Peter D. Maskell

Halogenated cytisine derivatives as agonists at human neuronal nicotinic acetylcholine receptor subtypes

Cytisine (cy) is a potent and competitive partial agonist at alpha4 subunit-containing nicotinic acetylcholine (nACh) receptors while at homomeric alpha7-nACh receptors it behaves as a full agonist with a relatively lower potency. In the present study, we assessed the effects of bromination or iodination of the pyridone ring of cy and N-methylcytisine (N-Me-cy) on the effects of these compounds on recombinant human (h) alpha7, halpha4beta2 and halpha4beta4 nACh receptors expressed in clonal cell lines and Xenopus oocytes. Halogenation at C(3) of cy or N-Me-cy usually brings about a marked increase in both affinity and efficacy at halpha7, halpha4beta2 and halpha4beta4 nACh, the extent of which depends on whether the halogen is bromine or iodine, and upon receptor subtype. The effects of halogenation at C(5) are strongly influenced by the specific halogen substituent so that bromination causes a decrease in both affinity and efficacy while iodination decreases affinity but its effects on efficacy range from a decrease (halpha7, halpha4beta4 nACh receptors) to a marked increase (halpha4beta2 nACh receptors). Based on these findings, which differ from those showing that neither the affinity nor efficacy of nicotine, 3-(2-azetidinylmethoxy)-pyridine or epibatidine are greatly affected by halogenation, dehalogenation or halogen exchange at equivalent positions, we suggest that cy, N-Me-cy and their halo-isosteres bind to neuronal nACh receptors in a different orientation allowing the halogen atom to interact with a hydrophobic halogen-accepting region within the predominantly hydrophobic agonist-binding pocket of the receptors.

Indirect sensing of insulin-induced hypoglycaemia by the carotid body in the rat

The most physiologically important sensors for systemic glucoregulation are located in extra‐cranial sites. Recent evidence suggests that the carotid body may be one such site. We assessed rat carotid body afferent neural output in response to lowered glucose, indirectly by measurement of ventilation, and directly by recording single or few‐fibre chemoafferent discharge, in vitro. Insulin (0.4Ukg−1min−1)‐induced hypoglycaemia (blood glucose reduced by ca 50% to 3.4 ± 0.1mmoll−1) significantly increased spontaneous ventilation in sham‐operated animals but not in bilateral carotid sinus nerve sectioned (CSNX) animals. In both groups, metabolic rate (measured as ) was almost doubled during hypoglycaemia. The ventilatory equivalent was unchanged in the sham group leading to a maintained control level of P  a, CO 2, but was significantly reduced in the CSNX group, giving rise to an elevation of 6.0 ± 1.3mmHg in P  a, CO 2. When pulmonary ventilation in sham animals was controlled and maintained, phrenic neural activity increased during hypoglycaemia and was associated with a significant increase in P  a, CO 2 of 5.1 ± 0.5mmHg. Baseline chemoreceptor discharge frequency, recorded in vitro, was not affected, and did not increase when the superfusate [glucose] was lowered from 10 mm to 2 mm by substitution with sucrose: 0.40 ± 0.20 Hz to 0.27 ± 0.15Hz, respectively (P > 0.20). We suggest therefore that any potential role of the carotid bodies in glucose homeostasis in vivo is mediated through its transduction of some other metabolically derived blood‐borne factor rather than glucose per se and that this may also provide the link between exercise, metabolic rate and ventilation.

Inhibition of human α7 nicotinic acetylcholine receptors by open channel blockers of N‐methyl‐D‐aspartate receptors

Human α7 nicotinic acetylcholine (ACh) receptors were expressed in Xenopus oocytes and the effects of the N‐methyl‐D‐aspartate (NMDA) receptor open channel blockers memantine and cerestat on this receptor were examined using two‐electrode voltage‐clamp recordings and 125I‐α‐bungarotoxin (125I‐α‐bgtx) binding. Memantine and cerestat produced complete inhibition of ACh‐induced inward currents with affinities similar to that reported for native NMDA receptors. Cerestat, IC50 1.7 (−1; +2) μM, was more potent than memantine, IC50 5 (−3;+8) μM, and the effects of both drugs were fully and rapidly reversible. Inhibition of α7 receptor function was voltage‐independent, and it occurred at concentrations far lower than those needed to inhibit (never completely) binding of 125I‐α‐bgtx to α7 receptors, suggesting that the effects of memantine or cerestat are noncompetitive. These results provide evidence that human α7 receptors are inhibited by memantine and cerestat and suggest that caution should be applied when using these compounds to study systems in which NMDA and nACh receptors co‐exist.

