An overview of the safety pharmacology society strategic plan.

Safety Pharmacology studies are conducted to characterize the confidence by which biologically active new chemical entities (NCE) may be anticipated as safe. Non-clinical safety pharmacology studies aim to detect and characterize potentially undesirable pharmacodynamic activities using an array of in silico, in vitro and in vivo animal models. While a broad spectrum of methodological innovation and advancement of the science occurs within the Safety Pharmacology Society, the society also focuses on partnerships with health authorities and technology providers and facilitates interaction with organizations of common interest such as pharmacology, physiology, neuroscience, cardiology and toxicology. Education remains a primary emphasis for the society through content derived from regional and annual meetings, webinars and publication of its works it seeks to inform the general scientific and regulatory community. In considering the future of safety pharmacology the society has developed a strategy to successfully navigate forward and not be mired in stagnation of the discipline. Strategy can be defined in numerous ways but generally involves establishing and setting goals, determining what actions are needed to achieve those goals, and mobilizing resources within the society to accomplish the actions. The discipline remains in rapid evolution and its coverage is certain to expand to provide better guidance for more systems in the next few years. This overview from the Safety Pharmacology Society will outline the strategic plan from 2016 to 2018 and beyond and provide insight into the future of the discipline which builds upon a previous strategic plan established in 2009.

Suppression of hypertension during chronic reduction of brain acetylcholine in spontaneously hypertensive rats.

Experiments were conducted to determine the effects of chronic depletion of brain acetylcholine (ACh) on the development and maintenance of hypertension in spontaneously hypertensive rats (SHR). Synthesis of brain ACh was inhibited by chronic infusion of hemicholinium-3 (HC-3) into the cerebral ventricles, and systolic blood pressure was monitored by tail cuff occlusion. In 5-week-old SHR, infusion of HC-3 (0.25 micrograms/h) suppressed development of hypertension when compared to saline-infused control SHR during the 21 days of infusion (140 versus 190 mmHg on day 21). Hypothalamic and brain-stem ACh during this period was reduced by 50% and by 60-75%, respectively. In 18-week-old SHR with established hypertension, HC-3 (0.25 and 0.5 micrograms/h) reduced systolic blood pressure by 35-40 mmHg for 8 days, after which pressures returned to control hypertensive levels (191 mmHg) by day 14. The increase in blood pressure was accompanied by recovery of hypothalamic ACh levels to 75% of control. The specificity and physiological effectiveness of HC-3 was shown by its ability to inhibit the centrally mediated pressor response to physostigmine but not to oxotremorine. Infusion of HC-3 did not affect body growth, water consumption, body temperature or gross behavior. From this study, it can be concluded that brain cholinergic neurons are an important component in the development and the maintenance of hypertension in the SHR.

Urea and 2-imidazolidone derivatives of the muscarinic agents oxotremorine and N-methyl-N-(1-methyl-4-pyrrolidino-2-butynyl)acetamide.

Some urea and 2-imidazolidone analogues of the muscarinic agents oxotremorine (1) and N-methyl-N-(1-methyl-4-pyrrolidino-2-butynyl)acetamide [10; BM-5] have been synthesized and assayed for muscarinic and antimuscarinic activity on the isolated guinea pig ileum. The new compounds (15-24) were found to be muscarinic agonists, partial agonists, or antagonists. The compounds were also tested for in vitro receptor binding to homogenates of the rat cerebral cortex using the muscarinic antagonist [3H]-3-quinuclidinyl benzilate ([3H]QNB) as the ligand. They were found to be less potent than 1 in this assay. On the guinea pig ileum, the N-3-methyl substituted imidazolidone analogue 20 was the most potent agonist of the new compounds studied; 20 was 5-fold more potent in inducing contractions of the ileum and had 4-fold higher affinity for ileal muscarinic receptors than the 3-methyl substituted 2-pyrrolidone 6. However, the N-3-unsubstituted urea and imidazolidone derivatives 15 and 19 were several-fold less potent than the parent acetamide N-methyl-N-(4-pyrrolidino-2-butynyl)acetamide [9; UH-5] and 1, respectively. The urea analogue (16) of the partial muscarinic agonist 10 was devoid of intrinsic activity and displayed 3-fold lower affinity than 10 for ileal muscarinic receptors.

