Evaluation of Tetrazine Tracers for Pretargeted Imaging within the Central Nervous System.

The pretargeting approach separates the biological half-life of an antibody from the physical half-life of the radioisotope label, providing a strategy for reducing the radiation burden. A widely explored pretargeting approach makes use of the bioorthogonal click reaction between tetrazines (Tzs) and trans-cyclooctenes (TCOs), combining the targeting specificity of monoclonal antibodies (mAbs) with the rapid clearance and precise reaction of Tzs and TCOs. Such a strategy can allow for the targeting and imaging (e.g., by positron emission tomography (PET)) of molecular markers, which cannot be addressed by solely relying on small molecules. Tz derivatives that undergo inverse electron-demand Diels-Alder (IEDDA) reactions with an antibody bearing TCO moieties have been investigated. This study describes the synthesis and characterization of 11 cold Tz imaging agent candidates. These molecules have the potential to be radiolabeled with 18F or 3H, and with the former label, they could be of use as imaging tracers for positron emission tomography studies. Selection was made using a multiparameter optimization score for the central nervous system (CNS) PET tracers. Novel tetrazines were tested for their pH-dependent chemical stability. Those which turned out to be stable in a pH range of 6.5-8 were further characterized in in vitro assays with regard to their passive permeability, microsomal stability, and P-glycoprotein transport. Furthermore, selected Tzs were examined for their systemic clearance and CNS penetration in a single-dose pharmacokinetic study in rats. Two tetrazines were successfully labeled with 18F, one of which showed brain penetration in a biodistribution study in mice. Another Tz was successfully tritium-labeled and used to demonstrate a bioorthogonal click reaction on a TCO-modified antibody. As a result, we identified one Tz as a potential fluorine-18-labeled CNS-PET agent and a second as a 3H-radioligand for an IEDDA-based reaction with a modified brain-penetrating antibody.

Antibody-Based In Vivo Imaging of Central Nervous System Targets-Evaluation of a Pretargeting Approach Utilizing a TCO-Conjugated Brain Shuttle Antibody and Radiolabeled Tetrazines.

Bioorthogonal pretargeted imaging using the inverse-electron-demand Diels-Alder (IEDDA) reaction between a tetrazine (Tz) and a trans-cyclooctene (TCO) represents an attractive strategy for molecular imaging via antibodies. The advantages of using a pretargeted imaging approach are on the one hand the possibility to achieve a high signal-to-noise ratio and imaging contrast; on the other hand, the method allows the uncoupling of the biological half-life of antibodies from the physical half-life of short-lived radionuclides. A brain-penetrating antibody (mAb) specific for ß-amyloid (Aß) plaques was functionalized with TCO moieties for pretargeted labeling of Aß plaques in vitro, ex vivo, and in vivo by a tritium-labeled Tz. The overall aim was to explore the applicability of mAbs for brain imaging, using a preclinical model system. In vitro clicked mAb-TCO-Tz was able to pass the blood-brain barrier of transgenic PS2APP mice and specifically visualize Aß plaques ex vivo. Further experiments showed that click reactivity of the mAb-TCO construct in vivo persisted up to 3 days after injection by labeling Aß plaques ex vivo after incubation of brain sections with the Tz in vitro. An attempted in vivo click reaction between injected mAb-TCO and Tz did not lead to significant labeling of Aß plaques, most probably due to unfavorable in vivo properties of the used Tz and a long half-life of the mAb-TCO in the blood stream. This study clearly demonstrates that pretargeted imaging of CNS targets via antibody-based click chemistry is a viable approach. Further experiments are warranted to optimize the balance between stability and reactivity of all reactants, particularly the Tz.

Small bowel homing T cells are associated with symptoms and delayed gastric emptying in functional dyspepsia.

