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(R)-[(18)F]MH.MZ ([(18)F]MH.MZ) is a promising positron emission tomography (PET) radiotracer for in vivo study of the 5-HT(2A) receptor. To facilitate clinical trials a fully automated radiosynthesis procedure for [(18)F]MH.MZ was developed using commercially available materials on the iPhase Flexlab module. The overall synthesis time was 100 minutes with a radiochemical yield of 7 ± 0.9% (n=3). The radiochemical purity was greater than 99% for [(18)F]MH.MZ with a molar activity of 361 ± 57 GBq/μmol (n=3). The protocol described herein reliably provides [(18)F]MH.MZ that meets all relevant release criteria for a GMP radiopharmaceutical.

We have previously identified that in the absence of MET amplification or mutation, RTK hyperphosphorylation, indicative of its active state, may contribute to resistance to the EGFR-directed therapeutic, cetuximab in colorectal cancer (CRC). In our in vitro 3D cultures and in vivo xenografts, we showed that inhibition of MET (and RON) by crizotinib can overcome cetuximab resistance in CRC lines and xenografts. We now show that MET may get hyperphosphorylated by the autocrine expression and maturation of its ligand, HGF. This non-genetic mode of cetuximab resistance contrasts with the usual genetic modes of CRC cetuximab resistance, with majority of them being RAS/RAF mutations. Exogenous addition as well as autocrine overexpression of HGF led to cetuximab resistance, which could be overcome by MET inhibition by crizotinib. Interestingly, HGF/HGFL are synthesized as inactive precursors and require cleavage by proteases (HGFA, Matriptase, and Hepsin) to be biologically active. A survey of TCGA datasets indicated that HGF/HGFL were overexpressed in several CRC CMS subtypes, while the endogenous inhibitor of HGF proteases, HAI-I, was downregulated across all CRC CMS subtypes. We next expressed human full-length HGF in cetuximab-sensitive CC cells and observed that HGF overexpression imparts cetuximab resistance. Moreover, cetuximab resistance induced by upstream HGF overexpression could be overcome by the downstream MET inhibition. Next, in a subset of KRAS WT CRC patient-derived xenografts (PDXs) we tested and found that at majority of PDXs exhibit elevated pMET/pRON levels. RNA-seq-based principal component analysis showed that the maximal differences in expression among PDXs could be explained by their pMET/pRON phosphorylation status. Next, we generated nude mice xenografts for one PDX with high pMET/pRON levels and showed that cetuximab inhibition alone was not sufficient to inhibit regrowth of tumors after cessation of drug treatment, but a combined cetuximab/crizotinib treatment was, indicating endogenous MET/RON activation contributes to cetuximab resistance. In a parallel tumor stratification strategy, we have optimized conditions to generate [18F]crizotinib and are testing its efficacy as a new non-invasive PET-based imaging probe to identify crizotinib-avid tumors with high MET/RON activity. Together, these studies may provide novel mechanistic insights into RTK cross-talk and provide a positive predictive biomarker for precision therapy in colorectal cancer. Citation Format: Vivian T. Jones, Ramona Graves-Deal, KyuOK Jeon, Adam J. Rosenberg, Zheng Cao, Galina T. Bogatcheva, Sarah J. Harmych, James N. Higginbotham, Kwangho Kim, James W. Janetka, Gary A. Sulikowski, Bhuminder Singh. Role of MET signaling in cetuximab resistance and generation of an imaging biomarker to identify MET-driven cetuximab resistance in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2562.

L-ergothioneine (ERGO) is a potent antioxidant with cytoprotective effects. To study ERGO biodistribution and detect oxidative stress in vivo, we report an efficient and reproducible preparation of [(11)C]-labeled ERGO PET radioligand based on protecting the histidine carboxylic group with a methyl ester. Overall, this new protection approach using methyl ester improved the chemical yield of a 4-step reaction from 14% to 24% compared to the previous report using t-butyl ester. The [(11)C]CH(3) methylation of the precursor provided the desired product with 55 ± 10% radiochemical purity and a molar activity of 450 ± 200 TBq/mmol. The [(11)C]ERGO radioligand was able to detect threshold levels of oxidative stress in a preclinical animal model of Alzheimer’s disease.

