Long-acting cocaine hydrolase for addiction therapy.

Cocaine abuse is a world-wide public health and social problem without a US Food and Drug Administration-approved medication. An ideal anticocaine medication would accelerate cocaine metabolism, producing biologically inactive metabolites by administration of an efficient cocaine-specific exogenous enzyme. Our recent studies have led to the discovery of the desirable, highly efficient cocaine hydrolases (CocHs) that can efficiently detoxify and inactivate cocaine without affecting normal functions of the CNS. Preclinical and clinical data have demonstrated that these CocHs are safe for use in humans and are effective for accelerating cocaine metabolism. However, the actual therapeutic use of a CocH in cocaine addiction treatment is limited by its short biological half-life (e.g., 8 h or shorter in rats). Here we demonstrate a novel CocH form, a catalytic antibody analog, which is a fragment crystallizable (Fc)-fused CocH dimer (CocH-Fc) constructed by using CocH to replace the Fab region of human IgG1. The CocH-Fc not only has a high catalytic efficiency against cocaine but also, like an antibody, has a considerably longer biological half-life (e.g., ∼107 h in rats). A single dose of CocH-Fc was able to accelerate cocaine metabolism in rats even after 20 d and thus block cocaine-induced hyperactivity and toxicity for a long period. Given the general observation that the biological half-life of a protein drug is significantly longer in humans than in rodents, the CocH-Fc reported in this study could allow dosing once every 2-4 wk, or longer, for treatment of cocaine addiction in humans.

Safety evaluation of a lipase enzyme (BD29241 Palmitase) preparation, expressed in Pseudomonas fluorescens, intended for removing palmitic acid from triacylglycerol.

The lipase enzyme, BD29241 Palmitase, can be used as a processing aid for removing palmitic acid from triacylglycerol in the production of refined oil. This enzyme was produced from a Pseudomonas fluorescens (P. fluorescens) production strain and was tested in acute, inhalation, and subchronic toxicity studies. In addition, this enzyme was also tested for its potential to induce genotoxicity. Dosages of the test article preparation ranged from 5000µg/plate for in vitro toxicity studies to 2000mg/kg/day for in vivo toxicity studies. The highest oral dose tested in vivo (NOAEL of 2000mg/kg/day) resulted in a safety margin of 2.442×10(3) based on a conservative estimate of the total human consumption of BD29241 Palmitase of 0.819mg/kg/day. There was no toxicity reported for any of these studies including additional safety studies. A review of the literature indicates that P. fluorescens fulfills recognized safety criteria pertinent to microbial production strains used in the manufacture of food enzyme preparations. The results of the toxicity studies presented herein attest to the safety of BD29241 Palmitase for its above-stated intended use.

The role of Janus kinase 2 (JAK2) activation in pneumococcal EstA protein-induced inflammatory response in RAW 264.7 macrophages.

In a previous study, we demonstrated pneumococcal EstA-induced inflammatory response through NF-κB and MAPK-dependent pathways. Herein, we tested the hypothesis that the Janus kinase 2 (JAK2) activation and associated signaling cascades may also be involved in EstA-induced inflammatory process in RAW 264.7 macrophages. Our immunoblot analysis indicated EstA-induced activation of JAK2, with the phosphorylated protein detected from 1 to 24 h post-stimulation. As type I interferon (IFN) signaling requires the JAK/STAT pathway, we investigated EstA-induced expression of INF-α4 and INF-ß by semi-quantitative and quantitative RT PCR. Our results indicated both concentration- and time-dependent increases in both IFN-α4 and IFN-ß mRNA expression after EstA challenge, with the highest fold-increases observed at 4 h and 6 h post-stimulation for IFN-α4 and IFN-ß mRNA, respectively. Furthermore, we applied a pharmacological approach to demonstrate the effect of JAK2 inhibition on EstA-induced nitric oxide (NO) and pro-inflammatory cytokine production. The JAK2 inhibitor AG-490 reduced significantly (P < 0.05) EstA-induced NO production and the expression of iNOS mRNA in a concentration-dependent manner. Similarly, EstA-induced IL-1ß and IL-6 production and their respective mRNA expression were markedly suppressed by AG-490. However, AG-490 had no inhibitory effect on both mRNA and protein levels of TNF-α. Taken together, we demonstrate that JAK2 activation and IFN I signaling are integral parts of EstA-induced inflammatory process. Further studies will elucidate the interaction of the different signaling pathways, the specific downstream targets of JAK2, the kinetics of cytokine release, and if EstA could induce the pro-inflammatory mediators to the same extent in alveolar macrophages.

Paralytic toxins in Taiwanese starfish Astropecten scoparius.

