Stability in Plasmas of Various Species of HPMA Copolymer–PGE<Subscript>1</Subscript> Conjugates

Purpose To determine the stability of HPMA copolymer–prostaglandin E₁ (PGE₁) conjugates in plasmas of different species and to identify the enzymes responsible for the cleavage of the ester bond. Methods The conjugates were incubated in human, rat, and mouse plasma at 37°C in the presence and absence of specific esterase inhibitors. The released PGE₁ was analyzed using an HPLC assay. To evaluate the effect of the conformation of the conjugate on the rate of PGE₁ release, its structure was modified by the attachment of hydrophobic side chains. Results The rate of PGE₁ release was strongly species dependent. Whereas the conjugate was stable in human plasma, the PGE₁ release in rat or mouse plasma was substantial. In rat plasma, the ester bond cleavage was mainly catalyzed by butyrylcholinesterase; in mouse plasma, in addition to butyrylcholinesterase, carboxylesterase also contributed to the cleavage. The formation of compact polymer coils stabilized the ester bond. Conclusions HPMA copolymer–PGE₁ conjugates are strong candidates as novel therapeutics for the treatment of osteoporosis. The observed species differences in plasma stability of ester bonds are of importance, because the ovariectomized rat model is recommended by the FDA for pre-clinical evaluation.     

Comparison of the subjective effects of Δ<Superscript>9</Superscript>-tetrahydrocannabinol and marijuana in humans

RATIONALE: There has been controversy about whether the subjective, behavioral or therapeutic effects of whole plant marijuana differ from the effects of its primary active ingredient, Delta(9)-tetrahydrocannabinol (THC). However, few studies have directly compared the effects of marijuana and THC using matched doses administered either by the smoked or the oral form.OBJECTIVE: Two studies were conducted to compare the subjective effects of pure THC to whole-plant marijuana containing an equivalent amount of THC in normal healthy volunteers. In one study the drugs were administered orally and in the other they were administered by smoking.METHODS: In each study, marijuana users (oral study: n=12, smoking study: n=13) participated in a double-blind, crossover design with five experimental conditions: a low and a high dose of THC-only, a low and a high dose of whole-plant marijuana, and placebo. In the oral study, the drugs were administered in brownies, in the smoking study the drugs were smoked. Dependent measures included the Addiction Research Center Inventory, the Profile of Mood States, visual analog items, vital signs, and plasma levels of THC and 11-nor-9-carboxy-THC.RESULTS: In both studies, the active drug conditions resulted in dose-dependent increases in plasma THC levels, and the levels of THC were similar in THC-only and marijuana conditions (except that at the higher oral dose THC-only produced slightly higher levels than marijuana). In both the oral study and the smoking study, THC-only and whole plant marijuana produced similar subjective effects, with only minor differences.CONCLUSION: These results support the idea that the psychoactive effects of marijuana in healthy volunteers are due primarily to THC.     

Role of β<Subscript>3</Subscript>-adrenoceptors in regulation of retinal vascular tone in rats

The aim of this study was to determine the role of β₃-adrenoceptors in the action of endogenous catecholamines (adrenaline and noradrenaline) on rat retinal arterioles in vivo. Using an original high-resolution digital fundus camera, the rat ocular fundus images were captured. The diameter of retinal arterioles contained in the images was measured. Both systemic blood pressure and heart rate were recorded continuously. Adrenaline (0.3–5.0 μg/kg/min, i.v.) increased the diameter of retinal arterioles, mean blood pressure and heart rate in a dose-dependent manner. Under blockade of β₁/β₂-adrenoceptors with propranolol (2 mg/kg, i.v. bolus followed by 100 μg/kg/min infusion), adrenaline decreased the diameter of retinal arterioles. Similar observation was made under treatment with the β₃-adrenoceptor antagonist L-748337 (50 μg/kg, i.v.). The pressor response to adrenaline was enhanced by propranolol, but not by L-748337. The positive chronotropic action of adrenaline was markedly prevented by propranolol, whereas it was unaffected by L-748337. Noradrenaline (0.03–1.0 μg/kg/min, i.v.) decreased the diameter of retinal arterioles but increased the mean blood pressure and heart rate. The effects of noradrenaline on retinal arteriolar diameter and blood pressure were unaffected by propranolol or L-748337. The positive chronotropic action of noradrenaline was almost completely abolished by propranolol. These results suggest that β₃-adrenoceptors play crucial roles in vasodilator responses to adrenaline of retinal arterioles but have minor or no effect on noradrenaline-induced responses. The results also indicate that the functional role of β₃-adrenoceptors may be more important than that in peripheral resistance vessels.     