Phenazepam: The drug that came in from the cold

In the past few years there has been concern in Western Europe and in the US about the rise in abuse of phenazepam, a benzodiazepine that was originally developed in the USSR in the mid- to late 1970s.(1-4) Although phenazepam is one of the most widely prescribed benzodiazepines in Russia and other commonwealth of independent state (CIS) countries, it has not been licensed elsewhere in the world. Due to very limited licensed geographical distribution, there is very little peer-reviewed literature that is not written in Russian. In this article, we review the current state of what is currently known about phenazepam. This information on phenazepam and how it can be detected in biological specimens should assist the forensic community in identifying phenazepam in routine toxicology screening and interpreting any phenazepam concentrations that are obtained.

Carbon dioxide sensitivity during hypoglycaemia-induced, elevated metabolism in the anaesthetized rat.

We have utilized an anaesthetized rat model of insulin‐induced hypoglycaemia to test the hypothesis that peripheral chemoreceptor gain is augmented during hypermetabolism. Insulin infusion at 0.4 U kg −1min−1 decreased blood glucose concentration significantly to 3.37 ± 0.12 mmol l−1. Whole‐body metabolism and basal ventilation were elevated without increase in P  a,CO 2 (altered non‐significantly from the control level, to 37.3 ± 2.6 mmHg). Chemoreceptor gain, measured either as spontaneous ventilatory airflow sensitivity to P  a,CO 2 during rebreathing, or by phrenic minute activity responses to altered P  a,CO 2 induced by varying the level of artificial ventilation, was doubled during the period of hypermetabolism. This stimulatory effect was primarily upon the mean inspiratory flow rate, or phrenic ramp component of breathing and was reduced by 75% following bilateral carotid sinus nerve section. In vitro recordings of single carotid body chemoafferents showed that reducing superfusate glucose concentration from 10 mm to 2 mm reduced CO2 chemosensitivity significantly from 0.007 ± 0.002 Hz mmHg−1 to 0.001 ± 0.002 Hz mmHg−1. Taken together, these data suggest that the hyperpnoea observed during hypermetabolism might be mediated by an increase in the CO2 sensitivity of the carotid body, and this effect is not due to the insulin‐induced fall in blood glucose concentration.

Naphyrone: analytical profile of the new "legal high" substitute for mephedrone.

Dear Editor, Mephedrone (4-methylmethcathinone) has been banned recently in several countries, including the UK as from April 2010. Banning of the drug in the UK followed a report from the Advisory Council on the Misuse of Drugs1 and newspaper reports of several deaths linked to mephedrone.2 Soon after mephedrone was banned, internet sites3 began to market a new legal alternative – naphyrone, which is also known as NRG-1. The structure of this new designer drug is similar to that of pyrovalerone, a monoamine uptake inhibitor, first synthesized in 1964.4 As with mephedrone, presently there is no safety or toxicity data available for naphyrone, which in its turn has now been banned in the UK as from July 12, 2010.5 Anticipating the problem of having to identify the drug in routine forensic toxicology drug screens, we purchased naphyrone from a website, since it is not available as a pure standard from certified sources. Analysis of he drug undertaken by HPLC-DAD, GC-MS and LC-MS-MS using our current standard laboratory drug screening methods has produced a useful analytical profile for naphyrone. Detailed analytical data and naphyrone’s UV and mass spectra can be found at: http://www.dundee.ac.uk/forensicmedicine/drugmonographs/naphyrone. This analytical data should assist the wider forensic community in identifying naphyrone in routine toxicology screening.