Dimethylsulfonium analogues of the muscarinic agent McN-A-343: [4-[[N-(3- or 4-halophenyl)carbamoyl]oxy]-2-butynyl] dimethylsulfonium perchlorates.

Some 3- and 4-bromophenyl and dimethylsulfonium analogues of the muscarinic agent [4-[[N-(3-chlorophenyl)-carbamoyl]oxy]-2-butynyl] trimethylammonium chloride (McN-A-343) (1) were synthesized. The new compounds were assayed for effects on arterial blood pressure in the pithed rat (ganglionic muscarinic activity). The dimethylsulfonium salts (13a-d) appeared to be partial agonists in relation to 1. The 4-bromophenyl-substituted trimethylammonium iodide 10d exceeded 1 in potency by 3-fold. The compounds retained the selectivity for ganglionic muscarinic receptors shown by 1 since they had only weak effects on the guinea pig ileum in vitro.

Amide, urea, and carbamate analogues of the muscarinic agent [4-[[N-(3-chlorophenyl)carbamoyl]oxy]-2-butynyl]trimethylammonium chloride.

A series of amide, urea, and carbamate analogues of the muscarinic (M1) ganglionic stimulant [4-[[N-(3-chlorophenyl)carbamoyl]oxy]-2-butynyl]trimethylammonium chloride (McN-A-343; 1) was prepared. The C1-methyl-substituted carbamates 8-11 were resolved into the enantiomers. In order to investigate the ganglionic stimulant activity and affinity of the new compounds we studied their ability to increase mean arterial blood pressure (MAP) in the pithed rat and their ability to displace the M1 receptor selective antagonist [3H]pirenzepine from rabbit sympathetic ganglia. The quaternary ammonium derivatives of 1, but not their corresponding tertiary amines, displayed ganglionic stimulant properties. The urea derivative 14 and the acetamide derivative 18 were almost equipotent to 1 as ganglionic agonists. In addition, 14 and 18 showed only 2- to 3-fold less affinity to ganglionic muscarinic receptors than 1. Introduction of a methyl group in the 1 position of the butynyl chain of 1 and its 4-chlorophenyl analogue increased ganglionic stimulant potency. The resulting (+/-)-9 and (+/-)-11 were the most potent analogues in this study. They were found to be partial agonists and showed 5- and 16-fold higher potency than 1, respectively, in increasing the MAP. They also displayed 6- and 18-fold higher affinity than 1 for ganglionic M1 receptors. The (S)-enantiomers of 9 and 11 were 1.5- and 4.9-fold more potent, respectively, than their antipodes as ganglionic muscarinic stimulants. The C1-methyl-substituted urea and acetamide derivatives (15 and 19) were 1.5- and 3-fold less potent than 1 and displayed several-fold lower affinity for ganglionic M1 receptors. The new quaternary analogues retained the selectivity for ganglionic muscarinic receptors since they produced weak partial agonist effects on the guinea pig ileum and showed several-fold lower nicotinic activity than 1 in the frog rectus abdominis assay.

Phenyl-substituted analogues of oxotremorine as muscarinic antagonists.

A series of phenyl-substituted analogues of the muscarinic agent oxotremorine (1) have been prepared. The new compounds (3b-11b and 9c) were assayed for antimuscarinic activity on the isolated guinea pig ileum and in intact mice. They were also evaluated for ability to inhibit the binding of the muscarinic antagonist (-)-[3H]-N-methylscopolamine to homogenates of the rat cerebral cortex. The phenyl-substituted derivatives were devoid of intrinsic muscarinic activity. Instead, they behaved as competitive muscarinic antagonists in these assays with similar or lower affinity for muscarinic receptors than the corresponding methyl-substituted analogues. The succinimide (8b) and the pyrrolidone (3b) derivatives of 1 substituted with a phenyl group at position 1 of the butynyl chain showed the highest antimuscarinic potency with dissociation constants (KD) of 0.10 and 0.20 microM, respectively, in the ileum assay. The phenyl-substituted analogues showed an approximately 10-fold lower in vivo antimuscarinic potency than their corresponding methyl-substituted positional isomers. A correlation was observed between in vitro and in vivo potency within subsets consisting of methyl- and phenyl-substituted derivatives.

