Abuse potential and dopaminergic effect of alkyl nitrites.

The abuse of alkyl nitrites is common among adolescents and young adults worldwide. However, the information regarding the effects of alkyl nitrites on the central nervous system and the associated psychological abuse potential is scarce. The abuse potential of 3 representative alkyl nitrites - isobutyl nitrite, isoamyl nitrite, and butyl nitrite - was evaluated in mice using conditioned place preference tests with an unbiased method. The dopamine levels released by synaptosomes extracted from the striatal region were measured using high performance liquid chromatography. Mice treated with the test substances (50mg/kg, i.p.) exhibited a significantly increased drug-paired place preference. Moreover, greater levels of dopamine were released by striatal region synaptosomes in response to isobutyl nitrite treatment in mice. Thus, our findings suggest that alkyl nitrites could lead to psychological dependence and dopaminergic effects. Furthermore, these results provide scientific evidence to support the regulation of alkyl nitrites as psychoactive substances in the future.

Neurotoxicity induced by alkyl nitrites: Impairment in learning/memory and motor coordination.

Although alkyl nitrites are used as recreational drugs, there is only little research data regarding their effects on the central nervous system including their neurotoxicity. This study investigated the neurotoxicity of three representative alkyl nitrites (isobutyl nitrite, isoamyl nitrite, and butyl nitrite), and whether it affected learning/memory function and motor coordination in rodents. Morris water maze test was performed in mice after administrating the mice with varying doses of the substances in two different injection schedules of memory acquisition and memory retention. A rota-rod test was then performed in rats. All tested alkyl nitrites lowered the rodents' capacity for learning and memory, as assessed by both the acquisition and retention tests. The results of the rota-rod test showed that isobutyl nitrite in particular impaired motor coordination in chronically treated rats. The mice chronically injected with isoamyl nitrite also showed impaired function, while butyl nitrite had no significant effect. The results of the water maze test suggest that alkyl nitrites may impair learning and memory. Additionally, isoamyl nitrite affected the rodents' motor coordination ability. Collectively, our findings suggest that alkyl nitrites may induce neurotoxicity, especially on the aspect of learning and memory function.

Rapid determination of isoamyl nitrite in pharmaceutical preparations by flow injection analysis with on-line UV irradiation and luminol chemiluminescence detection.

Isoamyl nitrite is used as a therapeutic reagent for cardiac angina and as an antidote for cyanide poisoning, but it is abused because of its euphoric properties. Therefore, a method to determine isoamyl nitrite is required in many fields, including pharmaceutical and forensic studies. In this study, a simple, rapid and sensitive method for the determination of isoamyl nitrite was developed using a flow injection analysis system equipped with a chemiluminescence detector and on-line photoreactor. This method is based on on-line ultraviolet irradiation of isoamyl nitrite and subsequent luminol chemiluminescence detection without the addition of an oxidant. A linear standard curve was obtained up to 1.0 µM of isoamyl nitrite with a detection limit (blank + 3SD) of 0.03 µM. The method was successfully applied to determine isoamyl nitrite content in pharmaceutical preparations.

Comparison of the relative propensities of isoamyl nitrite and sodium nitrite to ameliorate acute cyanide poisoning in mice and a novel antidotal effect arising from anesthetics.

Isoamyl nitrite has previously been considered acceptable as an inhaled cyanide antidote; therefore, the antidotal utility of this organic nitrite compared with sodium nitrite was investigated. To facilitate a quantitative comparison, doses of both sodium nitrite and isoamyl nitrite were given intraperitoneally in equimolar amounts to sublethally cyanide-challenged mice. Righting recovery from the knockdown state was clearly compromised in the isoamyl nitrite-treated animals, the effect being attributable to the toxicity of the isoamyl alchol produced during hydrolysis of the isoamyl nitrite to release nitrite anion. Subsequently, inhaled aqueous sodium nitrite aerosol was demonstrated to ameliorate sublethal cyanide toxicity, when provided to mice after the toxic dose, by the more rapid recovery of righting ability compared to that of the control animals given only the toxicant. Aerosolized sodium nitrite has thus been shown by these experiments to have promise as a better alternative to organic nitrites for development as an inhaled cyanide antidote. The inhaled sodium nitrite led to the production of NO in the bloodstream as determined by the appearance of EPR signals attributable to nitrosylhemoglobin and methemoglobin. The aerosol delivery was performed in an unmetered inhalation chamber, and in this study, no attempt was made to optimize the procedure. It is argued that administration of an effective inhaled aqueous sodium nitrite dose in humans is possible, though just beyond the capability of current individual metered-dose inhaler designs, such as those used for asthma. Finally, working at slightly greater than LD50 NaCN doses, it was fortuitously discovered that (i) anesthesia leads to significantly prolonged survival compared to that of unanesthetized animals and that (ii) the antidotal activity of nitrite anion was completely abolished under anesthesia. Plausible explanations for these effects in mice and their practical consequences in relation to testing putative cyanide antidotes are discussed.

