Preclinical Pharmacology and Pharmacokinetics of Inhaled Hexadecyl-Treprostinil (C16TR), a Pulmonary Vasodilator Prodrug
Michel R Corboz, Zhili Li, Vladimir Malinin, Adam J Plaunt, Donna M Konicek, Franziska G Leifer, Kuan-Ju Chen, Charles E Laurent, Han Yin, Marzena C Biernat, Dany Salvail, Jianguo Zhuang, Fadi Xu, Aidan Curran, Walter R Perkins and Richard W Chapman


Procedure for a better heart rate and temperature utilizing loggers read-out

IPST Research Paper, May 29 2017

Heart rate and body temperature recordings are extremely important for different kinds of studies, e.g. toxicology, metabolic or thermoregulation. Being able to measure heart rate and body core temperature without human interference reduces the stress placed on the animal – circumventing any consequential fluctuations in heart rate and temperature profile. This may also result in a reduction in the number of laboratory animals required with fewer animals needed to obtain reliable data.

Over the years, most scientists implanted those loggers subcutaneously to accelerate the surgery process (less invasive). However, we suspected that subcutaneous implantation wasn’t the best option for heart rate and body temperature recordings. Room temperature and non-spontaneous movement might cause some physiological artefacts measured by those loggers.

The aim of this study was to determine if subcutaneous implantation might be replaced by an intraperitoneal implantation to increase the physiological status and stability of recordings processed by the loggers….

2017.05.29.IPST. Logging.Article

12 Hour Hypothermic Oxygenated Machine Perfusion Preserves the Quality of Donor Hearts: A Biomarker Analysis.

The Journal of Heart and Lung Transplantation, April 2017, Volume 36, Issue 4, Supplement, Page S15.

S.G. Michel, M.L. Madariaga, S.M. Labbe, E. Dupre, L.M. Anderson, K. Shanmugarajah

“Purpose: Hypothermic oxygenated machine perfusion has been shown to be beneficial in clinical kidney and liver transplantation, but has not yet been used routinely in clinical heart transplantation….”

Article:  Loss of hepatic DEPTOR alters the metabolic transition to fasting

  • Whole-body DEPTOR KO mice are viable and do not display abnormalities.
  • Liver-specific DEPTOR KO mice are hypoglycemic when fasted.
  • Loss of DEPTOR promotes mTORC1 and increases oxidative metabolism.
  • Rapamycin corrects hypoglycemia in liver-specific DEPTOR KO mice.

The mechanistic target of rapamycin (mTOR) is a serine/threonine kinase that functions into distinct protein complexes (mTORC1 and mTORC2) that regulates growth and metabolism. DEP-domain containing mTOR-interacting protein (DEPTOR) is part of these complexes and is known to reduce their activity. Whether DEPTOR loss affects metabolism and organismal growth in vivo has never been tested.


We have generated a conditional transgenic mouse allowing the tissue-specific deletion of DEPTOR. This model was crossed with CMV-cre mice or Albumin-cre mice to generate either whole-body or liver-specific DEPTOR knockout (KO) mice.


Whole-body DEPTOR KO mice are viable, fertile, normal in size, and do not display any gross physical and metabolic abnormalities. To circumvent possible compensatory mechanisms linked to the early and systemic loss of DEPTOR, we have deleted DEPTOR specifically in the liver, a tissue in which DEPTOR protein is expressed and affected in response to mTOR activation. Liver-specific DEPTOR null mice showed a reduction in circulating glucose upon fasting versus control mice. This effect was not associated with change in hepatic gluconeogenesis potential but was linked to a sustained reduction in circulating glucose during insulin tolerance tests. In addition to the reduction in glycemia, liver-specific DEPTOR KO mice had reduced hepatic glycogen content when fasted. We showed that loss of DEPTOR cell-autonomously increased oxidative metabolism in hepatocytes, an effect associated with increased cytochrome c expression but independent of changes in mitochondrial content or in the expression of genes controlling oxidative metabolism. We found that liver-specific DEPTOR KO mice showed sustained mTORC1 activation upon fasting, and that acute treatment with rapamycin was sufficient to normalize glycemia in these mice.


We propose a model in which hepatic DEPTOR accelerates the inhibition of mTORC1 during the transition to fasting to adjust metabolism to the nutritional status.