Ergothioneine (ERGO) is a rare amino acid mostly found in fungi, including mushrooms, with recognized antioxidant activity to protect tissues from damage by reactive oxygen species (ROS) components. Prior to this publication, the biodistribution of ERGO has been performed solely in vitro using extracted tissues. The aim of this study was to develop a feasible chemistry for the synthesis of an ERGO PET radioligand, [11C]ERGO, to facilitate in vivo study. The radioligand probe was synthesized with identical structure to ERGO by employing an orthogonal protection/deprotection approach. [11C]methylation of the precursor was performed via [11C]CH3OTf to provide [11C]ERGO radioligand. The [11C]ERGO was isolated by RP-HPLC with a molar activity of 690 TBq/mmol. To demonstrate the biodistribution of the radioligand, we administered approximately 37 MBq/0.1 mL in 5XFAD mice, a mouse model of Alzheimer’s disease via the tail vein. The distribution of ERGO in the brain was monitored using 90-min dynamic PET scans. The delivery and specific retention of [11C]ERGO in an LPS-mediated neuroinflammation mouse model was also demonstrated. For the pharmacokinetic study, the concentration of the compound in the serum started to decrease 10 min after injection while starting to distribute in other peripheral tissues. In particular, a significant amount of the compound was found in the eyes and small intestine. The radioligand was also distributed in several regions of the brain of 5XFAD mice, and the signal remained strong 30 min post-injection. This is the first time the biodistribution of this antioxidant and rare amino acid has been demonstrated in a preclinical mouse model in a highly sensitive and non-invasive manner.

BACKGROUND: It is well known that following root surface debridement (RSD) residual deposits remain. Periodontal endoscopy has provided a method of directly visualizing root surfaces during periodontal debridement in an intact pocket without the need for surgical incision. The aim of this study was to determine if periodontal debridement using endoscopic visualization was more effective in improving clinical and radiographic parameters as compared to RSD. METHODS: Thirty-eight subjects were randomized into RSD with perioscope (n = 19) or RSD only (n = 19) groups. A full-mouth evaluation included probing pocket depths (PPDs), clinical attachment levels (CAL), bleeding on probing (BOP) and plaque scores (PI) recorded at baseline, 3 and 12 months and compared among groups. Radiographs were taken at sites with deepest pockets at baseline and 12-month and the change in radiographic bone levels (RBL) compared. An independent samples T-test was used to assess statistical significance. RESULTS: Both groups had significant improvements in clinical outcomes. The test (T) group had a significantly lower percentage of PPDs 7 to 9 mm at three (0.72 ± 1.2%) and 12 months (0.5 ± 1.0%) as compared with the control (C) group (2.25 ± 2.9%; 1.84 ± 2.3%). At 12 months, the test group recorded a significantly lower mean PPD (T: 2.70 + 0.2 mm; C: 2.98 ± 0.4 mm), BOP% (T: 4.3 ± 3.2%; C: 11.95 ± 7.1%), PI% (T: 25.61 ± 3.9%; C: 30.11 ± 6.3%) and less change in gingival recession (T: -0.13 ± 0.2 mm; C: -0.50 ± 0.6 mm) (P

