Improving the stability of 11C-labeled L-methionine with ascorbate.
ABSTRACT: Carbon-11 labeled L-methionine (11C-MET) is a popular tracer used in the clinic for imaging brain tumors with positron emission tomography. However, the stability of 11C-MET in its final formulation is not well documented in literature. Recently, we observed fast degradation of HPLC-purified 11C-MET over time, and systematic investigation was conducted to identify the cause.In this study, we verified the degraded product as 11C-labeled methionine sulfoxide (11C-METSO). To minimize oxidation, ascorbate (100 ppm) was added to the HPLC eluant, and the resulting HPLC-purified 11C-MET was stable in the final formulation solution without noticeable degradation for up to 1 h after the end of synthesis.Our data suggest that to minimize degradation, ascorbate can be added to the 11C-MET formulation solution especially if it is not administered into patients soon after the end of synthesis.
Project description:Occult bacterial infections represent a worldwide health problem. Differentiating active bacterial infection from sterile inflammation can be difficult using current imaging tools. Present clinically viable methodologies either detect morphologic changes (CT/ MR), recruitment of immune cells (111In-WBC SPECT), or enhanced glycolytic flux seen in inflammatory cells (18F-FDG PET). However, these strategies are often inadequate to detect bacterial infection and are not specific for living bacteria. Recent approaches have taken advantage of key metabolic differences between prokaryotic and eukaryotic organisms, allowing easier distinction between bacteria and their host. In this report, we exploited one key difference, bacterial cell wall biosynthesis, to detect living bacteria using a positron-labeled D-amino acid. After screening several 14C D-amino acids for their incorporation into E. coli in culture, we identified D-methionine as a probe with outstanding radiopharmaceutical potential. Based on an analogous procedure to that used for L-[methyl-11C]methionine ([11C] L-Met), we developed an enhanced asymmetric synthesis of D-[methyl-11C]methionine ([11C] D-Met), and showed that it can rapidly and selectively differentiate both E. coli and S. aureus infections from sterile inflammation in vivo. We believe that the ease of [11C] D-Met radiosynthesis, coupled with its rapid and specific in vivo bacterial accumulation, make it an attractive radiotracer for infection imaging in clinical practice.
Project description:S-[11C]-methyl-L-cysteine ([11C]MCYS) has been claimed to offer higher tumor selectivity than L-[methyl- 11C]methionine ([11C]MET). We examined this claim in animal models.Rats with implanted untreated (n = 10) or irradiated (n = 7, 1 × 25 Gy, on day 8) orthotopic gliomas were scanned after 6, 9, and 12 days, using positron emission tomography. Rats with striatal injections of saline (n = 9) or bacterial lipopolysaccharide (n = 9) were scanned after 3 days.Uptake of the two tracers in untreated gliomas was similar. [11C]MCYS was not accumulated in salivary glands, nasal epithelium, and healing wounds, in contrast to [11C]MET, but showed 40 % higher accumulation in the healthy brain. Both tracers showed a reduced tumor uptake 4 days after irradiation and minor accumulation in inflamed striatum. [11C]MCYS indicated higher lesion volumes than [11C]MET (untreated tumor + 47 %; irradiated tumor up to + 500 %; LPS-inflamed striatum + 240 %).[11C]MCYS was less accumulated in some non-tumor tissues than [11C]MET, but showed lower tumor-to-brain contrast.
Project description:Purpose: We developed a 11C-Methionine positron emission tomography/computed tomography (11C-MET PET/CT)-based nomogram model that uses easy-accessible imaging and clinical features to achieve reliable non-invasive isocitrate dehydrogenase (IDH)-mutant prediction with strong clinical translational capability. Methods: One hundred and ten patients with pathologically proven glioma who underwent pretreatment 11C-MET PET/CT were retrospectively reviewed. IDH genotype was determined by IDH1 R132H immunohistochemistry staining. Maximum, mean and peak tumor-to-normal brain tissue (TNRmax, TNRmean, TNRpeak), metabolic tumor volume (MTV), total lesion methionine uptake (TLMU), and standard deviation of SUV (SUVSD) of the lesions on MET PET images were obtained via a dedicated workstation (Siemens. syngo.via). Univariate and multivariate logistic regression models were used to identify the predictive factors for IDH mutation. Nomogram and calibration plots were further performed. Results: In the entire population, TNRmean, TNRmax, TNRpeak, and SUVSD of IDH-mutant glioma patients were significantly lower than these values of IDH wildtype. Receiver operating characteristic (ROC) analysis suggested SUVSD had the best performance for IDH-mutant discrimination (AUC = 0.731, cut-off ? 0.29, p < 0.001). All pairs of the 11C-MET PET metrics showed linear associations by Pearson correlation coefficients between 0.228 and 0.986. Multivariate analyses demonstrated that SUVSD (>0.29 vs. ? 0.29 OR: 0.053, p = 0.010), dichotomized brain midline structure involvement (no vs. yes OR: 26.52, p = 0.000) and age (? 45 vs. >45 years OR: 3.23, p = 0.023), were associated with a higher incidence of IDH mutation. The nomogram modeling showed good discrimination, with a C-statistics of 0.866 (95% CI: 0.796-0.937) and was well-calibrated. Conclusions: 11C-Methionine PET/CT imaging features (SUVSD and the involvement of brain midline structure) can be conveniently used to facilitate the pre-operative prediction of IDH genotype. The nomogram model based on 11C-Methionine PET/CT and clinical age features might be clinically useful in non-invasive IDH mutation status prediction for untreated glioma patients.