Open Access

Article: Loss of UCP2 impairs cold-induced non-shivering thermogenesis by promoting a shift toward glucose utilization in brown adipose tissue.

Uncoupling protein 2 (UCP2) was discovered in 1997 and classified as an uncoupling protein largely based on its homology of sequence with UCP1. Since its discovery, the uncoupling function of UCP2 has been questioned and there is yet no consensus on the true function of this protein. UCP2 was first proposed to be a reactive oxygen species (ROS) regulator and an insulin secretion modulator. More recently, it was demonstrated as a regulator of the mitochondrial fatty acid oxidation, which prompted us to investigate its role in the metabolic and thermogenic functions of brown adipose tissue. We first investigated the role of UCP2 in affecting the glycolysis capacity by evaluating the extracellular flux in cells lacking UCP2. We thereafter investigated the role of UCP2 in BAT thermogenesis with positron emission tomography using the metabolic tracers [(11)C]-acetate (metabolic activity), 2-deoxy-2-[(18)F]-fluoro-d-glucose ((18)FDG, glucose uptake) and 14(R,S)-[(18)F]fluoro-6-thia-heptadecanoic acid [(18)FTHA, non-esterified fatty acid (NEFA) uptake]. The effect of the β3-adrenoreceptor (ADRB3) selective agonist, CL316,243 (CL), on BAT (18)FDG and (18)FTHA uptakes, as well as (11)C-acetate activity was assessed in UCP2(KO) and UCP2(WT) mice exposed at room temperature or adapted to cold. Our results suggest that despite the fact that UCP2 does not have the uncoupling potential of UCP1, its contribution to BAT thermogenesis and to the adaptation to cold exposure appears crucial. Notably, we found that the absence of UCP2 promoted a shift toward glucose utilization and increased glycolytic capacity in BAT, which conferred a better oxidative/thermogenic activity/capacity following an acute adrenergic stimulation. However, following cold exposure, a context of high-energy demand, BAT of UCP2(KO) mice failed to adapt and thermogenesis was impaired. We conclude that UCP2 regulates BAT thermogenesis by favouring the utilization of NEFA, a process required for the adaptation to cold.

Caron, A., Labbé, S. M., Carter, S., Roy, M.-C., Lecomte, R., Ricquier, D., et al. (2017). Loss of UCP2 impairs cold-induced non-shivering thermogenesis by promoting a shift toward glucose utilization in brown adipose tissue. Biochimie.

Article: Dual antiplatelet and anticoagulant APAC prevents experimental ischemia–reperfusion-induced acute kidney injury – Online First – Springer

  • Raimo Tuuminen Affiliated with Transplantation Laboratory Haartman Institute, University of HelsinkiDepartment of Cardiothoracic Surgery, Helsinki University Hospital
  • Annukka Jouppila Affiliated with Helsinki University Hospital Research Institute
  • Dan Salvail Affiliated with IPS Therapeutique
  • Charles-E. Laurent Affiliated with IPS Therapeutique
  • Marie-Claude Benoit Affiliated with IPS Therapeutique
  • Simo Syrjälä Affiliated with Transplantation Laboratory Haartman Institute, University of HelsinkiDepartment of Cardiothoracic Surgery, Helsinki University Hospital
  • Heikki Helin Affiliated with Division of Pathology, HUSLAB and Helsinki University Hospital
  • Karl Lemström Affiliated with Transplantation Laboratory Haartman Institute, University of HelsinkiDepartment of Cardiothoracic Surgery, Helsinki University Hospital

The article – in its entirety – is available online at:



Renal ischemia–reperfusion predisposes to acute kidney injury (AKI) and mortality. APAC, mast cell heparin proteoglycan mimetic is a potent dual antiplatelet and anticoagulant inhibiting thrombosis in several vascular models.


Clinically relevant (0.06 and 0.13 mg/kg) and high (0.32 and 7.3 mg/kg) heparin doses of APAC and unfractionated heparin (UFH) were administered i.v. in pharmacological studies. Antithrombotic action of APAC and UFH was assessed with platelet aggregation to collagen, activated partial thromboplastin (APTT) and prothrombin (PT) times. Pharmacodynamics of [64Cu]-APAC or -UFH were monitored by PET/CT. Next, APAC and UFH doses (0.06 and 0.13 mg/kg) were i.v. administered 10 min prior to renal ischemia–reperfusion injury (IRI) in rats.


