Kirk A. Douglas

A simplified analysis of [18F]3′-deoxy-3′-fluorothymidine metabolism and retention

AbstractPurpose: [18F]3′-deoxy-3′-fluorothymidine (FLT) is a thymidine analog developed for imaging tumor proliferation with positron emission tomography (PET). To quantitatively assess images, the blood activities of FLT and its glucuronidated metabolite were measured and its kinetics analyzed. This study sought to limit the number of blood samples needed to measure FLT retention. Methods: Total FLT activity was measured from 18 venous samples obtained over the first hour and dynamic imaging performed on 33 patients (average dose 350 MBq/mmol). The 5-, 10-, 30- and 60-min samples were analyzed to measure the fraction of activity in FLT and its glucuronide. HPLC analysis was compared against a two-step column (Sep-Pak) and metabolic rates measured using full and limited sampling. Probenecid (2 g, oral) was given to two patients to determine whether imaging of the liver improved. Results: At 60 min, 74% of the blood activity was unmetabolized (range 57–85%). HPLC and Sep-Pak gave comparable results (r=0.97; average difference 2.1%). For kinetic analysis, eight venous samples were sufficient to accurately measure total activity; for metabolite analysis, a single sample at 60 min yielded data with mean errors of 2.2%. The metabolic rate correlated with average SUV (r2=0.85; p=0.0002). An aorta input function gave kinetic results comparable to venous blood (r2= 0.82). Probenecid did not improve imaging of the liver. Conclusion: Dynamic measurements of FLT retention can be used to calculate metabolic rates using a limited set of samples and correction for metabolites measured in a single sample obtained at 60 min.

Tumor Imaging Using 1-(2′-deoxy-2′-18F- Fluoro-β-d-Arabinofuranosyl)Thymine and PET

The kinetics of 1-(2′-deoxy-2′-fluoro-β-d-arabinofuranosyl)thymine (FMAU) were studied using PET to determine the most appropriate and simplest approach to image acquisition and analysis. The concept of tumor retention ratio (TRR) is introduced and validated. Methods: Ten patients with brain (n = 4) or prostate (n = 6) tumors were imaged using 18F-FMAU PET (mean dose, 369 MBq). Sixty-minute dynamic images were obtained; this was followed by whole-body images. Mean and maximum standardized uptake values (SUVmean and SUVmax, respectively) of each tumor were determined as the mean over 3 planes of each time interval. For kinetic analyses, blood activity was measured in 18 samples over 60 min. Samples were analyzed by high-performance liquid chromatography at 3 selected times to determine tracer metabolites. FMAU kinetics were measured using a 3-compartment model yielding the flux (K1 × k3/(k2 + k3)) (K1, k2, and k3 are rate constants) and compared with TRR measurements. TRR was calculated as the tumor 18F-FMAU uptake area under the curve divided by the product of blood 18F-FMAU AUC and time. A similar analysis was performed using muscle to estimate 18F-FMAU delivery. Results: SUVmean measurements obtained from 5 to 11 min correlated with those obtained from 30 to 60 min (r2 = 0.92, P < 0.0001) and 50 to 60 min (r2 = 0.92, P < 0.0001) due to the rapid clearance of 18F-FMAU. Similar results were obtained using SUVmax measurements (r2 = 0.93, P < 0.0001; r2 = 0.88, P < 0.0001, respectively). The measurement of TRR using either blood or muscle activity over 11 min provided results comparable to those of 60-min dynamic imaging and a 3-compartment model. This analysis required only 5 blood samples drawn at 1, 2, 3, 5, and 11 min without metabolite correction to produce comparable results. Conclusion: Tissue retention ratio measurements obtained over 11 min can replace flux measurements in 18F-FMAU imaging. The SUVmean and the SUVmax in 5–11 min images correlated well with those of images obtained at 50–60 min. The quality of the images and tissue kinetics in 11 min of imaging makes it a desirable and shorter tumor imaging option.

Analysis and Reproducibility of 3′-Deoxy-3′-[18F]Fluorothymidine Positron Emission Tomography Imaging in Patients with Non–Small Cell Lung Cancer

