Induction of resistance to the lipophilic cytarabine prodrug elacytarabine (CP-4055) in CEM leukemic cells.

The deoxynucleoside analogs cytarabine (Ara-C) and gemcitabine (dFdC) are widely used in the treatment of cancer. Due to their hydrophilic nature they need the equilibrative (hENT) and concentrative (hCNT) nucleoside transporters to enter the cell. To bypass drug resistance due to decreased uptake, lipophilic 5'elaidic acid esters were synthesized, elacytarabine (CP-4055, from ara-C) and CP-4126 (from gemcitabine), which are currently in clinical development for solid and hematological tumors. We investigated whether resistance can be induced in vitro, and treated the CEM leukemic cell line with weekly increasing elacytarabine concentrations, up to 0.28 microM (10 times IC(50)). The IC(50) of the resistant CEM/CP-4055 was 35 microM, about 1,000 times that of the wildtype CEM, and comparable to that of CEM/dCK- (deoxycytidine kinase deficient) (22 microM). CEM/CP-4055 was also cross-resistant to Ara-C, gemcitabine and CP-4126 (28 and 33 microM, respectively). A low level of mRNA dCK was observed, and similar to CEM/dCK-, CEM/CP-4055 did not accumulate Ara-CTP after exposure to Ara-C or elacytarabine, which is consistent with a deficiency in dCK. In conclusion, elacytarabine induced resistance similar to Ara-C. This resistance was caused by downregulation of dCK.

Cell cycle effects of fatty acid derivatives of cytarabine, CP-4055, and of gemcitabine, CP-4126, as basis for the interaction with oxaliplatin and docetaxel.

To bypass resistance due to limited entry into the cell derivatives of cytarabine (CP-4055, elacytarabine) and gemcitabine (CP-4126) containing a fatty acid chain at the 5' position of the nucleoside were developed. CP-4055 showed an increased retention of the active metabolite, the triphosphate. This characteristic was supposed to favor combinations, such as with the tubulin antagonist docetaxel, the platinum oxaliplatin and the antifolate pemetrexed. The role of the cell cycle effects of CP-4055 and CP-4126 on the efficacy of the combination with docetaxel or pemetrexed was determined. The combination of CP-4055 with oxaliplatin and docetaxel was also evaluated in a mouse xenograft model. CP-4055 induced a G2/M and S phase accumulation and CP-4126 an S phase accumulation. Both analogs induced a dose-dependent cell kill (apoptosis and necrosis). None of the docetaxel combinations induced a synergistic effect. The combination of docetaxel with CP-4055 or CP-4126 induced a G2/M accumulation in the A549 (lung cancer) cell line, but a G0/G1 accumulation in the WiDR (colon cancer) cell line. Preincubation with docetaxel induced an increased cell kill in both cell lines. The combination with oxaliplatin showed a synergistic effect in both cell lines. Combinations with pemetrexed were antagonistic in both cell lines. In the A549 cell line pemetrexed with CP-4055 led to an increase of the G0/G1 phase and the S phase. In WiDR the combination of pemetrexed with CP-4055 increased the G0/G1 phase and increased the cell kill. Pemetrexed with CP-4126 induced an increase in the G0/G1 phase and the S phase in the A549 cell line. In the xenograft study, on a colon cancer and a lung metastasis model, the combination of CP-4055 with docetaxel showed the best results. Treatment with CP-4055 followed by docetaxel after 4 h resulted in a reduction in metastasis in a lung metastasis model, and a favorable toxicity profile was observed. In conclusion, the combinations with oxaliplatin showed a synergistic effect in the combination studies. Although the combinations with docetaxel did not show an enhanced effect in the in vitro studies, this combination revealed an increased effect in the xenograft model.

Innovations and opportunities to improve conventional (deoxy)nucleoside and fluoropyrimidine analogs in cancer.

Many drugs that are currently used for the treatment of cancer have limitations, such as induction of resistance and/or poor biological half-life, which reduce their clinical efficacy. To overcome these limitations several strategies have been explored. Chemical modification by the attachment of lipophilic moieties to (deoxy)nucleoside analogs should enhance the plasma half live, change the biodistribution and improve cellular uptake of the drug. Attachment of a lipophilic moiety to a phosphorylated (deoxy)nucleoside analog will improve the activity of the drugs by circumventing the rate-limiting activation step of (deoxy)nucleoside analogs. Duplex and multiplex drugs consist of distinct active drugs with different mechanisms of action, which are linked to each other with either a lipid or a phosphodiester. Enzymatic cleavage of such a prodrug inside the cell releases the drug or the phosphorylated form of the drug. Antitumor activity of cytotoxic drugs can also be enhanced by the use of nanoparticles as carriers. Nanoparticles have the advantage of high stability, high carrier capacity, incorporation of hydrophobic and hydrophilic compounds and variable routes of administration. Encapsulating drugs in liposomes protects the drug against enzymatic breakdown in the plasma and makes it possible to get lipophilic compounds to the tumor site. Nanoparticles and liposomes can be used to target drugs either actively or passively to the tumor. In this review we discuss the considerable progress that has been made in increasing the efficacy of classic (deoxy)nucleoside and fluoropyrimidine compounds by chemical modifications and alternative delivery systems. We expect that combining different strategies could further increase the efficacy of these compounds.

