Comparison of efficacy and toxicity between botulinum toxin subtypes A1 and A2 in cynomolgus macaques.

Botulinum toxin type A (subtype A1) is used as therapeutic agent for some neurological disorders causing spasticity. The toxin products have an upper dosage limit, and their adverse events, such as side effects of diffusion following high-dose administration, have become serious issues. Therefore, a preparation with greater therapeutic efficacy at lower dosages and less diffusion in the body is desired. We have attempted to produce neurotoxin derived from subtype A2 (A2NTX), which has a different amino acid sequence from that of neurotoxin derived from subtype A1. In this study, to investigate whether A2NTX is applicable for treatment, we compared the muscle relaxation effects and the toxicity between A1LL and A2NTX in adult cynomolgus macaques. In the isometric muscle contraction test elicited by 30 Hz tetanus stimulation, the contractions observed in the 0.4 U/site A1LL-treated group were similar in value to those in the 0.13 U/site A2NTX-treated group. In the toxicity test, the 12 and 24 U/kg A1LL- and A2NTX-treated groups all exhibited similar signs of toxicity regarding symptoms, rate of weight loss, and decrease in the length of the right lower leg perimeter. Thus, A2NTX demonstrated approximately 3.0-times higher muscle relaxation activity than A1LL, and their toxicity was equivalent. This study suggested that A2NTX products are more suitable for the treatment of neurological disorders.

Generalization tests using different dosing routes from those of drug discrimination training in rats.

The purpose of this study was to investigate the discriminative stimulus properties of morphine and codeine using different administration routes to that used at drug discrimination training. Rats were trained to discriminate morphine at 3 mg/kg from saline by the intraperitoneal route in a standard two-lever drug discrimination paradigm. Generalization of morphine by the subcutaneous and the oral routes, and codeine by the intraperitoneal and the oral routes to the discriminative stimulus properties of the morphine training dose were investigated. Morphine at 3 mg/kg by the subcutaneous route generalized to the morphine training dose and 10 of 12 rats showed 80% or more morphine-lever responses. In the administration of morphine by the oral route, morphine at 30 mg/kg generalized to the morphine training dose and all rats showed 80% or more morphine-lever responses within the range of 3 to 30 mg/kg. In the administration of codeine by the intraperitoneal route, codeine at 20 mg/kg generalized to the morphine training dose and 14 of the 15 animals showed 80% or more morphine-lever responses within the range of 3 to 20 mg/kg. In the administration of codeine by the oral route, codeine at 60 mg/kg generalized to the morphine training dose and 14 of the 15 animals showed 80% or more morphine-lever responses within the range of 10 to 60 mg/kg. Thus, the discriminative stimulus properties of morphine and codeine were comparable when using different administration routes to those at discrimination training.

A new behavioral test for assessment of drug effects on attentional performance and its validity in cynomolgus monkeys.

The assessment of drug effects on attention is important in non-clinical pharmacology, for both evaluation of safety and therapeutic efficacy of medicinal products. In the present study, we have developed a two-lever choice behavioral test to assess drug effects on attentional performance in monkeys. In each trial of this experiment, one of two lamps in front of a monkey was randomly illuminated for a brief period of time and the monkey was required to press a lever beneath the lamp 30 times to obtain a food reward. The percentage of correct responses, response latency of correct choice responses and response speed were measured. Using this test, we examined the effects of three sedative drugs, diazepam (0.25, 1 and 4 mg/kg, i.g.), ethanol (0.5, 1 and 2 g/kg, i.g.), and pentobarbital (0.25, 1 and 4 mg/kg, i.v.). Diazepam and pentobarbital lengthened response latency without significantly affecting the percentage of correct responses, response and response speed, suggesting selective disruptive effects on attentional performance. In contrast, ethanol at the high dose tested caused deterioration in all three measurements, which is thought to reflect a general sedative effect including motor impairment as reflected by lengthening response speed. It is suggested that the present behavioral test method could detect drug effects on attentional performance in monkeys and could be a useful tool for safety assessment in drug development.

Caveolin-1 activates Rab5 and enhances endocytosis through direct interaction.

Caveolin-1, a constitutive protein of the caveolae, is implicated in processes of vesicular transport during caveolae-mediated endocytosis. However, the molecular mechanisms of caveolae-mediated endocytosis are not yet clearly defined. Here, we show the physiological role of the Rab5-caveolin-1 interaction during caveolae-mediated endocytosis. Rab5 was found in caveolae-enriched fractions and Rab5 directly bound to caveolin-1. Furthermore, binding sites of Rab5 to caveolin-1 were identified in the scaffold (SD), transmembrane (TM), and C-terminus (CC) domains, and the Rab5 binding domain of caveolin-1 was required for CTXB uptake. Subsequently, we performed a GST-R5BD pull-down assay to determine whether the Rab5 binding domain of caveolin-1 is involved in Rab5 activity or not. The results showed that overexpression of the Rab5 binding domain of caveolin-1 increase the amount of Rab5-GTP in Cos-1 cells. These findings imply that caveolin-1 controls the Rab5 activity during the caveolae-mediated endocytosis.