APAC in contrast to UFH inhibited platelet aggregation. During 0.06 and 0.13 mg/kg dose regimens APTT and PT remained at baseline, but at the high APTT prolonged fourfold to sixfold. Overall bio-distribution and clearance of APAC and UFH were similar. After bilateral 30-min renal artery clamping, creatinine, urea nitrogen and neutrophil gelatinase-associated lipocalin concentrations and histopathology indicated faster renal recovery by APAC (0.13 mg/kg). APAC, unlike UFH, prevented expression of innate immune ligand hyaluronan and tubulointerstitial injury marker Kim-1. Moreover, in severe bilateral 1-h renal artery clamping, APAC (0.13 mg/kg) prevented AKI, as demonstrated both by biomarkers and survival. Compatible with kidney protection APAC reduced the circulating levels of vascular destabilizing and pro-inflammatory angiopoietin-2 and syndecan-1. No tissue bleeding ensued.


APAC and UFH were similarly eliminated via kidneys and liver. In contrast to UFH, APAC (0.13 mg/kg) was reno-protective in moderate and even severe IRI by attenuating vascular injury and innate immune activation.


Ischemia–reperfusion injury Acute kidney injury Unfractionated heparin APAC

Abstract: Correlation between the thrombogenic Factor XIa and the thrombus score in the venous stasis rabbit model

Laurent C-E, Dupre E and Salvail D IPS Therapeutique, Sherbrooke, Canada. Journal of Thrombosis and Haemostasis, Special Issue: Abstracts of the 62nd Annual Meeting of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis May 25–28, 2016, 14 (Suppl. 1) (2016) 1–168.

Background: Activated factor XI (FXIa) has been identified as the probable causative agent associated with thromboembolic events (TEE) following intravenous (IV) IgG (IVIG) product administration. Although there is no official method required by the regulator to validate the therapeutic safety of new batches of IVIG, the rabbit venous stasis assay, a prothrombogenic model, remains the model of choice for in vivo assessment of potential TEE.

Aims: This study aimed at standardizing the in vivo venous stasis rabbit model and quantifying the formed thrombi on a ranking system to evaluate the amount of FXIa in new batches of IVIG, in order to predict potential TEE. Methods: Anesthetized rabbits (2.5 to 3 kg) were infused with 10 ml/kg of 200 mM glycine, 5% BSA (Gycine-BSA) solution spiked with increasing doses of FXIa followed by a jugular venous stasis of 2-cm long for 15-min. The formed thrombus was scored according to a ranking system from 0 to 4.

Results: Spiked Glycine-BSA solution with 4 increasing doses of FXIa (0, 1, 1.5, 2 and 7 mU/mL) showed a dose-dependent clot formation response of 0, 0.5, 1.0, 2.33, and 3.75 respectively. The IVIG Gamunex and spiked Gamunex with 1.5 mU/mL of FXIa scored 0.8 and 3.5 respectively. Concomitantly, the bleeding time in rabbits treated with the spiked Glycine-BSA decreased from 220 s to 130 s with increasing doses of FXIa.

Conclusions: The current study demonstrates that the standardized venous stasis rabbit model is very sensitive to FXIa and that the thrombus score correlates with the amount of FXIa present in the test solution. Furthermore, this standardized model can be used to predict the potential for a TEE in batches of IVIG.

Poster: RP5063 Prevents Monocrotaline Induced Pulmonary Arterial Hypertension in Rats. Bhat L, Salvail D, Bouchard A, Hawkinson J, Cantillon M

