Simulation of alcohol action upon a detailed Purkinje neuron model and a simpler surrogate model that runs >400 times faster.

BACKGROUND: An approach to investigate brain function/dysfunction is to simulate neuron circuits on a computer. A problem, however, is that detailed neuron descriptions are computationally expensive and this handicaps the pursuit of realistic network investigations, where many neurons need to be simulated. RESULTS: We confront this issue; we employ a novel reduction algorithm to produce a 2 compartment model of the cerebellar Purkinje neuron from a previously published, 1089 compartment model. It runs more than 400 times faster and retains the electrical behavior of the full model. So, it is more suitable for inclusion in large network models, where computational power is a limiting issue. We show the utility of this reduced model by demonstrating that it can replicate the full model's response to alcohol, which can in turn reproduce experimental recordings from Purkinje neurons following alcohol application. CONCLUSIONS: We show that alcohol may modulate Purkinje neuron firing by an inhibition of their sodium-potassium pumps. We suggest that this action, upon cerebellar Purkinje neurons, is how alcohol ingestion can corrupt motor co-ordination. In this way, we relate events on the molecular scale to the level of behavior.

Acute hemodynamic and renal effects of glucagon-like peptide 1 analog and dipeptidyl peptidase-4 inhibitor in rats.

BACKGROUND: Glucagon-like peptide 1 (GLP-1) analogs and dipeptidyl peptidase-4 (DPP4) inhibitors are a newer class of antidiabetics named as incretin-based therapy. In addition to the homeostatic control of glucose, the incretin-based therapy has shown beneficial effects on the cardiovascular system in preclinical and clinical studies. However, there is limited information on their renal effects. To this end, we assessed the acute hemodynamic and renal effects of a GLP-1 analog, Liraglutide, and a DPP4 inhibitor, MK-0626. METHODS: Experiments were performed in anesthetized male Sprague-Dawley rats. Three ascending doses of Liraglutide (3, 9, and 27 nmol/kg/h) or MK-0626 (1 mg/kg) with or without GLP-1 peptide (2.4, 4.8, or 9.6 pmol/kg/min) were administered. Blood pressure (BP) and heart rate (HR) were recorded from an indwelling catheter. Glomerular filtration rate (GFR) and renal blood flow (RBF) were assessed by inulin and para-aminohippurate clearance, respectively. Renal excretory function was assessed in metabolic studies. RESULTS: Both Liraglutide and MK-0626 plus GLP-1 evoked significant diuretic and natriuretic responses and increased GFR. MK-0626 alone increased RBF. Liraglutide at 27 nmol//kg/h and MK-0626 plus GLP-1 at 9.6 pmol/kg/min also increased HR, whereas BP was not affected. CONCLUSION: The results of the present study demonstrated that a GLP-1 analog and a DPP4 inhibitor may have beneficial effects on renal sodium and water handling. Additionally, the DPP4 inhibitor, MK-0626, favorably affects renal hemodynamics by increasing RBF. However, exceedingly high levels of GLP-1 receptor agonists may adversely affect the cardiovascular system in acute setting, as demonstrated by an acute increase in HR.

A potent and selective indole N-type calcium channel (Ca(v)2.2) blocker for the treatment of pain.

N-type calcium channels (Ca(v)2.2) have been shown to play a critical role in pain. A series of low molecular weight 2-aryl indoles were identified as potent Ca(v)2.2 blockers with good in vitro and in vivo potency.

Chronic antagonism of the mineralocorticoid receptor ameliorates hypertension and end organ damage in a rodent model of salt-sensitive hypertension.

We investigated the effects of chronic mineralocorticoid receptor blockade with eplerenone on the development and progression of hypertension and end organ damage in Dahl salt-sensitive rats. Eplerenone significantly attenuated the progressive rise in systolic blood pressure (SBP) (204 ± 3 vs. 179±3 mmHg, p < 0.05), reduced proteinuria (605.5 ± 29.6 vs. 479.7 ± 26.1 mg/24h, p < 0.05), improved injury scores of glomeruli, tubules, renal interstitium, and vasculature in Dahl salt-sensitive rats fed a high-salt diet. These results demonstrate that mineralocorticoid receptor antagonism provides target organ protection and attenuates the development of elevated blood pressure (BP) in a model of salt-sensitive hypertension.

