Long-term in vivo monitoring of mouse and human hematopoietic stem cell engraftment with a human positron emission tomography reporter gene.

Positron emission tomography (PET) reporter genes allow noninvasive whole-body imaging of transplanted cells by detection with radiolabeled probes. We used a human deoxycytidine kinase containing three amino acid substitutions within the active site (hdCK3mut) as a reporter gene in combination with the PET probe [(18)F]-L-FMAU (1-(2-deoxy-2-(18)fluoro-ß-L-arabinofuranosyl)-5-methyluracil) to monitor models of mouse and human hematopoietic stem cell (HSC) transplantation. These mutations in hdCK3mut expanded the substrate capacity allowing for reporter-specific detection with a thymidine analog probe. Measurements of long-term engrafted cells (up to 32 wk) demonstrated that hdCK3mut expression is maintained in vivo with no counter selection against reporter-labeled cells. Reporter cells retained equivalent engraftment and differentiation capacity being detected in all major hematopoietic lineages and tissues. This reporter gene and probe should be applicable to noninvasively monitor therapeutic cell transplants in multiple tissues.

Evaluation of 1-(2-deoxy-2-fluoro-1-D-arabinofuranosyl)-5-iodouracil (FIAU) as an ex vivo bacterial detection agent.

The nucleoside analogue, 1-(2-deoxy-2-fluoro-1-D-arabinofuranosyl)-5-iodouracil (FIAU) is a substrate for thymidine kinase (TK), which is commonly expressed in bacteria. It is currently being investigated in clinical studies as an in vivo bacterial infection detection agent. In developing countries where imaging facilities are not readily available, deploying such technology can be a big hurdle. However, a portable ex vivo system might provide a good alternative. In an in vitro system, [(125)I]-FIAU incubated with bacteria is phosphorylated by TK, and is trapped within the bacteria, which can be detected by radioscintography. The suitability of this agent to be utilized as part of an ex vivo bacterial detection system was evaluated. In the first part of this report, the optimization of the incubation and detection condition using E. coli as a test case is described. Samples were incubated in a growth promoting medium containing the label, then after filtering and washing, the amount of radioactivity trapped on the filter was quantitated by a scintillation counter. As a proof of concept demonstration, blinded urine samples from urinary tract infection (UTI) patients and normal donors were tested in the FIAU system. Of the 13 UTI positive and 15 normal urine samples tested, there were 2 false negatives and 1 false positive, respectively. Potential explanations for the false positive and negatives as well as the commercialization possibility of this system will be discussed.

Structure-guided engineering of human thymidine kinase 2 as a positron emission tomography reporter gene for enhanced phosphorylation of non-natural thymidine analog reporter probe.

Positron emission tomography (PET) reporter gene imaging can be used to non-invasively monitor cell-based therapies. Therapeutic cells engineered to express a PET reporter gene (PRG) specifically accumulate a PET reporter probe (PRP) and can be detected by PET imaging. Expanding the utility of this technology requires the development of new non-immunogenic PRGs. Here we describe a new PRG-PRP system that employs, as the PRG, a mutated form of human thymidine kinase 2 (TK2) and 2'-deoxy-2'-18F-5-methyl-1-ß-L-arabinofuranosyluracil (L-18F-FMAU) as the PRP. We identified L-18F-FMAU as a candidate PRP and determined its biodistribution in mice and humans. Using structure-guided enzyme engineering, we generated a TK2 double mutant (TK2-N93D/L109F) that efficiently phosphorylates L-18F-FMAU. The N93D/L109F TK2 mutant has lower activity for the endogenous nucleosides thymidine and deoxycytidine than wild type TK2, and its ectopic expression in therapeutic cells is not expected to alter nucleotide metabolism. Imaging studies in mice indicate that the sensitivity of the new human TK2-N93D/L109F PRG is comparable with that of a widely used PRG based on the herpes simplex virus 1 thymidine kinase. These findings suggest that the TK2-N93D/L109F/L-18F-FMAU PRG-PRP system warrants further evaluation in preclinical and clinical applications of cell-based therapies.

Noncompetitive inhibition of hepatitis B virus reverse transcriptase protein priming and DNA synthesis by the nucleoside analog clevudine.