Abstract Body
Rationale: Pulmonary arterial hypertension (PAH) is characterized by constriction and remodeling of the pulmonary vasculature. Changes in serotonin synthesis, receptor activation and uptake via the serotonin transporter (SERT) have been reported in experimental and clinical PAH. Acute vascular constriction by chronic exposure to high levels of serotonin (5-HT) can be mediated by 5-HT1B and 5-HT2A receptors and thickening of cardiac valves through 5-HT2B receptor. RP5063 is a novel multimodal modulator of dopamine and serotonin receptors with high affinity for 5-HT2B (Ki=0.19 nM),5-HT2A (Ki=2.5 nM),5-HT1A, 5-HT6 and 5-HT7 receptors. In this study, the possibility that RP5063 could bind to these serotonin receptors to alleviate the symptoms of experimental PAH in rats was quantified by hemodynamic monitoring and histological examination.
Methods: Adult male Sprague-Dawley rats were given a single s.c. injection of 50 mg/kg monocrotaline on Day 0. The animals were monitored for 28 days while PAH symptoms progressed. 10 animals per group received either vehicle, 1 mg/kg, 3 mg/kg, or 10 mg/kg RP5063, b.i.d. or 50 mg/kg sildenafil, b.i.d. by oral gavage for 28 days. Functional observations and body weight were monitored and blood samples were taken weekly. On Day 29, terminal surgery involving intra-arterial catheters (femoral and pulmonary), right ventricle, ECG recording and SO2 monitoring was performed to acquire a complete hemodynamic profile of the animal’s status. The lungs and heart were harvested, fixed, and embedded before being stained (H&E) and submitted to morphometric analysis to define the extent of the remodeling.

Results: Monocrotaline increased pulmonary diastolic and systolic pressures from 11.4 to 21.1 and 26.7 to 64.0 mmHg, resp. 10 mg/kg RP5063 significantly lowered the pulmonary diastolic and systolic pressures to 14.7 and 43.0 mmHg, resp. 10 mg/kg RP5063 reduced right-ventricle remodeling (Fulton Index decreased from 0.47 to 0.32) and lowered respiratory resistance from 20.9 to 10.7 mmHg/mL/sec, suggesting that RP5063 decreased pulmonary edema and fibrosis. Histology revealed a dose-dependent decrease of small vessel wall thickness from 41.9 to 25.3% (10 mg/kg RP5063) of the vessel diameter. Vascular remodeling produced 33.7% less muscular vessels in 10 mg/kg RP5063 -treated animals with significantly less alveolar macrophage infiltration in treated animals.

Conclusions: Combined with the morphometric evidence of limited lung remodeling, these results support a greater functional and structural benefit from RP5063 compared to a 5-fold higher dose of sildenafil, a current off-label treatment for clinical PAH.

Presented at the American Thoracic Society 2016 International Meeting in San Francisco May 13 to 18, 2016.

Article: Therapeutic antibody-induced vascular toxicity due to off-target activation of nitric oxide in cynomolgus monkeys. Pai R1, Ma N1, Connor AV1, Danilenko DM1, Tarrant JM1, Salvail D2, Wong L1, Hartley DP1, Misner D1, Stefanich E3, Wu Y4, Chen Y4, Wang H5, Dambach DM1Toxicol Sci. 2016 Feb 25. pii: kfw037. [Epub ahead of print]

  • 1* Safety Assessment, Genentech Inc., 1 DNA Way, South San Francisco, California 94080.
  • 2IPS Therapeutique, Sherbrooke, Quebec, Canada.
  • 3Preclinical & Translational Pharmacokinetics, Genentech Inc., 1 DNA Way, South San Francisco, California 94080.
  • 4Antibody Engineering, Genentech Inc., 1 DNA Way, South San Francisco, California 94080.
  • 5* Safety Assessment, Genentech Inc., 1 DNA Way, South San Francisco, California 94080

Abstract: PRO304186, a humanized monoclonal antibody targeting soluble interleukin-17 A and F, was developed for autoimmune and inflammatory disease indications. When administered to cynomolgus monkeys PRO304186 induced unexpected adverse effects characterized by clinical signs of hematemesis, hematochezia and moribundity. Pathology findings included hemorrhage throughout the gastrointestinal tract without any evidence of vascular wall damage or inflammatory cellular infiltration. Mechanistic investigation of these effects revealed mild elevations of serum MCP-1 and IL-12/23 but without a classical proinflammatory profile in PRO304186-treated animals. In vitro studies demonstrated off-target effects on vascular endothelial cells including activation of nitric oxide synthase leading to production of nitric oxide (NO) accompanied by increased mitochondrial membrane depolarization, glutathione depletion and increased paracellular permeability. Additionally, endothelial cell-PRO304186-conditioned medium reduced myosin light chain phosphorylation in vascular smooth muscle cells. Furthermore, an ex vivo study utilizing segments from cynomolgus aorta and femoral artery confirmed PRO304186-induced endothelium-dependent smooth muscle relaxation and vasodilation mediated via NO. Finally, a single dose of PRO304186 in cynomolgus monkeys induced a rapid and pronounced increase in NO in the portal circulation that preceded a milder elevation of NO in the systemic circulation and corresponded temporally with systemic hypotension; findings consistent with NO-mediated vasodilation leading to hypotension. These changes were associated with non-inflammatory, localized hemorrhage in the gastrointestinal tract consistent with hemodynamic vascular injury associated with intense local vasodilation. Together, these data demonstrate that PRO304186-associated toxicity in monkeys was due to an off-target effect on endothelium that involved regional NO release resulting in severe systemic vasodilation, hypotension and hemorrhage.

