Cancer chemotherapy and Drug resistanceK.Tejaswini M.pharm Ph.chem

Roll no :170112884002GPRCP

Contents

• Introduction

• Drug resistance

• Cancer chemotherapy

• Conclusion

• References

What is cancer ?Cancer is known medically as a malignant neoplasm, is a broad group of diseases involving unregulated cell growth.

In cancer, cells divide and grow uncontrollably, forming malignant tumors, and invade nearby parts of the body.

The cancer may also spread to more distant parts of the body through the lymphatic system or bloodstream.

Not all tumors are cancerous; benign tumors do not invade neighboring tissues and do not spread throughout the body.

Drug resistance

contents

• Introduction

• Mechanisms of resistance alterations indrug targets alterations in intracellular retention of drug alterations in drug detoxification pathways Increased DNA repair Defective apoptosis Epigenetic changes

introduction

• Because of baseline genetic alterations in tumor cells and the fact that malignant cells can acquire pleiotropic changes in the presence of chemotherapy. tumors may still become refractory to both drugs they have been exposed to as well as to drugs with which they have never been treated.

• The latter case, termed multidrug resistance, can involve compounds with completely unrelated structures and mechanisms of action .

• Clinical resistance occurs as the resistant clones are positively selected for during a course of chemotherapy .

• Despite the emergence of new classes of chemotherapeutic agents and the widespread use of rational protocol design, the study of the panoply of mechanisms of drug resistance continues to be essential for using this tool effectively for the most resistant subtypes of malignancies.

Mechanisms of resistance

• The cancer cell exhibits unique molecular properties, which render the cell unable to halt replication in the presence of DNA damage. Thus, cancer cells can continue to survive despite DNA damage, can rapidly incorporate new molecular configurations that confer a survival benefit, and can replicate rapidly and efficiently, increasing the speed at which molecular mechanisms of resistance can be incorporated into the population of cells.

• These changes include increased gene copy, mutations, altered transcription, and epigenetic changes.

• The overall phenotype of a cancer cell that has incorporated these changes in its genome is a cell that either no longer accumulates drug, no longer makes the drug's target protein, or alters the target protein in such a way that the protein no longer binds or is affected by the drug.

Alterations in drug targets

• One of the first mechanisms of acquired resistance to chemotherapy by the tumor cell was determined to be an alteration of the protein targeted by the drug, either by loss or gain of function.

• Additionally, some tumor cells can amplify copies of the genes encoding the drug's target and transcribe and translate more of the target molecules, overwhelming the drug's cytotoxic ability.

• Finally, some preexisting somatic mutations that are present in the tumor cell confer resistance to therapy and must be considered when choosing active agents for individuals.

Increased expression of drug target:Tumor cells having increased levals of intracellular DHFR after eposure to antifolate , methotrexate is an example .DHFR is target for 4-amino folate analogs or antifolate drugs.

Tumors which develop resistance to methotrexate were found to have increased DHFR gene & increased levals of expressed DHFR enzyme.Mutation of DHFR may also be the cause .

Increased Expression Of Biologic Target :

• Amplification of genes encoding oncogenic fusion proteins.• Inorder to avoid damage to normal host tissue , one strategy is to target oncogenic fusion proteins ,expressed in tumor cells with chromosomal translocations that result in expressed protein.

Eg : BCR/ABL –Expressed protein is a tyrosine kinase : result of unbalance translocation of chromosomes 9 & 22.

• Imatinibmesylate is a 2-phenyl amino pyrimidine derivative.

Marketed by Novartis as Gleevec (U.S.) & sometimes referred to by its investigational name STI-571 . Is a tyrosine-kinase inhibitor used in the treatment of multiple cancers.

•cells develop resistance due to either amplification of bcr/abl after prolonged exposure to ST1571 or due to a single amino acid substitution in the threonine residue of the Abl portion of the fusion protein that is required for hydrogen bond formation with the drug molecule .