OBJECTIVES: Immune activation may have an important pathogenic role in the irritable bowel syndrome (IBS). While little is known about immunologic function in functional dyspepsia (FD), we have observed an association between cytokine secretion by peripheral blood mononuclear cells (PBMCs) and symptoms in IBS. Upper gastrointestinal inflammatory diseases are characterized by enhanced small bowel homing α4-, ß7-integrin, chemokine receptor 9 (CCR9) positive T lymphocytes. We hypothesized that increased cytokine release and elevated circulating small bowel homing T cells are linked to the severity of symptoms in patients with FD. Thus, we aimed to (i) compare cytokine release in FD and healthy controls (HCs), (ii) quantify "gut homing" T cells in FD compared with HC and patients with IBS, and (iii) correlate the findings to symptom severity and gastric emptying. METHODS: PBMC from 45 (Helicobacter pylori negative) patients with FD (Rome II) and 35 matched HC were isolated by density gradient centrifugation and cultured for 24 h. Cytokine production (tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, IL-10) was measured by enzyme-linked immunosorbent assay. CD4+ α4ß7+CCR9+ T cells were quantified by flow cytometry in FD, HC and 23 patients with IBS. Gastric emptying was measured by scintigraphy. Symptom severity was assessed utilizing the standardized Gastrointestinal Symptom Score. RESULTS: FD patients had significantly higher TNF-α (107.2 ± 42.8 vs. 58.7 ± 7.4 pg/ml), IL-1ß (204.8 ± 71.5 vs. 80.2 ± 17.4 pg/ml), and IL-10 (218 ± 63.3 vs. 110.9 ± 18.5 pg/ml) levels compared with HC, and enhanced gut homing lymphocytes compared with HC or IBS. Cytokine release and CD4+α4ß7+CCR9+ lymphocytes were correlated with the symptom intensity of pain, cramps, nausea, and vomiting. Delayed gastric emptying was significantly associated (r = 0.78, P = 0.021) with CD4+α4ß7+CCR9+ lymphocytes and IL-1ß, TNF-α, and IL-10 secretion. CONCLUSIONS: Cellular immune activation with increased small bowel homing T cells may be key factors in the clinical manifestations of H. pylori-negative FD.

Immune activation in patients with irritable bowel syndrome.

BACKGROUND AND AIMS: We set out to test the hypothesis that irritable bowel syndrome (IBS) is characterized by an augmented cellular immune response with enhanced production of proinflammatory cytokines. We further aimed to explore whether symptoms and psychiatric comorbidity in IBS are linked to the release of proinflammatory cytokines. METHODS: We characterized basal and Escherichia coli lipopolysaccharide (LPS)-induced cytokine production in peripheral blood mononuclear cells (PBMCs) from 55 IBS patients (18 mixed-, 17 constipation-, 20 diarrhea-predominant) and 36 healthy controls (HCs). PBMCs were isolated by density gradient centrifugation and cultured for 24 hours with or without (1 ng/mL) LPS. Cytokine production (tumor necrosis factor [TNF]-alpha, interleukin [IL]-1beta, and IL-6) was measured by enzyme-linked immunosorbent assay. Abdominal symptoms and psychiatric comorbidities were assessed by using the validated Bowel Disease Questionnaire and the Hospital Anxiety and Depression Scale. RESULTS: IBS patients showed significantly (P < .017) higher baseline TNF-alpha, IL-1beta, IL-6, and LPS-induced IL-6 levels compared with HCs. Analyzing IBS subgroups, all cytokine levels were significantly (P < .05) higher in diarrhea-predominant IBS (D-IBS) patients, whereas constipation-predominant IBS patients showed increased LPS-induced IL-1beta levels compared with HCs. Baseline TNF-alpha and LPS-induced TNF-alpha and IL-6 levels were significantly higher in patients reporting more than 3 bowel movements per day, urgency, watery stools, and pain associated with diarrhea compared with patients without these symptoms (all P < .05). LPS-induced TNF-alpha production was associated significantly (r = 0.59, P < .001) with anxiety in patients with IBS. CONCLUSIONS: Patients with D-IBS display enhanced proinflammatory cytokine release, and this may be associated with symptoms and anxiety.