Discovery of betamethasone 17alpha-carbamates as dissociated glucocorticoid receptor modulators in the rat.

A series of betamethasone 17alpha-carbamates were designed, synthesized, and evaluated for their ability to dissociate the two main functions of the glucocorticoid receptor, that is, transactivation and transrepression, in rat cell lines. A number of alkyl substituted betamethasone 17alpha-carbamates were identified with excellent affinity for the glucocorticoid receptor (e.g., 7, GR IC(50) 5.1 nM) and indicated dissociated profiles in functional assays of transactivation (rat tyrosine aminotransferase, TAT, and rat glutamine synthetase, GS) and transrepression (human A549 cells, MMP-1 assay). Gratifyingly, the in-vivo profile of these compounds, for example, 7, also indicated potent anti-inflammatory activity with impaired effects on glucose, insulin, triglycerides, and body weight. Taken together, these results indicate that dissociated glucocorticoid receptor modulators can be identified in rodents.

Discovery of biaryl anthranilides as full agonists for the high affinity niacin receptor.

Biaryl anthranilides are reported as potent and selective full agonists for the high affinity niacin receptor GPR109A. The SAR presented outlines approaches to reduce serum shift and both CYPCYP2C8 and CYP2C9 liabilities, while improving PK and maintaining excellent receptor activity. Compound 2i exhibited good in vivo antilipolytic efficacy while providing a significantly improved therapeutic index over vasodilation (flushing) with respect to niacin in the mouse model.

Comparison of rat and dog models of vasodilatation and lipolysis for the calculation of a therapeutic index for GPR109A agonists.

INTRODUCTION: GPR109A is the receptor mediating both the antilipolytic and vasodilatory effects of nicotinic acid. In order to develop agonists for GPR109A with improved therapeutic indices we have sought to optimize animal models that evaluate both nicotinic acid-mediated inhibition of lipolysis and stimulation of vasodilatation. The rat and the dog have previously been used to study the antilipolytic effects of nicotinic acid, but no optimal vasodilatation model exits in either species. METHODS: We have developed a vasodilatation model in the rat that measures changes in ear perfusion using laser Doppler flowmetry. In the dog, we have developed a model of vasodilatation measuring changes in red color values in the ear, using a spectrocolorimeter. Effects of GPR109A agonists on lipolysis were measured in both species after oral dosing of compounds, and measuring plasma levels of free fatty acids. RESULTS: In both rat and dog, GPR109A agonists induce dose- and time-dependent vasodilatation, similar to that observed in humans. Vasodilatation is inhibited in both species with cyclooxygenase inhibitors or a specific DP1 receptor antagonist, indicating that, as in man, nicotinic acid-induced vasodilatation in rats and dogs is mainly mediated by the release of PGD(2). DISCUSSION: Our results show that both rat and dog are useful models for the characterization of GPR109A agonists. A therapeutic index for GPR109A agonists can be calculated in either species.

Chronic Inhibition of Renal Outer Medullary Potassium Channel Not Only Prevented but Also Reversed Development of Hypertension and End-Organ Damage in Dahl Salt-Sensitive Rats.