All currently approved antiviral drugs for the treatment of chronic hepatitis B virus (HBV) infection are nucleos(t)ide reverse transcriptase inhibitors (NRTI), which inhibit the DNA synthesis activity of the HBV polymerase. The polymerase is a unique reverse transcriptase (RT) that has a novel protein priming activity in which HP initiates viral DNA synthesis using itself as a protein primer. We have determined the ability of NRTI-triphosphates (TP) to inhibit HBV protein priming and their mechanisms of action. While entecavir-TP (a dGTP analog) inhibited protein priming initiated specifically with dGTP, clevudine-TP (a TTP analog) was able to inhibit protein priming independently of the deoxynucleoside triphosphate (dNTP) substrate and without being incorporated into DNA. We next investigated the effect of NRTIs on the second stage of protein priming, wherein two dAMP nucleotides are added to the initial deoxyguanosine nucleotide. The obtained results indicated that clevudine-TP as well as tenofovir DF-DP strongly inhibited the second stage of protein priming. Tenofovir DF-DP was incorporated into the viral DNA primer, whereas clevudine-TP inhibited the second stage of priming without being incorporated. Finally, kinetic analyses using the HBV endogenous polymerase assay revealed that clevudine-TP inhibited DNA chain elongation by HP in a noncompetitive manner. Thus, clevudine-TP appears to have the unique ability to inhibit HBV RT via binding to and distorting the HP active site, sharing properties with both NRTIs and nonnucleoside RT inhibitors.

In vivo trafficking and immunostimulatory potential of an intranasally-administered primary dendritic cell-based vaccine.

BACKGROUND: Coccidioidomycosis or Valley fever is caused by a highly virulent fungal pathogen: Coccidioides posadasii or immitis. Vaccine development against Coccidioides is of contemporary interest because a large number of relapses and clinical failures are reported with antifungal agents. An efficient Th1 response engenders protection. Thus, we have focused on developing a dendritic cell (DC)-based vaccine for coccidioidomycosis. In this study, we investigated the immunostimulatory characteristics of an intranasal primary DC-vaccine in BALB/c mouse strain that is most susceptible to coccidioidomycosis. The DCs were transfected nonvirally with Coccidioides-Ag2/PRA-cDNA. Expression of DC-markers, Ag2/PRA and cytokines were studied by flow cytometry, dot-immunoblotting and cytometric bead array methods, respectively. The T cell activation was studied by assessing the upregulation of activation markers in a DC-T cell co-culture assay. For trafficking, the DCs were co-transfected with a plasmid DNA encoding HSV1 thymidine kinase (TK) and administered intranasally into syngeneic mice. The trafficking and homing of TK-expressing DCs were monitored with positron emission tomography (PET) using 18F-FIAU probe. Based on the PET-probe accumulation in vaccinated mice, selected tissues were studied for antigen-specific response and T cell phenotypes using ELISPOT and flow cytometry, respectively. RESULTS: We found that the primary DCs transfected with Coccidioides-Ag2/PRA-cDNA were of immature immunophenotype, expressed Ag2/PRA and activated naïve T cells. In PET images and subsequent biodistribution, intranasally-administered DCs were found to migrate in blood, lung and thymus; lymphocytes showed generation of T effector memory cell population (T(EM)) and IFN-γ release. CONCLUSIONS: In conclusion, our results demonstrate that the intranasally-administered primary DC vaccine is capable of inducing Ag2/PRA-specific T cell response. Unique approaches utilized in our study represent an attractive and novel means of producing and evaluating an autologous DC-based vaccine.

Herpes simplex virus thymidine kinase imaging in mice with (1-(2'-deoxy-2'-[18F]fluoro-1-ß-D-arabinofuranosyl)-5-iodouracil) and metabolite (1-(2'-deoxy-2'-[18F]fluoro-1-ß-D-arabinofuranosyl)-5-uracil).