INTRODUCTION: The natural amino acid L-Glutamine (Gln) is essential for both cell growth and proliferation. In addition to glucose, cancer cells utilize Gln as a carbon source for ATP production, biosynthesis, and as a defense against reactive oxygen species. The utilization of [(11)C]Gln has been previously reported as a biomarker for tissues with an elevated demand for Gln, however, the previous reports for the preparation of [(11)C]Gln were found to be lacking several crucial aspects necessary for transition to human production. Namely, the presence of unreacted precursor and the use of noncommercialized, custom built, reaction platforms. Herein, we report the development and utilization of methodology for the automated production of [(11)C]Gln that meets institutional criteria for human use. METHODS: The preparation of [(11)C]Gln was carried out on the the GE FX(2)N platform. Briefly, after trapping of [(11)C]HCN with a solution of CsHCO(3) in DMF, the [(11)C]CsCN was reacted with a commercially available precursor. This intermediate was then purified by HPLC and deprotected/hydrolyzed under acidic conditions. Following pH adjustment, the product was filtered to give the desired [(11)C]Gln as a sterile injectable. The resulting product was then analyzed for quality assurance. RESULTS: Automated production by this method reliably provides over 3.7 GBq (100 mCi) of [(11)C]Gln. The resulting final drug product was found to have a >99% radiochemical purity

A novel method for radiolabeling perfluorinated alkyl substances (PFAS) with fluorine 18 has been developed, and after purification, the stability and biological distribution in healthy mice were evaluated. Three PFAS, [18F]PFOA (C8), [18F]PFHxA (C6), and [18F]PFBA (C4), were readily labeled and isolated in average yields between 12 and 31%. The stability of each compound was monitored in 0.1% ammonium hydroxide (NH4OH) in methanol, in saline, and in human, mouse, and rat sera. The amount of intact, radiolabeled PFAS was determined by radiometric instant thin layer chromatography and was calculated by the amount of free fluorine 18 observed over time. All compounds were highly stable in 0.1% NH4OH in methanol and saline, with

Upregulation of sphingosine-1-phosphate receptor 1 (S1PR1) expression in multiple sclerosis (MS) lesions is associated with neuroinflammatory response. This study investigated the correlation between neuroinflammation and S1PR1 expression in the spinal cord of an experimental autoimmune encephalomyelitis (EAE) rat model of MS, using the S1PR1 positron emission tomography (PET) radiotracer [(11)C]TZ3321.MicroPET imaging studies of [(11)C]TZ3321 were performed to measure uptake of [(11)C]TZ3321 in the spinal cord of EAE rats. Immunohistochemical staining was performed to confirm the overexpression of S1PR1 and other inflammatory biomarkers.MicroPET imaging demonstrated a 20-30 % increase in [(11)C]TZ3321 uptake in the lumbar spinal cord of EAE rats versus sham controls at 35-60 min post injection. The increased uptake of [(11)C]TZ3321 was correlated with the overexpression of S1PR1 in the lumbar spinal cord of EAE rats that was confirmed by immunohistochemical staining. Upregulated S1PR1 expression was associated with glial cell activation and immune cell infiltration.MicroPET imaging modality with a specific radioligand [(11)C]TZ3321 is able to assess the expression of S1PR1 in EAE rat lumbar spinal cord. This may provide a new approach to the assessment of neuroinflammatory response in MS and other inflammatory diseases.

A series of seventeen hydroxyl-containing sphingosine 1-phosphate receptor 1 (S1PR1) ligands were designed and synthesized. Their in vitro binding potencies were determined using [(32)P]S1P competitive binding assays. Compounds 10a, 17a, 17b, and 24 exhibited high S1PR1 binding potencies with IC(50) values ranging from 3.9 to 15.4 nM and also displayed high selectivity for S1PR1 over other S1P receptor subtypes (IC(50) > 1000 nM for S1PR2–5). The most potent compounds 10a, 17a, 17b, and 24 were subsequently radiolabeled with F-18 in high yields and purities. MicroPET studies in cynomolgus macaque showed that [(18)F]10a, [(18)F]17a, and [(18)F]17b but not [(18)F]24 crossed the blood brain barrier and had high initial brain uptake. Further validation of [(18)F]10a, [(18)F]17a, and [(18)F]17b in preclinical models of neuroinflammation is warranted to identify a suitable PET radioligand to quantify S1PR1 expression in vivo as a metric of an inflammatory response.