• Therefore, the early clinical results need to be viewed with caution, and additional investigation of combination therapies is currently underway.

SOMATIC GENETIC VARIATION :

Somatic variations of gene sequence affect adrug target –

Eg: somatic polymorphisms in the enhanser/promoter region of TS , involves a VNTRs ,shown to influence the cellular response to 5FU exposure .

• Cells whose promoters contain three repeats have higher TS activity than do cells with two repeats because of enhanced TS expression.

• Tumor cells derived from this somatic background(homozygous genotype for 2 repeats) will respond well to 5FU therapy.

• Therefore, prospective pharmacogenetic screening of the host's somatic TS promoter region may help predict both tumor response to conventional doses of 5FU, as well as consider either alternative drug regimens or increasing 5FU dosing for patients with higher TS activity.

Tumor mutations in drug target :Multiple mutations in topoisomerase II identified in tumor cells ,these induces resistance either through reduction in enzyme activity or alterations in protein structure that prevents binding of drug to topoisomerase molecule.

Topoisomerase II to remove supercoils. This involves a double-strand break (indicated by a short line), Without topoisomerases, the DNA cannot replicate normally.

Therefore, the inhibitors of topoisomerases(epipodophyllotoxins and anthracyclines) have been used as anti-cancer drugs to stop the proliferation of malignant cells.

Alterations in intracellular retention of drug

• By reducing the drug's ability to enter the cell (or) by rapid efflux of the drug from the cell, the cancer cell effectively removes the ability of the drug to exert its effect.

• This can be accomplished by either reducing copies of the genes for transporters required for the drug's influx into the cell or by increasing copy number of genes encoding transporters that rapidly efflux either the parent drug or the activated drug metabolite from the cell.

• Additionally, the cell can decrease metabolism of the parent drug, preventing the formation of toxic activated metabolites

Tumor decreases influx of drug :

• Altered influx of a drug as a mechanism of more resistance has been documented for therapy with methotrexate.

• The reduced folate carrier (RFC) is an ATP-dependent transporter that actively imports reduced folates in to the the cell against a concentration gradient for their use in DNA, RNA, and amino acid synthesis.

• Classic antifolates (e.g., methotrexate) are also transported via RFC into the cell.Mutations in RFC results in decreased accumulation of drug (less cytotoxicity when exposed to higher doses) & also decreases expression of RFC which confers marked resistance to antifolate therapy .

Resistance

Tumor increases efflux of drug :

• By increasing the efflux of drug molecules from the cell, resistance can be achieved.

• A set of ATP binding cassette (ABC) transporters are localized to the plasma membrane cells, and when expressed, confer resistance to multiple structurally unrelated drug compounds. These include the multidrug resistance protein (MDR), the multidrug resistance-related protein (MRP),& the breast cancer resistance protein(BCRP).

• Expression of these gene products is often turned on in tumor cells after they acquire alterations or deletions in the regulator genes normally controlling their expression (e.g., P53 or N-MYC) .

• Therefore, tumor cells can either increase expression of several ABC transporters after the transformation event, thus conferring de novo resistance to a broad spectrum of drugs or increase expression of these transporters after exposure to a drug in vivo.

• Because these transporters do transport a spectrum of compounds, up- regulation of ABC transporters in tumor cells not only yields cells that are resistant to drugs they have had previous exposure to, but also to drugs to which they have not yet been exposed.

• This limits the number of agents that can then be effectively used for eradication of these tumors in vivo .

Tumor affects pharmacologic path responsible for retention of drug :

• Methotrexate is an anionic compound with a mono- glutamate moiety, to which intracellular fclylpolyglutamate synthase (FPGS) adds additional glutamate molecules.

• Polyglutamylated methotrexate is highly anionic and is no longer a substrate for either passive or active efflux from the cell.

• These polyglutamylated metabolites are active inhibitors of all proteins in the folate metabolism pathway normally inhibited by the parent molecule and

provide prolonged toxicity when retained within the cell.

