Towards depersonalized abacavir therapy: chemical modification eliminates HLA-B*57 : 01-restricted CD8+ T-cell activation.

OBJECTIVE: Exposure to abacavir is associated with T-cell-mediated hypersensitivity reactions in individuals carrying human leukocyte antigen (HLA)-B57 : 01. To activate T cells, abacavir interacts directly with endogenous HLA-B57 : 01 and HLA-B57 : 01 expressed on the surface of antigen presenting cells. We have investigated whether chemical modification of abacavir can produce a molecule with antiviral activity that does not bind to HLA-B57 : 01 and activate T cells. DESIGN: An interdisciplinary laboratory study using samples from human donors expressing HLA-B57 : 01. Researchers were blinded to the analogue structures and modelling data. METHODS: Sixteen 6-amino substituted abacavir analogues were synthesized. Computational docking studies were completed to predict capacity for analogue binding within HLA-B57 : 01. Abacavir-responsive CD8 clones were generated to study the association between HLA-B57 : 01 analogue binding and T-cell activation. Antiviral activity and the direct inhibitory effect of analogues on proliferation were assessed. RESULTS: Major histocompatibility complex class I-restricted CD8 clones proliferated and secreted IFNγ following abacavir binding to surface and endogenous HLA-B57 : 01. Several analogues retained antiviral activity and showed no overt inhibitory effect on proliferation, but displayed highly divergent antigen-driven T-cell responses. For example, abacavir and N-propyl abacavir were equally potent at activating clones, whereas the closely related analogues N-isopropyl and N-methyl isopropyl abacavir were devoid of T-cell activity. Docking abacavir analogues to HLA-B57 : 01 revealed a quantitative relationship between drug-protein binding and the T-cell response. CONCLUSION: These studies demonstrate that the unwanted T-cell activity of abacavir can be eliminated whilst maintaining the favourable antiviral profile. The in-silico model provides a tool to aid the design of safer antiviral agents that may not require a personalized medicines approach to therapy.

Oxygen breathing affects 3'-deoxy-3'-18F-fluorothymidine uptake in mouse models of arthritis and cancer.

UNLABELLED: Noninvasive in vivo imaging of biologic processes using PET is an important tool in preclinical studies. We observed significant differences in 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) uptake in arthritic ankles and carcinomas between dynamic and static PET measurements when mice breathed oxygen. Thus, we suspected that air or oxygen breathing and the anesthesia protocol might influence (18)F-FLT tracer uptake. METHODS: We injected arthritic, healthy, and CT26 colon carcinoma-bearing mice with (18)F-FLT before static or dynamic small-animal PET measurements. The spontaneously oxygen- or air-breathing mice were kept conscious or anesthetized with ketamine and xylazine during (18)F-FLT uptake before the 10-min static PET measurements. For dynamic PET scans, mice were anesthetized during the entire measurement. (18)F-FLT uptake was reported in percentage injected dose per cubed centimeter by drawing regions of interest around ankles, carcinomas, and muscle tissue. Additionally, venous blood samples were collected before (18)F-FLT injection and after PET measurement to analyze pH, carbon dioxide partial pressure (pCO(2)), and lactate values. RESULTS: A significantly reduced (18)F-FLT uptake was measured in arthritic ankles and in CT26 colon carcinomas when the mice breathed oxygen and were conscious during tracer uptake, compared with mice that were anesthetized during (18)F-FLT uptake. Breathing air completely abolished this phenomenon. Analysis of blood samples that were obtained from the mice before (18)F-FLT injection and after the PET scan implicated respiratory acidosis that was induced by oxygen breathing and consciousness during tracer uptake. Acidosis was found to be the primary factor responsible for the reduced (18)F-FLT uptake, as reflected by increased pCO(2) and reduced pH and lactate values. CONCLUSION: Oxygen-breathing conscious mice sustained respiratory acidosis and, consequently, reduced cell proliferation and (18)F-FLT uptake in arthritic ankles and CT26 colon carcinomas. Thus, we suggest the use of air instead of oxygen breathing for (18)F-FLT PET measurements.

Early response assessment in prostate carcinoma by ¹8F-fluorothymidine following anticancer therapy with docetaxel using preclinical tumour models.

PURPOSE: The aim of the study was to assess the potential usefulness of 3-deoxy-3-(18)F-fluorothymidine (FLT) as a radiopharmaceutical for imaging the early therapeutic effects of docetaxel (DTX) on tumour proliferation in hormone-refractory prostate cancer (HRPC). METHODS: Cells of the androgen-independent human prostate tumour cell line, 22Rv1, were implanted in athymic male mice. Approximately 3 weeks after cell implantation, the mice were treated with DTX or vehicle. Before and after the treatment, the mice were imaged with a microPET-Focus-F120 scanner (Concorde Microsystems, Knoxville, TN, USA) using FLT and (18)F-fluorodeoxyglucose (FDG). Tracer accumulations in the tumours were then analysed and compared with the proliferation activity and apoptotic index of the tumours. In a separate cell study, 22Rv1 cells were treated with DTX, then incubated with FLT or FDG and examined for their tracer uptake. RESULTS: The microPET imaging showed a significant decrease of FLT uptake in tumours after administration of DTX, while the changes of FDG uptake were minimal. Immunohistochemical analysis of the tumours revealed that the changes of FLT uptake were well correlated with those of proliferation activity but not with the apoptotic index. In vitro studies demonstrated that the significant decrease of FLT uptake in the cells after incubation with DTX correlated with the % S-phase cell fraction, while there were only minimal changes in the prostate-specific antigen concentration of the cell medium and FDG uptake in the cells. CONCLUSION: These results indicate that FLT is a promising tracer for monitoring the early effects of anticancer therapy with DTX in patients with HRPC.

Deoxyadenosine-resistant human T lymphoblasts with elevated 5'-nucleotidase activity.

Although several different enzymes with 5'-nucleotidase activity have been described in mammalian cells, their functions in nucleotide metabolism have not been clearly distinguished. In the present experiments, a mutant human T lymphoblastoid cell line (CEM-dAdoR) was selected specifically for resistance to deoxyadenosine toxicity. Compared to parental CEM cells, the variant had 4-fold elevated ATP-activated cytosolic 5'-nucleotidase activity. Other enzymes of potential importance for deoxyadenosine metabolism were indistinguishable in the two cell types. In medium supplemented with the adenosine deaminase inhibitor deoxycoformycin, the T cells with increased 5'-nucleotidase accumulated less nucleotides from exogenously added deoxyadenosine, or 9-beta-D-arabinofuranosyladenine, than did parental T lymphocytes. These metabolic changes were associated with resistance to the growth inhibitory effects of these nucleosides, and also to deoxyguanosine and to 9-beta-D-arabinofuranosylguanine. The T cells with elevated 5'-nucleotidase activity formed more 2',3'-dideoxyadenosine than did parental cells, in deoxycoformycin-supplemented medium. The accumulation of 2',3'-dideoxyadenosine 5'-triphosphate from 2',3'-dideoxyinosine was similarly augmented in the mutant. These data establish the importance of the cytosolic 5'-nucleotidase for the metabolism of purine 2'-deoxyribonucleosides, arabinonucleosides and 2',3'-dideoxyribonucleosides in T lymphoblasts.