© The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email:

KEYWORDS: hemodynamic vascular injury; nitric oxide; non-human primates; off-target toxicity; splanchnic circulation; therapeutic antibody

Poster: LPS-induced Cytokine Storm Produces QTc Prolongation Which Can be Prevented by an Antiinflammatory Eutectic Blend. Annie Bouchard1, Peter Sordillo2, Lawrence Helson2, George Shopp3, Sabrina Baillargeon1, Walter Shaw4, Dany Salvail1

1IPS Therapeutique Inc., Sherbrooke, QC, Canada, 2SignPath Pharma Inc., Quakertown, PA, USA,
3Shopp NonClinical Consulting LLC., Boulder, CO, USA, 4Avanti Polar Lipids Inc., Alabaster, AL, USA

Abstract: There is increasing evidence that excess levels of pro-inflammatory cytokines play a major role in the pathogenesis of the prolonged QT syndrome. Inversely, blockers such as tocilizumab (IL-6), or anticytokine antibodies (TNFα) contribute to a shortening of the previously-prolonged QT interval. In this study, LPS and Kdo2-Lipid-A were used to induce cytokine release in guinea-pigs with concomitant ECG monitoring and blood draws, followed by Q-ELISA measurement of cytokine production. The guinea pig was selected because it yields reliable QTc prolongation as a result of pro-arrhythmic challenge, with consistently visible T-waves on the ECG. Male adult guinea pigs received 300 μg/kg LPS at time 0, and had ECGs analyzed at 1h, 2h, and 4 hours post-LPS, with simultaneous blood draw. Animals receiving LPS only exhibited a 8-msec increase in QTc after 1h post-LPS, when TNFα levels were maximal at 5.5-fold the pre-LPS values. A 29-msec QTc prolongation 2h post-LPS correlated with 7- and 9- fold increases in IL-1β and IL-6, respectively. The QTc prolongation remained (27 msec) after 4 hours post-LPS, when the animals were euthanized. When 9 mg/kg EU8120 (a lipid blend shown to prevent IKrchannel block by a variety of hERG blockers) was given 1 hour prior to LPS-induction, QTc prolongation was limited to 5 ms after 2 hours, and completely prevented at 1 and 4 hours post-LPS. Plasma levels of TNFα, IL1β, and IL-6 were significantly lower in EU8120-administered animals. It is suggested that EU8120 reduces QT prolongation through suppression of pro-inflammatory cytokines.

Presented at Safety Pharmacology Society (SPS) Annual Meeting in Prague on Tuesday, September 29th. 

Poster available for download as .pdf – Poster Bouchard SPS2015

Poster: Pharmacokinetics and pharmacodynamics of APAC, a dual novel anticoagulant and antiplatelet heparin complex. Jouppila Annukka, MSc, Marie-Claude Benoit, Charles Laurent, Dany Salvail, Lassila Riitta, MD, PhD. Helsinki University Central Hospital and Aplagon, Helsinki Finland, and IPS Therapeutique, Sherbrooke, Quebec. Presented at the XXV Congress of the International Society on Thrombosis and Haemostatis & 61st Annual SSC Meeting, June 20-25, 2015 in Toronto, Ontario.

Keywords. APAC2, UFH, antiplatelet, anticoagulant.

Background. APAC2 heparin-protein complex is inspired by mast cell-derived heparin proteoglycans which attenuate platelet-collagen interactions under blood flow and reduce platelet thrombosis. In addition of inhibiting platelets APAC2 is an anticoagulant and arrests thrombus growth in two arterial baboon models (Lassila and Jouppila, STH 2014).. Upon local administration APAC2 targets and remains at vascular injury site. In vitro, APAC2 inhibits collagen-induced platelet aggregation and procoagulant activity in CAT.