• Patients with T lymphoblastic ALL have lower overall formation of polyglutamylated MTX than do patients with B lineage ALL.

• Because T lineage ALL is known to have a worse prognosis than B lineage ALL, the mechanism by which this occurs was investigated in primary ALL blasts.

• T lineage blasts were found to have lower FPGS activity than B lineage blasts, providing the mechanism by which less active metabolite was formed, and the tumor cells were more resistant to therapy .

Alterations In Drug Detoxification Pathways

• Cells use secondary metabolism pathways (e.g., phase I1 metabolism) for the primary purpose of making drug molecules more water soluble in order to efflux them from the cell.

• By enhancing phase II metabolism, cancer cells can rapidly excrete drugs, thereby reducing their overall exposure time to the cytotoxic agents.

• For example, alterations in the glutathione conjugation pathway can alter a tumor cell's sensitivity to drugs that are significantly excreted via conjugation with glutathione.

• Drugs with convincing data for the impcrtance of glutathione conjugation in vivo include chlorambucil, melphalan, nitrogen mustard, acrolein, BCNU, hy- droxyalkenals, ethacrynic acid, and steroids .

Increased DNA repair

• By increasing either the rapidity of DNA repair or decreasing the efficiency of repair, cancer cells can overcome some of the DNA damage exerted by certain chemotherapeutic agents.

• One example is 06-alkyl guanine alkyl transferase (OGAT), a constitutively expressed DNA repair protein, which removes alkyl groups from the 06-position of guanine in DNA.

• Tumor cells that exhibit high OGAT activity are resistant to agents that form 06- alkyl adducts. such as the nitrosureas (e.g., BCNU) and triazene compounds (e.g., procar- bazine and temozolomide).

• Agents are currently in development that inactivate OGAT, rendering tumors sensitive to nitrosourea and triazene therapy .

Defective Apoptosis

• Apoptosis, or programmed cell death, is an energy-dependent process by which cells undergo an orderly series of intracellular events leading to cell death. • This process is initiated by a cell in response to specific stimuli, such as DNA damage.

• Apoptosis is required for maintaining appropriate function and structure of normal proliferating for renewable tissues.

• Disruption of the normal programmed cell death response prevents cells from self destructing when irreversible damage takes place, and they survive with this damage.

• Because they do not apoptose, these cells can continue to replicate unchecked.

• Two examples of genes that are commonly mutated in cancer cells are p53 and bc12 .

• p53 is a transcription factor, which in its wild-type form, both represses and initiates specific promoter transcription in addition to several other cellular functions.

• In normal cells,p53 is activated in response to DNA damage, triggering either growth arrest ,

or apoptosis depending on the current stage of the cell cycle.

• Greater than 50% of tumors have been documented to contain functional inactivation of p53.

• bcl2 is an oncogene, the expression of which inhibits apoptosis .

• In tumor cells ,bcl2 is activated, allowing cell to survive in the face of cellular damage that would normally signal for programmed cell death,thus making this cells more resistant to chemotherapy –indused cytotoxicity.

• In vitro models of cell lines with bcl-2 activation show that these cells continue to divide and grow despite high concentrations of drug in the media .

Epigenetic changes

• Epigenetic silencing of tumor suppressor genes has been documented in human cancers.

• Methylation of DNA occurs in eukaryotic cells. DNA meth- yltransferase catalyzes the reaction, whereby a methyl group is transferred to the carbon 5 position of the cytosine ring within a 5'-CG-3' sequence. Methylation of genes silences expression by preventing binding of transcrip- tion factors and recruiting binding of other proteins that limit the accessibility of the gene to the cell's transcription machinery. Demethylation of a gene activates its expression.

• The exact mechanism by which this occurs is not known, but investigators are currently determining whether this phenomenon extends to drug targets or enzymes that catalyze drug activation/detoxification pathways.

