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Taxol as an anti-cancer drug

Taxol as an anti-cancer drug . Florina Voica 10/02/04. Cancer cells.

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Taxol as an anti-cancer drug

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  1. Taxol as an anti-cancer drug Florina Voica 10/02/04

  2. Cancer cells • Cancer results from alterations in critical regulatory genes that control cell proliferation, differentiation and survival • Cancer cells divide on an uncontrolled pattern - don’t display density-dependent inhibition -autocrine growth stimulation -no contact inhibition -angiogenesis -no cell differentiation during division -failure to undergo apoptosis

  3. Cell cycle

  4. Cell cycle • Two phases: mitosisand interphase • Mitosis and cytokinesis last only about half an hour so 95% of the cell cycle takes place in the interphase • Interphase: -G1 –cell growth -S – DNA replication -G2 – cell growth checkpoints: G1,G2

  5. Mitosis • prophase– chromosomes start to condense, the mitotic spindle starts to from between the two centrosomes • Prometaphase- breakdown of the nuclear membrane, chromosomes start to attach to the mitotic spindle via their kinetochores • Metaphase- chromosomes attached to the spindle align at the equator of the cell • Anaphase- separation of chromatids, active movement of microtubules • Telophase- each set of chromatids arrives to the poles of the cell and a new nuclear envelope starts to re-assemble, the contractile ring starts to form • Cytokinesis- division of cytoplasm, total assemblage of the nuclear envelope

  6. microtubules • Dynamic structures that undergo continual assembly and disassembly • Together with actin filaments and intermediate filaments, they determine the cell shape and are involved in the intracellular movement of organelles • Originating from centrosomes they form the mitotic spindle • Appear as rigid hollow rod around 25nm in diameter • Assembled from tubulin, a dimer globular protein • Present polarity ( very important for their mechanism of action)

  7. Tubulin • A dimer protein consisting of two monomers(α, β tubulin) with very similar structures (Nature, 1998); γ tubulin was discovered in the structure of centrosomes where it initiates the microtubule assembly • Tubulin polymerizes to form polar microtubules( 13 linear protofilaments) • GTP binding sites on both subunits, however hydrolyzation of the GTP bound on the β tubulin follows polimerization

  8. Microtubules’ assembly and disassembly • When GTP is bound to tubulin ,the subunits have a higher affinity to polimerize • When GTP on the β subunit is hydrolyzed to GDP, the tubulin dimers start to depolymerize due to the decrease in their binding affinity • GDP-bound tubulin depolymerize at the (-) end of the microtubule, while GTP-bound tubulin attaches to the (+) end • When the microtubule is caped at one end( as in the mitotic spindle) this process takes place only at one end resulting in rapid shrinkage or elongation( dynamic instability) • Taxol inhibits microtubules dynamics

  9. Taxol(paclitaxel)

  10. Taxol-history • Julius Caesar mentions in his “Gallic Wars” that Catuvolcus, a cheiftain of the Eburones, committed suicide by taking extracts from the yew tree • Other more recent accounts report uses such as extracts as poison or as a cancer-healing folk medicine • In 1962, A.Barclay collected bark from Taxus brevifolia, the Pacific Yew tree, as part of a national project aimed at the discovery of new anticancer agents • in 1971, the molecular structure of taxol was reported based on an X-ray crytallographic analysis • In 1979, Horwitz reports the interaction of taxol with microtubules

  11. The taxol molecule • 1994, Nicolaou and coworkers reported the total synthesis of taxol • A challenge for chemists because of its - 6-8-6 tricyclic carbon framework - characteristic ester side chain - dense pattern of oxygenated functionality - large number of stereogenic centers

  12. Mechanism of action • Introduction - approved by FDA for the treatement of ovarian, breast, nonsmall cell lung carcinomas and Kaposi’s sarcoma - taxol binds to the tubulin polimer and inhibits its depolymerization ( other antimitotic drugs such as colchicine or vinca alkaloids are known) - in 2002, the only drug approved by FDA known to interact exclusively with a polimer form of tubulin

  13. Effects of taxol in vitro - in vitro it enhances the polymerization of tubulin, and those microtubules formed in the presence of the drug possess unusual stability and resist depolymerization by cold temperature, dilution and Ca2+ (they are organized in extremely stable bundles) - the drug is capable to polymerize tubulin even in the absence of GTP - it binds specifically and reversibly to the N-terminal 31 amino-acid of the β tubulin subunit with a stoichiometry, relative to the tubulin heterodimer, close to one - it alters the kinetics of the microtubule assembly - the microtubules formed in the presence of taxol are much shorter (1.4±0.7 mm). This brings the argument that taxol increases the number of nucleation sites

