Assessment Of Strategies In Cancer Chemotherapy Cytarabine Biology Essay

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Cytarabine also known as (Ara-C) is an injectable chemotherapeutic drug commonly used to treat acute myelocytic leukemia. This is a cancer situated on the myeloid line of blood cells, characterised by the rapid growth of abnormal white blood cells that accumulate in the bone marrow.

Ara-C attacks both cancerous and non-cancerous cells as they undergo DNA synthesis. [1] The mechanism of action of each drug is multifaceted but primarily involves inhibiting DNA synthesis. Ara-C is transported into the cell where it is activated by phosphorylation. [2]

DNA replication occurs at the leading strand of the replication fork by addition of the nucleotides in the 5′ --> 3′ direction to form one new daughter strand. The second daughter strand results from replication of the lagging DNA strand at the replication fork where the lagging strand replicates discontinuously and requires synthesis of a primer. Okazaki fragments are intermediates created from the lagging strand as a result.

The rate of replication for nuclear DNA is often elevated in malignant cells, particularly in aggressive tumors, relative to surrounding healthy tissues. [3] Disruption of DNA replication of malignant cells inhibits tumor growth and expansion. A mechanism through which this can be achieved is provided by the fact that Ara-C is an analogue of deoxycytidine, as shown in Figure 1.1, and becomes incorporated into during DNA replication causing alterations in the structure, stability or protein binding characteristics of DNA.

Cytarabine inhibits DNA elongation, with inhibition occurring predominantly at the lagging strand of the replication fork. [3] Ara-C causes inhibition of DNA polymerases and causes cell death as a result. This is because upon metabolism of Ara-C to Ara-CTP and incorporation into DNA, chain termination occurs. It is also an S-phase-specific agent upon activation is a competitive inhibitor of DNA polymerase alpha and beta. [4]

Figure 1.1 showing the similarity in structure between Ara-C (left) and cytidine(right), and hence as result becomes incorporated into DNA synthesis, especially useful for cancerous cells. [1]

Drug Discovery:

Cytarabine i.e. Ara-C was derived from knowledge gained from the close examination of bioactive marine nucleosides, found in the marine environment. Cytarabine is an example of a marine-derived drug. [5] The drug was first discovered in 1945 by a chemist named Werner Bergmann who collected sponges from the shallow waters from Elliot Key, Florida. This unidentified species called Cryptotethia crypta named by Dr. M. W. De Laubenfels discovered more of these sponges off the Islands in the Bahamas. Upon closer examination by the way of boiling the sponges in acetone in a Soxhlet extractor apparatus, Bergmann observed crystalline material separated from the acetone. This isolated material was found to be a component in a mixture of drugs used for the treatment for acute lymphoblastic leukemia. UV analysis in the form of an absorption spectrum that was similar to the structure of the nucleotide thymidine. This new compound similar to thymidine was named spongothymidine since the compound was derived from sponges.

Figure 2.1 Structure of spongothymidine. [5]This led to the discovery that a nucleoside has biological activity. The subsequent explosion of compounds and these discoveries led to the identification of a close analogue, cytosine arabinoside, as potent antileukemic agent and was subsequently commercialised by Upjohn (now known as Pharmacia) as Ara-C. [5] Other close compounds such as Ara-A were synthesised and commercialised by Burroughs Wellcome (now GSK).

Computer assisted drug design can be used to find the main active binding sites. These binding sites analogous to deoxycytidine identify significant bindings to functional groups also present within DNA in cancerous cells, i.e. the target enzymes involved in DNA synthesis. Computer assisted drug design allows the drug design to be similar in structure to cytidine i.e. be a cytidine analogue. This is shown in the diagrams of Figure 2.2.

Active site volumes of dCKc and dCKm were calculated by generating the binding pockets of the enzymes. These of which are similar to the structure of Ara-C: [6]


Figure 2.2 showing the binding sites of cytidine to major functional groups using computer assisted drug design for Ara-C, important to inhibition of DNA synthesis. Dotted (i)/yellow (ii) lines represent distances in Angstrom. [6]


Drug Synthesis: [7]

Figure 3.1 Synthesis of cytarabine (ara-C)

It is possible to synthesise cytarabine, ara-C using the following synthesis route. This route is also used and established for the synthesis of ara-AC, an ara-C derivative. The first step in the synthesis route involves hydrogenation of the initial compound in ethanolic hydrogen chloride leading to the reduction of the double bond.

The next stage in the synthesis route is treatment of the second compound at room temperature with bis-trimethylsilyl-trifluoroacetamide (BFTFA) in acetonitrile solution produced the pentakis-trimethylsilyl derivative. This is allowed to reflux for several hours until full conversion to the last product ara-C is completed. Evaporation of the reaction solution produces syrup where it is boiled in methanol to remove trimethylsilyl groups by solvolysis. Ara-C is then crystallised from the solution as a result.

Drug Selectivity, potency and SAR:

Cytarabine main target i.e. selectivity is identical to that of the nucleoside deoxycytidine due to the fact it is a nucleoside analogue. It acts mainly as a competitive inhibitor to cytidine triphosphate. Ara-C is metabolised in vivo to ara-CTP and competes with CTP during DNA synthesis with DNA polymerases both alpha and beta.

