Comparison of three radioligands for the labelling of human β-adrenoceptor subtypes

We have compared the ability of three radioligands, [¹²⁵I]-cyanopindolol, [³H]-CGP 12,177 and [³H]-dihydroalprenolol, to label the three human β-adrenoceptor subtypes. Saturation and competition binding experiments were performed using membrane preparations from Chinese hamster ovary cells stably transfected with the three subtypes. While [³H]-CGP 12,177 had very similar affinity for β₁- and β₂-adrenoceptors (about 40 pM), [¹²⁵I]-cyanopindolol and [³H]-dihydroalprenolol had 4- to 6-fold higher affinity for β₂- as compared to β₁-adrenoceptors (10 vs 45 and 187 vs 1,021 pM, respectively). The affinity of [¹²⁵I]-cyanopindolol at β₃-adrenoceptors was considerably lower (440 pM) than at the other two subtypes. The β₃-adrenoceptor affinity of [³H]-CGP 12,177 and [³H]-dihydroalprenolol was so low that it could not be estimated within the tested range of radioligand concentrations (up to 4,000 pM and 30,000 pM for [³H]-CGP 12,177 and [³H]-dihydroalprenolol, respectively). We conclude that all three radioligands are ill-suited to label β₃-adrenoceptors, particularly in preparations co-expressing multiple subtypes. In the absence of alternatives, [¹²⁵I]-cyanopindolol appears the least unsuitable to label β₃-adrenoceptors. There is a need for high-affinity radioligands which are either selective for β₃-adrenoceptors or reasonably non-selective among all three β-adrenoceptor subtypes.     

A contribution to a new test method for dandruff-inhibiting and “keratolytic” action of drugs

Summary Free cholesterol in lipids from the scalp and hair is predominantly a constituent of epidermal lipids. Therefore, a reduction in cholesterol content induced by a drug indicates a reduction in cell turnover in the epidermis. As, according to the literature, increased cell turnover in the epidermis results in formation of dandruff, a reduction in the proportion of cholesterol should indicate inhibition of the formation of dandruff. Conversely, an increase in free cholesterol should generally indicate a keratolytic effect. So unequivocal an interpretation has not so far been possible in persons with dandruff, as it was not known whether free cholesterol was increased or decreased. In addition, this interpretation was not possible after use of antimicrobial substances, as in vitro investigations had failed to exclude microbial esterification of cholesterol on the scalp. The present investigation has shown that correlation of free cholesterol level with cell turnover is permissible in patients with dandruff, even if antimicrobial drugs are being tested.     

Improved method for autoradiographic localization of ß-adrenoceptors using photoaffinity labelling

Summary Contact autoradiography of tissue sections, using emulsion coated coverslips or X-ray films, is widely used to provide information about the regional distribution of receptors. This easy to perform, standard technique has the disadvantage of an image spread due to the gap between the radioactive source and the film. The present study describes a new technique which combines photoaffinity labeling of ß-adrenoceptors with dipping autoradiography and a modified trichrome stain. Incubation of 16 m cryosections of rat lung tissue with the iodinated, photoaffintiy labeling, non-selective, ß-adrenergic agonist [¹²⁵I]-cyanopindololazide II ([¹²⁵I]-CYPA II) (100 pmol/1) in the absence or presence of 1 mol (±)-propranolol revealed strong, specific ß-adrenoceptor binding to alveolar parenchyma and bronchial epithelium of large and small bronchioles, lesser binding to smooth muscle bundles of large airways and only sparse binding to the smooth muscle of small bronchioles or peripheral branches of pulmonary artery. With standard autoradiographic techniques, a similiar distribution of the label was obtained, although resolution and sensitivity were inferior. Staining of tissue sections through the photo-emulison by means of a modified Mallory's trichrome dye facilitated the discrimination between alveolar and bronchial epithelium, muscular and collagenous tissues. In conclusion, the photoaffinity labeling of ß-adrenoceptors with [¹²⁵I]-CYPA II allows the use of dipping autoradiography. This technique, in combination with trichrome staining through the photoemulsion, results in an improved autoradiograpic image together with a better association of the label with distinct histological structures and the higher sensitivity of the method.Presented, in part, at the Frühjahrstagung der Deutschen Gesellschaft für Pharmakologie and Toxikologie in Mainz 1988 (Hesse and Erdmann, Naunyn Schiedebergs Arch Pharmacol 337 [Suppl]:R 100)     

A review of cannabidiol-containing electronic liquids—Current regulations and labelling accuracy