Antiproliferative activity and mechanism of action of fatty acid derivatives of gemcitabine in leukemia and solid tumor cell lines and in human xenografts.

UNLABELLED: Gemcitabine is a deoxycytidine analog, which can be inactivated by deamination catalyzed by deoxycytidine deaminase (dCDA). Altered transport over the cell membrane is a mechanism of resistance to gemcitabine. To facilitate accumulation, the fatty acid derivative CP-4125 was synthesized. Since, the fatty acid is acylated at the site of action of dCDA, a decreased deamination was expected. CP-4125 was equally active as gemcitabine in a panel of rodent and human cell lines and in human melanoma xenografts bearing mice. In contrast to gemcitabine, CP-4125 was not deaminated but inhibited deamination of deoxycytidine and gemcitabine. Pools of the active triphosphate of gemcitabine increased for over 20 hr after CP-4125 exposure, while these pools decreased directly after removal of gemcitabine. IN CONCLUSION: CP-4125 is an interesting new gemcitabine derivative.

Antiproliferative activity and mechanism of action of fatty acid derivatives of arabinosylcytosine (ara-C) in leukemia and solid tumor cell lines.

Resistance to, the hydrophilic drug ara-C, might be meditated by decreased membrane transport. Lipophilic prodrugs were synthesized to facilitate uptake. These compounds were equally active as ara-C, while the compounds with the shortest fatty-acid group and highest number of double bonds were the more active. These compounds also show a better retention profile, their effect is retained longer than for ara-C.

Antiproliferative activity and mechanism of action of fatty acid derivatives of arabinofuranosylcytosine in leukemia and solid tumor cell lines.

1-beta-D-arabinofuranosylcytosine (ara-C) is a deoxycytidine analog with activity in leukemia, which requires phosphorylation by deoxycytidine kinase (dCK) to allow formation of its active phosphate 1-beta-D-arabinofuranosylcytosine triphosphate, but can be deaminated by deoxycytidine deaminase. Altered membrane transport is also a mechanism of drug resistance. In order to facilitate ara-C uptake and prolong retention in the cell, lipophilic prodrugs were synthesized. Fatty acid groups with a varying acyl chain length and number of double bonds were esterified at the 5' position on the sugar moiety of ara-C. The compounds were tested in two pairs of ara-C resistant leukemic cell lines (murine L1210 and rat BCLO and their resistant variants L4A6 and Bara-C, respectively) and two pairs of cell lines with a resistance to gemcitabine, another deoxycytidine analog (human ovarian cancer A2780 and murine colon cancer C26-A and their resistant variants AG6000 and C26-G, respectively). L4A6, Bara-C and AG6000 have varying degrees of decreased dCK activity, while the mechanism for C26-G is not yet clear. In the parent cell lines, ara-C was more active, but in the resistant variants several of the analogs were more active, while the degree of cross-resistance varied. In AG6000 with a total dCK deficiency, all compounds were inactive. Structure-activity relation analysis showed that ara-C derivatives with shorter acyl chains and more double bonds were more active in the parental and drug resistant cells. Further mechanistic studies were performed with the elaidic acid derivative of ara-C (CP-4055). CP-4055 inhibited deamination of dCyd partly and induced DNA synthesis inhibition effectively in C26-A and C26-G cells, but the retention of inhibition was much longer for CP-4055 than for ara-C. In contrast to ara-C, CP-4055 inhibited RNA synthesis for 60% after drug exposure. In conclusion, CP-4055 seems to be a promising prodrug, whose effects were different and longer lasting than for the parent drug.

Antiviral activity of ganciclovir elaidic acid ester against herpesviruses.

A fatty acid derivative of ganciclovir (GCV), elaidic acid ganciclovir (E-GCV), has been evaluated for its inhibitory activity against laboratory and clinical strains of herpes simplex type 1 (HSV-1) and type 2 (HSV-2), varicella-zoster virus (VZV) and human cytomegalovirus (HCMV) in human embryonic lung fibroblasts. GCV, cidofovir, acyclovir (ACV), brivudin (BVDU) and foscarnet (PFA) were included as reference compounds. The viruses studied were wild-type, thymidine kinase-deficient (TK(-)) and PFA-resistant (PFA(r)) HSV strains. The IC(50) values obtained for E-GCV were 5- to 30-fold lower than those observed for GCV, the IC(50) value of E-GCV for HSV-1 strain KOS being 0.07 nM. A similarly increased activity of E-GCV (as compared to GCV) was noted for TK(-) and PFA(r) HSV-1 or HSV-2 strains. However, E-GCV did not exhibit superior activity over GCV to VZV or HCMV in vitro. The antiviral efficacy of E-GCV was also evaluated in vivo against intracerebral HSV-2 infection in NMRI mice. Animals were treated intraperitoneally or perorally with E-GCV, GCV or placebo once daily for 10 days, starting the day of infection. E-GCV compared to GCV at equimolar doses, proved markedly more efficacious than GCV in terms of reduction of mortality rate and delay of mean time of death. The elaidic acid ester of GCV should therefore be considered as a novel approach towards the treatment of HSV infections.