Resolved pyrrolidine, piperidine, and perhydroazepine analogues of the muscarinic agent N-methyl-N-(1-methyl-4-pyrrolidino-2-butynyl)acetamide.

A series of conformationally restricted analogues of the partial muscarinic agonist N-methyl-N-(1-methyl-4-pyrrolidino-2-butynyl)acetamide (BM 5; 1) was synthesized. Three of the racemic derivatives were resolved into the enantiomers. The compounds were investigated for muscarinic and antimuscarinic activity in the isolated guinea pig ileum. They were found to be fairly potent muscarinic antagonists or weak partial agonists. The new compounds were either equally or less potent than 1 in inhibiting (-)-[3H]-N-methylscopolamine binding in homogenates of the rat cerebral cortex. Thus, structural modifications to 1 in which the amide moiety and the methyl group in the butynyl chain have been joined to form a six- or seven-membered ring preserve affinity but abolish efficacy. The R enantiomers were found to have 14-79 times higher affinity to ileal muscarinic receptors than the respective antipodes. The enantiomeric affinity ratios were nearly identical in both preparations studied. As suggested by molecular mechanics calculations, the difference in affinity between the five-membered and the six- and seven-membered ring analogues may be rationalized in conformational terms.

Alpha 2-adrenoceptor antagonists potentiate acetylcholinesterase inhibitor effects on passive avoidance learning in the rat.

The cholinergic hypothesis of Alzheimer's disease (AD) has strongly influenced research on learning and memory over the last decade. However, there has been limited success treating AD dementia with cholinomimetics. Furthermore, there are indications that other neurotransmitter systems affected by this disease may be involved in cognitive processes. Animal studies have suggested that norepinephrine and acetylcholine may interact in learning and memory. The current experiments investigate this interaction in a step-down passive avoidance paradigm after coadministration of acetylcholinesterase inhibitors and alpha 2-adrenoceptor antagonists. Administration of acetylcholinesterase inhibitors heptylphysostigmine (0.625-5.0 mg/kg, IP), tacrine (2.5-10.0 mg/kg, PO), velnacrine (0.312-2.5 mg/kg, SC), and galanthamine (0.312-2.5 mg/kg IP) each enhanced retention of a passive avoidance response at selected moderate doses administered 30-60 min prior to training. The alpha 2-adrenoceptor antagonists idazoxan (0.312-2.5 mg/kg, IP), yohimbine (0.078-0.312 mg/kg, IP) and P86 7480 (0.156-0.625 mg/kg, IP) alone failed to enhance learning in this paradigm. Coadministration of a subthreshold dose of heptylphysostigmine (0.625 mg/kg, IP) with doses of idazoxan, yohimbine or P86 7480 enhanced passive avoidance learning. This synergistic interaction may represent effects of antagonism of presynaptic alpha 2-adrenoceptor since coadministration of heptylphysostigmine and the selective postsynaptic alpha 2-adrenoceptor antagonist SKF 104856 did not result in enhanced learning. Taken together these data suggest noradrenergic activation through pre-synaptic alpha 2-adrenoceptor blockade may potentiate cholinergic activity in the formation of a long-term memory trace. These observations may have implications for the treatment of AD with cholinergic and adrenergic agents.

Intracellular calcium release in N1E-115 neuroblastoma cells is mediated by the M1 muscarinic receptor subtype and is antagonized by McN-A-343.

Experiments using muscarinic receptor antagonists were done to determine which muscarinic receptor subtypes(s) mediate carbachol-evoked calcium release in N1E-115 cells. McN-A-343 and a new analog, (+/-)BN228, were weak antagonists and neither compound caused release on its own. The rank order of potency was 4-DAMP greater than pirenzepine greater than AFDX116 greater than (+/-)BN228 and McN-A-343. This profile, pirenzepine's high potency (19-fold greater than AFDX116) and its IC50 of 31 nM suggest that calcium release in this neuronal cell line is mediated by the M1 muscarinic receptor subtype.