Strategies to overcome resistance

• The future strategy to overcome resistance will be to individualize a patient's therapy prospectively, employing both knowledge of the patient's somatic mutations in drug metabolizing enzymes, drug receptors, and drug targets with knowledge of tumor specific changes that affect cytotoxicity of agents normally given systemically to patients.

• Some strategies used clinically are pharmacokinetic-based strategies, pharmacogenetic-based strategies, and basic tumor biology-directed strategies for dosing.

• The future of individualization, for patients lies in developing algorithms for dosing, based on a synthesis of all of these methods, to determine an overall phenotype for each patient prospectively.

Conclusion :Drug resistance continues to provide an obstacle for administering chemotherapy to patients. Although some patients respond verywell to chemotherapy, a significant number of treatment-refractory tumors are still under study. Ultimately, the combined knowledge of patient-specific factors that alter our ability to administer optimal doses of chemotherapy and tumor-specific factors that result in decreased efficacy of chemotherapy will be used to more efficiently dose patients. As new drugs emerge for bioselective treatment of tumors, understanding induction of resistance continues to be essential for providing the best therapy to patients. With the powerful genomics tools that are becoming more available, it willsoon be feasible to monitor both tumor and somatic variations in DNA sequence, expression of proteins, and in vitro response to therapy before initiating therapy in patients, thereby providing information required to reach the ultimate goals of reducing the incidence of side effects, improving response to therapy, and preventing the induction of resistant malignant clones in vivo.

IntroductionChemotherapy (often abbreviated to chemo) is the treatment of cancer with one or more cytotoxic antineoplastic drugs ("chemotherapeutic agents") as part of a standardized regimen.

• Dr. Sidney farber who is regarded as the father of modern chemotherapy

• Chemotherapy may be given with a curative intent or it may aim to prolong life or to palliate symptoms.

• It is often used in conjunction with other cancer treatments, such as radiation therapy or surgery.

• Certain chemotherapeutic agents also have a role in the treatment of other conditions, including ankylosing spondylitis, multiple sclerosis, Crohn's disease, psoriasis, psoriatic arthritis, systemic lupus erythematosus, rheumatoid arthritis, and scleroderma.

Contd…..

•Traditional chemotherapeutic agents act by killing cells that divide rapidly, one of the main properties of most cancer cells.

• This means that chemotherapy also harms cells that divide rapidly under normal circumstances: cells in the bonemarrow, Digestive tract & hair follicles

•This results in the most common side-effects of chemotherapy: myelosupression(decreased production of blood cells, hence also immunosupression), mucositis(inflammation of the lining of the digestive tract),&alopecia(hair loss).

General mode of action in cancer

Cell cycle specific agents cell cycle non-specfic agentsAntimetabolites Alkylating agentsBleomycin AntibioticsPodophyllin alkaloids Cisplatinplant alkaloids Nitroso ureas

• In the broad sense, most chemotherapeutic drugs work by impairing mitosis (cell division), effectively targeting fast-dividing cells. As these drugs cause damage to cells, they are termed cytotoxic.

• They prevent mitosis by various mechanisms including damaging DNA and inhibition of the cellular machinery involved in cell division.

• One theory as to why these drugs kill cancer cells is that they induce a programmed form of cell death known as apoptosis.

• As chemotherapy affects cell division, tumors with high growth rates(such as acute myelogenous leukemia and the aggressive lymphomas, including Hodgkin's disease) are more sensitive to chemotherapy, as a larger proportion of the targeted cells are undergoing cell division at any time.

• Malignancies with slower growth rates, such as indolent lymphomas, tend to respond to chemotherapy much more modestly .

