Does the well‐stirred model assess the intestinal first‐pass effect well?

The pre-systemic intestinal extraction ratio (E(g)) has been estimated by an equation based on the well-stirred model, which does not have a term of membrane transport. In this report, we have identified the application limitations of the well-stirred model equation to assess the pre-systemic intestinal extraction ratio. The E(g) of metoprolol (CYP2D6 substrate) was assessed by three methods. Intrinsic clearances for metoprolol metabolism in hepatic and gastrointestinal microsomes were from a published report. Method 1 (model-independent method): the E(g) of 0.228 was obtained according to the equation, F = F(f) x (1 - E(g)) x F(h), where F, F(f) and F(h) were the bioavailability, the fraction entering the intestinal tissue and the hepatic availability, respectively. Method 2: the E(g) of 0.0071 was calculated according to the well-stirred model equation, and was much lower than the value of 0.228. Method 3: the E(g) of 0.213 was obtained by the transport-metabolism-flow (TMF) model equation, and was much closer to the value of 0.228 obtained by the model-independent method than the E(g) of 0.0071 calculated by the well-stirred model equation. Therefore, we propose that the factor of membrane transport process be incorporated into the pharmacokinetic model for the assessment of the pre-systemic intestinal extraction ratio.     

Δ<Superscript>9</Superscript>-THC administered into the medial prefrontal cortex disrupts the spatial working memory

Rationale Δ⁹-Tetrahydrocannabinol (Δ⁹-THC) disrupts working memory. The prefrontal cortex (PFC) is involved in the processing of working memory, and its medial portion (mPFC) is part of a brain reward circuit as constituted by the mesocorticolimbic dopaminergic system.Objective This study examined the involvement of the mPFC in the effects of Δ⁹-THC on spatial working memory.Methods Ten male Wistar rats well-trained in a radial arm maze and with bilateral cannula implanted in the mPFC received Δ⁹-THC intracortically (Δ⁹-THC IC) at doses of 0 (VEH), 32, 100 or 180 μg, 5 min before a 5-s or a 1-h delayed task in order to measure a short- or long-term spatial working memory, respectively. By contrast, 11 other animals received Δ⁹-THC intraperitoneally (Δ⁹-THC IP) at doses of 0 (VEH), 0.32, 1 or 1.8 mg/kg, 30 min before a 5-s or a 1-h delayed task. Additionally, after a 15-day washout, the effect of an IP or IC pre-exposure of Δ⁹-THC was examined by repeating both dose–effect curves in a crossover order for the routes of administration.Results Δ⁹-THC IP produced significantly larger number of errors at doses of 0.32 or 1 mg/kg as compared to VEH in the 1-h post-delay performance. Δ⁹-THC 100 μg IC also produced significantly larger number of errors as compared to VEH and also to the other doses (32 or 180 μg) IC in the 1-h post-delay performance. Previous exposure to Δ⁹-THC IP or IC did not significantly affect the disruptive effect of this cannabinoid.Conclusions Δ⁹-THC administered directly in the mPFC impaired 1-h delayed task in the radial arm maze in a manner similar to that observed for its systemic administration, suggesting that the mPFC is involved in the disruptive effects of Δ⁹-THC on spatial working memory.     

Involvement of the βγ subunits of G proteins in the cAMP response induced by stimulation of the histamine H<Subscript>1</Subscript> receptor