Antitumor activity of P-4055 (elaidic acid-cytarabine) compared to cytarabine in metastatic and s.c. human tumor xenograft models.

The antineoplastic efficacy of P-4055, a 5'-elaidic acid (C18:1, unsaturated fatty acid) ester of cytarabine, a nucleoside antimetabolite frequently used in the treatment of hematological malignancies, was examined in several in vivo models for human cancer. In initial dose-finding studies in nude mice, the efficacy of P-4055 was highest when using schedules with repeated daily doses. In a Raji Burkitt's lymphoma leptomeningeal carcinomatosis model in nude rats, the control cytarabine- and saline-treated animals (five in each group) had a mean survival time of 13.2 days, whereas treatment with P-4055 resulted in three of five long-time survivors (>70 days). In a systemic Raji leukemia model in nude mice, 8 of 10 of the P-4055-treated animals survived (>80 days), compared with none of the cytarabine-treated animals (mean survival time, 34.2 days). In s.c. xenograft models, the effects of maximum tolerated doses of P-4055 and cytarabine, given in four weekly cycles of daily bolus i.v. injections for 5 subsequent days, against seven tumors (three melanomas, one lung adenocarcinoma, one breast cancer, and two osteogenic sarcomas) were investigated. P-4055 induced partial or complete tumor regression of the lung carcinoma, as well as of all three malignant melanomas. In two of the melanomas the activity was highly superior to that of cytarabine, and both P-4055 and cytarabine were, in general, more effective than several clinically established drugs previously tested in the same tumor models. In in vitro studies, inhibitors of nucleoside carrier-dependent transport, nitrobenzylmercaptopurine riboside and dipyridamol, reduced strongly the cellular sensitivity to cytarabine, but not to P-4055, indicating that P-4055 uses an alternative/additional mechanism of internalization into the cell compared with cytarabine. The results explain, at least in part, the observed differences between the two compounds in in vivo efficacy, and together the data strongly support the evaluation of P-4055 in clinical studies.

Evaluation of a novel, anti-herpes simplex virus compound, acyclovir elaidate (P-4010), in the female guinea pig model of genital herpes.

The antiviral effect of acyclovir elaidate in the female guinea pig model of genital herpes was investigated in a series of experiments. The antiherpesvirus effects of this novel compound, 9-(2'-[trans-9"-octadecenoyloxyl]ethoxymethyl)guanine (code no. P-4010), were studied in both primary and recurrent genital herpes in the female guinea pig, following oral gavage or intraperitoneal injection, with different formulations of the compound, and in comparison with acyclovir (ACV) or penciclovir (PCV). The results indicate that compound P-4010 has a greater capability than either ACV or PCV in reducing the clinical symptoms of primary genital herpes induced following the inoculation of herpes simplex virus type 2 (HSV-2) intravaginally into guinea pigs. In addition, the administration of P-4010 twice daily over a 10-day period by the intraperitoneal route (15 to 40 mg/kg of body weight/day) or by oral gavage (50 to 200 mg/kg/day), commencing 4 h subsequent to intravaginal HSV-2 infection, resulted in a degree of reduction in the incidence and severity of spontaneous, recurrent genital herpes in these animals. The findings are discussed in the light of the value and relevance of the female guinea pig model of genital herpes for the assessment of anti-herpes simplex virus compounds.

Frequency dependence of the shear moduli of spectrin studied using a multiple lumped resonator viscoelastometer.

The frequency dependence (119-7860 Hz) of the storage and loss shear moduli, G' and G'', of human erythrocyte spectrin dimer crude solutions at 22.5 degrees C has been measured using a Birnboim-Schrag multiple lumped resonator viscoelastometer. The measurements were carried out on solutions of ionic strength 1 mM containing 1.1-3.7 mg ml-1 spectrin. This corresponds to the terminal zone for G' and G''. Analysis of the data using the standard theory of hybrid relaxation spectra yields a relaxation time of 22.5 +/- 1 microseconds. The pure spectrin dimer relaxation time is estimated to be 16 +/- 3 microseconds. This result suggests that at an ionic strength of 1 mM, the spectrin dimers are extended and that the main relaxation process is simple end-over-end rotation.