Analysis of [11C]Choline and [13N]Ammonia using a single HPIC method.

A single HPIC method was developed and validated for the analysis of both [11C]Choline and [13N]Ammonia with the same setup. The HPIC system suitability tests were performed and [11C]Choline and [13N]Ammonia were used to verify their performance on this HPIC method. The HPIC setup and method provides qualitative and quantitative analysis information of [11C]Choline and [13N]Ammonia. The data suggested this HPIC method is validated for determining radiochemical/chemical purity and radiochemical identity of [11C]Choline and [13N]Ammonia products in CGMP compliant manufacturing process.

PET Imaging of Tumor Perfusion: A Potential Cancer Biomarker?

Perfusion, as measured by imaging, is considered a standard of care biomarker for the evaluation of many tumors. Measurements of tumor perfusion may be used in a number of ways, including improving the visual detection of lesions, differentiating malignant from benign findings, assessing aggressiveness of tumors, identifying ischemia and by extension hypoxia within tumors, and assessing treatment response. While most clinical perfusion imaging is currently performed with CT or MR, a number of methods for PET imaging of tumor perfusion have been described. The inert PET radiotracer 15O-water PET represents the recognized gold standard for absolute quantification of tissue perfusion in both normal tissue and a variety of pathological conditions including cancer. Other cancer PET perfusion imaging strategies include the use of radiotracers with high first-pass uptake, analogous to those used in cardiac perfusion PET. This strategy produces more visually pleasing high-contrast images that provide relative rather than absolute perfusion quantification. Lastly, multiple timepoint imaging of PET tracers such as 18F-FDG, are not specifically optimized for perfusion, but have advantages related to availability, convenience, and reimbursement. Multiple obstacles have thus far blocked the routine use of PET imaging for tumor perfusion, including tracer production and distribution, image processing, patient body coverage, clinical validation, regulatory approval and reimbursement, and finally feasible clinical workflows. Fortunately, these obstacles are being overcome, especially within larger imaging centers, opening the door for PET imaging of tumor perfusion to become standard clinical practice. In the foreseeable future, it is possible that whole-body PET perfusion imaging with 15O-water will be able to be performed in a single imaging session concurrent with standard PET imaging techniques such as 18F-FDG-PET. This approach could establish an efficient clinical workflow. The resultant ability to measure absolute tumor blood flow in combination with glycolysis will provide important complementary information to inform prognosis and clinical decisions.

Influential Factors in the Preparation of 68Ga-DOTATATE.

Acceptable and reproducible radiochemical purity (RP) for 68Ga-DOTATATE was difficult to obtain with the NETSPOT kit because the manufacturer instructions lacked details on the heater or needles used. Methods: The drug was prepared in an International Organization for Standardization (ISO) 5 environment. Multiple dry baths and needle types were used to investigate the effects of reaction temperature and metal contamination, respectively. Temperature curves were obtained with a calibrated thermocouple. The influence of the accuracy of the NETSPOT reagent volume and its effect on outcome were investigated. Results: The AccuBlock dry bath required recalibration for the ISO 5 environment; after calibration, the temperature was stable (only ±0.1°C from the set point). When we followed package insert recommendations (dry bath temperature set to 98°C, reaction time of 8 min), the reaction temperature was 90.6°C. When Becton Dickinson needles were used for reconstitution, 15 of 18 runs (83%) did not meet the RP specification. However, B. Braun Medical needles achieved satisfactory and stable RP. When the 68Ga generator was eluted with 5.0 mL of 0.1 M hydrochloric acid (HCl), only 3.8-3.9 mL of eluate reached the reaction vial; this volume did not impact labeling (final pH was 3.8). The labeling success rate increased markedly if the 68Ga eluate was passed through a conditioned silica gel cartridge or if no cartridge was used; then, RP was more than 99%. HCl contact with the septum of the labeling vial reduced RP. Conclusion: The needle type and the temperature setting of the dry bath have critical roles in 68Ga-DOTATATE preparation. The AccuBlock dry bath has excellent stability and accuracy and can be used for reliable preparation. By using a conditioned silica gel cartridge or by eliminating the cartridge altogether, the RP is reliably high and stable.

