Actividad 3: Orientación y ubicación con constelaciones y coordenadas

1.2.1.1 5-Fluorouracil and Leucovorin

For many years following fluoropyrimidine discovery, 5-FU remained the backbone of therapy for CRC and until recently was considered the standard first-line treatment for metastatic CRC (Goldberg, 2005). Fluoropyrimidines were developed following the observation that rat hepatomas utilised pyrimidine uracil more rapidly than normal tissues (Rutman et al., 1954), signifying that uracil metabolism may be a potential target for antimetabolite chemotherapy. 5-FU enters the cell using the same facilitated transport mechanism as uracil (Wohlhueter et al., 1980) and is converted intracellularly to several active metabolites: fluorodeoxyuridine monophosphate (FdUMP), fluorodeoxyuridine triphosphate (FdUTP) and fluorouridine triphosphate (FUTP). These active metabolites disrupt RNA synthesis triggering mis-incorporation of fluoro nucleotides into RNA and DNA alongside inhibition of the enzyme thymidylate synthase (Longley et al., 2003). Thymidylate synthase-catalysed reactions provide the sole source of thymidylate, a key molecule necessary for DNA replication and repair (Longley et al., 2003) (Figure 1.1).

5-FU has been used for more than 50 years in the treatment of CRC. Many early randomized studies of 5-FU in the adjuvant setting failed to show significant improvement in patient survival (Higgins et al., 1984;Buyse et al., 1988;Panettiere et al., 1988), with the overall response rate as a single agent in advanced CRC reported as approximately 10–15% (O'Connell, 1989). However, over the past 30 years important modulation strategies have been developed to reduce cytotoxicity, increase anti-cancer activity and overcome clinical resistance associated with 5-FU. Side-effects of 5- FU include stomatitis, esophagopharyngitis, diarrhoea, nausea, vomiting, dermatologic changes, alopecia, hematopoietic depression, fever, neurotoxicity, cardiotoxicity and death (Curreri et al., 1958;Diasio and Harris, 1989;Kennedy, 1999). Leukopenia is recognised as a major dose- limiting toxicity of 5-FU monotherapy with rates as high as 93% reported in

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some studies (O'Connell, 1989;Petrelli et al., 1989). However, dose reduction from 15 mg/kg/day to 12 mg/kg/day has resulted in tolerable toxicity (O'Connell, 1989), allowing 5-FU to remain a key agent for the treatment of both advanced and early-stage CRC (Longley et al., 2003).

Leucovorin (LV) (C20H23N7O7) is a folinic acid derivative that enhances the cytotoxic effects of 5-FU by inhibiting the production of thymidylate synthase (Longley et al., 2003). Thymidylate synthase catalyses the reductive methylation of deoxyuridine monophosphate (dUMP) to deoxythymidine

monophosphate (dTMP), using reduced folate 5, 10-

methylenetetrahydrofolate (CH2THF) as the methyl donor (Diasio and

Harris, 1989) (Figure 1.1). This reaction provides the sole de novo source

of thymidylate, which is necessary for DNA replication and repair (Longley et al., 2003).

High intracellular levels of the reduced folate CH2 THF are necessary for optimal binding of the 5-FU metabolite FdUMP to thymidylate synthase.

Leucovorin increases intracellular concentrations of CH2 THF and has been

shown to increase both in vitro (Matherly et al., 1990) and in vivo (Nadal et al., 1987) toxicity of 5-FU in many cancer cell lines. Synergistic administration of 5-FU/LV has been shown to improve response rates in CRC by up to 37% when compared to a single agent 5-FU bolus as well as increasing median and overall survival (Petrelli et al., 1987;Erlichman et al., 1988). Several studies have found the dose-limiting toxicity of both high and low dose 5-FU/LV regimens to be gastrointestinal (Petrelli et al., 1987;Petrelli et al., 1989) including severe ulcerative stomatitis occurring at rates as high as 30% of patients (Jebb et al., 1994). Although it has been reported that severe diarrhoea is not more frequent in patients treated with 5-FU/LV compared to patients treated with 5-FU alone (O'Connell, 1989), severe diarrhoea specifically has been described as a major treatment- related toxicity affecting approximately 40% of patients receiving 5-FU/LV, of these patients 52% not only required a dose reduction of 5-FU, but also hospitalization for intravenous hydration (Petrelli et al., 1987). Moreover,

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treatment related diarrhoea has been correlated with the demise of 5% of the patient cohort (Petrelli et al., 1989).