Discriminative stimulus effects of zolpidem in squirrel monkeys: role of GABA<Subscript>A</Subscript>/α<Subscript>1</Subscript> receptors

Abstract Rationale. The discriminative stimulus effects of zolpidem in squirrel monkeys trained at doses greater than or equal to 3.0 mg/kg differ from those of conventional benzodiazepines (BZs), but the extent to which these effects reflect the selectivity of zolpidem for GABA_A/α₁ receptors is not known. Objectives. The present study investigated the ability of GABA_A/α₁-preferring agonists to substitute for training doses of zolpidem greater than or equal to 3.0 mg/kg and the ability of GABA_A/α₁-preferring antagonists to block zolpidem's discriminative stimulus effects. Methods. Squirrel monkeys were trained to discriminate intravenous injections of zolpidem (3.0 or 5.6 mg/kg) from saline and tested with BZ agonists differing in selectivity and efficacy at GABA_A/α₁ receptors. Antagonism of the effects of zolpidem was studied using the GABA_A/α₁-preferring antagonists β-carboline-3-carboxylate-t-butyl ester (β-CCT) and 3-propyloxy-β-carboline (3-PBC). Results. Zolpidem and quazepam (GABA_A/α₁-preferring agonist) engendered full substitution for zolpidem, whereas CL 218,872 (GABA_A/α₁-preferring partial agonist) and the non-selective BZ agonists alprazolam and flunitrazepam engendered low and variable levels of zolpidem-lever responding (35–58%). Both β-CCT and 3-PBC antagonized the discriminative stimulus effects of zolpidem in a surmountable fashion. Conclusions. Our findings provide evidence for a key role of GABA_A/α₁ receptors in the discriminative stimulus effects of zolpidem at relatively high training doses, and suggest that selectivity and relatively high efficacy at GABA_A/α₁ receptors is required for BZ agonists to reproduce these discriminative stimulus effects. Electronic Publication     

Cross-talk between β<Subscript>1</Subscript>-adrenoceptors and ET<Subscript>A</Subscript> receptors in modulation of the slow component of delayed rectifier K<Superscript>+</Superscript> currents

Delayed rectifier K⁺ currents (I_K) play a critical role in determining cardiac action potential duration (APD). Modulation of I_K affects cardiac excitability critically. There are three components of cardiac delayed rectifier, and the slowly activating component (I_Ks) is influenced strongly by a variety of stimuli. Plasma levels of noradrenaline and endothelin are elevated in heart failure, and arrhythmias are promoted by such humoral abnormalities through modulation of ion channels. It has been reported that protein kinase A (PKA) and protein kinase C (PKC) modulate I_Ks from human minK in a complex manner. In the present study, we coexpressed human minK with the human ₁-adrenoceptor (h₁AR) and the endothelin receptor subtype A (hET_AR) in Xenopus oocytes and investigated the effects of receptor activation on the currents (I_Ks) flowing through the oocytes. ET-1 modulated I_Ks biphasically: a transient increase followed by a decrease. The PKC inhibitor chelerythrine completely inhibited the effects of ET-1. Intracellular EGTA abolished the transient increase by ET-1 and partially inhibited the subsequent decrease in the currents. When I_Ks was increased by 10^–6 M isoproterenol (ISO), ET-1 did not increase but rather decreased the current to an even greater extent than under control conditions. In addition, the effects of ISO on I_Ks were suppressed by ET_AR stimulation. These data indicate that I_Ks can be regulated by cross-talk between the ET_AR and ₁AR systems in addition to direct regulation by each receptor system.     