Aim. To study the pharmacokinetics (PK) and pharmacodynamics of APAC2 in rats.

Methods. Male Sprague-Dawley rats were dosed APAC2 or UFH at 0.13 or 7.3 mg/Kg. Blood samples were drawn pre- and post-dose (PD) at 60min, 90min, 6h, 24h and 48h for APTT and PT analysis. Distribution and retention were studied with 64Cu-labeled APAC2 or UFH and measured by gamma counter and corrected for 64Cu decay. Two rats per dose were scanned by PET. At 48h rats were euthanized and kidneys, liver, lungs and spleen harvested for 64Cu radioactivity.

Results. At both 0.13 and 7.3 mg/Kg (15-fold above target dose), APAC2 and UFH were mainly distributed to kidneys and liver and lesser extent to other organs or blood. APAC2 clearance (T1/2) was rapid, 15 min at 0.13 mg/Kg and 1 h at 7.3 mg/Kg while UFH T1/2 was 23-28 min at both doses. At 0.13 mg/Kg, APAC2 or UFH had no effect on APTT, but both prolonged APTT dose dependently and at 7,3 mg/Kg by 6- and 4-fold, respectively. APAC2 and UFH prolonged PT by only 1.5-fold at 7.3 mg/Kg. At the highest dose APTT remained elevated at 90min while PT reverted. Controlled at 6h APTT and PT were normal.

Conclusions. APAC2 and UFHwere mainly detected in kidney and liver following the heparin elimination route. APAC2 had 2.5-fold clearance time over UFH suggesting some retention.APAC2 prolonged APTT, while PT only modestly elevated referring to minimal role on extrinsic pathway. The PK profile of APAC2 appears beneficial.

Click on the link to download the .pdf file – jouppila et al isth 2015 (2)

Poster: Liposomal curcumin (LipocurcTM) and in vitro/in vivo surrogates for cytokine storm associated with uncontrolled EBOLA infection. Lawrence Helson1, Peter Sordillo1, George Shopp5, Dany Savail2, Annie Bouchard2, Walter A. Shaw3,Stephen W. Burgess3, Burkhard Kloesch4, Muhammed Majeed6 1.SignPath Pharma,Inc, USA 2.IPS Therapeutique,Inc, Canada 3.Avanti Polar Lipids,Inc USA 4.Ludwig Boltzmann Institute for Rheumatology, Austria 5.Shopp Nonclinical Consulting LLC., USA 6. Sabinsa Inc. USA. To be presented at the ‘Targeting Ebola World Congress’, Institut Pasteur, Paris France.

ABSTRACT: Massive over-production and persistent elevation of inflammatory cytokines over time by the body’s immune system can trigger a dangerous syndrome known as a cytokine storm. Frequently occurs in advanced or terminal stages of Ebola infection. Dysregulation of normal immune response characterized by high levels of circulating cytokines can induce potentially fatal pathologic changes in cells, tissues, and organs leading to multiple organ failure. Uncontrolled Ebola virus (EBOV) infection of peripheral blood mononuclear cells (PBMCs) results in induction of excessive IL-6 and TNF-α production designated as cytokine storm. Important pro-inflammatory cytokines: IL-1β, IL-6, IL-8, and TNF-α.

Click on the link to download the .pdf file – Poster-v6May-15

Poster: Pulmonary Arterial Hypertension is Insulin-Dependent in Type-1 and Type-2 Diabetic Rats. C.-E. Laurent, Ph.D.; M.-C. Benoit, DEC animal health; S. Baillargeon, DEC animal health; D. Salvail, Ph.D., Sherbrooke QC. Presented at the American Thoracic Society International Conference in Denver CO on Sunday, May 17 2015.