To exert its cytoxic effects, cytarabine is converted initially to the 5'-phosphate via rate limiting phosphorylation catalyzed by deoxycytidine kinase (dCK) in tumour cells and then ultimately to its 5'-triphosphate ara-CTP.

Ara-CTP presumably acts both by inhibiting the binding of 2'-deoxycytidine triphosphate to DNA polymerase and by incorporation into elongating DNA strands, resulting in defective ligation or incomplete synthesis of DNA fragments and ultimately cell death apoptosis. [9]

Figure 4.1 Metabolism of cytarabine (ara-C) in vivo within cancerous cells. [10]

Targets of this chemotherapeutic drug also include binding sites of the analogous molecule cytidine, and this is indicated earlier in Figure 2.2 i) and ii) and in Figure 4.2

Figure 4.2 Cytarabine (ara-C) binding within DNA double helix in vivo within cancerous cells, analogous to cytidine, right part of ara-C, left base guanine. [11]

The potency of the ara-C is however limited in its utility as an anti-cancer nucleoside because of poor intracellular transport characteristics. Also it has a weak activity as a substrate for tumour cell kinases. Therefore the development of drug resistance as a result from reduced nucleoside kinase activity as lowered the efficacy of this agent.

Tumour cells deficient in dCK (deoxycytidine kinase) are highly resistant to cytarabine, so intramolecular delivery of ara-CMP might be expected to circumvent resistance in these cells. [9] Ara-C and similar nucleoside analogues enter cells via specific transporters which are essential to ara-C cytotoxicity in human tumour cells. Hence nucleoside transporter deficient cells are highly resistant to cytarabine. Therefore the address this problem a prodrug of cytarabine used synthesised and can diffuse passively into cells where it is metalobolised into an active form, i.e. intracellular activation, and hence overcomes resistance problems such as limiting nucleoside transporters.

An example of a prodrug is cytarabine phosphoramidate. In an in-vitro study assay indicated the drug inhibited cell growth upto 50% of the control value where the stock drug was prepared in absolute ethanol. This procedure was undertaken in a 72h incubation period where data recorded fitted a sigmoid curve and the results were expressed as the IC50. [9]

The structure-activity relationship, i.e. the relationship between the chemical structure of the molecule and its biological activity is the biological reactivity with guanine in DNA replication, analogous to cytidine guanine interactions. The SAR components of cytarabine are thus as follows and derived from cytidine. The hydrogen bonding interaction is shown in Figure 4.2. Also similar in SAR and reactivity is gemcitabine which works in an equivalent fashion to both cytarabine and cytidine but targets lung cancer, pancreatic cancer and breast cancer cells.

Figure 4.3 SAR components of cytarabine and gemcitabine as highlighted in red.

[1] Pharmacology of Nucleotide Synthesis in Principles of Pharmacology:  The Pathophysiologic Basis of Drug Therapy, Golan DE, Tashjian, JR. AH, Armstrong EJ, Galanter JM, Armstrong AW, Arnaout RA, Rose HS, Lippincott Williams & Wilkins, Philadelphia, pp. 505-516, 2005.

[2] A. S. Prakasha Gowda, Joanna M. Polizzi, Kristin A. Eckert and Thomas E. Spratt Biochemistry, 2010, 49 (23), pp 4833-4840 Publication Date (Web): May 12, 2010 DOI: 10.1021/bi100200c

[3] William H. Gmeiner, David Konerding, and Thomas L. James Biochemistry, 1999, 38 (4), pp 1166 1175 Publication Date (Web): January 05, 1999 DOI: 10.1021/bi981702s

[4] Chu E, Sartorelli AC Cancer Chemotherapy in Basic and Clinical Pharmacology,  Chapter 55, 10th Edition, (Katzung BG, editor), McGraw-Hill, Lange Series, p.890, 2007.

[5] J. Nat. Prod. 2004, 67, 1216-123 David J. Newman* and Gordon M. Crag

[6] J. Johnsamuel et al. / Bioorg. Med. Chem. 13 (2005) 4160-416

[7] Drug Design and Chemistry Section, Laboratory of Medicinal Chemistry and Biology, NCI, NIH, Bethesda, Maryland 20014 USA September 1977, John A. Beislera, Mohamed M. Abbasia and John S. Driscolla

[8] Suckling, C. J. Chemical approaches to the Discovery of New Drugs. Sci. Prog. 1991, 75, 323-359

[9] J. American Chemical Society November 26, 2003, Molecular Pharmaceutics, 2004, 1 (2), pp 112-116, Sandra C. Tobias and Richard F. Borch, DOI: 10.1021/mp034019v

[10] Stam RW, Hubeek I, den Boer ML, Buijs-Gladdines JGCAM, Creutzig U, Kaspers GJL, Pieters R MLL Gene rearrangements have no direct impact on ARA-C sensitivity in infant acute lymphoblastic leukemia and childhood M4/M5 acute myeloid leukemia. Leukemia (2006) 20, 179-182, published on line November 2005.

[11] Lehninger, Nelson & Cox Principles of Biochemistry 5th Edition Pages 271-300.