Thirteen new sphingosine-1-phosphate receptor 1 (S1PR1) ligands were designed and synthesized by replacing azetidine-3-carboxylic acid moiety of compound 4 with new polar groups. The in vitro binding potency of these new analogs toward S1PR1 was determined. Out of 13 new compounds, four compounds 9a, 10c, 12b, and 16b displayed high S1PR1 binding potency with IC50 values of 13.2 ± 3.2, 14.7 ± 1.7, 9.7 ± 1.6, and 6.3 ± 1.3 nM, respectively; further binding studies of these four ligands toward S1PR2-5 suggested they are highly selective for S1PR1 over other S1PRs. The radiosynthesis of the lead radiotracer [18F]12b was achieved with good radiochemical yield (∼14.1%), high radiochemical purity (>98%), and good specific activity (∼54.1 GBq/μmol, decay corrected to the end of synthesis, EOS). Ex vivo autoradiography and initial biodistribution studies in rodents were performed, suggesting that [18F]12b was able to penetrate the blood-brain barrier (BBB) with high brain uptake (0.71% ID/g at 60 min post-injection) and no defluorination was observed. In vitro autoradiography study in brain slices of lipopolysaccharides (LPS)-induced neuroinflammation mice indicated that SEW2871, a specific S1PR1 ligand was able to reduce the uptake of [18F]12b, suggesting [18F]12b has S1PR1 specific binding. These initial results suggested that [18F]12b has potential to be an F-18 labeled radiotracer for imaging S1PR1 in the brain of the animal in vivo.

Nitric oxide is a free radical produced in host tissues by constitutive and inducible forms of the enzyme nitric oxide synthase. Nitric oxide plays physiological roles, but it is also involved in the pathophysiology of several inflammatory conditions, including arthritis, ulcerative colitis, and circulatory shock. Local increases in inducible nitric oxide synthase (iNOS) and reactive nitrogen products have also been demonstrated in humans and animals with periodontal disease. This masked, randomized, placebo-controlled preclinical investigation examined the effect of two mercaptoalkylguanidines, mercaptoethylguanidine (MEG) and guanidinoethyldisulfide (GED), which are iNOS inhibitors and reactive nitrogen scavenging compounds, on the development of experimental gingivitis in beagle dogs.Fifteen female, 1-year-old beagles first completed a 2-week dose-escalation experiment during which a maximum tolerated dose was determined for MEG and GED gels. Thereafter, all animals were brought to optimal gingival health by mechanical scaling, followed by rigorous daily toothbrushing over a 4-week washout period. Experimental gingivitis was then induced, with cessation of plaque control and institution of a soft diet over 8 weeks. Beagles randomly received 0.3% MEG, 0.3% GED, or placebo (vehicle) gels, topically applied twice daily to premolar teeth. Gingival inflammation, bleeding tendency, and supragingival plaque were clinically measured at baseline and at 2, 3, 4, 6, and 8 weeks. Comparisons among groups and between group pairs (active versus placebo) were made using Kruskal-Wallis tests.From baseline to day 7, all groups expressed similar indices. Thereafter, significant and time-dependent increases in the plaque index (PI), gingival index (GI), and percentage of bleeding on probing (%BOP) were observed in placebo-treated beagles. Mean GI scores for beagles treated with GED or MEG gels remained at or below baseline levels for the entire treatment period. At weeks 2, 3, 4, and 8, GI scores were significantly lower for MEG and GED groups compared to the placebo group (P0.05). In addition, MEG and GED gels significantly reduced gingival bleeding responses by 8 weeks (P0.05). Although placebo-treated beagles demonstrated %BOP scores of 43% at week 8, GED- and MEG-treated beagles exhibited %BOP scores of 21% and 26%, respectively. Since no statistical difference among PI scores was noted for any of the time points, neither mercaptoalkylguanidine appeared to affect supragingival plaque levels.The data from this preclinical study indicate that mercaptoalkylguanidines, topically administered, may significantly reduce experimental gingivitis in the beagle dog.