The renal outer medullary potassium (ROMK) channel mediates potassium recycling and facilitates sodium reabsorption through the Na+/K+/2Cl- cotransporter in the loop of Henle and potassium secretion at the cortical collecting duct. Evidence from the phenotype of humans and rodents with functional ROMK deficiency supports the contention that selective ROMK inhibitors (ROMKi) will represent a novel diuretic with potential of therapeutic benefit for hypertension. ROMKi have recently been synthesized by Merck & Co, Inc. The present studies were designed to examine the effects of ROMKi B on systemic hemodynamics, renal function and structure, and vascular function in Dahl salt-sensitive rats. Four experimental groups-control, high-salt diet alone; ROMKi B 3 mg·kg-1·d-1; ROMKi B 10 mg·kg-1·d-1; and hydrochlorothiazide 25 mg·kg-1·d-1-were included in prophylactic (from week 1 to week 9 on high-salt diet) and therapeutic studies (from week 5 to week 9 on high-salt diet), respectively. ROMKi B produced sustained blood pressure reduction and improved renal and vascular function and histological alterations induced by a high-salt diet. ROMKi B was superior to hydrochlorothiazide at reducing blood pressure. Furthermore, ROMKi B provided beneficial effects on both the plasma lipid profile and bone mineral density. Chronic ROMK inhibition not only prevented but also reversed the development of hypertension and end-organ damage in Dahl salt-sensitive rats. Our findings suggest a potential utility of ROMKi B as a novel antihypertensive agent, particularly for the treatment of the salt-sensitive hypertension patient population.

Immuno-suppressive effect of blocking the CD28 signaling pathway in T-cells by an active component of Echinacea found by a novel pharmaceutical screening method.

AFTIR (after flowing through immobilized receptor) is a novel method for screening herbal extracts for pharmaceutical properties. Using AFTIR, we identified Cynarin in Echinacea purpurea by its selective binding to chip immobilized CD28, a receptor of T-cells, which is instrumental to immune functioning. The results of surface plasma resonance show that binding between immobilized CD28 and Cynarin is stronger than the binding between CD28 and CD80, a co-stimulated receptor of antigen presenting cells. Cynarin's function was verified by its ability to downregulate CD28-dependent interleukin-2 (IL-2) expression in a T-cell culture line. AFTIR offers promise as an efficient screening method for herbal medicines.

Iatrogenic greenhouse gases: the role of anaesthetic agents.

The contribution of health-care activity to climate change is not negligible and is increasing. Anaesthetic greenhouse gases, in particular the fluranes, have a much more potent global warming capacity, volume for volume, than carbon dioxide, but their emissions remain completely unregulated.

Discovery of MK-7145, an Oral Small Molecule ROMK Inhibitor for the Treatment of Hypertension and Heart Failure.

ROMK, the renal outer medullary potassium channel, is involved in potassium recycling at the thick ascending loop of Henle and potassium secretion at the cortical collecting duct in the kidney nephron. Because of this dual site of action, selective inhibitors of ROMK are expected to represent a new class of diuretics/natriuretics with superior efficacy and reduced urinary loss of potassium compared to standard-of-care loop and thiazide diuretics. Following our earlier work, this communication will detail subsequent medicinal chemistry endeavors to further improve lead selectivity against the hERG channel and preclinical pharmacokinetic properties. Pharmacological assessment of highlighted inhibitors will be described, including pharmacodynamic studies in both an acute rat diuresis/natriuresis model and a subchronic blood pressure model in spontaneous hypertensive rats. These proof-of-biology studies established for the first time that the human and rodent genetics accurately predict the in vivo pharmacology of ROMK inhibitors and supported identification of the first small molecule ROMK inhibitor clinical candidate, MK-7145.

Discovery of a novel series of peroxisome proliferator-activated receptor alpha/gamma dual agonists for the treatment of type 2 diabetes and dyslipidemia.

A series of 2-aryloxy-2-methyl-propionic acid compounds and related analogues were designed, synthesized, and evaluated for their PPAR agonist activities. 2-[(5,7-Dipropyl-3-trifluoromethyl)-benzisoxazol-6-yloxy]-2-methylpropionic acid (4) was identified as a PPARalpha/gamma dual agonist with relative PPARalpha selectivity and demonstrated potent efficacy in lowering both glucose and lipids in animal models without causing body weight gain. The PPARalpha activity of 4 appeared to have played a significant role in lowering glucose levels in db/db mice.

Discovery of potent 3,5-diphenyl-1,2,4-oxadiazole sphingosine-1-phosphate-1 (S1P1) receptor agonists with exceptional selectivity against S1P2 and S1P3.

