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Cellular pharmacology of multi- and duplex drugs consisting of ethynylcytidine and 5-fluoro-2'-deoxyuridine.

Bijnsdorp IV, Schwendener RA, Schott H, Fichtner I, Smid K, Laan AC, Schott S, Losekoot N, Honeywell RJ, Peters GJ - Invest New Drugs (2009)

Bottom Line: Although the liposomal formulation was less active than ETC-L-FdUrd in FM3A/0 cells (IC(50):19.3 nM), resistance due to thymidine kinase (TK) deficiency was greatly reduced.ETC-L-FdUrd and the liposomal formulation were neither affected by transporter nor nucleotidase/phosphatase inhibition, indicating circumvention of active transporters.These formulations seem to be effective when a lipophilic linker is used combined with a liposomal formulation.

View Article: PubMed Central - PubMed

Affiliation: Department Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands.

ABSTRACT
Prodrugs can have the advantage over parent drugs in increased activation and cellular uptake. The multidrug ETC-L-FdUrd and the duplex drug ETC-FdUrd are composed of two different monophosphate-nucleosides, 5-fluoro-2'deoxyuridine (FdUrd) and ethynylcytidine (ETC), coupled via a glycerolipid or phosphodiester, respectively. The aim of the study was to determine cytotoxicity levels and mode of drug cleavage. Moreover, we determined whether a liposomal formulation of ETC-L-FdUrd would improve cytotoxic activity and/or cleavage. Drug effects/cleavage were studied with standard radioactivity assays, HPLC and LC-MS/MS in FM3A/0 mammary cancer cells and their FdUrd resistant variants FM3A/TK(-). ETC-FdUrd was active (IC(50) of 2.2 and 79 nM) in FM3A/0 and TK(-) cells, respectively. ETC-L-FdUrd was less active (IC(50): 7 nM in FM3A/0 vs 4500 nM in FM3A/TK(-)). Although the liposomal formulation was less active than ETC-L-FdUrd in FM3A/0 cells (IC(50):19.3 nM), resistance due to thymidine kinase (TK) deficiency was greatly reduced. The prodrugs inhibited thymidylate synthase (TS) in FM3A/0 cells (80-90%), but to a lower extent in FM3A/TK(-) (10-50%). FdUMP was hardly detected in FM3A/TK(-) cells. Inhibition of the transporters and nucleotidases/phosphatases resulted in a reduction of cytotoxicity of ETC-FdUrd, indicating that this drug was cleaved outside the cells to the monophosphates, which was verified by the presence of FdUrd and ETC in the medium. ETC-L-FdUrd and the liposomal formulation were neither affected by transporter nor nucleotidase/phosphatase inhibition, indicating circumvention of active transporters. In vivo, ETC-FdUrd and ETC-L-FdURd were orally active. ETC nucleotides accumulated in both tumor and liver tissues. These formulations seem to be effective when a lipophilic linker is used combined with a liposomal formulation.

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Related in: MedlinePlus

ETC nucleoside and nucleotide accumulation in human HT-29-xenograft (tumor) and liver tissues, 2 h after administration of the ETC-FdUrd and ETC-L-FdUrd. ETC and ETC nucleotides were measured with LC-MS/MS detection. Values represent means of three independent experiments ± SEM
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Fig4: ETC nucleoside and nucleotide accumulation in human HT-29-xenograft (tumor) and liver tissues, 2 h after administration of the ETC-FdUrd and ETC-L-FdUrd. ETC and ETC nucleotides were measured with LC-MS/MS detection. Values represent means of three independent experiments ± SEM

Mentions: After 2 h treatment with ETC-FdUrd, the ETC-nucleoside and especially the ETC-nucleotides accumulated to a high extent in the tumor tissue (Fig. 4). ETC derived from ETC-FdUrd accumulated in the liver tissue, although the phosphate-forms were present at lower levels than in the tumor tissue. After 4 h, ETC or ETC-nucleotides were hardly present in either the tumor or the liver tissue (data not shown). After 2 h treatment with the lipophilic ETC-L-FdUrd, both ETC-nucleoside and the active nucleotide were detectable at comparable levels in the tumor tissue. Both ETC and ETC-nucleotides were present in the liver tissue, but to a lower extent than after exposure to ETC-FdUrd. After 4 h, an increase in ETC-nucleotides was detectable in the liver tissue, however at lower levels than detected with ETC-FdUrd (data not shown). In addition, the difference in the presence of ETC and ETC-nucleotides between ETC-FdUrd and ETC-L-FdUrd did not result in a difference in anti-tumor response. This may be related to a more rapid and direct action by ETC-FdUrd and a slower action by the liphophilic ETC-L-FdUrd due to a slow release or cleavage mechanism.Fig. 4


Cellular pharmacology of multi- and duplex drugs consisting of ethynylcytidine and 5-fluoro-2'-deoxyuridine.