RATIONALE: Clinical evidence suggests that patients with Type-1 or Type-2 diabetes have a higher prevalence of pulmonary arterial hypertension (PAH). Recent preclinical observations have shown that diabetes induces pulmonary artery (PA) dysfunction that can lead to PAH in rats. The present study aimed to determine whether the reduced pulmonary arterial elasticity observed in isolated PA derived from diabetic rats translates into increased pulmonary arterial pressure in anesthetized diabetic animals. METHODS: Twelve (12) adult male Sprague-Dawley rats were randomly divided into control (saline), Type-1 and Type-2 diabetic groups. Type-1 diabetes was induced with dual i.v. injections of 45 mg/kg streptozotocin, and received stabilized insulin via implanted osmotic pumps for 4 weeks. Type-2 diabetes was induced with a single i.v. injection of 25 mg/kg streptozotocin, preceded by 4 weeks of high-fat diet, and followed by 3 weeks of sugar-enriched rat chow. Insulin levels, glycemia, and food/water consumption were monitored daily at first, and then weekly. Diabetic animals had glycemia levels greater than 30 mM. Twenty-eight (28) days post induction, the rats were instrumented and functional hemodynamics measured prior to organ harvesting for histological examination. RESULTS: Both diabetic models exhibited a 5-fold increase in glycemia. Insulin levels were below 5 µU/mL in Type-1, and approximately 20 µU/mL in Type-2 diabetics. Water and food consumption were 2-3-fold greater in diabetic animals than in control. Mean pulmonary arterial pressure (mPAP) was significantly increased in Type-1 rats when compared to control (22±0.21 vs 18±0.56 mmHg respectively, n=4, P≤0.05). Interestingly, no statistical difference in mPAP was calculated in Type-2 rats when compared to control (18±1.27 vs 18±0.56 mmHg, n=4). Oxygen saturation measured with a pulse oximeter showed a significant decrease in oxygen saturation in Type-1 when compared to control animals (92 vs 96% SO2 respectively, n=4, P≤0.05). Histological examination confirmed vascular remodeling in the lungs, with loss of functional endothelium associated with smooth muscle hypertrophy and hyperplasia. CONCLUSION: These results show that Type-1 diabetes-induced loss of PA elasticity reported from ex vivo experiments translates into PAH in Type-1 diabetic animals, but not in Type-2 animals, suggesting that hypoinsulinemia rather than hyperglycemia plays a major role in regulating PA elasticity in diabetic rats.

Am J Respir Crit Care Med 191;2015:A1964

Poster: Identifying a lead compound for mitigation of drug-induced PQTS.  L Helson, W. Shaw, S. Burgess, G Shopp, A Bouchard, D Salvail, M Majeed. SignPath Pharma, Inc. (Quakertown PA); Avanti Polar Lipids, Inc. (Alabaster AL); IPS Therapeutique, Inc. (Sherbrooke QC); Sabinsa Inc. (East Windsor NJ). Presented at the American Association for Cancer Research (AACR) Annual Meeting 2015 in Philadelphia PA on Monday, April 20 2015.

Abstract body: In addition to 170 approved therapeutic drugs whose adverse effects include QT prolongation, a prevalence of 1:2500 subjects in the US are genetically susceptible, or have a diabetic syndrome with increased risk of QT prolongation, torsade de pointes, and sudden death. To address this risk we determined the mitigating effect of a liposome and its components administered intravenously and orally on clinically approved QT-prolonging anticancer drugs, crizotinib and nilotinib, and an antibiotic moxifloxacin (MF) in vitro and in vivo in rats and guinea pigs. Intravenous MF at 20 mg/kg caused a statistically significant QTc prolongation of 35ms in guinea pigs. This observation is in line with peer-reviewed literature in which an oral dose of 400 mg (or approximately 6.1 mg/kg) in humans caused the FDA to black-label MF for causing QT interval prolongation in patients.

The mitigating effect of a liposome consisting of dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylglycerol (DMPG) on curcumin, a compound that inhibits the IKr (hERG) current, was initially discovered during preclinical testing with Lipocurc™. Additional patch-clamp studies with DMPC, DMPG, their metabolites myristoyl lysophosphatidylcholine (14:0 LPC) and myristoyl lysophosphatidylglycerol (14:0 LPG), and a synthetic compound, myristoyl ethyleneglycolphosphatidylglycerol (EGPG), revealed mitigation of crizotinib- and nilotinib-induced inhibition of the IKr current. Formulation of 14:0 LPG in a eutectic mixture with a monoglyceride and myristic acid (EU8120) given orally to both normal and diabetic rats prior to challenge with intravenous nilotinib resulted in significantly reduced QT prolongation.

