Characterization of the in vitro activity of AZD3409, a novel prenyl transferase inhibitor.

PURPOSE: AZD3409 is a novel DPTI that has potent activity against both FTase and GGTase-1. The in vitro inhibition profile of AZD3409 was characterized using three different cell lines: mouse embryogenic fibroblasts, transfected with H-Ras(V12) (MEF), A549 cells (Ki4B-Ras mutation) and MCF-7 cells (no Ras mutation). METHODS: Both cytotoxicity and levels of inhibition of farnesylation and geranylgeranylation were determined in different assays in relation to the concentration of AZD3409. Results were compared with those obtained with the first-generation FTase inhibitor lonafarnib or the GGTase-1 inhibitor GGTI-2147. RESULTS: The mean IC(50) for cytotoxicity of AZD3409 and lonafarnib was 510 and 15,200 nM in MEF cells, 10,600 and 2,740 nM in A549 cells and 6,170 and 9,490 nM in MCF7 cells, respectively. In these cells, the IC(50) for FTase activity of AZD3409 ranged from 3.0 to 14.2 nM and of lonafarnib from 0.26 to 31.3 nM. The inhibiting activity of AZD3409 and lonafarnib on general protein farnesylation was comparable with the specific farnesylation levels of HDJ-2. In vitro geranylgeranylation of Rap1a could be inhibited by GGTI-2147 in all three cell lines, but only in MCF-7 cells by AZD3409. These results are in agreement with the IC(50) values for GGTase-1 activity as the lowest IC(50) for AZD3409 was found in the MCF-7 cell line. CONCLUSIONS: AZD3409 inhibits farnesylation to a higher extent than geranylgeranylation. Both inhibition of farnesylation and geranylgeranylation could not be correlated to the antiproliferative activity of the drug.

Effect of the ATP-binding cassette drug transporters ABCB1, ABCG2, and ABCC2 on erlotinib hydrochloride (Tarceva) disposition in in vitro and in vivo pharmacokinetic studies employing Bcrp1-/-/Mdr1a/1b-/- (triple-knockout) and wild-type mice.

UNLABELLED: We tested whether erlotinib hydrochloride (Tarceva, OSI-774), an orally active epidermal growth factor receptor tyrosine kinase inhibitor, is a substrate for the ATP-binding cassette drug transporters P-glycoprotein (P-gp; MDR1, ABCB1), breast cancer resistance protein (BCRP; ABCG2), and multidrug resistance protein 2 (MRP2; ABCC2) in vitro and whether P-gp and BCRP affect the oral pharmacokinetics of erlotinib hydrochloride in vivo. In vitro cell survival, drug transport, accumulation, and efflux of erlotinib were done using Madin-Darby canine kidney II [MDCKII; wild-type (WT), MDR1, Bcrp1, and MRP2] and LLCPK (WT and MDR1) cells and monolayers as well as the IGROV1 and the derived human BCRP-overexpressing T8 cell lines. In vivo, the pharmacokinetics of erlotinib after p.o. and i.p. administration was studied in Bcrp1/Mdr1a/1b(-/-) (triple-knockout) and WT mice. In vitro, erlotinib was actively transported by P-gp and BCRP/Bcrp1. No active transport of erlotinib by MRP2 was observed. In vivo, systemic exposure (P = 0.01) as well as bioavailability of erlotinib after oral administration (5 mg/kg) were statistically significantly increased in Bcrp1/Mdr1a/1b(-/-) knockout mice (60.4%) compared with WT mice (40.0%; P = 0.02). CONCLUSION: Erlotinib is transported efficiently by P-gp and BCRP/Bcrp1 in vitro. In vivo, absence of P-gp and Bcrp1 significantly affected the oral bioavailability of erlotinib. Possible clinical consequences for drug-drug and drug-herb interactions in patients in the gut between P-gp/BCRP-inhibiting substrates and oral erlotinib need to be addressed.

New insights into the pharmacology and cytotoxicity of gemcitabine and 2',2'-difluorodeoxyuridine.