Indirect role of α<Subscript>2</Subscript>-adrenoreceptors in anti-ulcer effect mechanism of nimesulide in rats

Nimesulide, a non-steroidal, anti-inflammatory drug, produces ulcerogenic effects in adrenalectomized rats but is gastro-protective in intact rats. The objective of this study was to determine whether adrenal gland hormones are involved in the anti-ulcer effects of nimesulide. The results revealed that 100 mg/kg nimesulide produces gastric ulceration in adrenalectomized rats, which is prevented by prednisolone and adrenaline. The anti-ulcer effects of adrenaline and prednisolone in adrenalectomized rats were in turn antagonized by yohimbine, a selective α₂-receptor blocker, but not by doxazosine (α₁-receptor blocker) or propranolol (β-blocker). Adrenaline prevented the formation of indomethacin-induced ulcers in both adrenalectomized and intact rats, but prednisolone increased the indomethacin-induced ulcerous area in intact rats, whereas it decreased the size of the ulcers in adrenalectomized rats. In addition, prednisolone prevented ulcer formation in intact rats in which the adrenaline concentration had been decreased by metyrosine. These results suggest that glucocorticoids are anti-ulcerogenic in not only adrenalectomized rats but also in intact rats with diminished circulating levels of adrenaline. In the light of these data, the effect of nimesulide on plasma adrenaline concentrations was studied. In comparison to the adrenaline levels found in intact control rats, the administration of nimesulide at doses of 10, 20, 40 and 100 mg/kg decreased adrenaline concentrations by 12.8, 22.6, 30.4, and 58.2%, respectively, without affecting blood corticosterone concentrations. The anti-ulcer effect of nimesulide was observed to be dose-dependent, and the strength of this effect was directly correlated the decreasing concentration of adrenaline. The concentration of adrenaline was decreased by 60.9% in rats treated with 300 mg/kg metyrosine in which prednisolone produced anti-ulcer effects. In summary, we have shown that nimesulide produces its anti-ulcer effect by decreasing endogenous adrenaline concentrations and that glucocorticoids may induce anti-ulcer effects via α₂-adrenoreceptors, but not via their own receptors. This research was conducted in the Laboratory of Pharmacology at Ataturk University, Faculty of Medicine, Department of Pharmacology, 25240 Erzurum/Turkey.     

Effect of the blockade of μ<Subscript>1</Subscript>-opioid and 5HT<Subscript>2A</Subscript>-serotonergic/α<Subscript>1</Subscript>-noradrenergic receptors on sweet-substance-induced analgesia

Rationale Sweet-substance-induced analgesia has been widely studied, and the investigation of the neurotransmitters involved in this antinociceptive process is an important way for understanding the involvement of the neural system controlling this kind of antinociception.Objective The aim of this study was to investigate the involvement of opioid and monoaminergic systems in sweet-substance-induced analgesia.Methods The present work was carried out in an animal model with the aim of investigating whether acute (24 h) or chronic (14 days) intake of a sweet substance, such as sucrose (250 g/l), is followed by antinociception. Tail withdrawal latencies in the tail-flick test were measured before and immediately after this treatment. Immediately after the recording of baseline values, independent groups of rats were submitted to sucrose or tap-water intake and, after chronic treatment, they were pretreated with intraperitoneal administration of (1) naltrexone at 0.5, 1, 2 or 3 mg/kg; (2) naloxonazine at 5, 10, 20 or 30 mg/kg; (3) methysergide at 0.5, 1, 2 or 3 mg/kg; (4) ketanserin at 0.5, 1, 2 or 3 mg/kg; or (5) physiological saline.Results Naltrexone and methysergide at two major doses decreased sweet-substance-induced analgesia after chronic intake of a sweet substance. These effects were corroborated by peripheral administration of naloxonazine and ketanserin.Conclusions These data give further evidence for: (a) the involvement of endogenous opioids and a ₁-opioid receptor in the sweet-substance-induced antinociception; (b) the involvement of monoamines and 5HT_2A serotonergic/₁-noradrenergic receptors in the central regulation of the sweet-substance-produced analgesia.     