PURPOSE: FIAU, (1-(2'-deoxy-2'-fluoro-1-ß-D-arabinofuranosyl)-5-iodouracil) has been used as a substrate for herpes simplex virus thymidine kinases (HSV-TK and HSV-tk, for protein and gene expression, respectively) and other bacterial and viral thymidine kinases for noninvasive imaging applications. Previous studies have reported the formation of a de-iodinated metabolite of 18F-FIAU. This study reports the dynamic tumor uptake, biodistribution, and metabolite contribution to the activity of 18F-FIAU seen in HSV-tk gene expressing tumors and compares the distribution properties with its de-iodinated metabolite 18F-FAU. METHODS: CD-1 nu/nu mice with subcutaneous MH3924A and MH3924A-stb-tk+ xenografts on opposite flanks were used for the biodistribution and imaging studies. Mice were injected IV with either 18F-FIAU or 18F-FAU. Mice underwent dynamic imaging with each tracer for 65 min followed by additional static imaging up to 150 min post-injection for some animals. Animals were sacrificed at 60 or 150 min post-injection. Samples of blood and tissue were collected for biodistribution and metabolite analysis. Regions of interest were drawn over the images obtained from both tumors to calculate the time-activity curves. RESULTS: Biodistribution and imaging studies showed the highest uptake of 18F-FIAU in the MH3924A-stb-tk+ tumors. Dynamic imaging studies revealed a continuous accumulation of 18F-FIAU in HSV-TK expressing tumors over 60 min. The mean biodistribution values (SUV ± SE) for MH3924A-stb-tk+ were 2.07 ± 0.40 and 6.15 ± 1.58 and that of MH3924A tumors were 0.19 ± 0.07 and 0.47 ± 0.06 at 60 and 150 min, respectively. In 18F-FIAU injected mice, at 60 min nearly 63% of blood activity was present as its metabolite 18F-FAU. Imaging and biodistribution studies with 18F-FAU demonstrated no specific accumulation in MH3924A-stb-tk+ tumors and SUVs for both the tumors were similar to those observed with muscle. CONCLUSION: 18F-FIAU shows a continuous accumulation of activity in HSV-TK expressing tumors. 18F-FAU does not show any preferential accumulation in HSV-TK expressing tumors. In the 18F-FIAU treated mice, the 18F-FAU contribution to the total uptake seen in HSV-TK positive tumors is minimal.

5-[18F]Fluoroalkyl pyrimidine nucleosides: probes for positron emission tomography imaging of herpes simplex virus type 1 thymidine kinase gene expression.

INTRODUCTION: The preliminary in vivo evaluation of novel 5-[(18)F]fluoroalkyl-2'-deoxyuridines ([(18)F]FPrDU, [(18)F]FBuDU, [(18)F]FPeDU; [(18)F]1a-c, respectively) and 2'-fluoro-2'-deoxy-5-[(18)F]fluoroalkyl-1-beta-d-arabinofuranosyl uracils ([(18)F]FFPrAU, [(18)F]FFBuAU, [(18)F]FFPeAU; [(18)F]1d-f, respectively) as probes for imaging herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene expression is described. METHODS: [(18)F]1a-f were successfully synthesized by a rapid and efficient two-step one-pot nucleophilic fluorination reaction using 5-O-mesylate precursors and [(18)F]F(-). For in vivo studies, tumor xenografts were grown in nude mice by implanting RG2 cells stably expressing HSV1-tk (RG2TK+) and wild-type cells (RG2). RESULTS: Biodistribution studies at 2 h pi revealed that the uptake of [(18)F]1a-b and [(18)F]1d-e in RG2TK+ tumors was not significantly different from control tumors. However, [(18)F]1c and [(18)F]1f had an average 1.6- and 1.7-fold higher uptake in RG2TK+ tumors than control RG2 tumors. Blood activity curves for [(18)F]1c and [(18)F]1f highlight rapid clearance of radioactivity in the blood. Dynamic small animal PET (A-PET) imaging studies of tumor-bearing mice with [(18)F]1c and [(18)F]1f showed higher initial uptake (3.5- and 1.4-fold, respectively) in RG2TK+ tumors than in control tumors, with continued washout of activity from both tumors over time. CONCLUSIONS: Biological evaluations suggest that [(18)F]1c and [(18)F]1f may have limited potential for imaging HSV1-tk gene expression due to fast washout of activity from the blood, thus significantly decreasing sensitivity and specificity of tracer accumulation in HSV1-tk-expressing tumors.

Generation, biological consequences and repair mechanisms of cytosine deamination in DNA.

Base moieties in DNA are spontaneously threatened by naturally occurring chemical reactions such as deamination, hydrolysis and oxidation. These DNA modifications have been considered to be major causes of cell death, mutations and cancer induction in organisms. Organisms have developed the DNA base excision repair pathway as a defense mechanism to protect them from these threats. DNA glycosylases, the key enzyme in the base excision repair pathway, are highly conserved in evolution. Uracil constantly occurs in DNA. Uracil in DNA arises by spontaneous deamination of cytosine to generate pro-mutagenic U:G mispairs. Uracil in DNA is also produced by the incorporation of dUMP during DNA replication. Uracil-DNA glycosylase (UNG) acts as a major repair enzyme that protects DNA from the deleterious consequences of uracil. The first UNG activity was discovered in E. coli in 1974. This was also the first discovery of base excision repair. The sequence encoded by the ung gene demonstrates that the E. coli UNG is highly conserved in viruses, bacteria, archaea, yeast, mice and humans. In this review, we will focus on central and recent findings on the generation, biological consequences and repair mechanisms of uracil in DNA and on the biological significance of uracil-DNA glycosylase.