The use of cannabidiol in electronic liquids (e-liquids) is becoming increasingly widespread, and the current regulations enforced onto nicotine-containing e-liquids are not applicable to cannabidiol-based products. This has led to concerns about the quality of cannabidiol vapes. Articles investigating the reliability of product labelling were reviewed using systematic review criteria. Of 70 e-liquids, 77.1% of the e-liquids tested in the articles were found to have underestimated or overestimated the cannabidiol quantities stated in the product labelling. Statistical analysis confirmed that there was a significant difference between the labelled and analysed cannabidiol concentrations (p < 0.05, Mann-Whitney U and Wilcoxon Signed Rank). Inaccuracies in received cannabidiol dosages could lead to an increased risk of adverse reactions or limit the therapeutic effect received, highlighting the benefit of enforcing specific regulations on cannabidiol-based e-liquids to protect consumer safety and guarantee product efficacy.     

Electrointraperitoneography: A new method for testing the pharmacological effects of drugs at the intestine level—Preliminary note

A recent and nontraumatizing method, electrointraperitoneography (EIG), has been used in the anesthetized rat in order to test its usefulness for the pharmacological study of drug effects on the electrical activity of the intestine. The results show that such a study is possible for different drugs. Advantages and disadvantages of the method are discussed.     

Stability of drugs of abuse in synthetic oral fluid investigated using a simple “dilute and inject” method of analysis

Human oral fluid is well established as a matrix for drug screening, particularly in the workplace. The need to synthesise synthetic oral fluid (SOF) has been recognised in order to overcome human oral fluid's composition variability. We have used SOF spiked with six common drugs of abuse or their primary metabolites: morphine, amfetamine, benzoylecgonine, cocaine, diazepam, and (−)-Δ⁹-tetrahydrocannabinol (THC) in order to assess the suitability of this matrix for quality assurance purposes. For confirmation of a drug screening test, controls and spiked standards are normally required. All our analytes were detected by LC–MS/MS using a quick and easy “dilute and inject” sample preparation approach as opposed to relatively slower solid-phase extraction. The limit of detection (LOD) was 10 ng/ml for diazepam and THC and 5 ng/ml for morphine, amfetamine, benzoylecgonine and cocaine. Validation results showed good accuracy as well as inter- and intra-assay precision (CV [%] < 5). Our work highlighted the importance of adding Tween® 20 to the SOF and calibrants to reduce losses when handling THC. Furthermore, drug stability was tested at various temperatures (5°C, 20°C and 40°C), for a number of days or after freeze–thaw cycles. Recommendations regarding storage are provided, the spiked SOF being stable at 5°C for up to 1 week without significant drug concentration loss.     

Desire for information about drugs A multi‐method study in general medical inpatients

The purpose of this paper is to investigate patients' drug information preferences using a combination of quantitative and qualitative methods. Patient interviews (n=299) were conducted on general medical wards in three London teaching hospitals. The purpose was to refine and validate a quantitative 12item scale, the Intrinsic Desire for Information (IDI), by interfacing quantitative and qualitative data, and to explore the relationship between this scale score and patient demographics. The IDIscale was subjected to factor analysis. Two secondary factors were found in the IDI scale; a 5item factor describing the extent of information desired and a weaker 3item factor describing an inhibited desire for knowledge about illness/drugs. Reliability analysis and multiple regression analysis were undertaken. Responses to open answer questions during the qualitative interviews were transcribed at the bedside and imported into QSR NUD*IST software program for coding and analysis. The methodology employed in this study involved importing quantitative, summative data into a qualitative data base and reanalysing both the quantitative and qualitative data to validate the scale. Age was a predominant factor associated with patient desire for information, although the data suggest that educational and socioeconomic status are also influential. Factor 1, the extent of information desired, may have value in targeting receptive patients, or in identifying those who may be refractory to drug information. The refined tool could help health services to effectively target information provision based on evidence, rather than supposition.     

Mitochondria targeting drugs for neurodegenerative diseases—Design,mechanism and application

Neurodegenerative diseases (NDDs) such as Alzheimer's disease (AD) and Parkinson's disease (PD) are a heterogeneous group of disorders characterized by progressive degeneration of neurons. NDDs threaten the lives of millions of people worldwide and regretfully remain incurable. It is well accepted that dysfunction of mitochondria underlies the pathogenesis of NDDs. Dysfunction of mitochondria results in energy depletion, oxidative stress, calcium overloading, caspases activation, which dominates the neuronal death of NDDs. Therefore, mitochondria are the preferred target for intervention of NDDs. So far various mitochondria-targeting drugs have been developed and delightfully some of them demonstrate promising outcome, though there are still some obstacles such as targeting specificity, delivery capacity hindering the drugs development. In present review, we will elaborately address 1) the strategy to design mitochondria targeting drugs, 2) the rescue mechanism of respective mitochondria targeting drugs, 3) how to evaluate the therapeutic effect. Hopefully this review will provide comprehensive knowledge for understanding how to develop more effective drugs for the treatment of NDDs.     

