Effect of synthetic cathinones: mephedrone, butylone and 3,4 methylene-dioxypyrovalerone (MDPV) on social separation induced distress vocalization, vigilance and postural control of young domestic chicks.

Designer drugs have become a distinct social problem and health hazard in Europe and US, and their abuse has increased dramatically in the last decade. Selective effects of these agents on animal behavioral parameters may help in better understanding of the potential risks of human drug abuse. In the present study, the effects of three different abusive agents of the cathinone family, mephedrone, butylone and 3,4 methylene-dioxypyrovalerone (MDPV) were tested in young domestic chicks, following administration of single intraperitoneal injections (10mg/bwt). Early maturing (precocial) birds are particularly suited for investigation of isolation stress-related behavioral response and stereotypic or targeted pecking. Both mephedrone and MDPV increased the frequency of distress calls of socially isolated birds as measured over a period of 10min. While this effect of mephedrone was only evident in the first half of observation period, an increase with MDPV was more lasting. Though increased non-distress vocalization, butylone failed to enhance distress calls probably due to a general adverse effect on muscle tone. Apart from its effect on distress vocalization, mephedrone did not alter the behavior of chicks. However, both butylone and MDPV showed prominent behavioral changes, which were examined in another set of long term experiments, over a period of 120min. Butylone caused hyperventilation and a robust impairment of postural control, whereas neither the wakeful activity level, nor the pecking frequency was significantly affected. Conversely, no hyperventilation or postural disorder was observed with MDPV, however, both waking state and pecking were significantly enhanced. The results may be relevant to potentially different and specific effects of cathinone drugs under stress-related conditions, as well as on other physiological and behavioral parameters, even in case of closely related compounds.

Energy-1 ('NRG-1'): don't believe what the newspapers say about it being legal.

A 31-year-old man purchased the legal high Energy-1 (NRG-1) over the internet; this was advertised as containing the compound naphthylpyrovalerone (NPV), which at the time was currently legally available in the UK. He ingested 1 g of this substance and developed a prolonged high associated with palpitations, sweating and insomnia. Analysis of both the powder and serum samples from the patient demonstrated that he ingested two classified recreational drugs ß-keto-N-methylbenzodioxolylpropylamine (butylone) and methylenedioxypyrovalerone (MDPV) rather than the legal substance NPV. Users of legal highs need to be aware that legal highs purchased over the internet may contain illegal substances and therefore they may be liable for prosecution if found in possession of these substances. Future educational campaigns aimed at recreational drug and legal high users should include reference to the potential legal implications of buying these substances.

Immune-mediated inflammatory diseases (IMIDs) and biologic therapy: a medical revolution.

Targeted biologic therapies have revolutionised treatment of immune-mediated inflammatory diseases (IMIDs) due to their efficacy, speed of onset and tolerability. The discovery that clinically unrelated conditions, such as rheumatoid arthritis and Crohn's disease, share similar immune dysregulation has led to a shift in the management of IMIDs from one of organ-based symptom relief to mechanism-based treatment. The fact that anticytokine therapy has been effective in treating multiple orphan inflammatory conditions confirms the IMID paradigm. In this review we examine the biologic agents currently licensed for use in the US and Europe: infliximab, etanercept, adalimumab, rituximab, abatacept, anakinra, alefacept and efalizumab. We also discuss the rationale behind the management of IMIDs using rheumatoid arthritis, Crohn's disease, psoriasis and psoriatic arthritis as examples. For the medical profession, IMID represents a breakthrough in the way pathology is classified. In this burgeoning era of biologic therapy the prospect of complete disease remission is conceivable.

The role of N-hydroxyphenetidine in phenacetin-induced hemolytic anemia.