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.     

In vitro and in vivo safety evaluation of Nephure™

Nephure™ is a proprietary oxalate decarboxylase (OxDC) enzyme being developed as a food ingredient. In this study, the safety of Nephure™ was evaluated in a bacterial mutagenicity assay and in a sub-chronic (13-week) oral toxicity study in rats. Nephure™ did not show any mutagenic properties in the mutagenicity assay. In the 13-week sub-chronic oral toxicity study in which 10 Sprague Dawley rats per sex were administered 0, 118, 235 and 475 mg/kg bw/day (8260, 16450 and 33,250 Units/kg bw/day, respectively) of Nephure™ by gavage, male and female rats did not show any test article-related clinical observations or effects on body weight, body weight gain, food consumption, food efficiency, ophthalmology, functional observational battery parameters or motor activity. Furthermore, there were no changes in coagulation, clinical chemistry, urinalysis or hematology parameters, macroscopic/microscopic findings or organ weights that could be attributed to the test article. Based on these results, Nephure™ was not mutagenic and the no-adverse-effect level (NOAEL) in the 13-week study was determined to be 475 mg/kg bw/day (33,250 Units/kg bw/day). Evaluation of the estimated consumption of Nephure™, generation of the metabolite formate, and the current safety studies resulted in a conclusion of a tolerable upper limit of 3450 Units of OxDC activity/day (57.5 Units activity/kg bw/day), when Nephure™ is added to food to decrease dietary oxalate.     

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.     

Synthesis and anti-HIV activity of triterpene conjugates of α-<Emphasis Type="Italic">d</Emphasis>-glucosamine

This report describes new triterpene conjugates of α-D-glucosamine, i.e. modified glycyrrhizic acid (GA) analogs containing 18,19-dehydroglycyrrhetic acid 3-O-hemisuccinate and maleate and 11-deoxyglycyrrhetic acid 3-O-hemiphthalate fragments synthesized using N,N′-dicyclohexylcarbodiimide-N-hydroxybenzotriazole. 3-O-[3-(N-2-deoxy-α-D-glucopyranos-2-yl)-carbamoyl]-phthaloyl-11-deoxyglycyrrhetic acid was found to have marked anti-HIV activity (the CD₅₀ (50% cytotoxic concentration) was 150 μg/ml, the ID₅₀ (50% effective concentration) was 1.5 μg/ml, and the index of selectivity (IS, IC₅₀/ID₅₀) was 100) andwas more active than GA in terms of IS (IS = 9.6). __________ Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 42, No. 2, pp. 14–17, February, 2008.     

Mechanism-Based Pharmacokinetic–Pharmacodynamic Modeling of the Dopamine D<Subscript>2</Subscript> Receptor Occupancy of Olanzapine in Rats

Purpose  

A mechanism-based PK-PD model was developed to predict the time course of dopamine D₂ receptor occupancy (D₂RO) in rat striatum following administration of olanzapine, an atypical antipsychotic drug.     

Comparison of the α-adrenoceptor-mediated effects of β<Subscript>3</Subscript>-adrenoceptor ligands in rat pulmonary artery

We have recently shown that the -adrenoceptor ligands CGP 12177, bupranolol, and SR 59230A (aryloxypropanolamines), but not BRL 37344 and CL 316243 (phenylethanolamines), exhibit significant affinity for ₁-adrenoceptors and that CGP 12177 displays partial agonist properties at -adrenoceptors in rat pulmonary artery. In this study, bupranolol and SR 59230A were further evaluated for their potential -adrenoceptor mediated effects (i.e., agonist and/or antagonist properties) in rat intralobar pulmonary artery and compared with BRL 37344 and CL 316243. Bupranolol induced a relaxation in phenylephrine-precontracted arteries, but had no effect in prostaglandin -precontracted ones. SR 59230A also elicited a relaxation in phenylephrine-precontracted arteries. In -precontracted arteries, SR 59230A induced a contractile response that was insensitive to the irreversible -adrenoceptor antagonist phenoxybenzamine. BRL 37344 at high concentrations, but not CL 316243, produced slight relaxation in both phenylephrine- and -precontracted arteries. The contractile response to phenylephrine was antagonized by bupranolol and SR 59230A in a competitive manner (pA₂: 6.38 and 7.08 respectively). The concentration–response curve to phenylephrine was also shifted to the right by BRL 37344 (mean pK_b: 4.45), but not by CL 316243 (100 M). This study indicates that the aryloxypropanolamine derivatives bupranolol and SR 59230A exhibit competitive antagonist, but no agonist properties on ₁-adrenoceptors, SR 59230A also inducing -adrenoceptor-independent contraction. Among the phenylethanolamines, BRL 37344 but not CL 316243, also exerts an antagonist effect on ₁-adrenoceptors, with a much lower potency than the aryloxypropanolamines studied.     