Construction and validation of improved triple fusion reporter gene vectors for molecular imaging of living subjects.

Multimodality imaging using several reporter genes and imaging technologies has become an increasingly important tool in determining the location(s), magnitude, and time variation of reporter gene expression in small animals. We have reported construction and validation of several triple fusion genes composed of a bioluminescent, a fluorescent, and a positron emission tomography (PET) reporter gene in cell culture and in living subjects. However, the bioluminescent and fluorescent components of fusion reporter proteins encoded by these vectors possess lesser activities when compared with the bioluminescent and fluorescent components of the nonfusions. In this study, we first created a mutant (mtfl) of a thermostable firefly luciferase (tfl) bearing the peroxisome localization signal to have greater cytoplasmic localization and improved access for its substrate, d-luciferin. Comparison between the three luciferases [mtfl, tfl, and firefly luciferase (fl)] both in cell culture and in living mice revealed that mtfl possessed 6- to 10-fold (in vitro) and 2-fold (in vivo) higher activity than fl. The improved version of the triple fusion vector carrying mtfl as the bioluminescent reporter component showed significantly (P < 0.05) higher bioluminescence than the previous triple fusion vectors. Of the three different red fluorescent reporter genes (jred, hcred, and mrfp1, isolated from jellyfish chromophore, coral Heteractis crispa, and coral Discosoma, respectively) evaluated, mrfp1 was able to preserve highest expression as a component of the triple fusion reporter gene for in vivo fluorescence imaging. A truncated version of wild-type herpes simplex virus 1 (HSV1) thymidine kinase gene (wttk) retained a higher expression level than the truncated mutant HSV1-sr39 TK (ttk) as the third reporter component of this improved triple fusion vector. Multimodality imaging of tumor-bearing mice using bioluminescence and microPET showed higher luciferase activity [(2.7 +/- 0.1 versus 1.9 +/- 0.1) x (10(6) p/s/cm(2)/sr)] but similar level of fluorine-18-labeled 2'-fluoro-2'-deoxyarabinofuranosyl-5-ethyluracil (18F-FEAU) uptake (1.37 +/- 0.15 versus 1.37 +/- 0.2) percentage injected dose per gram] by mtfl-mrfp1-wttk-expressing tumors compared with the fl-mrfp1-wttk-expressing tumors. Both tumors showed 4- to 5-fold higher accumulation (P < 0.05) of 18F-FEAU than fluorine-18-labeled 9-(4-fluoro-3-hydroxymethylbutyl)guanine. This improved triple fusion reporter vector will enable high sensitivity detection of lower numbers of cells from living animals using the combined bioluminescence, fluorescence, and microPET imaging techniques.

Simultaneous determination of cytosine arabinoside and its metabolite uracil arabinoside in human plasma by LC-MS/MS: Application to pharmacokinetics-pharmacogenetics pilot study in AML patients.

Purine analogs like aracytine (AraC) are a mainstay for treating acute myeloid leukemia (AML). There are marked differences in drug dosing and scheduling depending on the protocols when treating AML patients with AraC. Large inter-patient pharmacokinetics variability has been reported, and genetic polymorphisms affecting cytidine deaminase (CDA), the liver enzyme responsible for the conversion of Ara-C to inactive uracil arabinoside (AraU) could be a culprit for either life-threatening toxicities or poor efficacy related to substantial changes in plasma exposure levels among patients. The quantitative determination of Ara-C in plasma is challenging due the required sensitivity because of the short half-life of this drug (i.e., <10 min) and the metabolic instability in biological matrix upon sampling possibly resulting in erratic values. We developed and validated a liquid chromatography tandem mass spectrometry method (UPLC-MS/MS) for the simultaneous determination of Ara-C and Ara-U metabolite in human plasma. After simple and rapid precipitation, analytes were successfully separated and quantitated over a 1-500 ng/ml range for Ara-C and 250-7500 ng/ml range for AraU. The performance and reliability of this method was tested as part of an investigational study in AML patients treated with low dose cytarabine and confirmed marked differences in drug exposure levels and metabolic ratio, depending on the CDA status of the patients. Overall, this new method meets the requirements of current bioanalytical guidelines and could be used to monitor drug levels in AML patients with respect to their CDA phenotypes.

Clevudine is efficiently phosphorylated to the active triphosphate form in primary human hepatocytes.