Radiation induced oxidation of [18F]fluorothia fatty acids under cGMP manufacturing conditions.

OBJECTIVE: The objectives of the present work were to optimize and validate the synthesis and stability of 14(R,S)-[18F]fluoro-6-thia-heptadecanoic acid ([18F]FTHA) and 16-[18F]fluoro-4-thia-palmitic acid ([18F]FTP) under cGMP conditions for clinical applications. METHODS: Benzyl-14-(R,S)-tosyloxy-6-thiaheptadecanoate and methyl 16-bromo-4-thia-palmitate were used as precursors for the synthesis of [18F]FTHA and [18F]FTP, respectively. For comparison, a fatty acid analog lacking a thia-substitution, 16-[18F]fluoro-palmitic acid ([18F]FP), was synthesized from the precursor methyl 16-bromo-palmitate. A standard nucleophilic reaction using cryptand (Kryptofix/K222, 8.1 mg), potassium carbonate (K2CO3, 4.0 mg) and 18F-fluoride were employed for the 18F-labeling and potassium hydroxide (0.8 M) was used for the post-labeling ester hydrolysis. The final products were purified via reverse phase semi-preparative HPLC and concentrated via trap and release on a C-18 plus solid phase extraction cartridge. The radiochemical purities of the [18F]fluorothia fatty acids and [18F]FP were examined over a period of 4 h post-synthesis using an analytical HPLC. All the syntheses were optimized in an automated TRACERlab FX-N Pro synthesizer. Liquid chromatography mass spectrometry (LCMS) and high resolution mass spectrometry (HRMS) was employed to study the identity and nature of side products formed during radiosynthesis and as a consequence of post-synthesis radiation induced oxidation. RESULTS: Radiosyntheses of [18F]FTHA, [18F]FTP and [18F]FP were achieved in moderate (8-20% uncorrected) yields. However, it was observed that the HPLC-purified [18F]fluorothia fatty acids, [18F]FTHA and [18F]FTP at higher radioactivity concentrations (>1.11 GBq/mL, 30 mCi/mL) underwent formation of 18F-labeled side products over time but [18F]FP (lacking a sulfur heteroatom) remained stable up to 4 h post-synthesis. Various radiation protectors like ethanol and ascorbic acid were examined to minimize the formation of side products formed during [18F]FTHA and [18F]FTP synthesis but showed only limited to no effect. Analysis of the side products by LCMS showed formation of sulfoxides of both [18F]FTHA and [18F]FTP. The identity of the sulfoxide side product was further confirmed by synthesizing a non-radioactive reference standard of the sulfoxide analog of FTP and matching retention times on HPLC and molecular ion peaks on LC/HRMS. Radiation-induced oxidation of the sulfur heteroatom was mitigated by dilution of product with isotonic saline to reduce the radioactivity concentration to <0.518 GBq/mL (14 mCi/mL). CONCLUSIONS: Successful automated synthesis of [18F]fluorothia fatty acids were carried out in cGMP facility for their routine production and clinical applications. Instability of [18F]fluorothia fatty acids were observed at radioactivity concentrations exceeding 1.11 GBq/mL (30 mCi/mL) but mitigated through dilution of the product to <0.518 GBq/mL (14 mCi/mL). The identities of the side products formed were established as the sulfoxides of the respective thia fatty acids caused by radiation-induced oxidation of the sulfur heteroatom.

Rapid Instant Thin-Layer Chromatography System for Determining the Radiochemical Purity of 68Ga-DOTATATE.