[Prevalence of new designer drugs and their legal status in Japan].

In recent years, many analogs of narcotics have been widely distributed as easily available psychotropic substances and have become a serious problem in Japan. To counter the spread of these non-controlled substances, the Pharmaceutical Affairs Law in Japan was amended in 2006 to establish a new category; Designated Substances in order to more strictly control these substances. In April 2007, 31 compounds and 1 plant were first controlled as Designated Substances. Before 2007, the major compounds distributed in the Japanese illegal drug market were tryptamines, phenethylamines and piperazines. Alkyl nitrites, such as isobutyl nitrite and isopentyl nitrite, were also widely distributed. After they were listed as Narcotics or Designated Substances in 2007, these compounds, especially the tryptamines, quickly disappeared from the market. In their place, cathinone derivatives have been widely distributed, as well as different phenethylamines and piperazines. Additionally, in recent years, new herbal products containing synthetic cannabinoids have appeared globally. As at July 2012, 78 substances (including 1 plant; Salvia divinorum) were listed in the category of Designated Substances. They were 13 tryptamines, 17 phenethylamines, 11 cathinones, 4 piperazines, 23 synthetic cannabinoids, 6 alkyl nitrites, 3 other compounds and 1 plant. In this review, we show our survey of the spread of new designer drugs in Japan, focusing especially on synthetic cannabinoids and cathinone derivatives. Also, the prevalence and legal status of these substances in other countries will be presented.

Synthesis of alpha-oximinoketones under ultrasound irradiation.

The reaction of 6-acylmethylphenanthridines with isoamyl nitrite results alpha-oximino-6-acylmethylphenanthridines in 73-95% yields in DMF under ultrasound irradiation. Compared with conventional methods, the main advantages of the present procedure are milder conditions, shorter reaction time and higher yields.

Synthesis of peroxynitrite using isoamyl nitrite and hydrogen peroxide in a homogeneous solvent system.

A method for the synthesis of peroxynitrite is described. It involves nitrosation of H2O2 at pH> or = 12.5 by isoamyl or butyl nitrite in mixed solvents of isopropyl alcohol (IPA) and water at 25+/-1 degrees C. Maximum yields of peroxynitrite are obtained after 15 min of incubation at IPA concentrations of 30-70% (v/v). The solutions of peroxynitrite are processed for removal of IPA and isoamyl alcohol by solvent extraction. Unreacted H2O2 is removed by catalytic decomposition on granular MnO(2). The post processed solutions of peroxynitrite are useful in several chemical and biochemical investigations where bolus additions are required. The method as reported is amenable for large scale synthesis as it involves sequential mixing of solvents (water and IPA) to alkali followed by the addition of H2O2 and alkyl nitrite.

Isoamyl nitrite depolymerized heparin as a universal calibrator for heparins and low molecular weight heparins.

Because the subcutaneous absorption of heparin depends on its molecular weight, high performance size exclusion chromatography methods, among others, have been developed for its determination. Because heparin consists of polymer chains of varying lengths, a large number of calibrators, 19 in our work, are required for these molecular weight analyses. Because the preparation of that many calibrants is both time-consuming and expensive, other methods requiring fewer calibrants are being sought. In pursuit of these aims, a heparinase-degraded heparin has been accepted as the first international reference preparation low molecular weight heparin for molecular weight calibration. We have previously proposed another calibrant, the Heparin Mass Calibrator. In this article, we introduce another calibrant, called the Heparin Molecular Mass Calibrant. It offers the advantage of a much wider availability because it is a commercial product. In addition to having a superior refractive index chromatography run profile, when used in combination with two Narrow Range Calibrators with molecular weights of 34.4 and 18.4 kDa, this new calibrant covers both the higher and the lower molecular weight ranges. Besides, ultraviolet detection is not needed for molecular weight calculations. Therefore, it is proposed that its feasibility as a universal reference calibrator for both heparins and low molecular weight heparins be considered.

Effects of acute inhalation exposure to isoamyl nitrite on the hypothalamo-pituitary-adrenal axis in male Sprague-Dawley rats.