  14. Effects of taxol in cells - at low drug concentration, the drug suppresses microtubule dynamics - at high conc it increases tubulin polimer mass and induces extensive formation of microtubule bundles - taxol is a potent inhibitor of eukaryotic cell division causing a block in the late G2/M phase, however after the disruption of microtubules , the precise means by which cell death occurs are not clear. In HeLa cells, low conc of taxol induces a cell-cycle block at the metaphase/anaphase transition of mitosis - if taxol is added to a cell at the beginning of the S phase, it has no influence on the progress through this phase - low concentrations of taxol have negligible effects on DNA, RNA and protein synthesis - it was found that taxol can cause alterations in cell shape and movement

  15. Structure-function studies • Hydrophobic nature and small water solubility • Some derivatives of taxol show no significant loss of activity( for ex. 7-OH can be esterified, epimerized or even removed) • Studies have shown that the side-chain at C13 has one of the requirements for biological activity(Baccatain III is not cytotoxic and is totally inactive in vitro toward microtubule assembly) • The C-2 benzoyl group and an intact oxetane ring are essential for cytoxicity and stabilization of microtubules • Substituents on the C-2 benzoyl group have profound effects on the biological activity of taxol

  16. Pharmacokinetics • Small water solubility. Present Taxol is formulated with a polyethoxulated castor oil (Cremphor EL) containing 50% ethanol. It seems that the serious hipersensitivity of taxol may be related to the Camphor EL vehicle. • Usually administered after premedication with antihistamines, corticosteroids etc. • Characteristics: - large volumes of distribution( in animals not distributed in testes and central nervous system) - rapid uptake by most tissues - long half-lives of elimination - substantial hepatic disposition • Linear pharmacokinetic behavior • Insignificant renal clearance

  17. Primary route of systematic elimination occurs via hepatic metabolism and biliary excretion(70% in feces) • In vitro studies showed that taxol is extensively metabolized in humans by liver cytochrome P450 enzymes marked by interindividual variability • Taxol is generally administered at a dose of 175mg/m2 over 3 h or 135-175 mg/m2 over 24 h every three weeks in patients with advanced cancers of the ovary and breast

  18. Toxicity • Neutropenia (manifestation of leukemia or bone marrow toxicity) • Hypersensitivity reactions • Peripheral neuroapathy • Cardiac rhythm disturbances • Reversible loss of hair

  19. Conclusions • In vitro and in vivo studies have shown that taxol is an effective antimitotic drug because of its high specificity, even in very low concentrations • It can be used to treat different types of cancers in both advanced and premature states • It is readily metabolized and eliminated • The side-effects are pretty much the same as for other anti-cancer drugs

  20. Finally… Let’s hope we’ll find a perfect cure…

  21. references • www.emc.maricopa.edu/.../ BIOBK/BioBookmito.html • micro.magnet.fsu.edu/.../ microtubules.html • George A. Orr, Lifeng He, Susan Band Horwitz, Taxol and other molecules that interact with microtubules, Encyclopedia of cancer, Second Edition, vol. 4, 319-327 • Rowinksy Eric K. , The development and clinical utility of the taxane class of antimicrotubule chemotherapy agents, Annu. Rev. Med. 1997. 48:353-74 • Monsarrat B. , Chatelut E, et al. , Modification of paclitaxel metabolism in a cancer patient by induction of cytochrome P450 3A4, Drug metabolism and disposition, 26:229-233 • Jordon MA, Wendell K, Gardiner S, et al.1996 Mitotic block induced in HeLa cells by low concetrations of paclitaxel results in abnormal mitotic exit and apoptic cell death. Cancer Res. 56:816-25 • Derry W, Wilson L et co. , Substoichiometric binding of taxol suppresses microtubule dynamics, Biochemistry 34:2203-2211 • Gligorov J, Lotz P, Preclinical pharmacology of the taxanes: implications of the differences, The oncologist 2004;9:3-8 • He L, Horwitz S et co. , A common pharmacophore for taxol and the epothilones based on the biological activity of a taxane molecule lacking a C-13 side chain, Biochemistry2000, 39, 3972-3978

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