Purpose: Imaging tumor proliferation with 3′-deoxy-3′-[18F]fluorothymidine (FLT) and positron emission tomography is being developed with the goal of monitoring antineoplastic therapy. This study assessed the methods to measure FLT retention in patients with non-small cell lung cancer (NSCLC) to measure the reproducibility of this approach. Experimental Design: Nine patients with NSCLC who were untreated or had progressed after previous therapy were imaged twice using FLT and positron emission tomography within 2 to 7 days. Reproducibility (that is, error) was measured as the percent difference between the two patient scans. Dynamic imaging was obtained during the first 60 min after injection. Activity in the blood was assessed from aortic images and the fraction of unmetabolized FLT was measured. Regions of interest were drawn on the plane with the highest activity and the adjacent planes to measure standardized uptake value (SUVmean) and kinetic variables of FLT flux. Results: We found that the SUVmean obtained from 30 to 60 min had a mean error of 3.6% (range, 0.6-6.9%; SD, 2.3%) and the first and second scans were highly correlated (r2 = 0.99; P < 0.0001). Using shorter imaging times from 25 to 30 min or from 55 to 60 min postinjection also resulted in small error rates; SUVmean mean errors were 8.4% and 5.7%, respectively. Compartmental and graphical kinetic analyses were also fairly reproducible (r2 = 0.59; P = 0.0152 and r2 = 0.58; P = 0.0175 respectively). Conclusion: FLT imaging of patients with NSCLC was quite reproducible with a worst case SUVmean error of 21% when using a short imaging time.

Biodistribution, PET, and Radiation Dosimetry Estimates of HSV-tk Gene Expression Imaging Agent 1-(2′-Deoxy-2′-18F-Fluoro-β-d-Arabinofuranosyl)-5-Iodouracil in Normal Dogs

FIAU is of interest as a potential reporter probe to monitor herpes simplex virus thymidine kinase (HSV-tk) gene expression and bacterial infections. This study investigates the biodistribution, metabolism, and DNA uptake of 1-(2′-deoxy-2′-18F-fluoro-β-d-arabinofuranosyl)-5-iodouracil (18F-FIAU) in normal dogs. Methods: Four normal dogs were intravenously administered 18F-FIAU. A dynamic PET scan was performed for 60 min over the upper abdomen; this was followed by a whole-body scan for a total of 150 min on 3 dogs. The fourth dog was not scanned and was euthanized at 60 min. Blood and urine samples were collected at stipulated time intervals and analyzed by high-performance liquid chromatography to evaluate tracer clearance and metabolism. Tissue samples collected from various organs were analyzed to evaluate tracer uptake and DNA incorporation. Dynamic accumulation of the tracer in different organs was derived from reconstructed PET images. Nondecay-corrected time–activity curves were used for residence time calculation and absorbed dose estimation. Results: At 60 min after injection, unmetabolized FIAU radioactivity in blood and urine samples was greater than 78% and 63%, respectively, demonstrating resistance to metabolism. The tissue-to-muscle ratio derived from image and tissue analysis showed a slightly higher uptake in proliferating organs (mean tissue-to-muscle values: small intestine, 1.97; marrow, 1.70) compared with nonproliferative organs (heart, 1.07; lung, 1.06). A high concentration of activity was seen in the bile (mean, 23.02), demonstrating hepatobiliary excretion of the tracer. Extraction analysis of tissue samples showed that >62% of the activity in the small intestine, 74% in marrow, and <21% in heart, liver, and muscle was incorporated into DNA. Conclusion: These results demonstrate that FIAU is resistant to metabolism and moderately incorporates into DNA in proliferating tissues. These results suggest that incorporation into the DNA of normal tissues may need to be considered when FIAU is used to track reporter gene activity. Studies in humans are needed to determine whether imaging properties differ in patients and are altered as a result of metabolism changes affected by gene therapies.

Restricted rotation analogs of decarboxylated S-adenosylmethionine as inhibitors of polyamine biosynthesis.

Abstract The synthesis and preliminary biological evaluation of decarboxylated S-adenosylmethionine analogs 1 and 2 is described. In an L1210 cultured cell assay, these compounds, designed as conformationally restricted active site probes for spermidine and spermine synthase, exhibit LD 50 values of 0.1 and 1.0 mM, respectively. In addition, these compounds have significant effects on levels of cellular polyamines and S-adenosylmethionine.

Restricted rotation analogs of S-adenosylmethionine: synthesis, evaluation as inhibitors of S-adenosylmethionine decarboxylase, and potential use as selective antitrypanosomal agents

Abstract A series of restricted rotation analogs of S-adenosylmethionine were synthesized and evaluated as inhibitors of S-adenosylmethionine decarboxylase from Escherichia coli. These analogs were also evaluated for inhibitory activity against cultured mammalian cells, and against the parasite Trypanosoma brucei brucei. All analogs tested appeared to selectively inhibit trypanosomal growth. In particular, compound 5 (AdoMac) inhibits the growth of Trypanosoma brucei brucei with an IC50 of 5.2μM.