Isoamyl nitrite (IAN) is a member of the family of volatile organic nitrites that exert vasodilatory effects and have recently exhibited a considerable potential for inhalation abuse. In an effort to provide mechanistic insight into the neurotoxic effects and abuse potential of these agents, the present study was designed to evaluate the acute effects of IAN on the hypothalamo-pituitary-adrenal (HPA) axis. Attempts were also made to correlate the neuroendocrine effects of IAN with its pharmacokinetic profile. Male Sprague-Dawley rats were exposed to 600 or 1200 ppm IAN by inhalation for 10 or 30 min. Following exposure, adrenocorticotropic hormone (ACTH) and corticosterone in plasma and corticotropin-releasing factor (CRF) in three brain regions (hypothalamus, hippocampus, and frontal cortex) were determined by radioimmunoassay. Levels of IAN in the three brain regions as well as in blood were measured by gas chromatography to determine the target tissue concentrations responsible for neuroendocrine changes. Uptake of IAN into blood and all brain regions was very rapid, as stable concentrations were achieved within 10 min of exposure and maintained for 30 min of continuous inhalation. Plasma corticosterone decreased significantly after 10 min inhalation of both IAN doses, and returned to control levels after 30 min. Moreover, plasma ACTH was significantly increased by 10 and 30 min of exposure to 600 and 1200 ppm IAN, while hypothalamic CRF increased significantly after 30 min of exposure to the 600 ppm dose. These latter findings suggest activation of the hypothalamus and pituitary due to a reduction in negative feedback resulting from the initial decrease in corticosterone. Although plasma ACTH was greatly increased after 30 min, plasma corticosterone levels were unchanged, indicating that IAN primarily acts to inhibit the synthesis or secretion of adrenal steroids and that activation of the HPA axis is not involved in the behavioral manifestations of IAN inhalation. These compensatory effects of HPA axis regulation, and possibly the vasodilatory properties of IAN, also likely precluded the establishment of definitive relationships between observed changes in hormone levels and blood or regional brain concentrations of the inhalant.

Vascular and hemodynamic differences between organic nitrates and nitrites.

Because nitroglycerin (NTG, an organic nitrate) and isoamyl nitrite have similar chemical structures and a common mechanism of vascular relaxation (i.e., conversion to nitric oxide in vascular tissues and activation of guanylyl cyclase), it has often been assumed that organic nitrates and nitrites have identical pharmacologic actions. Because recent studies have shown that the vascular enzymes responsible for nitric oxide generation from organic nitrates and nitrites are distinct, we hypothesized that the in vitro vascular actions, in vivo hemodynamic effects and tolerance properties (both in vitro and in vivo) would be different as well. Isolated blood vessel studies showed that NTG provided more stable relaxation effects than ISAN, was more potent and caused greater in vitro vascular tolerance. Because the mechanism(s) of vascular tolerance in vitro may not be the same as those occurring in vivo, we also compared the left ventricular hemodynamic effects and tolerance properties of NTG vs. isoamyl nitrite and in congestive heart failure rats. Constant NTG infusion (10 micrograms/min) caused initial reductions in left ventricular end-diastolic pressure of 45 to 55%, which returned to baseline within 10 hr (tolerance development). In contrast, isobutyl nitrite and isoamyl nitrite (45 micrograms/min) caused initial reductions in left ventricular end-diastolic pressure similar to NTG (42-58%), but these hemodynamic effects of organic nitrites were maintained even when infusions were carried out to 22 hr. These results show that organic nitrites and organic nitrates are not pharmacologically identical (in vitro or in vivo), and may suggest a therapeutic advantage for organic nitrites in the treatment of some cardiovascular diseases.

Synthesis of peroxynitrite in a two-phase system using isoamyl nitrite and hydrogen peroxide.

A new method for the preparation of high concentrations of peroxynitrite (up to 1 M) is described. The synthesis uses a two-phase system and involves a displacement reaction by the hydroperoxide anion (in the aqueous phase) on isoamyl nitrite (in the organic phase). The product peroxynitrite remains in the aqueous phase, whereas isoamyl alcohol forms a new organic phase along with the unreacted isoamyl nitrite. The aqueous phase contains some 0.15 M isoamyl alcohol and the unreacted hydrogen peroxide, but no isoamyl nitrite. Removal of isoamyl alcohol or traces of isoamyl nitrite is accomplished by washing the aqueous phase with dichloromethane, chloroform, or hexane. A near total removal of hydrogen peroxide is then achieved by passing the solutions through a short column of manganese dioxide. The peroxynitrite in these postprocessed solutions has broad absorption spectrum with a maximum around 302 nm, follows a characteristic first-order decomposition at pH 7.2 and 25 degrees C (k = 0.34 +/- 0.1 s-1), and reacts with organic compounds to give either nitrated or one-electron transfer products. When stored frozen at -20 degrees C, these peroxynitrite solutions decompose at a rate of about 1.7 % per day and should be used within 2-4 weeks. For short-term storage of about 1 week or less, these solutions can be stored at refrigerator temperatures (approximately 5 degrees C) where peroxynitrite has a half-life of about 7 days.

Molecular mechanisms of nitrovasodilator bioactivation.