Project description:Cytosolic glutathione S-transferases (GSTs) were isolated from 1-day-old Leghorn chick livers by glutathione (GSH)-affinity chromatography. After sample loading and extensive washing with 0.2 M NaCl, the column was sequentially eluted with 5 mM GSH and 1 mM S-hexylglutathione. The isolated GSTs were subjected to reverse-phase HPLC, electrospray ionization-MS, N-terminal and internal peptide sequencing analyses. The proteins recovered from the 5 mM GSH eluant were predominantly cGSTM1. A protein (cGSTM1') with an N-terminal amino acid sequence identical to that of cGSTM1 but with the initiator methionine retained and a novel class-mu isozyme (cGSTM2*) were also recovered from this fraction. Nine class-alpha isozymes with distinctive molecular masses were identified from the 1 mM S-hexylglutathione eluant. Three of these proteins are probably variants with minor amino acid substitutions of other isozymes. Of the six remaining class-alpha isozymes, three of them have had their complete (cGSTA1 and cGSTA2) or partial (cGSTA3) cDNA sequences reported previously in the literature. A chicken liver cDNA library was screened with oligonucleotides generated from the cGSTA2 sequence as probes. Clones that encompass the complete coding regions of cGSTA3 and cGSTA4 were obtained. A clone encoding the C-terminal 187 residues of cGSTA5 was also isolated.
Project description:11C-methionine (11C-MET) is a new positron emission tomography (PET) tracer for the assessment of disease activity in multiple myeloma (MM) patients, with preliminary data suggesting higher sensitivity and specificity than 18F-fluorodeoxyglucose (18F-FDG). However, the value of tumor burden biomarkers has yet to be investigated. Our goals were to corroborate the superiority of 11C-MET for MM staging and to compare its suitability for the assessment of metabolic tumor burden biomarkers in comparison to 18F-FDG. Twenty-two patients with newly diagnosed, treatment-naïve symptomatic MM who had undergone 11C-MET and 18F-FDG PET/CT were evaluated. Standardized uptake values (SUV) were determined and compared with total metabolic tumor volume (TMTV) for both tracers: total lesion glycolysis (TLG) and total lesion 11C-MET uptake (TLMU). PET-derived values were compared to Revised International Staging System (R-ISS), cytogenetic, and serologic MM markers such as M component, beta 2 microglobulin (B2M), serum free light chains (FLC), albumin, and lactate dehydrogenase (LDH). In 11 patients (50%), 11C-MET detected more focal lesions (FL) than FDG (p < 0.01). SUVmax, SUVmean, SUVpeak, TMTV, and TLMU were also significantly higher in 11C-MET than in 18F-FDG (p < 0.05, respectively). 11C-MET PET biomarkers had a better correlation with tumor burden (bone marrow plasma cell infiltration, M component; p < 0.05 versus p = n.s. respectively). This pilot study suggests that 11C-MET PET/CT is a more sensitive marker for the assessment of myeloma tumor burden than 18F-FDG. Its implications for prognosis evaluation need further investigation.
Project description:Currently, there exists no accurate, noninvasive clinical imaging method to detect living bacteria in vivo. Our goal is to provide a positron emission tomography (PET) method to image infection by targeting bacteria-specific metabolism. Standard of care methodologies detect morphologic changes, image immunologic response to infection, or employ invasive tissue sampling with associated patient morbidity. These strategies, however, are not specific for living bacteria and are often inadequate to detect bacterial infection during fever workup. As such, there is an unmet clinical need to identify and validate new imaging tools suitable for noninvasive, in vivo (PET) imaging of living bacteria. We have shown that d-[methyl-11C]methionine (d-[11C]Met) can distinguish active bacterial infection from sterile inflammation in a murine infection model and is sensitive to both Gram-positive and Gram-negative bacteria. Here, we report an automated and >99% enantiomeric excess (ee) synthesis of d-[11C]Met from a linear d-homocysteine precursor, a significant improvement over the previously reported synthesis utilizing a d-homocysteine thiolactone hydrochloride precursor with approximately 75-85% ee. Furthermore, we took additional steps toward applying d-[11C]Met to infected patients. d-[11C]Met was subject to a panel of clinically relevant bacterial strains and demonstrated promising sensitivity to these pathogens. Finally, we performed radiation dosimetry in a normal murine cohort to set the stage for translation to humans in the near future.