Sphingosine-1-phosphate receptors (S1PRs) are important regulators of vascular permeability, inflammation, angiogenesis and vascular maturation. Identifying a specific S1PR PET radioligand is imperative, but it is hindered by the complexity and variability of current for binding affinity measurement procedures. Herein, we report a streamlined protocol for radiosynthesis of [(32)P]S1P with good radiochemical yield (36-50%) and high radiochemical purity (>99%). We also report a reproducible procedure for determining the binding affinity for compounds targeting S1PRs in vitro.

Sphingosine-1-phosphate receptor 1 (S1PR1) is highly expressed in vascular smooth muscle cells from intimal lesions. PET imaging using S1PR1 as a biomarker would increase our understanding of its role in vascular pathologies including in-stent restenosis. The S1PR1 compound TZ3321 was synthesized for in vitro characterization and labeled with Carbon-11 for in vivo studies. The biodistribution of [11C]TZ3321 was evaluated in normal mice; microPET and immunohistochemistry (IHC) studies were performed using a murine femoral artery wire-injury model of restenosis. The high potency of TZ3321 for S1PR1 (IC 50 = 2.13 ± 1.63 nM), and high selectivity (>1000 nM) for S1PR1 over S1PR2 and S1PR3 were confirmed. Biodistribution data revealed prolonged retention of [11C]TZ3321 in S1PR1-enriched tissues. MicroPET imaging of [11C]TZ3321 showed higher uptake in the wire-injured arteries of ApoE−/− mice than in injured arteries of wild-type mice (SUV 0.40 ± 0.06 vs 0.28 ± 0.04, n = 6, P .05). Post-PET autoradiography showed >4-fold higher [11C]TZ3321 retention in the injured artery of ApoE−/− mice than in wild-type mice. Subsequent IHC staining confirmed higher expression of S1PR1 in the neointima of the injured artery of ApoE−/− mice than in wild-type mice. This preliminary study supports the potential use of PET for quantification of the S1PR1 expression as a biomarker of neointimal hyperplasia.

Imidazo[4,5-c]pyridines were synthesized in three steps utilizing a palladium-catalyzed amidation/cyclization strategy. N-Aryl substrates were synthesized using copper-catalyzed amidation of 3-amino-N-Boc-4-chloropyridine. Complementary protocols for the selective chlorination of imidazo[4,5-c]pyridines at the C2 and C7 positions were also developed.

Imidazo[4,5-c]pyridines were synthesized in three steps utilizing a palladium-catalyzed amidation/cyclization strategy. N-Aryl substrates were synthesized using copper-catalyzed amidation of 3-amino-N-Boc-4-chloropyridine. Complementary protocols for the selective chlorination of imidazo[4,5-c]pyridines at the C2 and C7 positions were also developed.

The presented approach is completely regioselective, procedurally simple, and provides rapid access to previously difficult to access moieties.

The C2 amination of imidazo[4,5-b]pyridines was accomplished through C2 halogenation followed by substitution (SNAr) with functionalized primary and secondary amines. This regioselective sequence is operationally simple and provides an easy access to derivatives of protected imidazo[4,5-b]pyridines.

Pentosidine, a biologically important advanced glycation endproduct, has been accessed in a rapid, high-yielding manner. The synthesis was accomplished via a six-step sequence starting with 3-amino-2-chloropyridine and features a palladium-catalyzed tandem cross-coupling/cyclization to construct the imidazo[4,5-b]pyridine core.

The process allows a quick access to various natural products and biologically interesting title compounds with substituents at N-1 and C-2.

A facile synthesis of imidazo[4,5-b]pyridines and -pyrazines is described using a Pd-catalyzed amide coupling reaction. This reaction provides quick access to products with substitution at N1 and C2. A model system relevant to the natural product pentosidine has been demonstrated, as well as the total synthesis of the mutagen 1-Me-5-PhIP.