Stimulation of the histamine H₁ receptor has been shown to enhance adenosine 3′, 5′-cyclic monophosphate (cAMP) accumulation in various cell types but, to date, the mechanism by which this occurs is still unclear. In the present study, we examined the possibility that the βγ subunits of G proteins (Gβγ) are involved in this process in cultured Chinese hamster ovary cells transfected with the human histamine H₁ receptor (CHO-H₁). Histamine increased intracellular cAMP levels in a concentration-dependent manner in CHO-H₁ cells, and this histamine action was abolished by pyrilamine (1 μM). Inhibition of histamine H₁ receptor-G_q protein coupling by stable expression of the C-terminal peptide of Gα_q protein significantly attenuated the cAMP accumulation induced by histamine. By comparison, neither BAPTA/AM (50 μM), an intracellular Ca²⁺ chelator, nor GF 109203X (1 μM), an inhibitor of protein kinase C, influenced the cAMP response. Histamine H₁ receptor-mediated cAMP accumulation was significantly inhibited by transient transfection of CHO-H₁ cells with the C-terminal peptide of β-adrenoceptor kinase I (residues 542–685), a scavenger of Gβγ. Stable expression of the C-terminal peptide of the Gα_s protein, but not treatment with pertussis toxin (200 ng/ml for 24 h), attenuated the histamine H₁ receptor-mediated cAMP accumulation. These results suggest that stimulation of histamine H₁ receptors activates adenylyl cyclase through the release of Gβγ subunits from G proteins, thereby elevating intracellular cAMP levels.     

Molecular structure of the rabbit α<Subscript>2A</Subscript>-adrenoceptor: a contribution to the α<Subscript>2A</Subscript>-adrenoceptor versus I<Subscript>1</Subscript> imidazoline receptor controversy

In view of the high structural and pharmacological similarities between the alpha(2A)-adrenoceptors of humans and other mammalian species, it has been concluded, in particular, from experiments in rabbits that the (2A)-adrenoceptor is the exclusive site of action of central antihypertensive drugs, although the amino acid sequence of the alpha(2A)-adrenoceptor of just this species was unknown. Therefore, the aim of the present investigation was to determine the complete nucleotide sequence of the coding region of the rabbit alpha(2A)-adrenoceptor gene. Degenerate oligonucleotides corresponding to regions of the alpha(2A)-adrenoceptor conserved between rat and man were used in a polymerase chain reaction with genomic DNA prepared from rabbit. A 1,356-base pair product with an open reading frame of 1,353 base pairs was obtained that encodes a protein of 451 amino acids which is similar to the alpha(2A)-adrenoceptors of other mammals (man, pig, rat, mouse, guinea-pig and cattle) but not to their alpha(2B)- and alpha(2C)-adrenoceptor subtypes suggesting its classification as an alpha(2A)-adrenoceptor. However, the degree of amino acid sequence identity is, at best, only 80% and, thus, about 10% less than between the other mammalian species. Compared with the human sequence there are 81 substantial changes of amino acids. In conclusion, rabbit and human alpha(2A)-adrenoceptors substantially differ in their amino acid sequence which may explain the opposite pharmacodynamic properties of the central antihypertensive drug rilmenidine (alpha(2)-adrenoceptor agonism and antagonism, respectively) reported in the literature. Hence, the present study supports the view that experiments with central antihypertensive drugs in rabbits are not reliably predictive for the site of action of such drugs in man.     

Aspartate138 is required for the high-affinity ligand binding site but not for the low-affinity binding site of the β<Subscript>1</Subscript>-adrenoceptor

The ₁-adrenoceptor two-site ligand binding hypothesis was investigated by comparing the pharmacological activities of the receptor with an Asp to Glu mutation of amino acid 138 after transient transfection into CHO cells. The high-affinity binding of (–)-[³H]-CGP12177 (pK_D=9.4) and binding inhibition by (–)-isoprenaline (pK_i=6.2), observed with Asp138-₁-adrenoceptors, were absent at Glu138-₁-adrenoceptors. (–)-[³H]-CGP12177 bound with a pK_D=7.6 to Glu138-₁-adrenoceptors and (–)-isoprenaline enhanced binding, probably allosterically. Glu138-₁-adrenoceptors compared with Asp138-₁-adrenoceptors showed a 500,000-fold decrease in cyclic AMP-enhancing potency by (–)-isoprenaline and antagonism by (–)-bupranolol (1 M) was abolished. At Glu138-₁-adrenoceptors, the agonist potency of (–)-CGP12177, compared with (–)-isoprenaline was reduced five-fold, but the antagonism by (–)-bupranolol (pK_B=7.1) was not significantly changed, compared with Asp138-₁-adrenoceptor. Thus, Asp138 of the ₁-adrenoceptor is essential for the binding of (–)-isoprenaline, (–)-bupranolol and (–)-CGP12177 to a high-affinity site, but not for the binding of (–)-CGP12177 and (–)-bupranolol to a low-affinity site.     

