Selective protection of murine cerebral Gi/o-proteins from inactivation by parenterally injected pertussis toxin.

Pertussis toxin (PTX) is a potent virulence factor in patients suffering from whooping cough, but in its detoxified version, it is applied for vaccination. It is thought to contribute to the pathology of the disease including various CNS malfunctions. Based on its enzymatic activity, PTX disrupts GPCR-dependent signaling by modifying the α-subunit of heterotrimeric Gi/o-proteins. It is also extensively used as a research tool to study neuronal functions in vivo and in vitro. However, data demonstrating the penetration of PTX from the blood into the brain are missing. Here, we examined the Gαi/o-modifying activity of PTX in murine brains after its parenteral application. Ex vivo biodistribution analysis of [124I]-PTX displayed poor distribution to the brain while relatively high concentrations were visible in the pancreas. PTX affected CNS and endocrine functions of the pancreas as shown by open-field and glucose tolerance tests, respectively. However, while pancreatic islet Gαi/o-proteins were modified, their neuronal counterparts in brain tissue were resistant towards PTX as indicated by different autoradiographic and immunoblot SDS-PAGE analyses. In contrast, PTX easily modified brain Gαi/o-proteins ex vivo. An attempt to increase BBB permeability by application of hypertonic mannitol did not show PTX activity on neuronal G proteins. Consistent with these findings, in vivo MRI analysis did not point to an increased blood-brain barrier (BBB) permeability following PTX treatment. Our data demonstrate that the CNS is protected from PTX. Thus, we hypothesize that the BBB hinders PTX to penetrate into the CNS and to deliver its enzymatic activity to brain Gαi/o-proteins. KEY MESSAGES: i.p. applied PTX is poorly retained in the brain while reaches high concentration in the pancreas. Pancreatic islet Gαi/o- but not cerebral Gαi/o-proteins are modified by i.p. administered PTX. Gαi/o-proteins from isolated cerebral cell membranes were easily modified by PTX ex vivo. CNS is protected from i.p. administered PTX. PTX does not permeabilize the BBB.

Quantification of fat fraction in lumbar vertebrae: correlation with age and implications for bone marrow dosimetry in molecular radiotherapy.

Absorbed dose to active bone marrow is a predictor of hematological toxicity in molecular radiotherapy. Due to the complex composition of bone marrow tissue, the necessity to improve the personalized dosimetry has led to the application of non-conventional imaging methods in nuclear medicine. The aim of this study is to apply magnetic resonance imaging (MRI) for quantification of the fat fraction in lumbar vertebrae and to analyze its implications for bone marrow dosimetry. First, a highly accelerated two-point Dixon MRI sequence for fat-water separation was validated in a 3T system against the magnetic resonance spectroscopy (MRS) gold standard. The validation was performed in a fat-water phantom composed of 11 vials with different fat fractions between 0% and 100%, and subsequently repeated in the lumbar vertebrae of three healthy volunteers. Finally, a retrospective study was performed by analyzing the fat fraction in five lumbar vertebrae of 44 patients scanned with the two-point Dixon sequence. The two-point Dixon phantom acquisition showed a good agreement (maximum difference = 2.9%) between the nominal fat fraction and MRS. In the volunteers, a statistical analysis showed a non-significant difference (p = 0.19) between MRI and MRS. In the patients, gender-specific linear fits for female and male data indicated that the age-dependent marrow conversion (red → yellow marrow) is slower in males (0.3% per year) than in females (0.5% per year). Lastly, the fat fraction values showed a considerable variability in patients of similar ages and the same gender. Two-point Dixon MRI enables a non-invasive and spatially resolved quantification of the fat fraction in bone marrow. Our study provides important evidence on the differences in marrow conversion between females and males. In addition, differences were observed in the cellularity values of the International Commission on Radiological Protection (ICRP) reference man (0.7) and the median values obtained in our patient group. These observations lead to the conclusion that the fat fraction in bone marrow should be considered as a patient-specific variable in clinical dosimetry procedures.

64Cu antibody-targeting of the T-cell receptor and subsequent internalization enables in vivo tracking of lymphocytes by PET.