Phenacetin is well known to cause hemolytic anemia and methemoglobinemia in humans. Early mechanistic studies clearly established a causal role for active/reactive drug metabolites in the process but did not unequivocally identify these metabolite(s) or resolve the question of whether these two hemotoxicities are mechanistically linked. As part of ongoing studies on the mechanism underlying arylamine-induced hemotoxicities, we have recently shown that the arylhydroxylamine metabolites of aniline and dapsone mediate the hemolytic activity of aniline and dapsone, respectively. The present study was undertaken to determine if N-hydroxyphenetidine (PNOH), the known arylhydroxylamine metabolite of phenacetin, is responsible for phenacetin-induced hemolytic anemia. As measured by decreased survival of 51Cr-labeled erythrocytes in rats, phenacetin, p-phenetidine, and PNOH were all hemolytic in vivo, with PNOH being significantly the most potent of the three. In vitro exposure of 51Cr-tagged erythrocytes to PNOH, followed by transfusion into isologous rats, resulted in a concentration-dependent reduction in erythrocyte survival, indicating that PNOH is a direct-acting hemolytic agent. Phenacetin and p-phenetidine were inactive. Phenacetin, p-phenetidine, and PNOH all produced dose-dependent methemoglobinemia in rats. In parallel in vitro studies, PNOH elevated methemoglobin levels, p-phenetidine and phenacetin did not. However, attempts to identify PNOH in the blood of phenacetin- and p-phenetidine-treated rats were unsuccessful, despite the use of a highly sensitive analytical method. Hemotoxic concentrations of PNOH were found to be highly unstable in the presence of red cells, though relatively stable in the buffer vehicle alone. Inhibitors of acetylation (p-aminobenzoic acid [PABA]) and deacetylation (bis-[p-nitrophenyl]phosphate [BNPP]), used to alter the cyclic interconversion of phenacetin and p-phenetidine, caused changes in phenacetin hemotoxicity that indicated the hemotoxin was a deacetylated metabolite distal to p-phenetidine. These data are consistent with the hypothesis that PNOH, formed during the metabolic clearance of phenacetin, mediates phenacetin-induced hemolytic anemia and methemoglobinemia through direct toxic actions in the erythrocyte.

Carcinogenicity of bucetin in (C57BL/6 X C3H)F1 mice.

The carcinogenicity of bucetin [(3-hydroxy-p-butyrophenetidide) CAS: 1083-57-4], an antipyretic analgesic drug, was examined in 300 (C57BL/6 X C3H)F1 mice. Groups of 50 mice of each sex were treated with 1.5 or 0.75% bucetin in their basal diet for 76 weeks and then fed a basal diet for 8 weeks. Control groups were given a basal diet for 84 weeks. In 10 of 46 (22%) male mice given the high dose of bucetin and in 6 of 45 (13%) given the low dose, renal cell tumors were induced. Dysplastic lesions of the proximal tubules were frequently seen in the males given bucetin in a dose-related fashion. Neither tumorous nor preneoplastic lesions developed in the kidneys of bucetin-treated female mice and control animals. Papilloma of the urinary bladder in 1 male mouse and papillary or nodular hyperplasia in 9 mice of both sexes were observed in groups given the high dose of bucetin.

Mutagenicity of the phenacetin metabolites: N-hydroxy-p-phenetidine and nitrosophenetol in S. typhimurium TA100 and derivatives deficient in nitroreductase or O-acetylase: probes for testing intrabacterial metabolic activation.

Two mutagenic metabolites of phenacetin, p-nitrosophenetol and N-hydroxy-p-phenetidine, were tested in S. typhimurium strains TA100, its nitroreductase-deficient derivative TA100NR, and O-acetylase-deficient strains TA100 Tn5-1,8-DNP1011 and -DNP1012 in the presence or absence of an exogenous metabolic activation system. The results indicate that bacterial nitroreductase(s) and O-acetylase(s), shown to be involved in the conversion of certain nitroarenes, are not required for the intrabacterial activation of the two phenacetin metabolites to bacterial mutagens. In view of the low reactivity of nitrosoarenes towards nucleophiles at neutrality, the mechanism by which they exert such a high mutagenic effect in S. typhimurium strains remains to be clarified, but is discussed.

Reaction of mutagenic phenacetin metabolites with glutathione and DNA. Possible implications for toxicity.