In a clinical study with oral gemcitabine (2',2'-difluorodeoxycytidine, dFdC), 2',2'-difluorodeoxyuridine (dFdU) was extensively formed and accumulated after multiple oral dosing. Here, we have investigated the in vitro cytotoxicity, cellular uptake, efflux, biotransformation, and nucleic acid incorporation of dFdC and dFdU. Short-term and long-term cytotoxicity assays were used to assess the cytotoxicity of dFdC and dFdU in human hepatocellular carcinoma HepG2, human lung carcinoma A549, and Madin-Darby canine kidney cell lines transfected with the human concentrative or equilibrative nucleoside transporter 1 (hCNT1 or hENT1), or empty vector. Radiolabeled dFdC and dFdU were used to determine cellular uptake, efflux, biotransformation, and incorporation into DNA and RNA. The compounds dFdC, dFdU, and their phosphorylated metabolites were quantified by high-performance liquid chromatography with UV and radioisotope detection. dFdU monophosphate, diphosphate, and triphosphate (dFdU-TP) were formed from dFdC and dFdU. dFdU-TP was incorporated into DNA and RNA. The area under the intracellular concentration-time curve of dFdC-TP and dFdU-TP and their extent of incorporation into DNA and RNA inversely correlated with the IC(50) of dFdC and dFdU, respectively. The cellular uptake and cytotoxicity of dFdU were significantly enhanced by hCNT1. dFdU inhibited cell cycle progression and its cytotoxicity significantly increased with longer duration of exposure. dFdU is taken up into cells with high affinity by hCNT1 and phosphorylated to its dFdU-TP metabolite. dFdU-TP is incorporated into DNA and RNA, which correlated with dFdU cytotoxicity. These data provide strong evidence that dFdU can significantly contribute to the cytotoxicity of dFdC.

The effect of low pH on breast cancer resistance protein (ABCG2)-mediated transport of methotrexate, 7-hydroxymethotrexate, methotrexate diglutamate, folic acid, mitoxantrone, topotecan, and resveratrol in in vitro drug transport models.

Some cellular uptake systems for (anti)folates function optimally at acidic pH. We have tested whether this also applies to efflux from cells by breast cancer resistance protein (BCRP; ABCG2), which has been reported to transport folic acid, methotrexate, and methotrexate di- and triglutamate at physiological pH. Using Spodoptera frugiperda-BCRP membrane vesicles, we showed that the ATP-dependent vesicular transport of 1 muM methotrexate by BCRP is 5-fold higher at pH 5.5 than at physiological pH. The transport of methotrexate was saturable at pH 5.5, with apparent Km and Vmax values of 1.3 +/- 0.2 mM and 44 +/- 2.5 nmol/mg of protein/min, respectively, but was linear with drug concentration at pH 7.3 up to 6 mM methotrexate. In contrast to recent reports, we did not detect transport of methotrexate diglutamate at physiological pH, but we did find transport at pH 5.5. We also found that 7-hydroxy-methotrexate, the major metabolite of methotrexate, is transported by BCRP both at physiological pH and (more efficiently) at low pH. The pH effect was also observed in intact BCRP-overexpressing cells: we found a 3-fold higher level of resistance to both methotrexate and the prototypical BCRP substrate mitoxantrone at pH 6.5 as at physiological pH. Furthermore, with MDCKII-BCRP monolayers, we found that resveratrol, which is a neutral compound at pH < or = 7.4, is efficiently transported by BCRP at pH 6.0, whereas we did not detect active transport at pH 7.4. We conclude that BCRP transports substrate drugs more efficiently at low pH, independent of the dissociation status of the substrate.

Absence of N-linked glycosylation does not affect plasma membrane localization of breast cancer resistance protein (BCRP/ABCG2).

Breast cancer resistance protein (BCRP/ABCG2) is an ATP-binding cassette (ABC) multidrug transporter that confers resistance to various anticancer drugs like topotecan and mitoxantrone. To obtain more insight in its cellular functioning, we investigated phosphorylation and N-linked glycosylation of BCRP. In the epithelial Madin-Darby canine kidney (MDCK) cell line, we did not detect phosphorylation of BCRP, in contrast to MRP2, which was phosphorylated. In the ovarian carcinoma cell line T8 also no phosphorylated BCRP was detected. As BCRP in both lines effectively transports drugs, it appears that phosphorylation of BCRP (if it occurs at all) is not needed for drug transport. We further mutated the asparagine residues 418, 557 and 596 in three putative N-linked glycosylation motifs of BCRP to alanines. Mutant proteins were expressed in CHO9 and MDCKII cells by transient transfection and characterized by Western blot and immunofluorescence analysis. We found that only BCRP-N596A and a mutant with all three asparagines mutated (triple mutant) were not glycosylated anymore, indicating that only asparagine 596 is normally glycosylated. The mutation of asparagine 596 (or 418) had little effect on the subcellular localization of BCRP, indicating that N-linked glycosylation is not essential for routing to the plasma membrane. However, BCRP-N557A and the triple mutant were mainly localized intracellularly, probably in the endoplasmic reticulum, suggesting that this mutation disrupted proper routing of BCRP.