All nitrovasodilators act intracellularly by a common molecular mechanism. This is characterized by the release of nitric oxide (NO). They are, thus, prodrugs or carriers of the active principle NO, responsible for endothelial controlled vasodilation. The rate of NO-formation strongly correlates with the activation of the soluble guanylate cyclase in vitro, resulting in a stimulation of cGMP synthesis. Nitrovasodilators thus are therapeutic substitutes for endogenous EDRF/NO. The pathways of bioactivation, nevertheless, differ substantially, depending on the individual chemistry of the nitrovasodilator. Besides NO, numerous other reaction products such as nitrite and nitrate anions are formed. The guanylate cyclase is only activated if NO is liberated. In the case of organic nitrates such as GTN, NO is only formed if certain thiol compounds are present as an essential cofactor. The rate of NO-formation correlates with the number of nitrate ester groups and proceeds with a simultaneous nitrite formation (with a ratio of 1:14 in the presence of cysteine). Nitrosamines such as molsidomine do not need thiol compounds for bioactivation. They directly liberate NO from the ring-open A-forms. This process basically depends on the presence of oxygen as electron acceptor from the sydnonimine molecule. Therefore, besides NO also superoxide radicals are formed, which may react with the generated NO under formation of nitrate ions. Organic nitrites (such as amyl nitrite) require the preceding interaction with a mercapto group to form a S-nitrosothiol intermediate, from which finally NO radicals are liberated. Nitrosothiols (like S-nitroso-acetyl-penicillamine) and sodium nitroprusside spontaneously release NO. The molecules themselves do not possess a direct enzyme activating potency. In the presence of thiol compounds organic nitrites (e.g., amyl nitrite) and nitrosothiols may act as intermediary products of NO generation.

Electron paramagnetic resonance and spin trapping study of radicals formed during reaction of aromatic amines with isoamyl nitrite under aprotic conditions.

Diazotization of primary aromatic amines with isoamyl nitrite in benzene at room temperature was studied employing EPR and spin trapping techniques. Nitrosodurene (ND), 2-methyl-2-nitrosopropane (MNP), and 5,5-dimethyl-pyrroline N-oxide (DMPO) were used as spin trapping agents. Aryl radicals were detected employing ND and MNP. Using DMPO as a spin trap most of the amines produced EPR spectra ascribed to adducts with aniline-type radicals (N-centred radicals). The assignments were verified using 15N-labeled anilines. Similar spectra of DMPO adducts were recorded from amines treated with benzoyl peroxide or benzophenone plus UV. Possible mechanisms of formation of these adducts (radical trapping versus nucleophilic addition to DMPO followed by oxidation) during treatment of the amines with isoamyl nitrite are discussed.

Pentobarbital-like discriminative stimulus properties of halothane, 1,1,1-trichloroethane, isoamyl nitrite, flurothyl and oxazepam in mice.

Volatile inhalants represent a diverse group of chemicals which pose a public health problem because of their abuse potential and neurobehavioral toxicity. Although relatively little is known about their pharmacology, they share certain pharmacological properties with classic central nervous system depressants. Drug discrimination procedures were used to compare the effects produced by pentobarbital (PB), oxazepam and several inhalants. Mice were trained to discriminate PB from saline injections in a two-lever operant task. Stimulus generalization was examined after 20-min inhalation exposures to halothane (500-8000 ppm), 1,1,1-trichloroethane (500-12000 ppm), isoamyl nitrite (150-1050 ppm), flurothyl (562-1300 ppm) and injections of oxazepam (0.1-20 mg/kg). Halothane, 1,1,1-trichloroethane and oxazepam produced discriminative stimulus effects similar to those produced by PB. Isoamyl nitrite and flurothyl, while decreasing rates of responding, were not consistently generalized from PB. Differences are apparent in the PB-like discriminative effects of inhalants. Toluene, as shown in a previous study, as well as halothane and 1,1,1-trichloroethane have PB-like discriminative effects which are also shared by oxazepam and may be another common effect of central nervous system depressants. Other chemicals, like isoamyl nitrite and flurothyl, are representative of qualitatively different classes of behaviorally active inhalants.

Acute effects of some volatile nitrites on motor performance and lethality in mice.

Mice were examined for effects on lethality and motor performance on an inverted screen test following inhalation exposure to isoamyl, n-butyl and isobutyl nitrite. All three nitrites produced concentration-related effects on both measures, with EC50s for motor performance only about one-half of the LC50s for lethality. Isoamyl and isobutyl nitrite were equally potent on both lethality and motor performance measures, whereas n-butyl nitrite was significantly more potent than isoamyl and isobutyl for lethality. Slope estimates of the concentration-effect curves for lethality were significantly greater than those of the motor performance measure. The steepness of the concentration-effect curves and low LC50/EC50 ratios relative to abused organic solvents suggest that behaviorally active concentrations of volatile nitrites may put users at risk to other health consequences.