Legend

Drug Class

Sub-class

Prototype Drug

Mechanism of action• Alkylate nucleophilic group of DNA bases

N7 Guanine N1 ,N3 Adenine

N3 Cytosine• Abnormal base pairing, cross linking of

bases & DNA strand breakage

Alkylating agents -MOA

Cyclophosphamide, Ifosfamide

Wegener’s granulomatosis, ALL, CLL, NHL,HL Multiple myeloma (MM), breast, ovary, lung ,cervix, testis

Mechlorethamine Hodgkin’s diseaseMelphalan Multiple myeloma, breast, ovaryChlorambucil CLL, HL, NHL

A. Nitrogen Mustards:

Currently used drugs:

Drug related side effects:

Cyclophosphamide, Ifosfamide: Hemorrhagic cystitis, SIADH

Cyclophosphamide metabolism

Mesna( sodium 2 mercaptoethane sulfonate) and n-acetyl cysteine removes toxic compounds .

SAR:

Structure differs only in third group (R) attached to nitrogen.

If R is – aliphatic pushes electrons towards N,this electronic enrichment enhances the nucleophyllic character of lone pair of electrons and increases the speed at which the beta carbon of the mustard will be attacked.

Conversely ,if R is –aromatic –stabilizes the lone pair of electrons through resonance.which slows down the intramolecular nucleophyllic attack,aziridinum ion formation.

R

NEWER AGENTS :

Troposamide : prodrug of ifosamide

Prednimustine : ester of prednisolone and chlorambucil

Uramustine :Derivative of nitrogen mustard and uracil

Nitrosoureas: Carmustine (bis-chloroethylnitrosourea)

Lomustine(Chloroethylcyclohexylnitrosourea)

Semustine Streptozocin-naturally occuring sugar

containing

BCNU CCNU

MOA : alkylation ,crosses BBB Newer agents Fotemustine ranimustine nimustin

Platinum derivatives :

Testicular Ca, Ovarian Ca, Head and neck Ca, bladder Ca, Oesophagous cancer.

Cisplatin 1st Generation Highly nephrotoxic

Carboplatin 2nd Generation Less nephrotoxic

Oxaliplatin 3rd Generation Cisplatin/ Carboplatin Resistant

Newer agents : nadaplatin ,triplatin tetranitrate

These compounds alkylate N7 of guanine. They cause nephro- and ototoxicity.

To counteract the effects of nephrotoxicity, give mannitol as an osmotic diuretic,

or induce chloride diuresis with 0.1% NaCl.

Side effects: nausea and vomiting (5HT3 antagonist- ondansetron ,granisetron)

Oxaliplatin

Alkyl sulphonates :Busulfan

MOA - Methyl sulphonate portion directly get sustituted by nucleophyllic groups of DNA .

USE : CGL

Newer agents :MannosulfanTreosulfan

Ethelenimines :Thiotepa (tris (1-aziridinyl)phospholine)

MOA : Hydrolysis of any aziridene groups monofunctional

USE : Testicular liver cancers .

D. Other Alkylating agents

Currently used:

Procarbazine→ Hodgkin’s, brain tumors

Dacarbazine→ malignant melanoma, hodgkin’s lymphoma

Temozolomide→ malignant gliomas

Procarbazine Dacarbazine

Antimetabolites

LegendDrug ClassSub-classPrototype Drug

Folic Acid Analogs

MethotrexateTrimetrexatePemetrexed

An essential dietary factor, from which THF cofactors are formed which provide single carbon groups for the synthesis of precursors of DNA and RNATo function as a cofactor folate must be reduced by DHFR to THF

Folate:

Structures

The enzyme DHFR is the 1º site of action MTX prevents the formation of THF, causing an intracellular deficiency of folate

coenzymes and accumulation of the toxic inhibitory substrate, DHF polyglutamate The one carbon transfer reactions for purine and thymidylate synthesis cease,

interrupting DNA and RNA synthesis

Methotrexate-

psoriasis,rheumatoid arthritis,acutelymphoblastic leukemia, meningeal leukemia, choriocarcinoma, osteosarcoma, mycosis fungoides, Burkitt’s and non-Hodgkin’slymphomas, cancers of the breast, head and neck, ovary, and bladder .

