Head-to-head comparison of DFO* and DFO chelators: selection of the best candidate for clinical 89Zr-immuno-PET.

PURPOSE: Almost all radiolabellings of antibodies with 89Zr currently employ the hexadentate chelator desferrioxamine (DFO). However, DFO can lead to unwanted uptake of 89Zr in bones due to instability of the resulting metal complex. DFO*-NCS and the squaramide ester of DFO, DFOSq, are novel analogues that gave more stable 89Zr complexes than DFO in pilot experiments. Here, we directly compare these linker-chelator systems to identify optimal immuno-PET reagents. METHODS: Cetuximab, trastuzumab and B12 (non-binding control antibody) were labelled with 89Zr via DFO*-NCS, DFOSq, DFO-NCS or DFO*Sq. Stability in vitro was compared at 37 °C in serum (7 days), in formulation solution (24 h ± chelator challenges) and in vivo with N87 and A431 tumour-bearing mice. Finally, to demonstrate the practical benefit of more stable complexation for the accurate detection of bone metastases, [89Zr]Zr-DFO*-NCS and [89Zr]Zr-DFO-NCS-labelled trastuzumab and B12 were evaluated in a bone metastasis mouse model where BT-474 breast cancer cells were injected intratibially. RESULTS: [89Zr]Zr-DFO*-NCS-trastuzumab and [89Zr]Zr-DFO*Sq-trastuzumab showed excellent stability in vitro, superior to their [89Zr]Zr-DFO counterparts under all conditions. While tumour uptake was similar for all conjugates, bone uptake was lower for DFO* conjugates. Lower bone uptake for DFO* conjugates was confirmed using a second xenograft model: A431 combined with cetuximab. Finally, in the intratibial BT-474 bone metastasis model, the DFO* conjugates provided superior detection of tumour-specific signal over the DFO conjugates. CONCLUSION: DFO*-mAb conjugates provide lower bone uptake than their DFO analogues; thus, DFO* is a superior candidate for preclinical and clinical 89Zr-immuno-PET.

Haplotype diversity generated by ancient recombination-like events in the MHC of Indian rhesus macaques.

The Mamu-A, Mamu-B, and Mamu-DRB genes of the rhesus macaque show several levels of complexity such as allelic heterogeneity (polymorphism), copy number variation, differential segregation of genes/alleles present on a haplotype (diversity) and transcription level differences. A combination of techniques was implemented to screen a large panel of pedigreed Indian rhesus macaques (1,384 individuals representing the offspring of 137 founding animals) for haplotype diversity in an efficient and inexpensive manner. This approach allowed the definition of 140 haplotypes that display a relatively low degree of region variation as reflected by the presence of only 17 A, 18 B and 22 DRB types, respectively, exhibiting a global linkage disequilibrium comparable to that in humans. This finding contrasts with the situation observed in rhesus macaques from other geographic origins and in cynomolgus monkeys from Indonesia. In these latter populations, nearly every haplotype appears to be characterised by a unique A, B and DRB region. In the Indian population, however, a reshuffling of existing segments generated "new" haplotypes. Since the recombination frequency within the core MHC of the Indian rhesus macaques is relatively low, the various haplotypes were most probably produced by recombination events that accumulated over a long evolutionary time span. This idea is in accord with the notion that Indian rhesus macaques experienced a severe reduction in population during the Pleistocene due to a bottleneck caused by geographic changes. Thus, recombination-like processes appear to be a way to expand a diminished genetic repertoire in an isolated and relatively small founder population.

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.

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.