A class of 3,5-diphenyl-1,2,4-oxadiazole based compounds have been identified as potent sphingosine-1-phosphate-1 (S1P1) receptor agonists with minimal affinity for the S1P2 and S1P3 receptor subtypes. Analogue 26 (S1P1 IC50 = 0.6 nM) has an excellent pharmacokinetics profile in the rat and dog and is efficacious in a rat skin transplant model, indicating that S1P3 receptor agonism is not a component of immunosuppressive efficacy.

Platelet transfusion reverses bleeding evoked by triple anti-platelet therapy including vorapaxar, a novel platelet thrombin receptor antagonist.

Vorapaxar is a novel protease-activated receptor-1 (PAR-1) antagonist recently approved for the reduction of thrombotic cardiovascular events in patients with a history of myocardial infarction or with peripheral arterial disease. Patients who received vorapaxar in addition to standard of care antiplatelet therapy had an increased incidence of major bleeding events compared with placebo. To assess whether platelet transfusion can restore hemostasis in primates on triple antiplatelet therapy, template bleeding times were assessed concurrently in the buccal mucosa, finger pad, and distolateral tail of anesthetized cynomolgus macaques to evaluate bleeding with vorapaxar as either monotherapy or in combination with aspirin or aspirin and clopidogrel. Aspirin (5mg/kg, IV) or vorapaxar (1mg/kg, PO) alone had no significant effect on bleeding times in the three vascular beds examined. A modest (<2-fold) increase in bleeding time was achieved in the three beds with the dual combination of aspirin and vorapaxar. Major increases in bleeding time were achieved in the three beds with the triple combination of aspirin (5mg/kg, IV), vorapaxar (1mg/kg, PO), and clopidogrel (1mg/kg, PO). Transfusion of fresh human platelet rich plasma, but not platelet poor plasma, reversed the increase in bleeding time in the triple therapy group. Transfusion of human platelets may be a viable approach in situations requiring a rapid reversal of platelet function in individuals treated with triple anti-platelet therapy that includes vorapaxar.

Discovery of a Potent and Selective ROMK Inhibitor with Pharmacokinetic Properties Suitable for Preclinical Evaluation.

A new subseries of ROMK inhibitors exemplified by 28 has been developed from the initial screening hit 1. The excellent selectivity for ROMK inhibition over related ion channels and pharmacokinetic properties across preclinical species support further preclinical evaluation of 28 as a new mechanism diuretic. Robust pharmacodynamic effects in both SD rats and dogs have been demonstrated.

Novel N-arylpyrazolo[3,2-c]-based ligands for the glucocorticoid receptor: receptor binding and in vivo activity.

A novel series of selective ligands for the human glucocorticoid receptor (hGR) are described. Preliminary structure-activity relationships were focused on substitution at C-1 and indicated a preference for 3-, 4-, and 5-substituted aromatic and benzylic groups. The resulting analogues, e.g., 18 and 34, exhibited excellent affinity for hGR (IC(50) 1.9 nM and 2.8 nM, respectively) and an interesting partial agonist profile in functional assays of transactivation (tyrosine aminotransferase, TAT, and glutamine synthetase, GS) and transrepression (IL-6). The most potent compounds described in this study were the tertiary alcohol derivatives 21 and 25. These candidates showed highly efficacious IL-6 inhibition versus dexamethasone. The thiophenyl analogue 25 was evaluated in vivo in the mouse LPS challenge model and showed an ED(50) = 4.0 mg/kg, compared to 0.5 mg/kg for prednisolone in the same assay.

Skeletal muscle: a dual system to measure glucocorticoid-dependent transactivation and transrepression of gene regulation.

The use of chronic glucocorticoid (GC) therapy for the treatment of inflammatory diseases is limited by associated metabolic side effects, including muscle atrophy. Therefore, selective glucocorticoid receptor-(GR)-binding ligands that maintain anti-inflammatory activity and demonstrate diminished side-effect profiles would have great therapeutic utility. In this work, we use Taqman PCR and ELISA methods to show that GCs can inhibit basal, and lipopolysaccharide (LPS)-stimulated levels of cytokines IL-6 and TNFalpha, and also the chemokine MCP-1 in a non-inflammatory system such as primary human skeletal muscle cells. In the murine C2C12 skeletal muscle cell line we observe a similar effect of GCs on IL-6 and MCP-1; however, in contrast to previous reports, we observe a time-dependent repression of TNFalpha. Furthermore, in skeletal muscle cells, concomitant with cytokine repression, GCs transcriptionally induce glutamine synthetase (GS), a marker for muscle wasting, in an LPS independent manner. Similarly, administration of dexamethasone to mice, previously administered LPS, results in an increase in GS and an inhibition of TNFalpha and MCP-1 in skeletal muscle tissue. Thus, skeletal muscle cells and tissues present a novel system for the identification of selective GR-binding ligands, which simultaneously inhibit cytokine expression in the absence of GS induction.