The Role of the Acidity of N-Heteroaryl Sulfonamides as Inhibitors of Bcl-2 Family Protein–Protein Interactions

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Stimulatory effect of Coca-Cola<Superscript>TM</Superscript> on gastroduodenal HCO<Stack><Subscript>3</Subscript><Superscript>−</Superscript></Stack> secretion in rats

We examined the effect of various carbonated beverages, especially Coca-Cola^TM, on the HCO₃⁻ secretion in the rat stomach and duodenum. Under urethane anaesthesia, a chambered stomach or a proximal duodenal loop was perfused with saline, and HCO₃⁻ secretion was measured at pH 7.0 using a pH-stat method and by adding 2 mM HCl. The amount of CO₂ contained in these beverages was about 4–7 g/mL. Coca-Cola^TM topically applied to the mucosa for 10 min significantly increased the HCO₃⁻ secretion in both the stomach and the duodenum. The HCO₃⁻ response in the duodenum was totally abolished by indomethacin and also partially inhibited by acetazolamide, an inhibitor of carbonic anhydrase. Likewise, the response in the stomach was also markedly inhibited by either acetazolamide or indomethacin. The mucosal application of Coca-Cola^TM increased the PGE₂ contents in both the stomach and the duodenum. Other carbonated beverages, such as sparkling water, Fanta Grape^TM or cider, also increased the HCO₃⁻ secretion in these tissues. These results suggest that Coca-Cola^TM induces HCO₃⁻ secretion in both the stomach and the duodenum, and these responses may be attributable to both the intracellular supply of HCO₃⁻ generated via carbonic anhydrase, and endogenous PGs, probably related to the acidic pH of the solution. Received 4 August 2006; accepted 10 November 2006     

Attenuation of <Emphasis Type="Italic">d</Emphasis>-amphetamine-induced disruption of conditional discrimination performance by α-flupenthixol

Rationale Previous evidence suggests that manipulation of forebrain dopamine (DA) systems may impair the use of conditional information to inform goal-directed performance, and this may be related to impairments in the ability to use task-setting cues in schizophrenia.Objective To investigate, using the indirect DA agonist d-amphetamine and the D₁/D₂ receptor antagonist -flupenthixol, the influence of DAergic manipulation on discrimination performance that requires the use of conditional information to inform goal-directed performance.Methods Both instrumental and Pavlovian conditional discriminations were employed in which rats learned to respond appropriately according to the presence of auditory conditional stimuli, and results from these experiments were contrasted with a control Pavlovian-instrumental transfer task.Results Experiment 1 showed a disruption of instrumental conditional discrimination performance by d-amphetamine at 1.5 mg/kg and attenuation of correct responding following 1.0 mg/kg. Disruption with both doses was observed in experiment 2 using a conditional discrimination based on Pavlovian, conditioned responding. Results from a control Pavlovian-instrumental transfer task (experiment 3) revealed that d-amphetamine (0.5, 1.0 and 1.5 mg/kg) did not have any detrimental effect on subjects basic sensory, motor or motivational processes. Experiment 4 showed that d-amphetamine disruption of instrumental conditional discrimination was attenuated by pre-treatment with the D₁/D₂ receptor antagonist -flupenthixol.Conclusion These results demonstrate that tasks dependent on conditional relationships are highly sensitive to manipulation of DAergic systems.