Bijnsdorp IV, Schwendener RA, Schott H, Fichtner I, Smid K, Laan AC, Schott S, Losekoot N, Honeywell RJ, Peters GJ - Invest New Drugs (2009)

ETC nucleoside and nucleotide accumulation in human HT-29-xenograft (tumor) and liver tissues, 2 h after administration of the ETC-FdUrd and ETC-L-FdUrd. ETC and ETC nucleotides were measured with LC-MS/MS detection. Values represent means of three independent experiments ± SEM
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3037477&req=5

Fig4: ETC nucleoside and nucleotide accumulation in human HT-29-xenograft (tumor) and liver tissues, 2 h after administration of the ETC-FdUrd and ETC-L-FdUrd. ETC and ETC nucleotides were measured with LC-MS/MS detection. Values represent means of three independent experiments ± SEM
Mentions: After 2 h treatment with ETC-FdUrd, the ETC-nucleoside and especially the ETC-nucleotides accumulated to a high extent in the tumor tissue (Fig. 4). ETC derived from ETC-FdUrd accumulated in the liver tissue, although the phosphate-forms were present at lower levels than in the tumor tissue. After 4 h, ETC or ETC-nucleotides were hardly present in either the tumor or the liver tissue (data not shown). After 2 h treatment with the lipophilic ETC-L-FdUrd, both ETC-nucleoside and the active nucleotide were detectable at comparable levels in the tumor tissue. Both ETC and ETC-nucleotides were present in the liver tissue, but to a lower extent than after exposure to ETC-FdUrd. After 4 h, an increase in ETC-nucleotides was detectable in the liver tissue, however at lower levels than detected with ETC-FdUrd (data not shown). In addition, the difference in the presence of ETC and ETC-nucleotides between ETC-FdUrd and ETC-L-FdUrd did not result in a difference in anti-tumor response. This may be related to a more rapid and direct action by ETC-FdUrd and a slower action by the liphophilic ETC-L-FdUrd due to a slow release or cleavage mechanism.Fig. 4

Bottom Line: Although the liposomal formulation was less active than ETC-L-FdUrd in FM3A/0 cells (IC(50):19.3 nM), resistance due to thymidine kinase (TK) deficiency was greatly reduced.ETC-L-FdUrd and the liposomal formulation were neither affected by transporter nor nucleotidase/phosphatase inhibition, indicating circumvention of active transporters.These formulations seem to be effective when a lipophilic linker is used combined with a liposomal formulation.

View Article: PubMed Central - PubMed

Affiliation: Department Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands.

ABSTRACT
Prodrugs can have the advantage over parent drugs in increased activation and cellular uptake. The multidrug ETC-L-FdUrd and the duplex drug ETC-FdUrd are composed of two different monophosphate-nucleosides, 5-fluoro-2'deoxyuridine (FdUrd) and ethynylcytidine (ETC), coupled via a glycerolipid or phosphodiester, respectively. The aim of the study was to determine cytotoxicity levels and mode of drug cleavage. Moreover, we determined whether a liposomal formulation of ETC-L-FdUrd would improve cytotoxic activity and/or cleavage. Drug effects/cleavage were studied with standard radioactivity assays, HPLC and LC-MS/MS in FM3A/0 mammary cancer cells and their FdUrd resistant variants FM3A/TK(-). ETC-FdUrd was active (IC(50) of 2.2 and 79 nM) in FM3A/0 and TK(-) cells, respectively. ETC-L-FdUrd was less active (IC(50): 7 nM in FM3A/0 vs 4500 nM in FM3A/TK(-)). Although the liposomal formulation was less active than ETC-L-FdUrd in FM3A/0 cells (IC(50):19.3 nM), resistance due to thymidine kinase (TK) deficiency was greatly reduced. The prodrugs inhibited thymidylate synthase (TS) in FM3A/0 cells (80-90%), but to a lower extent in FM3A/TK(-) (10-50%). FdUMP was hardly detected in FM3A/TK(-) cells. Inhibition of the transporters and nucleotidases/phosphatases resulted in a reduction of cytotoxicity of ETC-FdUrd, indicating that this drug was cleaved outside the cells to the monophosphates, which was verified by the presence of FdUrd and ETC in the medium. ETC-L-FdUrd and the liposomal formulation were neither affected by transporter nor nucleotidase/phosphatase inhibition, indicating circumvention of active transporters. In vivo, ETC-FdUrd and ETC-L-FdURd were orally active. ETC nucleotides accumulated in both tumor and liver tissues. These formulations seem to be effective when a lipophilic linker is used combined with a liposomal formulation.

Show MeSH
Related in: MedlinePlus