The objective of this study was to develop instant thin-layer chromatography (ITLC) conditions for the determination of radiochemical purity of 68Ga-DOTATATE in a shorter time period than those stated in the NETSPOT (Advanced Accelerator Applications, Saint-Genis-Pouilly, France; AAA) kit package insert (PI). A faster ITLC system is needed to reduce the 48- to 50-min development time so that more radioactivity is available for single patient use and wait times are shorter in the event of kit failure. Methods: Variations of the PI mobile system were evaluated with microfiber chromatography paper impregnated with silica gel (ITLC-SG). After a more suitable mobile system was identified, evaluation began by attempting to shorten the 10-cm development distance to 7, 8, and 9 cm. Results: Experiments using variations of PI mobile phase showed that increasing the proportion of methanol in the mobile phase decreased development time. Additionally, if the ratio of 1 M ammonium acetate was reduced to 10% or less, retention factor values fall outside specification. Reducing the development distance shortened development time as expected; however, it also affected the resolution aspect of the radiochromatogram. Conclusion: The fastest developing ITLC system, which maintained resolution and peak shape, was methanol:1 M ammonium acetate (80:20 V/V) with ITLC-SG using a development distance of 8 cm.

Preparation and preliminary evaluation of 63Zn-zinc citrate as a novel PET imaging biomarker for zinc.

UNLABELLED: Abnormalities of zinc homeostasis are indicated in many human diseases. A noninvasive imaging method for monitoring zinc in the body would be useful to understand zinc dynamics in health and disease. To provide a PET imaging agent for zinc, we have investigated production of (63)Zn (half-life, 38.5 min) via the (63)Cu(p,n)(63)Zn reaction using isotopically enriched solutions of (63)Cu-copper nitrate. A solution target was used for rapid isolation of the (63)Zn radioisotope from the parent (63)Cu ions. Initial biologic evaluation was performed by biodistribution and PET imaging in normal mice. METHODS: To produce (63)Zn, solutions of (63)Cu-copper nitrate in dilute nitric acid were irradiated by 14-MeV protons in a low-energy cyclotron. An automated module was used to purify (63)Zn from (63)Cu in the target solution. The (63)Cu-(63)Zn mixture was trapped on a cation-exchange resin and rinsed with water, and the (63)Zn was eluted using 0.05 N HCl in 90% acetone. The resulting solution was neutralized with NaHCO3, and the (63)Zn was then trapped on a carboxymethyl cartridge, washed with water, and eluted with isotonic 4% sodium citrate. Standard quality control tests were performed on the product according to current good manufacturing practice, including radionuclidic identity and purity, and measurement of nonradioactive Zn(+2), Cu(+2), Fe(+3), and Ni(+2) by ion-chromatography high-performance liquid chromatography. Biodistribution and PET imaging studies were performed in B6.SJL mice after intravenous administration of (63)Zn-zinc citrate. (63)Cu target material was recycled by eluting the initial resin with 4N HNO3. RESULTS: Yields of 1.07 ± 0.22 GBq (uncorrected at 30-36 min after end of bombardment) of (63)Zn-zinc citrate were obtained with a 1.23 M (63)Cu-copper nitrate solution. Radionuclidic purity was greater than 99.9%, with copper content lower than 3 µg/batch. Specific activities were 41.2 ± 18.1 MBq/µg (uncorrected) for the (63)Zn product. PET and biodistribution studies in mice at 60 min showed expected high uptake in the pancreas (standard uptake value, 8.8 ± 3.2), liver (6.0 ± 1.9), upper intestine (4.7 ± 2.1), and kidney (4.2 ± 1.3). CONCLUSION: A practical and current good manufacturing practice-compliant preparation of radionuclidically pure (63)Zn-zinc citrate has been developed that will enable PET imaging studies in animal and human studies. (63)Zn-zinc citrate showed the expected biodistribution in mice.

Synthesis and preliminary evaluation of N-(16-18F-fluorohexadecanoyl)ethanolamine (18F-FHEA) as a PET probe of N-acylethanolamine metabolism in mouse brain.