BACKGROUND: Clevudine (CLV) is a nucleoside analogue of the unnatural L-configuration that has demonstrated potent activity against hepatitis B virus (HBV) in vitro and in Phase III clinical studies. Human hepatoma cell lines are the only liver-derived cells in which CLV metabolism has been investigated. Here, we examine CLV metabolism in primary human hepatocytes, which better represent CLV metabolism in the liver. METHODS: HPLC analysis of primary human hepatocyte extracts incubated with radiolabelled CLV was used to determine the time course of CLV phosphorylation. Effects of the exogenous cell concentration of CLV on phosphorylation were assessed and the half-life of the CLV phosphorylated forms was determined. RESULTS: The major CLV metabolite formed in human primary hepatocytes was 5'-monophosphate, whereas in the hepatoma cell lines the major metabolite was 5'-triphosphate. The level of CLV 5'-triphosphate was similar in both cell types. In primary hepatocytes the conversion of CLV 5'-monophosphate to the corresponding 5'-diphosphate was the rate-limiting step in CLV phosphorylation; the level of CLV phosphorylation was dependent upon exogenous drug concentration and exposure time. CLV 5'-triphosphate accumulated rapidly with peak levels observed after approximately 8 h. When cells were incubated with 1 microM CLV, the approximate maximal plasma concentration achieved in individuals receiving the 30 mg dose, the average intracellular concentration of CLV 5'-triphosphate was 41.3 +/- 8.4 pmols/10(6) cells (approximately 10 microM). The initial half-life of CLV triphosphate was approximately 11 h. CONCLUSIONS: These results are consistent with once daily CLV dosing currently being used in Phase III clinical studies.

Imaging of gene expression in live pancreatic islet cell lines using dual-isotope SPECT.

UNLABELLED: We are combining nuclear medicine with molecular biology to establish a sensitive, quantitative, and tomographic method with which to detect gene expression in pancreatic islet cells in vivo. Dual-isotope SPECT can be used to image multiple molecular events simultaneously, and coregistration of SPECT and CT images enables visualization of reporter gene expression in the correct anatomic context. We have engineered pancreatic islet cell lines for imaging with SPECT/CT after transplantation under the kidney capsule. METHODS: INS-1 832/13 and alphaTC1-6 cells were stably transfected with a herpes simplex virus type 1-thymidine kinase-green fluorescent protein (HSV1-thymidine kinase-GFP) fusion construct (tkgfp). After clonal selection, radiolabel uptake was determined by incubation with 5-(131)I-iodo-1-(2-deoxy-2-fluoro-beta-d-arabinofuranosyl)uracil ((131)I-FIAU) (alphaTC1-6 cells) or (123)I-FIAU (INS-1 832/13 cells). For the first set of in vivo experiments, SPECT was conducted after alphaTC1-6/tkgfp cells had been labeled with either (131)I-FIAU or (111)In-tropolone and transplanted under the left kidney capsule of CD1 mice. Reconstructed SPECT images were coregistered to CT. In a second study using simultaneous acquisition dual-isotope SPECT, INS-1 832/13 clone 9 cells were labeled with (111)In-tropolone before transplantation. Mice were then systemically administered (123)I-FIAU and data for both (131)I and (111)In were acquired simultaneously. RESULTS: alphaTC1-6/tkgfp cells showed a 15-fold greater uptake of (131)I-FIAU, and INS-1/tkgfp cells showed a 12-fold greater uptake of (123)I-FIAU, compared with that of wild-type cells. After transplantation under the kidney capsule, both reporter gene expression and location of cells could be visualized in vivo with dual-isotope SPECT. Immunohistochemistry confirmed the presence of glucagon- and insulin-positive cells at the site of transplantation. CONCLUSION: Dual-isotope SPECT is a promising method to detect gene expression in and location of transplanted pancreatic cells in vivo.

Human breast tumor cells express multimodal imaging reporter genes.