Paralytic toxicity was seasonally detected in every 10 specimens of the starfish Astropecten scoparius from Pingtung in Taiwan from December 1995 to November 1996. The highest toxicity and the average toxicity of total specimens, expressed as tetrodotoxin (TTX), were 5,938 and 1,679 mouse unit (MU), respectively. The highest value of monthly average toxicity in viscera and other parts were 354 and 247 MU/g, respectively. The toxin was partially purified by YM-2 membrane ultrafiltration and Bio-Gel P-2 column chromatography. HPLC and GC-MS analyses showed that the starfish toxin was mainly composed of tetrodotoxin (88%), along with minor gonyautoxin 2-3 and saxitoxin (12%).

Delayed neurotoxicity caused by a single massive dose of dichlorvos to adult hens.

A moderate neuropathic response was obtained in hens 2 weeks after being given a single massive s.c. dose of dichlorvos (100 mg/kg of active ingredient in a commercial 50% formulation). 1 day after dosing inhibition of neurotoxic esterase in peripheral nerve was 79-90%, in spinal cord 70-81% and in brain 89-92%. It is unlikely that repeated exposure of man to dichlorvos could cause neuropathy except at doses causing severe cholinergic effects at every exposure.

Safety evaluation of tannase enzyme preparation derived from Aspergillus oryzae.

Tannase is an acylhydrolase enzyme preparation from Aspergillus oryzae that can be used as a processing aid for the manufacture of cold water-soluble tea beverages. A 91-day oral toxicity study in the rat and a gene mutation study in Salmonella typhimurium were performed to establish the safety of the enzyme preparation for the consumer. General toxicity was low, with no adverse effects observed at the highest dose tested, 1% in the diet. There was no evidence of mutagenic potential with or without metabolic activation. These results, together with knowledge of the production organism and the chemical and microbiological characterization of the enzyme preparation, indicate that tannase can be regarded as safe for its intended use in processing tea.

Quantitative structure-activity relationships predict the delayed neurotoxicity potential of a series of O-alkyl-O-methylchloroformimino phenylphosphonates.

Inhibition of acetylcholinesterase (AChE) versus inhibition and aging of neuropathy target esterase (NTE) by organophosphorus (OP) compounds in vivo can give rise to distinct neurological consequences: acute cholinergic toxicity versus OP compound-induced delayed neurotoxicity (OPIDN). Previous work has shown that the relative potency of an OP compound to react with NTE versus AChE in vitro may predict its capability to produce OPIDN. The present study was conducted to evaluate further the validity of such predictions and to enhance them with quantitative structure-activity relationships (QSAR) using a homologous series of alkyl phenylphosphonates (RO)C6H5P(O)ON = CCICH3 (PhP; R = alkyl). Neuropathic potential of PhP was assessed by measuring ki(NTE)ki(AChE) ratios in vitro and comparing these with ED50 ratios in vivo. Selectivity for NTE increased with rising R-group hydrophobicity. The ki(NTE)/ki(AChE) ratios were 0.42 (methyl), 3.6 (ethyl), 15 (isopropyl), 36 (propyl), 69 (isobutyl), 105 (butyl), and 124 (pentyl). Ratios > 1 suggest the potential to produce OPIDN at doses lower than the LD50. Inhibition of NTE and AChE in hen brain in vivo was studied 24 h after i.m. injection of hens with increasing doses of methyl and butyl derivatives. Analysis of dose-response curves yielded ED50(AChE)/ED50(NTE) ratio of 0.86 for methyl PhP and 22.1 for butyl PhP. These results predict that the butyl derivative should be more neuropathic than the methyl analogue. Excellent correspondence between in vivo and in vitro predictions of neuropathic potential indicate that valid predictive QSAR models may be based on the in vitro approach. Adoption of this system would result in reducing experimental animal use, lowering costs, accelerating data production, and enabling standardization of a biochemically based risk assessment of the neuropathic potential of OP compounds.

[Studies on hemorrhagic toxins from the venoms of Trimeresurus mucrosquamatus, Crotalus ruber ruber, Vipera aspis aspis and Agkistrodon acutus and arginine ester hydrolases from Trimeresurus mucrosquamatus venom].

Venom samples were corrected from several poisonous snakes, such as Bungarus multicinctus, Trimeresurus mucrosquamatus, T. gramineus, T. flavoviridis, and Agkistrodon acutus, and stored in a desiccator at room temperature for 25 to 31 years. Then they were compared with fresh venoms as to their biological activities. The characteristic local symptoms produced by the bite of venomous snakes of Crotalidae and Viperidae are hemorrhage, necrosis and muscular degeneration. Hemorrhagic toxins were purified from Trimeresurus mucrosquamatus, Crotalus ruber ruber, Vipera aspis aspis, and Agkistrodon acutus venoms and their biological, biochemical, and pathological properties were investigated. Arginine ester hydrolases are present in the venoms of Crotalidae and Viperidae, but are not found in the venoms of Elapidae and Hydrophiidae. In this paper we describe the enzymatic and biological activities of arginine ester hydrolases from a Trimeresurus mucrosquamatus venom.