Project description:The role of metabolic imaging in the diagnosis, treatment, and response assessment of diffuse intrinsic pontine glioma (DIPG) is poorly defined. We investigated the uptake of 11C-methionine in pediatric patients with newly diagnosed DIPG and evaluated the associations of 11C-methionine PET metrics with conventional MRI indices and survival outcomes. Methods: Twenty-two patients with newly diagnosed DIPG were prospectively enrolled on an institutional review board-approved investigational study of 11C-methionine PET. All patients underwent baseline 11C-methionine PET/CT, and initial treatment-response scans after chemotherapy or radiation therapy were obtained for 17 patients. Typical and atypical DIPGs were assessed clinically and radiographically and defined by multidisciplinary consensus. Three-dimensional regions of interest, reviewed by consensus between a nuclear medicine physician and a radiation oncologist, were delineated after coregistration of PET and MR images. Associations of 11C-methionine uptake intensity and uniformity with survival, along with associations between 11C-methionine uptake and conventional MRI tumor indices over time, were evaluated. 11C-methionine PET voxel values within regions of interest were assessed as threshold values across proportions of the study population, and 11C-methionine uptake at baseline was assessed relative to MRI-defined tumor progression. Results: 11C-methionine uptake above that of uninvolved brain tissue was observed in 18 of 22 baseline scans (82%) and 15 of 17 initial response scans (88%). 11C-methionine avidity within MRI-defined tumor was limited in extent, with 11 of 18 positive baseline 11C-methionine PET scans (61%) showing less than 25% 11C-methionine-avid tumor. The increase in total tumor volume with 11C-methionine PET was relatively limited (17.2%; interquartile range, 6.53%-38.90%), as was the extent of 11C-methionine uptake beyond the MRI-defined tumor (2.2%; interquartile range, 0.55%-10.88%). Although baseline 11C-methionine PET intensity and uniformity metrics did not correlate with survival outcomes, initial 11C-methionine avidity overlapped with recurrent tumor in 100% of cases. A clinical diagnosis of atypical DIPG was associated with borderline significantly prolonged progression-free survival (P = 0.07), yet 11C-methionine PET indices at diagnosis did not differ significantly between atypical and typical DIPGs. Conclusion: Most newly diagnosed DIPGs are successfully visualized by 11C-methionine PET. Baseline 11C-methionine uptake delineates regions at increased risk for recurrence, yet intensity and uniformity metrics did not correlate with treatment outcomes in children with DIPG in this study.
Project description:Positron emission tomography (PET) is being increasingly utilized for the management of brain tumors. Herein, we primarily review our previous studies on the use of PET in glioma that utilize three types of tracers: 11C-methionine (MET), 11C-choline, and 18F-fluorodeoxyglucose. These studies included aspects such as tumor behavior, diagnosis, grade of malignancy, spread and invasion, viability, and genetic deletions; moreover, they also evaluated PET as a tool for planning radiation therapy (RT) and determining its outcome. MET-PET in particular is considered to be the most informative for diagnosis and therapeutic decision-making for glioma patients; it is therefore considered crucial for brain tumor therapy. MET-PET is expected to be widely used for brain tumor patients going forward.
Project description:This work describes the formulation design and development of a novel protein based adjuvant, a double mutant of heat labile toxin (dmLT), based on knowledge of the protein's structural integrity and physicochemical degradation pathways. Various classes of pharmaceutical excipients were screened for their stabilizing effect on dmLT during exposure to thermal and agitation stresses as monitored by high throughput analytical assays for dmLT degradation. Sucrose, phosphate, sodium chloride, methionine and polysorbate-80 were identified as potential stabilizers that protected dmLT against either conformational destabilization, aggregation/particle formation or chemical degradation (e.g., Met oxidation and Lys glycation). Different combinations and concentrations of the selected stabilizers were then evaluated to further optimize dmLT stability while maintaining pharmaceutically acceptable ranges of solution pH and osmolality. The effect of multiple freeze-thaw (FT) cycles on the physical stability of candidate bulk formulations was also examined. Increasing the polysorbate-80 concentration to 0.1% in the lead candidate bulk formulation mitigated the loss of protein mass during FT. This formulation development study enabled the design of a new bulk formulation of the dmLT adjuvant and provides flexibility for future use in combination with a variety of different vaccine dosage forms with different antigens.
Project description:For patients with type 1 diabetes mellitus, a bihormonal artificial endocrine pancreas system utilizing glucagon and insulin has been found to stabilize glycemic control. However, commercially available formulations of glucagon cannot currently be used in such systems because of physical instability characterized by aggregation and chemical degradation. Storing glucagon at pH 10 blocks protein aggregation but results in chemical degradation. Reductions in pH minimize chemical degradation, but even small reductions increase protein aggregation. We hypothesized that common pharmaceutical excipients accompanied by a new excipient would inhibit glucagon aggregation at an alkaline pH.As measured by tryptophan intrinsic fluorescence shift and optical density at 630?nm, protein aggregation was indeed minimized when glucagon was formulated with curcumin and albumin. This formulation also reduced chemical degradation, measured by liquid chromatography with mass spectrometry. Biological activity was retained after aging for 7 days in an in vitro cell-based bioassay and also in Yorkshire swine.Based on these findings, a formulation of glucagon stabilized with curcumin, polysorbate-80, l-methionine, and albumin at alkaline pH in glycine buffer may be suitable for extended use in a portable pump in the setting of a bihormonal artificial endocrine pancreas.