PET imaging of the dopamine transporter with 18F-FECNT: a polar radiometabolite confounds brain radioligand measurements.

18F-2beta-Carbomethoxy-3beta-(4-chlorophenyl)-8-(2-fluoroethyl)nortropane (18F-FECNT), a PET radioligand for the dopamine transporter (DAT), generates a radiometabolite that enters the rat brain. The aims of this study were to characterize this radiometabolite and to determine whether a similar phenomenon occurs in human and nonhuman primate brains by examining the stability of the apparent distribution volume in DAT-rich (striatum) and DAT-poor (cerebellum) regions of the brain. METHODS: Two rats were infused with 18F-FECNT and sacrificed at 60 min. Extracts of brain and plasma were analyzed by high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometric (LC-MS) techniques. Two human participants and 3 rhesus monkeys were injected with 18F-FECNT and scanned kinetically, with serial arterial blood analysis. RESULTS: At 60 min after the injection of rats, 18F-FECNT accumulated to levels about 7 times higher in the striatum than in the cortex and cerebellum. The radiometabolite was distributed at equal concentrations in all brain regions. The LC-MS techniques identified N-dealkylated FECNT as a major metabolite in the rat brain, and reverse-phase HPLC detected an equivalent amount of radiometabolite eluting with the void volume. The radiometabolite likely was 18F-fluoroacetaldehyde, the product expected from the N-dealkylation of 18F-FECNT, or its oxidation product, 18F-fluoroacetic acid. The distribution volume in the cerebellum increased up to 1.7-fold in humans between 60 and 300 min after injection and 2.0 +/- 0.1-fold (mean +/- SD; n = 3) in nonhuman primates between 60 and 240 min after injection. CONCLUSION: An 18F-fluoroalkyl metabolite of 18F-FECNT originating in the periphery confounded the measurements of DAT in the rat brain with a reference tissue model. Its uniform distribution across brain regions suggests that it has negligible affinity for DAT (i.e., it is an inactive radiometabolite). Consistent with the rodent data, the apparent distribution volume in the cerebellum of both humans and nonhuman primates showed a continual increase at late times after injection, a result that may be attributed to entry of the radiometabolite into the brain. Thus, reference tissue modeling of 18F-FECNT will be prone to more errors than analysis with a measured arterial input function.     

In-Vitro Neutralization of the Neurotoxicity of Coastal Taipan Venom by Australian Polyvalent Antivenom: The Window of Opportunity

Coastal taipan (Oxyuranus scutellatus) envenoming causes life-threatening neuromuscular paralysis in humans. We studied the time period during which antivenom remains effective in preventing and arresting in vitro neuromuscular block caused by taipan venom and taipoxin. Venom showed predominant pre-synaptic neurotoxicity at 3 µg/mL and post-synaptic neurotoxicity at 10 µg/mL. Pre-synaptic neurotoxicity was prevented by addition of Australian polyvalent antivenom before the venom and taipoxin and, reversed when antivenom was added 5 min after venom and taipoxin. Antivenom only partially reversed the neurotoxicity when added 15 min after venom and had no significant effect when added 30 min after venom. In contrast, post-synaptic activity was fully reversed when antivenom was added 30 min after venom. The effect of antivenom on pre-synaptic neuromuscular block was reproduced by washing the bath at similar time intervals for 3 µg/mL, but not for 10 µg/mL. We found an approximate 10–15 min time window in which antivenom can prevent pre-synaptic neuromuscular block. This time window is likely to be longer in envenomed patients due to the delay in venom absorption. Similar effectiveness of antivenom and washing with 3 µg/mL venom suggests that antivenom most likely acts by neutralizing pre-synaptic toxins before they interfere with neurotransmission inside the motor nerve terminals.     

In vitro and in silico biological studies of novel thiazolo[3,2-a]pyrimidine-6-carboxylate derivatives

In the present study, we report the synthesis, characterization of new series of thiazolo[3,2-a]pyrimidine-6-carboxylate derivatives 3a–f and 4a–f. The newly synthesized compounds were screened for in vitro antimicrobial and antiviral activities. The probable mode of action of these active compounds was determined through in silico docking study by docking the receptor methionyl-tRNA synthetase and human inosine-5′-monophosphate dehydrogenase (IMPDH) for antibacterial and antiviral activities, respectively. Among the compounds, 4c exhibited excellent in vitro antimicrobial activity against all tested strains with binding and docking energies ?35.6 and ?12.4 kcal/mol, respectively. The antiviral studies were carried out for the selected compounds in which 4a exhibited 73.69 and 54.42 % of inhibition of buffalopox and camelpox viruses, respectively. Furthermore, compound 4a showed minimum docking and binding energy along with the maximum hydrogen/hydrophobic interaction with IMPDH. The study contributes towards identification and screening of potential antimicrobial and antiviral agent’s against the pathogens.     