The emergence of new psychoactive substance (NPS) benzodiazepines: a review

The market for new psychoactive substances has increased markedly in recent years and there is now a steady stream of compounds appearing every year. Benzodiazepines consist of only a fraction of the total number of these compounds but their use and misuse has rapidly increased. Some of these benzodiazepines have only been patented, some of them have not been previously synthesised, and the majority have never undergone clinical trials or tests. Despite their structural and chemical similarity, large differences exist between the benzodiazepines in their pharmacokinetic parameters and metabolic pathways and so they are not easily comparable. As benzodiazepines have been clinically used since the 1960s, many analytical methods exist to quantify them in a variety of biological matrices and it is expected that these methods would also be suitable for the detection of benzodiazepines that are novel psychoactive substances. Illicitly obtained benzodiazepines have been found to contain a wide range of compounds such as opiates which presents a problem since the use of them in conjunction with each other can lead to respiratory depression and death. This review collates the available information on these benzodiazepines and provides a starting point for the further investigation of their pharmacokinetics which is clearly required.

Seven fatalities associated with ethylphenidate

Ethylphenidate is a stimulant novel psychoactive substance that is an analogue of the prescription drug methylphenidate (Ritalin(®)). Methylphenidate is used commonly for the treatment of attention deficit hyperactivity disorder. Due to its stimulant effects ethylphenidate is being abused. There is a single case report of a death associated with ethylphenidate in Germany, and a case series of 19 deaths in the East of Scotland, but otherwise, the contribution of ethylphenidate to death is poorly documented. We report the analytical results of 7 cases (between February 2013 and January 2015) in which ethylphenidate was detected and quantitated with a validated liquid chromatography tandem mass spectrometry method (LC-MS/MS). The individuals (all male) ranged in age from 23 to 49 years (median 25 years). The concentration of ethylphenidate in the cases ranged from 0.026mg/L to 2.18mg/L in unpreserved post-mortem femoral blood. Only one case had ethylphenidate present as a sole drug. All other cases had at least 2 other drug classes present (benzodiazepines, heroin, methadone antipsychotics, other new psychoactive compounds). Ethylphenidate toxicity was the sole contribution to the cause of death in one case. Hanging was the cause of death in 2 cases, with the other 4 cases being reported as having occurred due to mixed drug toxicity. These data will further help with the interpretation of post-mortem ethylphenidate levels.

Analysis of phenazepam and 3-hydroxyphenazepam in post-mortem fluids and tissues.

Phenazepam is a benzodiazepine that is predominantly used clinically in the former Soviet states but is being abused throughout the wider world. This study reports the tissue distribution and concentration of both phenazepam and 3-hydroxyphenazepam in 29 cases quantitated by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in a variety of post-mortem fluids (subclavian blood, femoral blood, cardiac blood, urine, vitreous humour) and tissues (thalamus, liver and psoas muscle). In 27 cases, the cause of death was not directly related to phenazepam (preserved (fluoride/oxalate) femoral blood phenazepam concentrations 0.007 mg/L to 0.360 mg/L (median 0.097 mg/L). In two cases, phenazepam was either a contributing factor to, or the certified cause of death (preserved (fluoride/oxalate) femoral blood 0.97 mg/L and 1.64 mg/L). The analysis of phenazepam and 3-hydroxyphenazepam in this study suggests that they are unlikely to be subject to large post-mortem redistribution and that there is no direct correlation between tissues/fluid and femoral blood concentrations. Preliminary investigations of phenazepam stability comparing femoral blood phenazepam concentrations in paired preserved (2.5% fluoride/oxalate) and unpreserved blood show that unpreserved samples show on average a 14% lower concentration of phenazepam and we recommend that phenazepam quantitation is carried out using preserved samples wherever possible.

Phenazepam is currently being misused in the UK

Phenazepam is a benzodiazepine not currently controlled in the United Kingdom, mainland Europe, or the United States. Developed in the 1970s for the treatment of epilepsy, alcohol withdrawal syndrome, insomnia, and anxiety,1 2 it is currently prescribed only in the former Soviet Bloc. However, recent reports from Sweden, Finland, and the …