Does the Environment or the Source of the Population Define Stress Status and Energy Supply in the Freshwater Amphipod, <Emphasis Type="Italic">Gammarus fossarum</Emphasis>?

To investigate the effects of acclimation and/or adaptation on the stress protein (hsc/hsp70) response, adenylate energy charge (ACE), ATP/ADP ratio and both lipid and glycogen supply, specimens of four different populations of the freshwater amphipod Gammarus fossarum (Koch, 1835) were transplanted and exposed at sites with various levels of pollution. Induction of the stress protein response was highest in those gammarids transplanted from nearly unpolluted or just moderately polluted sites to severely polluted stream portions. The lowest hsc/hsp70 levels were found in animals transplanted from the polluted sites to the less polluted sites. In all cases the adenylic energy charge (AEC) and ATP/ADP ratio did not show any deficiency in the cellular energy supply. The amount of energy storage substrates, lipid droplets and glycogen in the hepatopancreas, the main metabolic tissue, was similar in all resident populations. In all these amphipod populations, tolerant phenotypes which had diverged genetically were not revealed; rather, the stress and recovery effects derived from the physiologically regulated, cellular stress response.     

Chemical allergens—What are the issues?

Chemical allergy describes the adverse health effects that may result when exposure to a chemical elicits an immune response. Allergy develops in two phases. In the first phase, exposure of an inherently susceptible subject results in stimulation of an immune response or immunological priming. If the then sensitised subject is exposed on a subsequent occasion to the same chemical then an accelerated and more aggressive secondary immune response will be provoked resulting in inflammation and the signs and symptoms of a clinically discernible allergic reaction. The two forms of chemical allergy of greatest relevance for occupational toxicology are skin sensitisation resulting in allergic contact dermatitis, and sensitisation of the respiratory tract associated with occupational rhinitis and asthma. In this brief survey we identify what we believe currently represent the key issues and key challenges in these areas.     

Predicting the Oxidative Metabolism of Statins: An Application of the <Emphasis Type="Italic">MetaSite</Emphasis>® Algorithm

Purpose This study was undertaken to examine the MetaSite algorithm by comparing its predictions with experimentally characterized metabolites of statins produced by cytochromes P450 (CYPs). Methods Seven statins were investigated, namely atorvastatin, cerivastatin, fluvastatin, pitavastatin and pravastatin which are (or were) used in their active hydroxy-acid form, and lovastatin and simvastatin which are used as the lactone prodrug. But given the fast lactone-hydroxy-acid equilibrium undergone by statins, both forms were investigated for each of the seven drugs. The MetaSite version 2.5.3 used here contains the homology 3D-models of CYP1A2, CYP2C19, CYP2C9, CYP2D6 and CYP3A4. In addition, we also used the crystallographic 3D-structure of human CYP2C9 and CYP3A4. To allow a better interpretation of results, the probability function P _SM i calculated by MetaSite (namely the probability of atom i to be a site of metabolism) was explicitly decomposed into its two components, namely a recognition score Ei (the accessibility of atom i) and the chemical reactivity Ri of atom i toward oxidation reactions. Results The current version of MetaSite is known to work best with prior experimental knowledge of the cytochrome(s) P450 involved. And indeed, experimentally confirmed sites of oxidation were correctly given a high priority by MetaSite. In particular 77% of correct predictions (including false positive but, as discussed, this is not necessarily a shortcoming) were obtained when considering the first five metabolites indicated by MetaSite. Conclusion To the best of our knowledge, this is the first independent report on the software. It is expected to contribute to the development of improved versions, but above all it demonstrates that the usefulness of such softwares critically depends on human experts. Electronic Supplementary Material The online version of this article (doi: ) contains supplementary material, which is available to authorized users.     