‘Old' and ‘new' drugs for the treatment of cancer pain

Introduction: More than 20 years ago the World Health Organization (WHO) published the booklet ‘Cancer Pain Relief', including the fundamentals and clear principles, which was summarized in five simple sentences: ‘by mouth', ‘by the clock', ‘by the ladder', ‘for the individual' and ‘attention to detail'. Over the years, several modifications to the analgesic ladder have been proposed, as the addition of two further steps, related to the switch of opioid and/or non-invasive route of administration, and to the use of invasive approaches, or again the skip of the second step; nevertheless the educational value and benefits related to the worldwide dissemination are of paramount importance.

Areas covered: To date, all the guidelines are inspired by the strategy of WHO; below some of the most important international guidelines published in the last two years are compared, particularly as regards the criteria of choice of opioids for moderate/severe pain.

Expert opinion: The discussion on the role of the second step of the WHO analgesic ladder is still open. The challenge for new formulations of ‘old' opioids will be to better manage cancer pain, with more tailored efficacy and possibly less side effects.     

Are hormones relevant for the search and design of anti-parasitic drugs?

Over the last years the biology of many parasites that infect humans and domestic animals has been intensively studied. Considerable efforts were addressed to obtain information on the parasite-host immune relationship. However, the knowledge of the endocrine physiology of parasites and the consequences of the local hormone production on the host tissues needs further investigation. We review here literature and our own studies on endocrine parasite capacities with special emphasis on cysticercosis. Besides the biological interest, these investigations may contribute to identify in the future alternative treatments for the disease.     

Development and validation of an EI–GC–MS method for the determination of benzodiazepine drugs and their metabolites in blood: Applications in clinical and forensic toxicology

Benzodiazepines are used widely in daily clinical practice, due to their multiple pharmacological actions. The frequent problems associated with the wide use of benzodiazepines, as well as the multiple incidents of poisonings, led to the necessity for the development of a precise, sensitive and rapid method for the simultaneous determination of the 23 most commonly used benzodiazepines (diazepam, nordiazepam, oxazepam, bromazepam, alprazolam, lorazepam, medazepam, flurazepam, fludiazepam, tetrazepam, chlordiazepoxide, clobazam, midazolam, flunitrazepam, 7-amino-flunitrazepam, triazolam, prazepam, nimetazepam, nitrazepam, temazepam, lormetazepam, clonazepam, camazepam) in blood. A gas chromatographic method combined with mass spectrometric detection was developed, optimized and validated for the determination of the above substances. This method includes liquid–liquid extraction with chloroform at pH 9 and two stages of derivatization using tetramethylammonium hydroxide and propyliodide (propylation), as well as a mixture of triethylamine:propionic anhydride (propionylation). The recoveries were higher than 74% for all the benzodiazepines. The calibration curves were linear within the dynamic range of each benzodiazepine with a correlation coefficient higher than 0.9981. The limits of detection and quantification for each analyte were statistically calculated from the relative calibration curves. Accuracy and precision were also calculated and were found to be less than 8.5% and 11.1%, respectively. The developed method was successfully applied for the investigation of both forensic and clinical toxicological cases of accidental and suicidal poisoning.     

Evaluation of a screening method by liquid chromatography-tandem mass spectrometry for estimating effect of drugs on the activation and β-oxidation of fatty acids in mitochondria

Objectives Fatty acid metabolism is controlled not only by the acyl‐coenzyme A (CoA) synthetases but by some enzymes in the β‐oxidation cycle. Medium‐chain and long‐chain acyl‐CoA esters are key metabolites in fatty acid metabolism. We have developed an enzymatic assay method for determining chain shortening of the acyl‐CoAs via β‐oxidation from palmitic and octanoic acids in liver mitochondria. We have evaluated the assay method for detecting whether drugs influence the activation or the β‐oxidation of fatty acids. Methods Liver mitochondria were used for investigating the effect of drugs on fatty acid metabolism. The drugs selected were salicylic acid, diclofenac, valproic acid and paracetamol. Each acyl‐CoA formed was analysed by liquid chromatography–tandem mass spectrometry. Key findings After less than 5 min of incubation, the levels of acyl‐CoAs reflected the acyl‐CoA synthetase activity, whereas after 60‐min incubation they reflected the activity of some enzymes in the β‐oxidation cycle. Salicylic acid, diclofenac and valproic acid inhibited the medium‐chain acyl‐CoA synthetases, whereas valproic acid only exhibited a weak inhibitory activity toward the β‐oxidation of the medium‐chain fatty acids. In the case of long‐chain fatty acid metabolism, salicylic acid and diclofenac inhibited both the activation and β‐oxidation, whereas valproic acid was a weak inhibitor for only the β‐oxidation activity. Paracetamol showed hardly any influence on the metabolism of medium‐chain and long‐chain fatty acids. Conclusions These findings suggest that salicylic acid, diclofenac, valproic acid and paracetamol exert a different influence on fatty acid metabolism depending on the length of the acyl chain. This assay allows sensitive and selective analysis for predicting the pathways by which drugs exert a greater influence over fatty acid metabolism.     