[Long-term toxicity test of arginine esterase from Agkistrodon halys ussuriensis venom].

To study the long-term toxicity of arginine esterase from Agkistrodon halys ussuriensis venom for the clinical application of arginine esterase in the future. Beagle dogs were used as experimental animals and were divided into control group, arginine esterase low dose group (0.06 u/kg), the middle dose group (0.18 u/kg) and high dose group (0.36 u/kg). Every group consisted of four dogs. The arginine esterase was given intravenously once a day for 180 days. Then three dogs in each group were sacrificed and the fourth one was fed without injecting arginine esterase for 15 days. The toxic reactions during treatment and recovery period were determined by evaluating and comparing the general criteria ( including locomotor activity, growth rate, appetite and death rate), clinical criteria (including blood test and urine test), pathological dissection and viscera coefficient of the treated animals and the control animals. There were no significant differences in general criteria. The clinical criteria of the treated animals were the same as those of the control animals except liver function. There were no significant differences in pathological dissection and viscera coefficient between the treated animals and the control animals except livers. The livers in high dose arginine esterase treated animals were swollen and vacuolated and there was significant difference in liver coefficient between them (P<0.05). The toxic symptom of liver disappeared after withdrawal of treatment. From these results, the non-toxic dose of arginine esterase for dogs was estimated to be 0.18 u/kg under the present study conditions and is about 15 times the clinical dosage for using the drug "Qingshuanmei" of which the main component is arginine esterase. The long-term toxicity test result indicates that the toxicity of pure arginine esterase is lower than that of "Qingshuanmei", suggesting that clinical use of the arginine esterase is safe.

Safety evaluation of a fungal pectinesterase enzyme preparation and its use in food.

The Aspergillus aculeatus pectinesterase enzyme is used to modify the texture of plant derived products. It is produced by A. oryzae transformed with the cloned full length cDNA of A. aculeatus encoding pectinesterase. It was subjected to a series of toxicological tests to document safety in use. The enzyme preparation was not found to be mutagenic in the Ames test, and did not cause chromosomal damage in a human lymphocyte assay. In a 13-week oral-toxicity study in rats, with daily dosages up to 10 g enzyme preparation kg body weight (b.w.), there were no adverse effects on mortality, clinical signs, body weight, food or water consumption, ophthalmoscopic findings, haematology or clinical chemistry. There were also no notable necropsy or histological findings. Statistically significant increases in heart weight were noted in male animals treated with 5 or 10 g enzyme preparation/kg b.w./day, following covariance analysis. However, this was not considered to be related to treatment with the enzyme preparation. The issue of the levels of free liberation of methanol in products processed with pectinesterase is addressed, and it is concluded that, from a nutritional and physiological point of view, free as well as bound methanol must be considered.

Neuromuscular effects of chronic exposure to fenthion in dogs and predictive value of electromyography.

Chronic exposure to organophosphates (OP) can result in nonspecific neurologic signs in both man and animals. Improved methods are needed to predict toxicity and to better characterize neuromuscular effects. In this study, dogs were exposed to an OP (fenthion) by weekly dermal application of a 20% solution at a dosage of 44 mg/kg. This dosage does not produce signs of acute OP toxicity in dogs, although plasma cholinesterase (ChE) levels are significantly decreased. Electromyograms (EMG) were used to monitor motor unit potential (MUP) activity at minimal and submaximal contractile effort in four different muscles. At 1-month intervals, muscle biopsies were obtained and plasma ChE levels were determined. At 3 months, hyperreflexia and/or mild proprioceptive deficits were observed. The dosage was reduced to 22 mg/kg for the remaining 3 months of the study. At the end of this 6-month study, nerve and muscle biopsies were obtained. Mean plasma ChE levels were decreased (preexposure value of 1775 IU/liter to low of 310 IU/liter) and correlated with duration of exposure and change in dosage level. Fourier analysis of EMG indicated some increase in higher frequency components of the power spectrum with time, and analysis of individual MUPs revealed a significant (p less than 0.05) increase in the product of the amplitude times the duration of the potentials in all muscles examined. Biopsy results were supportive of EMG findings of altered neuromuscular function and loss of small motor units. The EMG changes were most consistent in the gastrocnemius muscle and were detected prior to development of clinical signs. These results indicate that EMG can be useful in monitoring OP exposure and predicting toxicity.