Synthesis and antimicrobial study of novel heterocyclic compounds from hydroxybenzophenones

The triazolothiadiazine analogues 6a-e were obtained via a multistep synthesis sequences beginning with the hydroxybenzophenones 1a-e. Hydroxybenzophenones on reaction with ethyl chloroacetate affords ethyl (2-aroylaryloxy)acetates 2a-e which on treatment with hydrazine hydrate yields 2-(2-aroylaryloxy)acetohydrazides 3a-e. Intramolecular cyclization of 3a-e with carbon disulfide affords 5-(2-aroylaryloxy)methyl-1,3,4-oxadiazole-2-(3H)thiones 4a-e, which on treatment with hydrazine hydrate yields 4-amino-5-(2-aroyl aryloxy)methyl-1,2,4-triazole-3-(2H)thiones 5a-e. Condensation of 5a-e with alpha-halocarbonyl compound results in 3-(2-aroylaryloxy)methyl-6-phenyl-1,2,4-triazolo[3,4-b][1,3,4] thiadiazine 6a-e analogues. The compounds 4a-e, 5a-e and 6a-e were tested against variety of fungal and bacterial strains in comparison to fluconazole and chloramphenicol, respectively.     

Brain uptake and anticancer activities of vincristine and vinblastine are restricted by their low cerebrovascular permeability and binding to plasma constituents in rat

Summary Unidirectional blood-brain barrier transfer of the lipophilic anticancer agents vincristine and vinblastine was studied in anesthetized rats, using an isolated, in situ brain perfusion technique. Drug binding to plasma constituents was also measured. Despite the high lipophilicity of these agents (the log octanol/physiological saline partition coefficient equalled 2.14 and 1.68, respectively), the cerebrovascular permeability-surface area product, PA, of vincristine in plasma was only 0.49 × 10⁻⁴ ml s⁻¹ g⁻¹ for parietal cerebral cortex, whereas that of vinblastine was too low for determination. These values are similar to those of water-soluble, poorly diffusible nonelectrolytes. The PAs were significantly higher in the absence of plasma protein, being 1.24 × 10⁻⁴ and 5.36 × 10⁻⁴ ml s⁻¹ g⁻¹, respectively. Even these values, determined by brain perfusion of protein-free buffer, were lower than would be expected from the lipophilicity of the agents. The results suggest that additional factors, such as steric hindrance and molecular charge distribution, related to the chemical and geometric structure and the large size of vincristine and vinblastine (molecular weight, 825 and 814 daltons, respectively) restrict their passage across the blood-brain barrier. As a consequence of their paradoxically low permeability at the blood-brain barrier and restrictive binding to plasma and blood constituents, doses of both agents that cause significant inhibition of extracerebral Walker 256 carcinosarcoma tumor implants in rat have no effect on tumor located in the brain.     

Phospholipid composition and levels are altered in down syndrome brain

Phospholipid composition (mol %) and levels (nmol/mg protein) were determined in postmortem frontal cortical and cerebellar gray matter from older Down Syndrome (DS) patients (age range 38–68 years) and from control subjects. Neither DS nor control tissue exhibited any age-dependent alteration in phospholipid composition or levels. Total phospholipid content was significantly reduced approximately 20% in DS frontal cortex and cerebellum relative to these regions in control tissue. Individual phospholipid levels were also reduced in DS frontal cortex and cerebellum, including a specific 37% decrease in phosphatidylinositol (PtdIns) and a nearly 35% decrease in ethanolamine plasmalogen. Because of the large decrease in phospholipid content in DS brain, the cholesterol/phospholipid ratio was calculated for each group. There was no significant difference in this ratio between groups, indicative of compensatory changes to keep the cholesterol/phospholipid ratio constant. Despite the large changes in DS brain phospholipid levels, significant changes in composition were limited to a 18% decrease in PtdIns mol % and a 22% increase in the mol % of sphingomyelin. These results suggest either a decrease in membrane phospholipids due to a loss of dendrites and dendritic spines, or a general defect in brain lipid metabolism in older DS subjects. The proportionally greater alterations in PtdIns and PlsEtn levels, indicate that the metabolism of these two phospholipids was affected to a greater extent than the other phospholipids. Further, because these changes are found in both the frontal cortical and cerebellar gray matter, they likely are related to the Down syndrome condition rather than to Alzheimer neuropathology.     

Therapeutic Applications of Mesenchymal Stem Cells

The past decade has seen tremendous growth in the clinical application of cell-based therapies, and the number of planned human clinical trials to evaluate these therapies continues to increase in number and scope at a rapid pace. A considerable effort on this front has been devoted to evaluating the therapeutic potential of mesenchymal stem cells (MSCs), which were initially characterized as connective tissue progenitors resident in bone marrow. MSCs are now known to possess potent tissue reparative properties that have been linked to secretion of paracrine-acting angiogenic, trophic, anti-inflammatory, and immunomodulatory factors. Accordingly, MSC-based therapies are being evaluated for the treatment of a broad array of ischemic, inflammatory, and immunological disorders. Nevertheless, knowledge regarding how the wide-ranging activities of MSCs vary between and are specified within populations remains largely unexplored. Lack of such knowledge makes it difficult to predict and/or control how sampling bias and ex vivo expansion of populations alters their biological activity and therapeutic potency. Herein, we discuss how heterogeneity of MSC populations may explain, in part, disparate outcomes in both experimental animal and human clinical trial data, and discuss several strategies to achieve more reproducible and efficacious outcomes for MSC-based therapies.     