N-Acylethanolamines are lipid signaling molecules found throughout the plant and animal kingdoms. The best-known mammalian compound of this class is anandamide, N-arachidonoylethanolamine, one of the endogenous ligands of cannabinoid CB1 and CB2 receptors. Signaling by N-acylethanolamines is terminated by release of the ethanolamine moiety by hydrolyzing enzymes such as fatty acid amide hydrolase (FAAH) and N-acylethanolamine-hydrolyzing amidase (NAAA). Herein, we report the design and synthesis of N-(16-(18)F-fluorohexadecanoyl)ethanolamine ((18)F-FHEA) as a positron emission tomography (PET) probe for imaging the activity of N-acylethanolamine hydrolyzing enzymes in the brain. Following intravenous administration of (18)F-FHEA in Swiss Webster mice, (18)F-FHEA was extracted from blood by the brain and underwent hydrolysis at the amide bond and incorporation of the resultant (18)F-fluorofatty acid into complex lipid pools. Pretreatment of mice with the FAAH inhibitor URB-597 (1 mg/kg IP) resulted in significantly slower (18)F-FHEA incorporation into lipid pools, but overall (18)F concentrations in brain regions were not altered. Likewise, pretreatment with a NAAA inhibitor, (S)-N-(2-oxo-3-oxytanyl)biphenyl-4-carboxamide (30 mg/kg IV), did not significantly affect the uptake of (18)F-FHEA in the brain. Although evidence was found that (18)F-FHEA behaves as a substrate of FAAH in the brain, the lack of sensitivity of brain uptake kinetics to FAAH inhibition discourages its use as a metabolically trapped PET probe of N-acylethanolamine hydrolyzing enzyme activity.

Comparison of FASTlab 18F-FDG production using phosphate and citrate buffer cassettes.

UNLABELLED: The objective of this research is to determine whether there are significant differences in the (18)F-FDG produced by either the phosphate or the citrate buffer cassettes in the FASTlab synthesizer. METHODS: Forty batches of (18)F-FDG were produced with each cassette and analyzed retrospectively. The analysis consisted of determining the mean radiochemical yield (RCY)-uncorrected and corrected for decay-radiochemical purity (RCP), pH, and residual solvent content (ethanol and acetonitrile). An independent t test (alpha error [α], 0.05) was performed to determine whether the differences were statistically significant. RESULTS: The mean decay-corrected RCYs for (18)F-FDG produced by phosphate and citrate cassettes were 82.9% ± 17.4% and 79.2% ± 5.0%, respectively. The uncorrected RCY was 57.5% ± 16.7% for phosphate- and 58.8% ± 6.0% for citrate-buffered (18)F-FDG, leading to a difference of 4.4% and P value of 0.11 for corrected RCY and a difference of 2.2% and P value of 0.32 for uncorrected RCY. Thus, the RCY differences are neither statistically nor clinically significant. The mean RCPs were 99.4% ± 0.2% for the phosphate-buffered (18)F-FDG and 99.0% ± 1.1% for the citrate-buffered (18)F-FDG. There was a 0.5% difference and a P value of 0.021, meaning that the difference was statistically significant. The average pHs for (18)F-FDG produced by phosphate and citrate buffer cassettes were 5.9 ± 0.1 and 5.3 ± 0.2, respectively, resulting in a 9.6% difference and a P value close to zero (2.6 × 10(-19))-a statistically significant difference. The difference between ethanol content was also dramatic. Phosphate-buffered (18)F-FDG contained 0.08% ± 0.02% ethanol, whereas the citrate-buffered (18)F-FDG contained 0.20% ± 0.07%. No difference was found in the acetonitrile content of the 2 cassettes. CONCLUSION: The differences in yield between cassettes are due to statistical variability. The results confirm our hypothesis that there is no significant difference in RCY. The differences seen in the statistically significant data (those with a P value > 0.05) turn out to be insignificant in a real-world setting because all values fell within the limits set by the United States Pharmacopeia and Food and Drug Administration. Therefore, determining which cassette to use is a matter of the preference of the institution.

Stability evaluation of (18)F-FDG at high radioactive concentrations.