64- methotrexate65-aminopterin66-edatrexate

Methotrexate Toxicity

Bone marrow suppressionRescue with leucovorin (folinic acid)

Nephrotoxic give sodium bicarbonate to alkalinize the urine

Purine Antagonists

MercaptopurineThioguanine

Fludarabine PhosphateCladribine

The purine analogs 6-mercaptopurine(76) and 6-thioguanine (77) were among the first anticancer drugs to be used,registered in 1953 and 1966, respectively.

Later, the purine nucleoside analogs fludarabine (78) and pentostatin (79; 2'-deoxycoformycin) were registered in 1991, and cladribine (80; 2-chloro-2'-deoxyadenosine) was registered in 1992.

MOA

Cytosine arabinoside,fludarabine, and cladribine are taken into cells through a specific nucleoside transporter protein . phosphorylated to the mono-,di-, and triphosphates, with the first phosphorylation mainly by deoxycytidine kinase. The active triphosphate derivatives are incorporated into DNA, blockingpolymerase function and thus DNA synthesis .

Uses

Newer drugs : clofarabine Paediatric patients for Relapsed or Refractory ALL

FDA Approved in 2004

6 Mercaptopurine AML

6 Thioguanine AML , ALL

Cladribine Hairy cell leukamia, CLL, NHL

Fludarabine CLL, NHL

Pyrimidine AntagonistsFluorouracil - S-phase

Cytarabine GemcitabineCapecitabine

MOA:

Cytarabine activated to arabinoside CTP→ Inhibit DNA polymeraseα/β 5-FU converted to 5-dUMP→ Inhibit Thymidylate synthase Capecitabine prodrug of 5-FU Gemcitabine Phosphorylated to GDP→ Inhibit Ribonucleotide Reducatase GTP→ Inhibit DNA polymerase α/β, incorporated in DNA Azacytidine & DecitabineDNA hypomethylation by inhibiting DNA methyl transferase

Effect of 5-FU

Figure 2. This figure illustrates the effects of MTX and 5-FU on the biochemical pathway for reduced folates.

X5-FU

MTX

Cytarabine AML, ALL,CML in blast crises

5-FU Colorectal Ca, Anal Ca, Breast Ca, Gastro-esophageal Ca, Head & Neck Ca, hepatocellular Ca

Capecitabine Breast Ca, Colorectal Ca, Gastro-esophageal Ca, Hepatocellular Ca, Pancreatic Ca

Gemcitabine Pancreatic Ca, Bladder Ca, NSCL Ca, Ovary Ca, Soft tissue Sarcoma

Azacytidine & Decitabine Pancreatic Ca, lung Ca, ovarian Ca, Myelodysplasia

Newer agents : tegafur uracil , carmofur

MOA

VINCA ALKALOIDS : mitotic metaphase arrest

PODOPHYLLO TOXINS : inhibition of topoisomerase II

CAMPTOTHESINS : inhibition of topoisomerase I

TAXANES : Mitotic “spindle poison” through the enhancement of tubulin polymerization .

ANTIBIOTICS

Anthracyclines- Doxorubicin & Daunorubicin

DactinomycinPlicamycinMitomycinBleomycin

Miscellaneous AntiCancer Agents

AsparaginaseHydroxurea

MitoxantroneMitotane

Retinoic Acid DerivativesAmifostine

Mechanisms & Actions of Useful Chemotherapeutic Drugs in Neoplastic Disease

Conclusion :

The period from 1980 to the present has seen a remarkable growth in the understanding of many of the cellular and molecular mechanisms underlying malignant transformation of a cell.

Given our increasing knowledge about the biology of cancer, it is

clear that no single therapy will serve as a panacea & it is most likely that in the near future, agents directed against the molecular events will have to be combined with the existing standard chemotherapies for the desired outcome

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T h a n k y o u