Liposomal 64Cu-PET Imaging of Anti-VEGF Drug Effects on Liposomal Delivery to Colon Cancer Xenografts

Liposomes (LP) deliver drug to tumors due to enhanced permeability and retention (EPR). LP were labeled with 64Cu for positron emission tomography (PET) to image tumor localization. Bevacizumab (bev), a VEGF targeted antibody, may modify LP delivery by altering tumor EPR and this change can also be imaged. Objective: Assess the utility of 64Cu-labeled LP for PET in measuring altered LP delivery early after treatment with bev. Methods: HT-29 human colorectal adenocarcinoma tumors were grown subcutaneously in SCID mice. Empty LP MM-DX-929 (Merrimack Pharmaceuticals, Inc. Cambridge, MA) were labeled with 64CuCl2 chelated with 4-DEAP-ATSC. Tumor-bearing mice received ~200-300 μCi of 64Cu-MM-DX-929 and imaged with microPET. All mice were scanned before and after the treatment period, in which half of the mice received bev for one week. Scans were compared for changes in LP accumulation during this time. Initially, tissues were collected after the second PET for biodistribution measurements and histological analysis. Subsequent groups were divided for further treatment. Tumor growth following bev treatment, with or without LP-I, was assessed compared to untreated controls. Results: PET scans of untreated mice showed increased uptake of 64Cu-MM-DX-929, with a mean change in tumor SUVmax of 43.9%±6.6% (n=10) after 7 days. Conversely, images of treated mice showed that liposome delivery did not increase, with changes in SUVmax of 7.6%±4.8% (n=12). Changes in tumor SUVmax were significantly different between both groups (p=0.0003). Histology of tumor tissues indicated that short-term bev was able to alter vessel size. Therapeutically, while bev monotherapy, LP-I monotherapy, and treatment with bev followed by LP-I all slowed HT-29 tumor growth compared to controls, combination provided no therapeutic benefit. Conclusions: PET with tracer LP 64Cu-MM-DX-929 can detect significant differences in LP delivery to colon tumors treated with bev when compared to untreated controls. Imaging with 64Cu-MM-DX-929 is sensitive enough to measure drug-induced changes in LP localization which can have an effect on outcomes of treatment with LP.

Integrating Dynamic Positron Emission Tomography and Conventional Pharmacokinetic Studies to Delineate Plasma and Tumor Pharmacokinetics of FAU, a Prodrug Bioactivated by Thymidylate Synthase

FAU, a pyrimidine nucleotide analogue, is a prodrug bioactivated by intracellular thymidylate synthase to form FMAU, which is incorporated into DNA, causing cell death. This study presents a model‐based approach to integrating dynamic positron emission tomography (PET) and conventional plasma pharmacokinetic studies to characterize the plasma and tissue pharmacokinetics of FAU and FMAU. Twelve cancer patients were enrolled into a phase 1 study, where conventional plasma pharmacokinetic evaluation of therapeutic FAU (50–1600 mg/m2) and dynamic PET assessment of 18F‐FAU were performed. A parent‐metabolite population pharmacokinetic model was developed to simultaneously fit PET‐derived tissue data and conventional plasma pharmacokinetic data. The developed model enabled separation of PET‐derived total tissue concentrations into the parent drug and metabolite components. The model provides quantitative, mechanistic insights into the bioactivation of FAU and retention of FMAU in normal and tumor tissues and has potential utility to predict tumor responsiveness to FAU treatment.

Using Radiolabeled 3′-Deoxy-3′-18F-Fluorothymidine with PET to Monitor the Effect of Dexamethasone on Non–Small Cell Lung Cancer

Non–small cell lung cancer (NSCLC) is a leading cause of cancer mortality in the United States, and pemetrexed-based therapies are regularly used to treat nonsquamous NSCLC. Despite widespread use, pemetrexed has a modest effect on progression-free survival, with varying efficacy between individuals. Recent work has indicated that dexamethasone, given to prevent pemetrexed toxicity, is able to protect a subset of NSCLC cells from pemetrexed cytotoxicity by temporarily suppressing the S phase of the cell cycle. Therefore, dexamethasone might block treatment efficacy in a subpopulation of patients and might be contributing to the variable response to pemetrexed. Methods: Differences in retention of the experimental PET tracer 3′-deoxy-3′-fluorothymidine (FLT) were used to monitor S-phase suppression by dexamethasone in NSCLC cell models, animals with tumor xenografts, and patients with advanced cancer. Results: Significant reductions in tracer uptake were observed after 24 h of dexamethasone treatment in NSCLC cell lines and xenograft models expressing high levels of glucocorticoid receptor α, coincident with pemetrexed resistance visualized by attenuation of the flare effect associated with pemetrexed activity. Two of 4 patients imaged in a pilot study with 18F-FLT PET after dexamethasone treatment demonstrated reductions in tracer uptake from baseline, with a variable response between individual tumor lesions. Conclusion: 18F-FLT PET represents a useful method for the noninvasive monitoring of dexamethasone-mediated S-phase suppression in NSCLC and might provide a way to individualize chemotherapy in patients receiving pemetrexed-based regimens.