Binding affinity and antimuscarinic activity of sigma and phencyclidine receptor ligands.

The characterization of the sigma receptor has been hampered by the lack of a functional bioassay system. Drugs that bind to sigma receptors have been reported to inhibit carbachol-induced phosphatidylinositol turnover in rat brain; however, these drugs might directly affect muscarinic acetylcholine receptors. The purpose of the present study was to determine the affinity for muscarinic receptors and the antimuscarinic activity of sigma and phencyclidine receptor ligands. All of the drugs tested inhibited the binding of [3H]N-methylscopolamine to guinea pig cerebral cortical membranes with KI values in the micromolar range and also inhibited carbachol-induced contractions in the guinea pig ileum. These results demonstrate that these compounds have substantial antimuscarinic activity which might limit the use of the inhibition of carbachol-induced phosphatidylinositol turnover as a functional assay system for studying sigma ligands. Furthermore, this antimuscarinic activity must be considered when evaluating the effects of these compounds after in vivo administration.

Vascular alpha 1D-adrenoceptors have a role in the pressor response to phenylephrine in the pithed rat.

In rats, the pressor response to intravenous (i.v.) phenylephrine is mediated by vascular alpha 1A- and alpha 1B-adrenoceptors, but the role of alpha 1D-adrenoceptors is uncertain. These studies evaluated the effect of a selective alpha 1D-adrenoceptor antagonist, BMY 7378 (8-[2-[4-(2- methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspirol[4,5]decane-7,9-dio ne 2HCl), on the pressor effect to i.v. phenylephrine (alpha 1A/B/D-adrenoceptor agonist) and (R)A-61603 (alpha 1A-adrenoceptor agonist; N-[5-(4,5-dihydro-1 H-imidazol-2yl)-2-hydroxy-5,6,7,8-tetrahydronaphthalen-l -yl] methanesulfonamide HBr) in pithed rats. Pretreatment with BMY 7378 (0.1-1 mg/kg) competitively antagonized the phenylephrine pressor response, but not the (R)A-61603 pressor curve. At 10 mg/kg, BMY 7378 antagonized the (R)A-61603 response, indicating the non-selective blockade of alpha 1A-adrenoceptors. These findings demonstrate that i.v. phenylephrine can activate vascular alpha 1D-adrenoceptors in the pithed rat.

Physiological release of nitric oxide is dependent on the level of vascular tone.

The pressor effect of NG-methyl-L-arginine (NMA) was tested in urethane-anesthetized rats which were untreated (control) or devoid of sympathetic tone. In contrast with controls, the NMA response was attenuated by pithing or ganglionic blockade. In pithed rats, the induction of moderate or intense vasoconstriction with constant phenylephrine infusion restored or augmented, respectively, the NMA pressor response. Our data suggest that vascular tone may physiologically regulate the release of nitric oxide in vivo.

Vascular alpha-1 adrenergic receptor subtypes in the regulation of arterial pressure.

Alpha 1 (alpha 1)-adrenoceptors can be found at numerous end organs in the autonomic nervous system, especially vascular smooth muscle. The tonic sympathetic activation of vascular alpha 1-adrenoceptors maintains vascular resistance and is vital to the regulation of arterial pressure. Recent evidence clearly demonstrates that alpha 1-adrenoceptors are a heterogenous class of receptors and that each subtype may subserve specific cardiovascular functions. Elucidation of the physiological role of each subtype in the regulation of vascular resistance and arterial pressure will enhance our understanding of the cardiovascular system and may facilitate the development of therapeutics with improved efficacy and tolerability.

Antimuscarinic potency and functional selectivity of oxotremorine analogs at muscarinic receptor subtypes in the rat.

The potency of six antimuscarinic oxotremorine analogs at sympathetic ganglionic M1 and brainstem M2 muscarinic receptors was compared in the rat. Inhibition of the pressor effects of McNA343 or physostigmine was used as functional indicators of M1 and M2 blockade, respectively. 50% inhibitory doses (ID50) were calculated against both cholinomimetics. Of the analogs, PCA-10 was the most potent, with ID50 values of 0.16 and 0.11 mumol/kg versus McNA343 and physostigmine, respectively. A correlation analysis indicated that these analogs did not functionally discriminate between responses mediated by neuronal M1 or M2 muscarinic receptors in vivo. In contrast, these analogs antagonized M1 ganglionic responses at doses which produced negligible antagonism at cardiac and vascular M2 receptors.