Although modest, the improvements resulting from the combination of 5-FU with LV offered the possibility of enhancing the anti-cancer efficacy of 5-FU. Increased understanding of the mechanisms of action of 5-FU in the years to come led to development of strategies to successfully increase its anti- cancer activity (Longley et al., 2003), with 5-FU now used in clinical combinations with several new generation anti-cancer chemotherapeutics.

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Figure 1.1 5-Flourouracil, Capecitabine and Leucovorin.

Capecitabine is an oral prodrug that is converted to its only active metabolite, 5-Fluorouracil (5-FU) by thymidine phosphorylase. The chief mechanism of 5-FU activation is conversion of 5-FU to fluorouridine monophosphate (FUMP). FUMP is phosphorylated to fluorouridine diphosphate (FUDP), which can either be further phosphorylated to the active metabolite fluorouridine triphosphate (FUTP), or be converted to fluorodeoxyuridine diphosphate (FdUDP). In turn, FdUDP can either be phosphorylated or dephosphorylated to generate the active metabolites fluorodeoxyuridine triphosphate (FdUTP) and fluorodeoxyuridine monophosphate (FdUMP), respectively. An alternative activation pathway involves the thymidine phosphorylase catalysed conversion of 5-FU to fluorodeoxyuridine (FUDR), which is then phosphorylated to FdUMP. Thymidylate synthase (TS) catalyses the conversion of

deoxyuridine monophosphate (dUMP) to deoxythymidine

monophosphate (dTMP) with 5,10-methylene tetrahydrofolate

(CH2THF) as the methyl donor. The active metabolite FdUMP binds to TS and CH2THF, blocking access of dUMP to the nucleotide-binding site and inhibiting dTMP synthesis. This results in imbalances to deoxynucleotide (dNTP) levels and accumulation of deoxyuridine triphosphate (dUTP). Accumulating dUTP triggers misincorporation of uracil into the DNA via DNA polymerase. Although the DNA repair mechanism can excise the misincorporated DNA, the existing dUMP is just re-phosphorylated to dUTP and re-incorporated back into the DNA. This process is known as futile cycling. DNA strand breakage and damage occur as repair mechanisms ultimately fail.

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1.2.1.2 Capecitabine

Capecitabine (CAPE) (C15H22FN3O6) is an oral prodrug that is enzymatically converted to 5-FU by thymidine phosphorylase. Derived from a predecessor prodrug of 5-FU, doxifluridine, CAPE is metabolized to FU through 3 activation steps: i) CAPE is absorbed through the intestine and converted to 5'-deoxy-S-fluorocytidine (5'-DFCR) by carboxylesterase and then ii) to 5'-deoxy-S-fluorouridine (5'-DFUR) by cytidine deaminase, in the liver; iii) thymidine phosphorylase then converts 5'-DFUR to the active drug, FU (Figure 1.1). This occurs in both neoplastic and healthy tissues. However, thymidine phosphorylase is present at higher levels in tumour cells than in healthy tissues, allowing for selective activation of the drug and less systemic toxicity (Budman et al., 1998;Miwa et al., 1998;Walko and Lindley, 2005).

Initially approved in 1998 for use in patients with metastatic breast cancer whose tumours were resistant to standard chemotherapy with paclitaxel (Taxol) and an anthracycline-containing regimen, CAPE was first trialled for the use in metastatic CRC in the early 2000’s. Results demonstrated preferential conversion of CAPE to 5-FU in colorectal tumours after oral administration to patients (Schüller et al., 2000). Several clinical trials have demonstrated that single agent CAPE administration has a significantly superior response rate compared to 5-FU/LV, even in patient subgroups with poor prognostic indicators. CAPE, compared with bolus 5-FU/LV treatment also demonstrated improved safety producing a significantly lower incidence of diarrhoea, stomatitis, nausea, alopecia and myelosuppression (Hoff et al., 2001;Twelves et al., 2001;Van Cutsem et al., 2004). However, severe hand-foot syndrome and hyperbilirubinemia were significantly more frequent in CAPE-treated patients compared to those treated with 5-FU/LV (Hoff et al., 2001). Regardless of this, CAPE patients required substantially fewer hospital visits following drug administration than 5-FU/LV patients and spent fewer days in hospital for the management of treatment-related adverse events than did patients treated with 5-FU/LV (Hoff et al., 2001;Twelves et al., 2001).

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