T cells are key players in inflammation, autoimmune diseases, and immunotherapy. Thus, holistic and noninvasive in vivo characterizations of the temporal distribution and homing dynamics of lymphocytes in mammals are of special interest. Herein, we show that PET-based T-cell labeling facilitates quantitative, highly sensitive, and holistic monitoring of T-cell homing patterns in vivo. We developed a new T-cell receptor (TCR)-specific labeling approach for the intracellular labeling of mouse T cells. We found that continuous TCR plasma membrane turnover and the endocytosis of the specific (64)Cu-monoclonal antibody (mAb)-TCR complex enables a stable labeling of T cells. The TCR-mAb complex was internalized within 24 h, whereas antigen recognition was not impaired. Harmful effects of the label on the viability, DNA-damage and apoptosis-necrosis induction, could be minimized while yielding a high contrast in in vivo PET images. We were able to follow and quantify the specific homing of systemically applied (64)Cu-labeled chicken ovalbumin (cOVA)-TCR transgenic T cells into the pulmonary and perithymic lymph nodes (LNs) of mice with cOVA-induced airway delayed-type hypersensitivity reaction (DTHR) but not into pulmonary and perithymic LNs of naïve control mice or mice diseased from turkey or pheasant OVA-induced DTHR. Our protocol provides consequent advancements in the detection of small accumulations of immune cells in single LNs and specific homing to the sites of inflammation by PET using the internalization of TCR-specific mAbs as a specific label of T cells. Thus, our labeling approach is applicable to other cells with constant membrane receptor turnover.

Pharmacokinetics and PET imaging properties of two recombinant anti-PSMA antibody fragments in comparison to their parental antibody.

BACKGROUND: Radioimmunoimaging with disease-specific tracers can be advantageous compared to that with nonspecific tracers for the imaging of glucose metabolism and cell proliferation. Monoclonal antibodies (mAbs) or their fragments are excellent tools for immuno-positron emission tomography (PET). In this study, PSMA-specific mAb 3/F11 and its recombinant fragments were compared for the imaging of prostate cancer in xenografts. METHODS: Recombinant anti-PSMA antibody fragments D7-Fc and D7-CH3 were constructed by genetically fusing the binding domains of mAb 3/F11 (D7) to the human IgG3 CH3 or CH2-CH3 (Fc) domain. The fragments and the mAb 3/F11 were DOTA conjugated, tested in vitro, and radiolabeled with (64) Cu. PSMA-positive C4-2 and PSMA-negative DU 145 prostate cancer xenografts were used for PET-MR imaging and for ex vivo biodistribution. RESULTS: The constructs showed strong and specific binding to PSMA-positive C4-2 cells in vitro which did not decrease after DOTA conjugation. Both tested fragments showed stable accumulation in PSMA-positive C4-2 tumors at all measured time points but reduced uptake compared to the full-length antibody. Other organs and PSMA-negative tumors showed a very low tracer uptake only 3 hr after injection, with the exception of the kidneys, which demonstrated high radioactivity uptake due to rapid renal clearance of the mAb fragments. CONCLUSION: Stable tumor uptake and fast serum clearance of the tested radiolabeled fragments was observed in this preclinical study compared to the full length mAb. Since the fragments show rapid and specific tumor uptake, the tested fragments might serve as tools for theranostic imaging with suitable isotopes for radioimmunotherapy.

[68Ga]NODAGA-RGD for imaging αvß3 integrin expression.

PURPOSE: A molecular target involved in the angiogenic process is the α(v)ß(3) integrin. It has been demonstrated in preclinical as well as in clinical studies that radiolabelled RGD peptides and positron emission tomography (PET) allow noninvasive monitoring of α(v)ß(3) expression. Here we introduce a (68)Ga-labelled NOTA-conjugated RGD peptide ([(68)Ga]NODAGA-RGD) and compare its imaging properties with [(68)Ga]DOTA-RGD using small animal PET. METHODS: Synthesis of c(RGDfK(NODAGA)) was based on solid phase peptide synthesis protocols using the Fmoc strategy. The (68)Ga labelling protocol was optimized concerning temperature, peptide concentration and reaction time. For in vitro characterization, partition coefficient, protein binding properties, serum stability, α(v)ß(3) binding affinity and cell uptake were determined. To characterize the in vivo properties, biodistribution studies and microPET imaging were carried out. For both in vitro and in vivo evaluation, α(v)ß(3)-positive human melanoma M21 and α(v)ß(3)-negative M21-L cells were used. RESULTS: [(68)Ga]NODAGA-RGD can be produced within 5 min at room temperature with high radiochemical yield and purity (> 96%). In vitro evaluation showed high α(v)ß(3) binding affinity (IC(50) = 4.7 ± 1.6 nM) and receptor-specific uptake. The radiotracer was stable in phosphate-buffered saline, pH 7.4, FeCl(3) solution, and human serum. Protein-bound activity after 180 min incubation was found to be 12-fold lower than for [(68)Ga]DOTA-RGD. Biodistribution data 60 min post-injection confirmed receptor-specific tumour accumulation. The activity concentration of [(68)Ga]NODAGA-RGD was lower than [(68)Ga]DOTA-RGD in all organs and tissues investigated, leading to an improved tumour to blood ratio ([(68)Ga]NODAGA-RGD: 11, [(68)Ga]DOTA-RGD: 4). MicroPET imaging confirmed the improved imaging properties of [(68)Ga]NODAGA-RGD compared to [(68)Ga]DOTA-RGD. CONCLUSION: The introduced [(68)Ga]NODAGA-RGD combines easy accessibility with high stability and good imaging properties making it an interesting alternative to the (18)F-labelled RGD peptides currently used for imaging α(v)ß(3) expression.