Oligosilane chain-length dependence of electron transfer of zinc porphyrin-oligosilane-fullerene molecules.

A new series of zinc porphyrin-fullerenes bridged by flexible oligosilane chains ZnP-[Sin]-C60 (n = 1-5) was synthesized, and the photophysical properties of these molecules were investigated using steady-state and time-resolved spectroscopic methods. The spectral observations can be well explained by assuming the coexistence of extended conformers and folded conformers, that is, the observed emissions are from the extended conformers while the folded conformers form very short lifetime nonfluorescent excited-state charge-transfer (CT) complexes. Time-resolved transient absorption spectra suggest the generation of intramolecular radical-ion pairs that have sub-microsecond lifetimes. With the number of silicon atoms of the bridged oligosilane, the lifetimes of the radical-ion pairs do not vary regularly, indicating that intramolecular collision of the radical-cation moiety with the radical-anion moiety controls the charge-recombination rate. The attenuation factor of the electron transfer of the silicon chain was evaluated by the bridge-length dependence of charge-separation rate to be 0.16 A-1 on the basis of the oligosilane chain-length dependence of fluorescence lifetimes. This is the first evaluation of the attenuation factor for the one-dimensional Si-Si chain to the best of our knowledge.

Effects of ifenprodil on the discriminative stimulus effects of cocaine in rhesus monkeys.

Ifenprodil is a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist which prefers NR2B-containing NMDA receptors to NR2A-containing NMDA receptors. It has been reported that ifenprodil suppresses morphine-induced place preference in mice. In this study, the effects of ifenprodil on the discriminative stimulus effects of cocaine were examined in rhesus monkeys. Five monkeys were trained to discriminate cocaine at 0.25 or 0.5 mg/kg im from saline using a standard two-lever drug-discrimination paradigm under a fixed-ratio schedule of food reinforcement. A single dose of cocaine (0.06-0.5 mg/kg) produced a dose-dependent increase in cocaine-appropriate response, and training doses produced 100% cocaine-lever response in each monkey. Pretreatment with ifenprodil (1 or 2 mg/kg, i.v.) blocked the cocaine-appropriate response when low doses of cocaine were used. The results suggest that NR2B-containing NMDA receptor-mediated mechanisms modulate the discriminative stimulus effects of cocaine in rhesus monkeys.

Strained enamines as versatile intermediates for stereocontrolled construction of nitrogen heterocycles.

This contribution assesses the synthetic utility of molecules that impose conformational constrains onto aziridine-derived enamines. Synthetically versatile [3.1.0] and [4.1.0] bicyclic enamines have been prepared by intramolecular oxidative cycloamination of aziridine-containing olefins. This process is initiated by N-bromosuccinimide followed by base-mediated elimination of HBr to afford highly strained exo-bicyclic enamines. In addition, intramolecular aziridine addition to aldehyde functionality was found to afford the [3.1.0] and [4.1.0] bicyclic hemiaminals. These routes highlight possibilities for chemoselective oxidative transformations of aziridine-containing precursors without nitrogen protection/deprotection steps. The resulting products provide straightforward synthetic entries into a wide range of pyrrolidine- and piperidine-containing heterocycles that are positioned toward subsequent transformations via aziridine ring opening.

Synthesis of the axially substituted titanium Pc-C60 dyad with a convenient method.

[structure: see text] We successfully synthesized the axially substituted titanium Pc-C(60) dyad with a convenient method that improves on the traditional asymmetrical phthalocyanine routine to covalently linked phthalocyanines with other functional molecules. The intramolecular photoinduced process between phthalocyanine donor and fullerene acceptor was preliminarily studied.

Oxidative cycloamination of olefins with aziridines as a versatile route to saturated nitrogen-containing heterocycles.

Highly reactive [5,3] and [6,3] bicyclic aziridines can be readily prepared from the corresponding NH aziridines and N-bromosuccinimide by intramolecular oxidative cycloamination of olefins. These compounds, including surprisingly stable exo-methylene bicyclic aziridines, provide versatile synthetic entries into a wide range of pyrrolidine- and piperidine-containing heterocycles.

N-arylation of aziridines.

A range of N-arylaziridines were prepared by the palladium or copper catalyzed amination reaction between N-H aziridines and aryl bromides or arylboronic acids. These results showcase the synthetic utility of metal-bound aziridine species in nitrogen transfer processes.