OBJECTIVE: Human ZR75-1 cells were among the first few characterized estrogen-dependent mammary gland carcinoma cell lines and had been utilized in various studies for the pro- or antitumor effect of xenoestrogens and antiestrogens. The objective of this study was to establish a breast tumor model in ZR75-1 cells bearing multimodal reporter genes to allow noninvasive imaging of tumor growth using fluorescence and nuclear imaging platforms. METHODS AND RESULTS: Enhanced green fluorescent protein (eGFP) cDNA was fused at the C-terminus with herpes simplex virus type 1 thymidine kinase (HSV1-tk) to form the fusion reporter gene (eGFP-tk). In vitro proliferation, migration, and invasion assays revealed that eGFP-tk-transfected ZR75-1 cells exhibited decreased proliferation rate, migratory activity, and invasion ability compared to the wild-type cells. The functional HSV1-tk enzymatic activity in stably transfected cells were confirmed by in vitro ganciclovir (GCV) sensitivity and [123I]2-fluoro-2-deoxy-1-beta-D-arabinofuranosyl-5-iodouracil (FIAU) accumulation assays. In vivo fluorescence and nuclear imaging were performed on nude mice bearing multiple subcutaneous xenografts established from ZR75-1-eGFP-tk and wild-type cells. Optical imaging was able to detect the green fluorescence of eGFP-tk tumor. The eGFP-tk reporter gene-specific imaging was achieved by single photon emission computed tomography (SPECT) using [123I]FIAU as a radiotracer and demonstrated decreased FIAU uptake in eGFP-tk tumor by GCV treatment. Probably due to a flare reaction after GCV treatment, micro-positron emission tomography (micro-PET) imaging using 2-deoxy-2-[18F]fluoro-D-glucose (FDG) could not demonstrate decreases in FDG uptake. However, in vitro metabolic assay also revealed that eGFP-tk cells transiently increased [3H]-deoxyglucose uptake in response to GCV treatment. CONCLUSIONS: This study confirmed the usefulness of eGFP-tk in many applications by providing, in vitro and in vivo, the sensitive and reporter gene-specific imaging. ZR75-1-eGFP-tk cells that are ready to incorporate in various imaging platforms constitute a useful model in breast cancer research.

In vivo measurement of cell proliferation in canine brain tumor using C-11-labeled FMAU and PET.

INTRODUCTION: Noncatabolized thymidine analogs are being developed for use in imaging DNA synthesis. We sought to relate a labeling index measured by immunohistochemical staining bromodeoxyuridine (BUdR) technique to the uptake of (11)C 2'-fluoro-5-methyl-1-beta-d-arabinofuranosyluracil (FMAU) measured with positron emission tomography (PET) in a brain tumor model. METHODS: Adult beagles (n=8) with implanted brain tumors received [(11)C]FMAU and dynamic imaging with arterial sampling. Six dogs were then infused with BUdR (200 mg/m(2)) and sacrificed. Tumor time-activity curves (TACs) obtained from computed-tomography-defined regions of interest were corrected for partial volume effects and crosstalk from brain tissue. Tissue was analyzed for the percentage of tumor volume occupied by viable cells and by viable cells in S-phase as identified by BUdR staining. PET/[(11)C]FMAU and BUdR were compared by linear regression analysis and analysis of variance, as well as by a nonparametric rank correlation test. RESULTS: Tumor standardized uptake values (SUVs) and tumor-to-contralateral-brain uptake ratios at 50 min were 1.6+/-0.4 and 5.5+/-1.2 (n=8; mean+/-S.E.M.), respectively. No (11)C-labeled metabolites were observed in the blood through 60 min. Tumor TACs were well described with a three-compartment/four-parameter model (k(4)=0) and by Patlak analysis. Parametric statistical analysis showed that FMAU clearance from plasma into tumor Compartment 3 (K(FMAU)) was significantly correlated with S-phase percent volume (P=.03), while tumor SUV was significantly correlated with both S-phase percent volume and cell percent volume (P=.02 and .03, respectively). Patlak slope, K(FMAU) and tumor SUV were equivalent with regard to rank correlation analysis, which showed that tumor uptake and trapping of FMAU were correlated with the volume density of dividing cells (P=.0003) rather than nondividing cells (P=.3). CONCLUSIONS: Trapping of [(11)C]FMAU correlated with tumor growth rate, as measured by direct tissue analysis with BUdR in a canine brain tumor model, suggesting that [(11)C]FMAU is useful for the imaging of cell proliferation in cancers.

Cellular and molecular events leading to mitochondrial toxicity of 1-(2-deoxy-2-fluoro-1-beta-D-arabinofuranosyl)-5-iodouracil in human liver cells.

