the “on” position, leading to malignant proliferation

Subtypes of enzyme implicated in cancers include Platelet derived growth factor receptor Kit: a growth factor receptor of type III

tyrosine kinase family ABL-Kinase

Tyrosine kinase inhibitors

Three agents have obtained FDA approval: Imatinib

CML (ABL positive) GIST (Kit Mutation positive)

Gefitinib Erlotinib

Thalidomide

A number of mechanisms have been proposed for the effects Direct cytotoxic/proapoptotic effects Inhibition of cytokine production, release

and signaling, leading to antiangiogenic effects

Immunostimulatory effects, enhaning natural killer cells cell-mediated cytotoxicity

Used in multiple myeloma treatment

Other agents

Interleukin-2 Monoclonal antibodies

Naked Trastuzumab (herceptin) for the treatment

of breast cancer Conjugated to cytotoxic agents

Gemtuzumab Treatment of acute myelocytic leukemia

Hormones

Glucocorticoids In acute lymphoblastic leukemia in children Malignant lymphomas in children

Progestins Metastatic hormone dependent breast

cancer Anti-androgen therapy

Metastatic prostatic cancer Anti-estrogen therapy

Tamoxifen for breast cancer

Research in our lab

Spinal-Z Two polymethoxy

flavones Can inhibit cell growth in

vitro Can inhibit tumor growth

in vivo

Research in our lab

Found them to be anti-angiogenic

Summary of mode of action of antineoplastic agents