UNLABELLED: The objective of our study was to determine the concentration of ethanol, a known radiolytic stabilizer, needed to maintain stability for 12 h at an (18)F-FDG concentration of 19.7-22.6 GBq/mL (533-610 mCi/mL) at the end of synthesis (EOS). METHODS: (18)F(-) was formed by the (18)O(p, n)(18)F reaction using 16.5-MeV protons on a cyclotron. (18)F-FDG was synthesized using a synthesis platform. The final product was formulated in 15 mL of phosphate buffer. The synthesis took 22 min, delivering up to 336.7 GBq (9.1 Ci) of (18)F-FDG at the EOS. A series of 9 runs, 19.7-22.6 GBq/mL (533-610 mCi/mL), was completed. Three runs were doped with 0.1% ethanol, 3 with 0.2% ethanol, and 3 with no ethanol added. The radiochemical purity (RCP) was tested at about 1-h increments over a 12-h period. RCP was found by radio-thin-layer chromatography using aluminum-backed silica gel plates, acetonitrile, and water 90:10. An (18)F-FDG standard of 1 mg/mL was used to confirm radiochemical identity. The chromatography plates were analyzed on a radio-thin-layer chromatograph using a ß-detector. Residual solvents were also tested using gas chromatography with flame ionization detection and a capillary column. Other quality control measurements performed were pH and appearance. RESULTS: The 3 runs doped with 0.1% ethanol failed RCP after 5 h. The 3 runs using an ethanol concentration of 0.2% maintained stability through 12 h beyond the EOS. For these 3 runs, the radiolytic impurities were relatively constant at 6.1% ± 0.7% after 3 h. The runs using no ethanol failed RCP at 1 h. The pH varied between 5.3 and 6.1. Visual inspection was always clear and particulate-free. For the runs with 0.2% and 0.1% ethanol, the residual solvents were 0.21% ± 0.02% and 0.10% ± 0.02%, respectively. Regardless of ethanol concentration, chemical purity and identity passed quality control measurements. CONCLUSION: With the addition of 0.2% ethanol, (18)F-FDG (19.7-22.6 GBq/mL [533-610 mCi/mL]) kept stability through 12 h beyond the EOS. Each run passed stability parameters related to radiolysis-that is, radiochemical identity and RCP, chemical purity and identity, appearance, pH, and residual solvents.

Radiolysis of 2-[18F]fluoro-2-deoxy-D-glucose ([18F]FDG) and the role of ethanol and radioactive concentration.

Radiolysis is the process by which radioactively labeled compounds degrade. Many positron emission tomography (PET) radiopharmaceuticals produced with high radioactive concentrations and specific activities exhibit low radiochemical purity because of radiolysis. Little data exist that describe the radiolytic decomposition of 2-[(18)F]fluoro-2-deoxy-d-glucose ([(18)F]FDG). The objective of our study was to profile the degradation of [(18)F]FDG at various radioactive concentrations by measuring radiochemical purity at different time intervals and to study the effects of ethanol, a well-known reductant stabilizer of [(18)F]FDG preparations.

Using fluorodeoxythymidine to monitor anti-EGFR inhibitor therapy in squamous cell carcinoma xenografts.

BACKGROUND: 3'-18F-fluoro-3'-deoxy-fluorothymidine (18F-FLT), a nucleoside analog, could monitor effects of molecularly targeted therapeutics on tumor proliferation. METHODS: We tested whether (18)F-FLT positron emission tomography (PET) uptake changes are associated with antitumor effects of erlotinib in A431 xenografts or cetuximab in SCC1 xenografts. RESULTS: Compared with pretreatment FLT PET scans, 3 days of erlotinib in A431 reduced the standardized uptake value (SUV) by 18%, whereas placebo increased SUV by 1% (p = .005). One week of cetuximab in SCC1 reduced SUV by 62%, whereas placebo reduced SUV by 16% (p = .005). FLT uptake suppression following anti-epidermal growth factor receptor (EGFR) treatment was associated with reduced tumor thymidine kinase-1 (TK1) activity. In vitro TK1 knockdown studies confirmed the importance of TK1 activity on intracellular FLT accumulation suppression. CONCLUSIONS: 18F-FLT PET imaging detects tumor responses to EGFR-inhibitors within days of starting therapy. This technique may identify patients likely to benefit from EGFR-inhibitors early in their treatment course.