We have explored the mechanism(s) related to FIAU-induced liver toxicity, particularly focusing on its effect on mitochondrial function in a human hepatoma cell line-HepG2. The potential role of FMAU and FAU, metabolites detected in FIAU-treated patients were also ascertained. FIAU and FMAU inhibited cell growth and were effectively phosphorylated. A substantial increase in lactic acid production in medium of cells incubated with 1-10 microM FIAU or FMAU was consistent with mitochondrial dysfunction. Slot blot analysis demonstrated that a two week exposure to 10 microM FIAU or FMAU was not associated with a decrease in total mitochondrial (mt) DNA content. However, FIAU and FMAU were incorporated into nuclear and mtDNA and relative values suggest that both compounds incorporate at a much higher rate into mtDNA. Electron micrographs of cells incubated with 10 microM FIAU or FMAU revealed the presence of enlarged mitochondria with higher cristae density and lipid vesicles. In conclusion, these data suggest that despite the lack of inhibition of mtDNA content, incorporation of FIAU and FMAU into mtDNA of HepG2 cells leads to marked mitochondrial dysfunction as evidenced by disturbance in cellular energy metabolism and detection of micro- and macrovesicular steatosis.

Cerebrospinal fluid and plasma pharmacokinetics of high doses of 1-beta-D-arabinofuranosylcytosine in nonhuman primates.

The pharmacokinetics of 1-beta-D-arabinofuranosylcytosine (ara-C) in plasma, cerebrospinal fluid (CSF), and urine was studied in nonhuman primates. Conventional and high-dose schedules of ara-C were administered i.v. and intraventricularly through indwelling Ommaya reservoirs. ara-C and its metabolite 1-beta-D-arabinofuranosyluracil (ara-U) were measured by high-pressure liquid chromatography. Due to rapid peripheral deamination (230 nm ara-C/hr/ml plasma), the half-life of ara-C in plasma after a 140-mg/kg i.v. 1-hr infusion was short (3.7 min). Peak plasma concentrations of ara-C, ranging between less than 0.1 and 49 micrograms/ml, were dose dependent (ara-C, 15 to 140 mg/kg). Under these conditions, ara-C was undetectable in CSF. About 53% of the administered dose (140 mg/kg) was excreted in urine during the first 5 hr mostly as ara-U. Intraventricular administration of 50 and 250 mg of ara-C resulted in peak lumbar CSF levels of 435 and 2235 micrograms/ml, respectively, at about 155 and 80 min postinjection. Concomitant ara-U levels were one-tenth of those of ara-C and increased progressively, suggesting deamination of the drug in the central nervous system. The half-life of ara-C in CSF ranged between 50 and 60 min. Administration of 50 mg of ara-U intraventricularly resulted in peak lumbar ara-U levels of 595 micrograms/ml at about 180 min with a prolonged clearance. Concomitant plasma levels throughout the study were less than 0.1 micrograms/ml, suggesting slower equilibrium. No hematological or nervous system toxicity was observed during these studies. The clinical implications of these findings are discussed.

A radioimmunoassay for 1-beta-D-arabinofuranosyluracil with reference to cross-reactivity of 1-beta-D-arabinofuranosylcytosine with an antibody.

Antibodies directed against 1-beta-D-arabinofuranosyluracil have been produced in rabbits by immunization with a conjugate of 1-(5-O-succinyl-beta-D-arabinofuranosyl)uracil with human serum albumin. Two of four antibodies so obtained showed high specificity for 1-beta-D-arabinofuranosyluracil and allowed the development of a sensitive and reliable radioimmunoassay for this substrate. On the other hand, one antibody had a high affinity for 1-beta-D-arabinofuranosylcytosine. The binding of 1-beta-D-arabinofuranosylcytosine to this antibody was practically constant between pH 5.2 and 9.0, whereas 1-beta-D-arabinofuranosyluracil binding was affected drastically by pH. The pH-binding profile for 1-beta-D-arabinofuranosylcytosine and 1-beta-D-arabinofuranosyluracil was reminiscent of the specificity of ara-C-specific antibodies, which we previously obtained after immunization of rabbits with 1-(5-O-succinyl-beta-D-arabinofuranosyl)cytosine as a hapten.

Pharmacokinetics of N4-behenoyl-1-beta-D-arabinofuranosylcytosine in patients with acute leukemia.