Platinated DNA adducts enhance poisoning of DNA topoisomerase I by camptothecin.

Camptothecins constitute a novel class of chemotherapeutics that selectively target DNA topoisomerase I (Top1) by reversibly stabilizing a covalent enzyme-DNA intermediate. This cytotoxic mechanism contrasts with that of platinum drugs, such as cisplatin, which induce inter- and intrastrand DNA adducts. In vitro combination studies using platinum drugs combined with Top1 poisons, such as topotecan, showed a schedule-dependent synergistic activity, with promising results in the clinic. However, whereas the molecular mechanism of these single agents may be relatively well understood, the mode of action of these chemotherapeutic agents in combination necessitates a more complete understanding. Indeed, we recently reported that a functional homologous recombination pathway is required for cisplatin and topotecan synergy yet represses the synergistic toxicity of 1-beta-D-arabinofuranosyl cytidine in combination with topotecan (van Waardenburg, R. C., de Jong, L. A., van Delft, F., van Eijndhoven, M. A., Bohlander, M., Bjornsti, M. A., Brouwer, J., and Schellens, J. H. (2004) Mol. Cancer Ther. 3, 393-402). Here we provide direct evidence for Pt-1,3-d(GTG) poisoning of Top1 in vitro and demonstrate that persistent Pt-DNA adducts correlate with increased covalent Top1-DNA complexes in vivo. This contrasts with a lack of persistent lesions induced by the alkylating agent bis[chloroethyl]nitrosourea, which exhibits only additive activity with topotecan in a range of cell lines. In human IGROV-1 ovarian cancer cells, the synergistic activity of cisplatin with topotecan requires processive DNA polymerization, whereas overexpression of Top1 enhances yeast cell sensitivity to cisplatin. These results indicate that the cytotoxic activity of cisplatin is due, in part, to poisoning of Top1, which is exacerbated in the presence of topotecan.

Homologous recombination is a highly conserved determinant of the synergistic cytotoxicity between cisplatin and DNA topoisomerase I poisons.

Phase I and II clinical trails are currently investigating the antitumor activity of cisplatin and camptothecins (CPTs; DNA topoisomerase I poisons), based on the dramatic synergistic cytotoxicity of these agents in some preclinical models. However, the mechanistic basis for this synergism is poorly understood. By exploiting the evolutionary conservation of DNA repair pathways from genetically tractable organisms such as budding and fission yeasts to mammalian cells, we demonstrate that the synergism of CPT and cisplatin requires homologous recombination. In yeast and mammalian cell lines defective for RAD52 and XRCC2/3, respectively, the combination of these agents proved antagonistic, while greater than additive activity was evident in isogenic wild-type cells. Homologous recombination appears to mediate a similar interaction of X-rays and CPT, but antagonizes the synergism of cytarabine (Ara-C) with CPT. These findings suggest that homologous recombination comprises an evolutionarily conserved determinant of cellular sensitivity when CPTs are used in combination with other therapeutics.

DNA repair mechanisms involved in gemcitabine cytotoxicity and in the interaction between gemcitabine and cisplatin.

The influence of DNA repair mechanisms on the interaction between gemcitabine and cisplatin was studied using a panel of Chinese hamster ovary (CHO) cell lines deficient in one of the following repair pathways: base excision repair (BER), nucleotide excision repair (NER), homologous recombination (HR) and non-homologous end joining (NHEJ). NER and HR are known to be involved in platinum-DNA adduct repair. Single agent experiments demonstrated that each of the repair deficient cell lines had a similar sensitivity towards gemcitabine as the parental cell lines, whereas the NER- and HR-deficient lines showed increased sensitivity towards cisplatin. Furthermore, in the parental cell lines, the administration sequence cisplatin followed by gemcitabine was synergistic, whereas the reversed schedule showed additivity and simultaneous administration revealed antagonistic cytotoxicity. In the repair deficient cell lines, using this synergistic schedule of cisplatin followed by gemcitabine, loss of synergy was observed in the NER- and HR-deficient cell lines. However, the magnitude of the effect in the NER-deficient cells was small. The sensitivity to the combination of cisplatin and gemcitabine shown by the BER- and NHEJ-deficient cell lines did not differ significantly from that of the parental cell line. Cellular accumulation of platinum as well as the formation of GG- and AG-intrastrand adducts in the parental line and in the HR-deficient line were not affected by gemcitabine. In conclusion, our results indicate that BER, NER, HR, and NHEJ are most likely incapable of modulating the cytotoxicity of gemcitabine, and that HR is involved in the synergistic interaction between cisplatin and gemcitabine in our cell system.