Inhibition evaluation for a 20-min endotoxin limit test on FDG.

OBJECTIVES: In chemical quality control tests for 2-[18F]fluoro-2-deoxy-D-glucose (18F-FDG), gel time is inversely related to endotoxin concentration. Solutions for positive product control (PPC) and positive water control (PWC) should contain a highly concentrated endotoxin level. The aims of our study were to derive an endotoxin concentration that causes PPC and PWC to gel and to evaluate inhibitory effects caused by FDG on gel formation in PPC and PWC test solutions. METHODS: At expiration, the maximum administered total dose (in millilitres) of FDG should contain fewer than 175 endotoxin units (EU). Our average batch volume of FDG is 15 ml; thus, the minimum endotoxin limit should be 12 EU x ml(-1) . Twelve tubes were tested for each of four concentrations. Inhibition was assessed using Limulus amoebocyte lysate reagent, depyrogenated vials, and pyrogen-free sterile micropipette tips. Each study was performed with four groups of solutions: decayed 18F-FDG, negative control, PPC and PWC. RESULTS: In the study of undiluted FDG, seven of 50 PPC vials at 12 EU x ml(-1) did not gel. The lowest endotoxin concentration that consistently gelled at 20 min was 4 EU x ml(-1) . In the sample tested for inhibition using PPC tubes at 4 EU x ml(-1) , 49 tubes gelled at 20 min, and one did not. CONCLUSION: The inhibition rate improved between undiluted (i.e., 7/50 (14%)) and diluted (i.e., 1/50 (2%)) FDG PPC trials (P=0.03). However, given that 1 of 50 trials failed for diluted FDG PPC, we conclude that inhibition still occurs and there is a 95% chance that the inhibition rate could be as high as 11% or as low as 0% on repetition of the experiment.

The planning and design of a new PET radiochemistry facility.

The objectives of the Mayo positron emission tomography (PET) radiochemistry facility are the production of PET drugs for clinical service of our in-house patients, commercial distribution of PET drug products, and development of new PET drugs. The factors foremost in the planning and design phases were the current regulatory climate for PET drug production, radiation safety issues, and effective production flow. A medium-energy cyclotron was preferred for its small footprint to allow a compact vault, its high-proton energy to offer a higher product radioactivity; and its research capabilities. A vault installation was chosen instead of a self-shielded machine for improved access and ease of maintenance. Adjacent to the cyclotron is an area that houses the support equipment and a large dedicated workshop to support machine maintenance and targetry development. The total floor area of the PET radiochemistry facility is 344.2 m(2) (3,705.5 ft(2)), of which the radiochemistry laboratory occupies 130.7 m(2) (1,407 ft(2)). To reduce environmental contamination of PET drug products, the laboratory contains a controlled-air environment class 10,000 (M5.5) clean room with access via an interlocking entry change area. A fully shielded isolator (class 100 [M3.5]) is located in the clean room. The PET drugs are delivered via shielded tubing between the synthesizer and isolator. Inside the isolator, there is an automated device for dispensing the PET drug into either a bulk-activity vial or a unit-dose syringe. The dispensed PET radiopharmaceutical then passes through a hatch to a dedicated area where it is packaged for in-house use or commercial distribution. Unit doses for in-house patients are transported via pneumatic tube to the PET imaging area 76.2 m (250 ft) away. There is extensive radiation area monitoring throughout the facility that continuously measures radiation levels. We believe that our new PET radiochemistry facility not only meets overall objectives, but also provides an ergonomic, efficient working environment for the production and development of PET drugs.