Elevation of cerebrospinal fluid choline levels by nicotinamide involves the enzymatic formation of N1-methylnicotinamide in brain tissue.

Nicotinamide administration can elevate plasma and brain choline levels and produce a marginal increase in striatal acetylcholine levels in the rat. We now report that subcutaneous nicotinamide produces a substantial and long-lasting rise in cisternal cerebrospinal fluid (CSF) levels of choline in free-moving rats, possibly through the enzymatic formation of N1-methylnicotinamide (NMN) in brain. CSF choline levels peaked 2 hours after nicotinamide administration and were accompanied by increases in striatal, cortical, hippocampal and plasma choline levels. The enzymatic formation of [3H]NMN in rat brain was evaluated by incubating aliquots of rat brain cytosol with unlabelled nicotinamide and the methyl donor [3H]S-adenosylmethionine. High performance liquid chromatography and radiochemical detection demonstrated that [3H]NMN was specifically formed by a brain cytosolic enzyme. The production of [3H]NMN was dependent on exogenous nicotinamide and could be prevented by denaturing the cytosol. The metabolism of nicotinamide to NMN in rat brain may explain the rise in CSF choline levels since NMN, a quaternary amine, can inhibit choline transport at the choroid villus and reduce choline clearance.

Detection of QTc interval prolongation using jacket telemetry in conscious non-human primates: comparison with implanted telemetry.

BACKGROUND AND PURPOSE: During repeat-dose toxicity studies, ECGs are collected from chemically or physically-restrained animals over a short timeframe. This is problematic due to cardiovascular changes caused by manual restraint stress and anesthesia, and limited ECG sampling. These factors confound data interpretation, but may be overcome by using a non-invasive jacket-based ECG collection (JET). The current study investigated whether a jacketed external telemetry system could detect changes in cardiac intervals and heart rate in non-human primates (NHPs), previously implanted with a PCT transmitter. EXPERIMENTAL APPROACH: Twelve male cynomolgus monkeys were treated weekly with vehicle or sotalol (8, 16, 32 mg kg⁻¹) p.o. ECGs were collected continuously for 24 hours, following treatment, over 4 weeks. A satellite group of six NHPs was used for sotalol toxicokinetics. KEY RESULTS: Sotalol attained Cmax values 1-3 hours after dosing, and exhibited dose-proportional exposure. In jacketed NHPs, sotalol dose-dependently increased QT/QTc intervals, prolonged PR interval, and reduced heart rate. Significant QTc prolongation of 27, 54 and 76 msec was detected by JET after 8, 16, and 32 mg kg⁻¹ sotalol, respectively, compared with time-matched vehicle-treated animals. Overall, JET-derived PR, QT, QTc intervals, QRS duration, and heart rate correlated well with those derived from PCT. CONCLUSIONS AND IMPLICATIONS: The current findings clearly support the use of JET to quantify cardiac interval and rhythm changes, capable of detecting QTc prolongation caused by sotalol. JET may be a preferred method compared to restraint-based ECG because high-density ECG sampling can be collected in unstressed conscious monkeys, over several weeks.

Centrally active antimuscarinic analogs of oxotremorine selectively block physostigmine-induced hypertension, but not peripheral muscarinic vasodepression.