The direct-acting mutagens, N-hydroxy-p-phenetidine and p-nitrosophenetole, are known to be metabolites of the analgesic phenacetin and may be responsible for its carcinogenic activity. In this study, the potential detoxification of these metabolites by glutathione was examined. Glutathione reacted rapidly with p-nitrosophenetole, which was quantitatively converted to a single product as determined by high-pressure liquid chromatography. The analysis of the product by fast atom bombardment mass spectrometry and 500-MHz 1H-NMR spectroscopy established its structure as N-(glutathion-S-yl)-p-phenetidine. The same glutathione conjugate was also formed when N-hydroxy-p-phenetidine was incubated with glutathione. However, since conjugate formation from N-hydroxy-p-phenetidine occurred slowly and was decreased in the presence of an argon atmosphere as well as by higher levels of glutathione, it was concluded that the conjugate resulted from oxidation of the N-hydroxy arylamine to the nitrosoarene, which subsequently reacted with glutathione. N-(Glutathion-S-yl)-p-phenetidine was semistable in water (half-life, 6-7 hr) and very unstable in the presence of nucleophiles such as 10 mM glutathione (half-life, 7 min), quantitatively decomposing to p-phenetidine. The conjugate was also very unstable in acidic buffers (half-life, 17 min, pH 5). Radiolabeled N-hydroxy-p-phenetidine, but not p-nitrosophenetole, was shown to bind covalently to calf thymus DNA in vitro, and 4 times more binding was detected at pH 5 than at pH 7. Glutathione did not significantly decrease binding of the N-hydroxy derivative at either pH, nor did purified ring-radiolabeled N-(glutathion-S-yl)-p-phenetidine significantly bind to DNA at either pH. Thus, we hypothesize that an important detoxification pathway for phenacetin in vivo could involve the facile oxidation of N-hydroxy-p-phenetidine to p-nitrosophenetole, which then reacts rapidly with glutathione to form an excretable conjugate.

The synthesis and mutagenicity of the N-formyl analog of N-hydroxyphenacetin.

The synthesis and purification of N-hydroxy-N-formyl-p-phenetidine (N-OH-FP) is described. This new compound was subjected to mutagenicity testing using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 both in the presence and absence of the post-mitochondrial fraction of rat liver homogenate. Simultaneous mutagenicity testing of the known phenacetin metabolite, N-hydroxyphenacetin (N-OH-AP), was conducted with the same tester strains. The N-formyl derived hydroxamic acid (N-OH-FP) was found to be a much stronger mutagen than N-hydroxy-phenacetin (N-OH-AP). Furthermore, N-OH-FP also behaved as a direct-acting mutagen unlike N-OH-AP. The chemical stabilities of N-OH-AP and N-OH-FP were studied in phosphate buffer in the pH range of 3-8; and both the hydroxamic acids were found to be stable to the conditions employed. The results of this study support the hypothesis that enzymatic deacylation is an activation process for the expression of mutagenicity by hydroxamic acids.

Immunogenicity of p-phenetidine, 2-hydroxy-p-phenetidine and their protein conjugates in guinea pigs, rabbits and man.

Guinea pigs were sensitized by p-phenetidine (PT), 2-hydroxy-p-phenetidine (HPT) as well as by conjugates prepared by reacting PT and HPT with proteins in vitro. Sensitization was evaluated by delayed skin reactivity and in vitro antigen-induced lymphocyte proleferation. HPT and HPT-protein conjugates were found to be the most effective sensitizing agents. Reaginic antibodies could be raised in both guinea pigs and rabbits by immunizing with PT- and HPT-protein conjugates but not by PT and HPT alone: these PCA antibodies showed strong cross-reactivity and could be elicited equally well with either the PT- or HPT-protein derivatives. By contrast, no precipitating antibodies could be raised in either species even after repeated immunizations over a period of 4 months. Peripheral blood lymphocytes, from a few patients who gave a positive patch test with PT, could be stiumlated in vitro with phenacetin and to a lesser degree with PT and with a HPT-derivative of human serum albumin.