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

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

Cellular pharmacology of multi- and duplex drugsconsisting of ethynylcytidine and 5-fluoro-2′-deoxyuridine

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 (IC50 of 2.2 and 79 nM) in FM3A/0 and TK− cells, respectively. ETC-L-FdUrd was less active (IC50: 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 (IC50:19.3 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

Mutations in TBL1X Are Associated With Central Hypothyroidism

Context: Isolated congenital central hypothyroidism (CeH) can result from mutations in TRHR, TSHB, and IGSF1, but its etiology often remains unexplained. We identified a missense mutation in the transducin ␤-like protein 1, X-linked (TBL1X) gene in three relatives diagnosed with isolated CeH. TBL1X is part of the thyroid hormone receptor-corepressor complex. Objective: The objectives of the study were the identification of TBL1X mutations in patients with unexplained isolated CeH, Sanger sequencing of relatives of affected individuals, and clinical and biochemical characterization; in vitro investigation of functional consequences of mutations; and mRNA expression in, and immunostaining of, human hypothalami and pituitary glands. Design: This was an observational study. Setting: The study was conducted at university medical centers. Patients: Nineteen individuals with and seven without a mutation participated in the study. Main Outcome Measures: Outcome measures included sequencing results, clinical and biochemical characteristics of mutation carriers, and results of in vitro functional and expression studies. Results: Sanger sequencing yielded five additional mutations. All patients (n ϭ 8; six males) were previously diagnosed with CeH (free T 4 [FT4] concentration below the reference interval, normal thyrotropin). Eleven relatives (two males) also carried mutations. One female had CeH, whereas 10 others had low-normal FT4 concentrations. As a group, adult mutation carriers had 20%-25% lower FT4 concentrations than controls. Twelve of 19 evaluated carriers had hearing loss. Mutations are located in the highly conserved WD40-repeat domain of the protein, influencing its expression and thermal stability. TBL1X mRNA and protein are expressed in the human hypothalamus and pituitary. C entral hypothyroidism (CeH) is characterized by suboptimal thyroid hormone (TH) secretion due to insufficient stimulation by TSH of an otherwise normal thyroid gland. CeH may be caused by congenital or acquired disorders of the pituitary gland or hypothalamus (1). The diagnosis is based on a plasma free T 4 (FT4) concentration below the reference interval in combination with an inappropriately normal TSH. Congenital CeH has an esti- Conclusion

Metabolism and accumulation of the lipophilic deoxynucleoside analogs elacytarabine and CP-4126

Cytarabine (ara-C) and gemcitabine (dFdC) are commonly used anticancer drugs, which depend on the equilibrative (ENT) and concentrative-nucleoside-transporters to enter the cell. To bypass transport-related drug resistance, lipophilic derivatives elacytarabine (CP-4055), ara-C-5′elaidic-acid-ester, and CP-4126, (CO 1.01) gemcitabine-5′elaidic-acid-ester, were investigated for the entry into the cell, distribution, metabolism and retention. The leukemic CEM-cell-line and its deoxycytidine-kinase deficient variant (CEM/dCK-) were exposed for 30 and 60 min to the radiolabeled drugs; followed by culture in drug-free medium in order to determine drug retention in the cell. The cellular fractions were analyzed with thin-layer-chromatography and HPLC. Elacytarabine and CP-4126 were converted to the parent compounds both inside and outside the cell (35–45%). The ENT-inhibitor dipyridamole did not affect their uptake or retention. Inside the cell Elacytarabine and CP-4126 predominantly localized in the membrane and cytosolic fraction, leading to a long retention after removal of the medium. In contrast, in cells exposed to the parent drugs ara-C and dFdC, intracellular drug concentration increased during exposure but decreased to undetectable levels after drug removal. In the dCK- cell line, no metabolism was observed. The concentrations of ara-CTP and dFdCTP reached a peak at the end of the incubation with the drugs, and decreased after drug removal; peak levels of dFdCTP were 35 times higher than ara-CTP and was retained better. In contrast, after exposure to elacytarabine or CP-4126, ara-CTP and dFdCTP levels continued to increase not only during exposure but also during 120 min after removal of the elacytarabine and CP-4126. Levels of ara-CTP and dFdCTP were higher than after exposure to the parent drugs. In conclusion, the lipophilic derivatives elacytarabine and CP-4126 showed a nucleoside-transporter independent uptake, with long retention of the active nucleotides. These lipophilic nucleoside analogues are new chemical entities suitable for novel clinical applications