The cobalt way to angucyclinones: asymmetric total synthesis of the antibiotics (+)-rubiginone B2, (-)-tetrangomycin, and (-)-8-O-methyltetrangomycin.

A cobalt(I)-mediated convergent and asymmetric total synthesis of angucyclinones with an aromatic B ring has been developed. In the course of our research, we synthesized three naturally occurring anguclinone derivatives, namely, (+)-rubiginone B(2) (1), (-)-8-O-methyltetrangomycin (2), and (-)-tetrangomycin (3). By combining 3-hydroxybenzoic acid, 3-methoxybenzoic acid, citronellal, and geraniol as starting materials in a convergent way, we were able to synthesize chiral triyne chains, which were cyclized with [CpCo(C(2)H(4))(2)] (Cp=cyclopentadienyl) by means of an intramolecular [2+2+2] cycloaddition to their corresponding tetrahydrobenzo[a]anthracenes. Successive oxidation and deprotection steps led to the above-mentioned natural products 1-3.

Reactions at the Ru-S bonds of coordinatively unsaturated ruthenium complexes with tethered 2,6-dimesitylphenyl thiolate.

Coordinatively unsaturated ruthenium complexes with a tethered SDmp (Dmp=2,6-dimesitylphenyl) ligand, [(DmpS)Ru(PR(3))][BAr(F) (4)] (3 a: R=Et; 3 b: R=Ph; Ar(F)=3,5-(CF(3))(2)C(6)H(3)), were synthesized by the reactions of [{(p-cymene)RuCl}(2)(mu-Cl)(2)], LiSDmp, phosphines, and NaBAr(F) (4). The Ru--S bonds in 3 a and 3 b were found to serve as the polarized reactive site in reactions with alkyl halides, diazoalkanes, (p-tosyliminoiodo)benzene, phenylacetylene, and H(2). Alkylation of 3 a and 3 b with methyl iodide or ethyl bromide occurred instantaneously to give the thioether complexes [(DmpSR')RuX(PR(3))][BAr(F) (4)] (4 a: R=Et, R'=Me, X=I; 4 b: R=R'=Et, X=Br; 4 c: R=Ph, R'=Me, X=I; 4 d: R=Ph, R'=Et, X=Br). Treatment of 3 a with diazoalkanes N(2)CHR (R=CO(2)Et, SiMe(3)) led to the cycloaddition of carbenes to the Ru--S bond to form [DmpS(CHR)Ru(PEt(3))][BAr(F) (4)] (5 a: R=CO(2)Et; 5 b: R=SiMe(3)), whereas the reaction with (p-tosyliminoiodo)benzene gave rise to [DmpS{NS(O)(C(6)H(4)-4-CH(3))O}Ru(PEt(3))][BAr(F) (4)] (6), which contains a five-membered ruthenacycle of RuSNSO. Addition of phenylacetylene to the Ru--S bond occurred reversibly to produce the vinyl sulfide complexes [DmpS(PhCCH)Ru(PR(3))][BAr(F) (4)] (7 a: R=Et; 7 b: R=Ph). On the other hand, the phenylacetylene at ruthenium slowly isomerized to vinylidene and bridged Ru and S in the products, [DmpS{C(CHPh)}Ru(PR(3))][BAr(F) (4)] (8 a: R=Et; 8 b: R=Ph). Complex 3 a catalyzed the hydrogenation of acetophenone, in which the heterolytic H-H splitting at the Ru-S site is suggested to be involved in the mechanism.

Total synthesis of (-)-8-O-methyltetrangomycin (MM 47755).

A stereoselective total synthesis of the natural antibiotic (-)-8-O-methyltetrangomycin 1 is reported. The essential steps for this convergent synthesis are the transformation of a geraniol epoxide into a chiral octadiyne derivative, which was converted into a triyne. The cobalt-mediated [2+2+2] cycloaddition of the triyne led to a benz[a]anthracene system, which was oxidized with Ag(Py)(2)MnO(4) to a benz[a]anthraquinone. Deprotection with aqueous HF in acetonitrile and photooxidation afforded the desired product (-)-1. [reaction: see text]