Nandrolone treatment decreases the level of rat kidney α<Subscript>1B</Subscript>-adrenoceptors

Abuse of anabolic androgenic steroids (AAS) is associated with serious side effects, such as hypertension and fluid retention. Renal ₁- and ₂-adrenoceptors are implicated in the regulation of blood pressure and fluid balance. In the present study, the levels of renal _1A-, _1B-, _2A- and _2B-adrenoceptors, and spleen _1B-adrenoceptors, were quantified in tissue membranes from rats treated with the AAS nandrolone decanoate (15 mg/kg) for 14 days. The radioligands used were [³H]-prazosin and [³H]-RX821002. The nandrolone treatment caused a 50% reduction of kidney _1B-adrenoceptors (from 15 fmol/mg protein in control rats to 6.5 fmol/mg protein in treated rats). In contrast, the levels of kidney _1A-, _2A- and _2B-, and spleen _1B-adrenoceptors were unaffected. These results raise the possibility that a decreased level of kidney _1B-adrenoceptors may cause some of the effects observed on blood pressure and fluid balance in heavy abuse of AAS.     

Accessibility of ‘essential’ alcohol in the time of COVID‐19: Casting light on the blind spots of licensing?

Among the Australian and UK governments' responses to the COVID‐19 pandemic has been the designation of outlets selling alcohol for off‐premise consumption as ‘essential’ services, allowing them to remain open while pubs, hotels and restaurants have been forced to close. In a context of restrictions on movement outside the home in both countries, and where alcohol providers are trying to find new ways to reach their customers, this may lead to an intensification of the social and health harms associated with home drinking. By examining the current situation in both Australia and the UK, we argue that heightened risks from home drinking amid COVID‐19 bring into sharp focus long‐standing weaknesses within licensing systems in both countries: the regulation of off‐premise outlets to minimise harms from drinking at home. We call for critical conversations on how licensing systems should be revised to take more responsibility for protecting people from the health and social harms associated with home drinking, both under COVID‐19 and in the future.     

Selective antagonism of the ataxic effects of zolpidem and triazolam by the GABA<Subscript>A</Subscript>/α<Subscript>1</Subscript>-preferring antagonist β-CCt in squirrel monkeys

Abstract Rationale. Delineation of the receptor mechanisms underlying the behavioral effects of benzodiazepines should allow for the development of drugs with improved clinical utility and reduced side effects. Objectives. The purpose of the present study was to investigate the role of GABA_A/α₁ receptors in the sedative and motor-impairing effects of benzodiazepines. Methods. Squirrel monkeys were tested with the GABA_A/α₁-preferring agonist zolpidem and the nonselective benzodiazepine agonist triazolam alone and in combination with the GABA_A/α₁-preferring antagonist β-CCt and the nonselective benzodiazepine antagonist flumazenil. During 30-min experimental sessions, all occurrences of normal behaviors like locomotion, environment- and self-directed behaviors, as well as side effects such as ataxia, rest and procumbent postures were scored. Results. Zolpidem and triazolam produced dose-dependent reductions in locomotion and environment-directed behavior and increased ataxia and procumbent posture. Triazolam, but not zolpidem, also engendered species-typical rest posture at some doses. Flumazenil antagonized all of the behavioral effects of zolpidem and triazolam, whereas β-CCt antagonized only zolpidem- and triazolam-induced ataxia. Conclusions. GABA_A/α₁ receptor mechanisms appear to play a key role in the ataxic effects of benzodiazepine agonists in squirrel monkeys, similar to recent results with transgenic mice. In contrast to the findings of these recent studies, GABA_A mechanisms other than or in addition to those mediated at the α₁ subunit may play a more important role in the sedative/hypnotic effects of benzodiazepines in squirrel monkeys. Electronic Publication