N4-Behenoyl-1-beta-D-arabinofuranosylcytosine (BHAC), a lipophilic and deaminase-resistant derivative of 1-beta-D-arabinofuranosylcytosine (ara-C), was studied pharmacologically in patients with acute leukemia. The concentrations of BHAC, ara-C, and 1-beta-D-arabinofuranosyluracil were measured by high-performance liquid chromatography, bioassay, and gas chromatography-mass spectrometry-mass fragmentography, respectively. The data of plasma BHAC concentrations were analyzed by a MULTI computer program. In seven patients given BHAC (200 mg/body weight; 2.97 to 4.26 mg/kg) i.v. for 90 min, the plasma disappearance curve of BHAC was biphasic with a mean initial half-life of 0.37 hr and a mean second half-life of 5.27 hr. The apparent volume of the central compartment and the apparent volume of distribution were 0.047 and 0.316 liter/kg, respectively; the systemic clearance was 0.051 liter/hr/kg. BHAC concentrations in erythrocytes were significantly higher (p less than 0.01) than those in plasma at 4 to 22.5 hr after infusion, suggesting that the erythrocytes may act as a reservoir for the drug. The plasma 1-beta-D-arabinofuranosyluracil level increased to 603 ng/ml at 4 hr after infusion, and it was over 129 ng/ml for at least 22.5 hr after infusion. Plasma ara-C levels, which could be detected in only 2 of 11 patients examined, were maintained (over 0.08 micrograms/ml) for 8 hr after infusion. Urinary BHAC excretion was less than 0.2 micrograms/ml of the sensitivity limit in all samples. Prolonged urinary ara-C excretion was detected, but it was only 0.5% of the administered BHAC for 24 hr. At 12 hr after a 200-mg infusion of BHAC, BHAC level in bone marrow fluid was significantly higher (p less than 0.01) than that in plasma. In spite of the lipophilic nature of the agent, the BHAC concentration in cerebrospinal fluid was less than 0.2 micrograms/ml in 8 of 9 patients without meningeal involvement. These findings were thought to indicate a restricted and prolonged BHAC distribution including plasma, blood cells, and bone marrow fluids, which may be of importance in the administration of BHAC in the chemotherapy of hematological cancers.

Development of resistance to 1-beta-D-arabinofuranosylcytosine after high-dose treatment in childhood lymphoblastic leukemia: analysis of resistance mechanism in established cell lines.

Cell lines PER-163 and PER-164 are derived from a patient with acute lymphoblastic leukemia who developed resistance to 1-beta-D-arabinofuranosylcytosine (ara-C) after high-dose (HD) therapy. Both lines are highly resistant to ara-C and have maintained stable resistance for more than 18 mo. The resistance in PER-164 cells is the result of a selection process in vivo only, while PER-163 cells have in addition been exposed to ara-C in culture. Comparison with cell line PER-145, which is sensitive to ara-C and was established from the same patient before HDara-C therapy, revealed no differences with respect to surface markers, morphology, cytochemical stains, or requirements for growth in vitro. The leukemic origin of the three cell lines is indicated by the close similarities of all three cell lines to the patient's fresh cells. The analysis of the two resistant cell lines shows that resistance to ara-C is not due to lower ara-C transport capacity nor to cytokinetic reasons, since the percentage of cells in S-phase is similar in all three cell lines. In addition, the resistant cell lines do not show any increased cytidine deaminase activity. PER-164 cells show a markedly reduced deoxycytidine kinase activity, 4.8 nmol/h/mg of protein, compared to PER-145 cells with an enzyme activity of 21.48 nmol/h/mg of protein. In PER-163 cells, no deoxycytidine kinase activity could be detected. Furthermore, the two resistant cell lines show significantly different dCTP levels. The sensitive PER-145 cells generated 97.9 pmol of 1-beta-D-arabinofuranosylcytosine triphosphate (ara-CTP)/10(7) cells during a 45-min incubation period in the presence of 10(-6) M ara-C. This contrasts with 0.16 and 12 pmol of ara-CTP/10(7) cells for PER-163 and PER-164 cells, respectively. These investigations suggest that cell phenotypes with distinct features can be generated after HDara-C treatment and that decreased deoxycytidine kinase activity appears to be one of the major mechanisms of resistance.

A radioimmunoassay method for 1-beta-D-arabinofuranosyluracil using antibodies directed against 1-beta-D-arabinofuranosylcytosine.

Above pH 7.0 1-beta-D-arabinofuranosyluracil (ara-U) shows marked pH-dependent cross-reactivity with antibodies directed towards 1-beta-D-arabinofuranosylcytosine. Since this peculiar phenomenon has not been observed with other nucleosides and nucleotides thus far tested, it is probably the result of base-catalyzed tautomerism of ara-U to its enolic form which renders it more structurally similar to 1-beta-D-arabinofuranosylcytosine. By performing the radioimmunoassay at both pH 6.2 and 8.6 we could determine 1-beta-D-arabinofuranosylcytosine and ara-U simultaneously. This method for ara-U assay is simple, fairly reliable, and applicable to blood level studies.