The metabolism of steroids, toxins and drugs by 11β-hydroxysteroid dehydrogenase 1

11β-Hydroxysteroid dehydrogenase isoform 1 (11β-HSD1) is a member of the alcohol short-chain family enzyme, and catalyzes the interconversion between active glucocorticoid cortisol (in human) and inactive cortisone. The redox ratio of NADP+/NADPH determines its direction with NADPH favoring its reductase activity and NADP+ favoring its oxidase activity. In many tissues such as the liver and lung, 11β-HSD1 behaves primarily as a reductase because of the intracellular enzyme coupling with hexose-6-phosphate dehydrogenase, which uses the NADP+ as cofactor to supply NADPH driving 11β-HSD1 to function as a reductase. 11β-HSD1 catalyzes the metabolism of 7- or 11-keto steroids as well as many toxins and drugs. Some steroids, drugs and toxins are metabolically activated. The present review discusses the steroid, drug and toxin metabolism by 11β-HSD1.     

Astrocytic control of synaptic transmission and plasticity: a target for drugs of abuse?

It is well recognized that drugs of abuse lead to plastic changes in synapses and that these long-term modifications have the potential to underlie adaptive changes of the brain that lead to substance abuse. However the variety of molecular mechanisms involved in these responses are not completely defined. We are just beginning to understand some of the roles of glial cells that are associated with synapses. At many synapses an astrocyte process is associated with pre- and postsynaptic neuron processes leading to the naming of this synaptic structure as the Tripartite Synapse. Therefore, these glial cells are positioned so that they influence synaptic transmission and thus could potentially regulate the actions of some drugs of abuse. In mammalian systems there are correlations between long-term structural changes in astrocytes and responses to drugs of abuse. However, whether such changes in glia impact brain function and subsequent behaviors associated with addiction is poorly understood. Studies using Drosophila show important roles of fly glia in mediating responses to cocaine pointing to the potential for the involvement of mammalian glia in the brain's responses to this as well as other drugs. In agreement with this possibility three receptor systems known to be important in substance abuse, mGluR5, GABA(B) and CB-1 receptors, are all expressed by astrocytes and the activation of these glial receptors is now known to impact neuronal excitability and synaptic transmission. Given our new knowledge about the presence of reciprocal signaling between astrocytes and synapses we are now at a time when it becomes appropriate to determine how glial cells respond to drugs of abuse and whether they contribute to the changes in brain function underlying substance abuse.     

A review of nicotine-containing electronic cigarettes—Trends in use,effects, contents,labelling accuracy and detection methods

Electronic cigarettes (ECs) are thought to be less harmful than traditional combustible cigarettes and were originally intended to help smokers quit. Over the past two decades, they have especially gained popularity with the younger generation. To date, there are over 7000 unique e-liquid flavours available and over 400 different e-cigarette brands. The accuracy of nicotine strength labelling in e-liquids was assessed in this work. Twenty-three studies from around the world were chosen to assess the level and frequency of nicotine mislabelling in 545 e-liquid products. Nicotine strengths were most commonly mislabelled by between 5% and 20%, with the majority testing lower than what the label indicated. Fifteen European e-liquids that were assessed were labelled as 20 mg/ml or less, yet when tested, they contained more than 20 mg/ml of nicotine. One e-liquid that was supposed to contain no nicotine in fact contained 23.91 mg/ml of nicotine. Furthermore, the difference between the medians of the available labelled and experimental nicotine concentrations was significant (p < 0.001, Wilcoxon signed rank test). Preliminary studies show that high nicotine levels delivered via aerosol increase the risk for nicotine poisoning and cause airway inflammation. Other EC ingredients, such as flavourings, contribute to EVALI and ‘popcorn lung’. There is evidence that certain flavourings, such as menthol, reinforce the effects of nicotine and modify drug absorption and metabolism. There is a global need for better quality control in EC products in order to make these safe for consumers.