Investigation of the Metabolites of (S,S)-[11C]MeNER in Humans, Monkeys and Rats

Introduction  (S,S)-[¹¹C]MeNER ((S,S)-2-(α-(2-[¹¹C]methoxyphenoxy)benzyl)morpholine) is a positron emission tomography (PET) radioligand recently applied in clinical studies of norepinephrine transporters (NETs) in the human brain in vivo. In view of further assessment of the suitability of (S,S)-[¹¹C]MeNER as a NET radioligand, its metabolism and the identity of the in vivo radiometabolites of (S,S)-[¹¹C]MeNER are of great interest. Materials and Methods  Thus, PET studies were used to measure brain dynamics of (S,S)-[¹¹C]MeNER, and plasma reverse-phase radiochromatographic analysis was performed to monitor and quantify its rate of metabolism. Eighteen healthy human volunteers, five cynomolgus monkeys, and five rats were studied. Results and Discussion  In human subjects, the plasma radioactivity representing (S,S)-[¹¹C]MeNER decreased from 88 ± 5% at 4 min after injection to 82 ± 7% at 40 min, while a polar radiometabolite increased from 3 ± 3% to 16 ± 7% at the same time-points, respectively. A more lipophilic radiometabolite than (S,S)-[¹¹C]MeNER decreased from 9 ± 5% at 4 min to 1 ± 2% at 40 min. In monkeys, plasma radioactivity representing (S,S)-[¹¹C]MeNER decreased from 97 ± 2% at 4 min to 74 ± 7% at 45 min, with a polar fraction as the major radiometabolite. A more lipophilic radiometabolite than (S,S)-[¹¹C]MeNER, constituted 3 ± 2% of radioactivity at 4 min and was not detectable later on. In rats, 17 ± 4% of plasma radioactivity was parent radioligand at 30 min with the remainder comprising mainly a polar radiometabolite. (S,S)-[¹¹C]MeNER in rat brain and urine at 30 min after injection were 90% and 4%, respectively. On a brain regional level, parent radioligand ranged from 87.5 ± 3.9% (57.2 ± 14.2% SUV [standard uptake values, %injected radioactivity per mL multiplied with animal weight (in g)]; cerebellum) to 92.9 ± 1.8% (36.1 ± 4.7% SUV; striatum), with differential distribution of the radiometabolite in the cerebellum (6.7 ± 0.3% SUV) and the striatum (2.5 ± 0.3% SUV). Liquid chromatography-mass spectrometry analysis of rat urine identified a hydroxylation product of the methoxyphenoxy ring of (S,S)-MeNER as the main metabolite. In the brain, the corresponding main metabolite was the product from O-de-methylation of (S,S)-MeNER. PET measurements were performed in rats as well as in wild-type and P-gp-knock-out mice. In rats, the brain peak level of radioactivity was found to be very low (65%SUV). In mice, there was only a small difference in peak brain accumulation between P-gp knock-out and wild-type mice (145 vs. 125%SUV) with the following rank order of regional brain radioactivity: cerebellum × thalamus > cortical regions > striatum. Conclusion  It can be concluded that radiometabolites of (S,S)-[¹¹C]MeNER are of minor importance in rat and monkey brain imaging. The presence of a transient lipophilic radiometabolite in peripheral human plasma may induce complications with brain imaging, but its kinetics appear favorable in relation to the slow kinetics of (S,S)-[¹¹C]MeNER in humans.     

Development and validation of H11B2C2 monoclonal antibody-reactive hyaluronic acid binding protein: overexpression of HABP during human tumor progression

Informative biomarkers of tumor progression have been elusive. The interaction between hyaluronic acid (HA) and its binding proteins (HABP) plays a pivotal role during malignancy. In the present study, we have developed a monoclonal antibody (mAb, termed as H11B2C2 mAb) and showed that this mAb specifically reacts with overexpressed HABP from a wide variety of malignant tumors as compared with benign tumors. In Western blot analysis, H11B2C2 mAb detected a major 80-kDa protein from human cancer cell lines, and the overexpression of 55–57- and 30-kDa proteins in malignant tumors compared with benign tumors. Furthermore, immunohistochemical analysis of different types of benign and malignant tumors with different grades showed higher expression of HABP in all the malignant tumors when compared with the benign tumors. HABP overexpression was specific to tumor cells when compared with the surrounding stroma and localized on the cell surface as well as in the intracellular region. The competitive inhibition experiments using HA polymer and its oligosaccharides in the Western blot and immunohistopathology experiments suggested that the H11B2C2 mAb-reactive protein is HABP. Altogether, the present study showed overexpression of the H11B2C2 mAb-reactive HABP in various malignant tumors as compared with benign tumors. Thus, H11B2C2 mAb-reactive HABP can be used as a potential biomarker during tumor progression.