Some tertiary antimuscarinic amines related to oxotremorine were compared with atropine and scopolamine for their ability to block physostigmine-induced hypertension and acetylcholine-induced hypotension in rats. These cardiovascular responses are mediated via muscarinic receptors in the brainstem and on the vasculature, respectively. In urethane-anesthetized rats, physostigmine (77 nmol/kg i.v.) increased mean arterial pressure 40 +/- 5 mm Hg. One hour later, each rat received a single dose of antimuscarinic, then a second bolus of physostigmine 10 min later. Linear regression analysis of antagonist dose-percent inhibition curves showed that all the agents inhibited physostigmine's pressor effect, thus indicating an ability to antagonize brain muscarinic receptors. BM-5 and BoK-1 were estimated to be equipotent with atropine, all having 50% inhibitory doses (ID50) in the 1.5 to 1.8 mumol/kg range. BR-370 and DKJ-21 were 3- and 30-fold less potent, respectively, than atropine in this assay, while scopolamine was the most potent (ID50: 0.039 mumol/kg). In contrast with atropine and scopolamine, the oxotremorine analogs possessed selective central antimuscarinic effects and were completely unable to antagonize the peripheral depressor response. Like atropine, only BM-5 showed significant tachycardiac effects, which suggested that this agent possessed affinity for myocardial muscarinic receptors. These findings support the concept that congeners of oxotremorine behave as centrally selective antimuscarinics at doses which have low peripheral antimuscarinic activity in vivo. This unique pharmacological profile makes them novel probes to study central cholinergic muscarinic mechanisms in cardiovascular regulation.

Role of vascular alpha-1 adrenoceptor subtypes in the pressor response to sympathetic nerve stimulation in the pithed rat.

Previous studies suggest that systemic arterial pressure is tonically regulated by the interaction of peripheral sympathetic nerves with vascular alpha-1A adrenoceptors in vivo. To explore this relationship further, the present study examined the inhibitory effect of selective alpha-1A [5-methylurapidil (5-MU) and nifedipine (NIF)] and alpha-1B [chloroethylclonidine (CEC)] antagonists on the pressor response to electrical stimulation (ES) of the spinal cord in pithed rats. Diastolic pressure changes were measured in the presence of 5-MU or CEC and compared with control responses. Pretreatment with 5-MU (0.5 mg/kg i.v.) significantly suppressed the ES pressor response (50-80% inhibition) at all stimulation frequencies. Likewise, NIF (inhibitor of calcium influx associated with alpha-1A adrenoceptor activation) selectively inhibited the pressor response to ES to the same degree as did 5-MU. CEC (25 mg/kg i.v.) also significantly shifted the ES response curve; however, this effect was mediated by activation of presynaptic alpha-2 receptors on sympathetic terminals because prior administration of idazoxan (5 mg/kg) prevented the inhibitory effect of CEC. Based on the potent inhibitory effects of 5-MU and NIF on the ES pressor response in the pithed rat, it was concluded that vascular alpha-1A adrenoceptors reside in the synaptic region of neurovascular junction where they are primarily activated by neuronal norepinephrine release.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of the inhibitory potencies of N(G)-methyl-, N(G)-nitro- and N(G)-amino-L-arginine on EDRF function in the rat: evidence for continuous basal EDRF release.

The relative potencies of the argininolytic agents NG-methyl-L-arginine (L-NMA), NG-nitro-L-arginine (L-NNA) and NG-amino-L-arginine (L-NAA) were assayed by their inhibitory effect on both basal and stimulated release of endothelium-derived NO in vitro and in vivo. Basal NO release was indirectly assessed by the ability of the analogs to contract phenylephrine-preconstricted rat aortic rings and their ability to produce a hypertensive response in awake, unanesthetized normotensive rats. In aortic rings, the three analogs induced vasocontraction and inhibited the vasorelaxation mediated by ACh-stimulated endothelial NO release. In this latter assay, L-NNA was 30 times more potent than either L-NMA or L-NAA. In free-moving rats, the agents caused dose-dependent increases in arterial pressure due to the blockade of endogenous NO formation. Dose-response analysis indicated that L-NNA was 87 and 230 times more potent than L-NMA and L-NAA, respectively. Pretreatment with L-NNA was also found to selectively inhibit, but not abolish, the depressor effects of acetylcholine in unanesthetized and phenylephrine- or vasopressin-infused normotensive-pithed rats. These studies indicate that L-NNA is a potent antagonist of endothelium-derived relaxing factor formation in vitro and in vivo. The contractile and hypertensive effects of the argininolytic agents clearly demonstrates that a continuous basal release of endothelium-derived relaxing factor/NO occurs in both isolated vascular rings and whole animals.