The surgical management of consecutive exotropia.

PURPOSE: To review the results and techniques of surgical treatment of consecutive exotropia. METHODS: We performed a retrospective chart review of all patients who underwent surgery for consecutive exotropia in a pediatric ophthalmology practice between 1992 and 2001. Patients were excluded if follow-up lasted < 6 weeks or if exotropia was caused by other ocular disorders such as previous trauma or congenital cataracts. RESULTS: Fifty-nine patients were identified. The procedure performed in the majority of cases was unilateral lateral rectus recession and medial rectus advancement to the original insertion. Seven patients underwent bilateral lateral rectus recession, and 6 underwent lateral rectus recession combined with medial rectus resection. The mean interval between original surgery and surgery for consecutive exotropia was 14.1 years (range 4 months to 47.5 years). The mean preoperative distance exodeviation was 31.7 prism diopters (PD). Satisfactory alignment (ie, within 10 PD of orthophoria) was achieved in 36 patients (61%) at week 1 and 42 patients (71%) at final follow-up. Mean follow up was 16.0 months. Thirty-nine patients (66%) demonstrated an exodrift after surgery (mean 7.6 PD). CONCLUSION: Consecutive exotropia may occur many years, even decades, after esotropia surgery. Lateral rectus recession with advancement of the previously recessed medial rectus is an effective treatment. An exotropic drift occurs after consecutive exotropia surgery, usually within the first 6 weeks. A suitable ocular alignment immediately after surgery for consecutive exotropia is a small-angle esotropia of 5 to 10 PD.

Comparison of Cardiovascular Parameters and/or Serum Chemistry and Hematology Profiles in Conscious and Anesthetized Rhesus Monkeys (Macaca mulatta).

The rhesus monkey is often used in pre-clinical research, and such studies frequently involve a variety of anesthetic conditions. Therefore, it is important to determine baseline physiologic blood chemistry and cardiovascular parameters in anesthetized animals to facilitate appropriate comparisons. The present study compares the cardiovascular parameters, hematology, serum chemistry, and blood gas levels of rhesus monkeys anesthetized with pentobarbital, isoflurane, ketamine, and propofol. Hematology, serum chemistry, and blood gas levels were unaffected by the four anesthetic regimens. However, because of its formulation, propofol is inappropriate for use in animals in which changes in tryglycerides will be evaluated. Compared to those in conscious, unrestrained monkeys, heart rates were higher in anesthetized animals, but the rates of anesthetized animals were similar regardless of anesthetic agent used. In contrast, mean arterial blood pressure was lower in animals anesthetized with pentobarbital, propofol, or isoflurane than in the conscious monkeys. However, mean arterial pressure of ketamine-anesthetized monkeys was similar to that of the conscious monkeys.

Intracellular calcium dynamics permit a Purkinje neuron model to perform toggle and gain computations upon its inputs.

Without synaptic input, Purkinje neurons can spontaneously fire in a repeating trimodal pattern that consists of tonic spiking, bursting and quiescence. Climbing fiber input (CF) switches Purkinje neurons out of the trimodal firing pattern and toggles them between a tonic firing and a quiescent state, while setting the gain of their response to Parallel Fiber (PF) input. The basis to this transition is unclear. We investigate it using a biophysical Purkinje cell model under conditions of CF and PF input. The model can replicate these toggle and gain functions, dependent upon a novel account of intracellular calcium dynamics that we hypothesize to be applicable in real Purkinje cells.