We quantified and compared the conduction delays on QT intervals in guinea pigs induced by MF alone with those of MF preceded by oral administration of EU8120, 14:0 LPG, 16:0 LPG, EGPG and DMPG. Three ratios of phospholipids:MF were tested for mitigation of conduction delays: 3:1, 1:1 and 0.3:1. At the 3:1 ratio, all the compounds tested mitigated MF-induced prolongation of QT intervals. While EGPG induced the most protection it caused bradycardia which limited its beneficial effects at that dose level. DMPG was the least potent. At lower ratios of 1:1 and 0.3:1 the mitigating effects of EU8120, EGPG and DMPG were maintained with equal potency. Lipid:MF ratios of 0.1:1 and 0.03:1 are currently being tested and will identify a lead compound for drug development, and offer insight into its mechanism of action.

SignPath AACR 2015 v2

Poster: A 14-Day Assessment of the Tolerability and Pharmacokinetics (Pk) with a Nanoparticle Formulation of Hexadecyl-Treprostinil, a Long-Acting Pulmonary Vasodilator, In Rats [Publication Number: A1948]

Rationale: Studies in both rats and dogs demonstrate that single dose inhalation with a nanoparticle formulation of the prodrug, hexadecyl-treprostinil (C16TR) is well tolerated, produces long acting pulmonary vasodilation and sustained levels of treprostinil (TRE) in the plasma and C16TR in the lungs. To assess whether repeated dosing with inhaled C16TR is well tolerated and alters PK, rats were exposed to C16TR for 14-consecutive days.

V. Malinin, PhD1, Z. Li, PhD1, R.W. Chapman, PhD1, F. Leifer, PhD1, D. Konicek, PhD1, D. Salvail, PhD2, C. Laurent, PhD2, H. Yin, MS2, W. Perkins, PhD1
1Bridgewater, NJ/US, 2Sherbrooke, QC/CA, Am J Respir Crit Care Med 191;2015:A1948

Poster: Incorporation Into Lipid Nanoparticles Extends the Duration of Activity of Treprostinil in an Acute Hypoxia Rat Model of Pulmonary Arterial Hypertension

D Omiatek1, F Leifer1, V Malinin1, J Ong1, T Henn1, Z Li1, RW Chapman1, D Salvail2, CE Laurent2, WR Perkins1
1 Insmed, Inc., Bridgewater, NJ, USA
2 IPS Therapeutique Inc., Sherbrooke, QC, Canada


Poster: Prolonged Activity of Inhaled Treprostinil Prodrug Nanoparticles in a Rat Model of Pulmonary Arterial Hypertension

F Leifer1, D Omiatek1, V Malinin1, J Ong1, Z Li1, P Klecha1, RW Chapman1, D Salvail2, C-E Laurent2, WR Perkins1
1 Insmed, Inc., Bridgewater, NJ, USA
2 IPS Therapeutique Inc., Sherbrooke, QC, Canada

Poster: Treprostinil Pharmacokinetics in Rats Are Extended Using Inhaled Prodrug Formulations

V Malinin1, Z Li1, RW Chapman1, F Leifer1, D Omiatek1, J Ong1, D Salvail2, C-E Laurent2, WR Perkins1
1 Insmed, Inc., Bridgewater, NJ, USA
2 IPS Therapeutique Inc., Sherbrooke, QC, Canada

Poster : Increased risk of Torsades de Pointes in streptozotocin-induced diabetic rats

Annie Bouchard*, Marie-Claude Benoit*, Sabrina Baillargeon*, Laurence Helson, George Shopp, Dany Salvail*

* iPS Therapeutique Inc., Sherbrooke, QC, Canada

SignPath Pharma, Inc., Quakertown, PA, USA

Poster Bouchard Diabetes QT v4 Final

Presentation : Increased risk of Torsades de Pointes in streptozotocin-induced diabetic rats

Annie Bouchard*, Marie-Claude Benoit*, Sabrina Baillargeon*, Laurence Helson, George Shopp, Dany Salvail*

* iPS Therapeutique Inc., Sherbrooke, QC, Canada

SignPath Pharma, Inc., Quakertown, PA, USA

Salvail Diabetes Presentation SPS2014 Final

Pierre Sirois has been fascinated with “the basics of life” since he was a child. An entrepreneurial scientist excited by the challenges of tackling difficult problems in the lab, Pierre completed his studies (B.A., B.Sc., M.Sc., Ph.D) at Laval University, Université de Sherbrooke, Hospital for Sick Children at University of Toronto, Royal College of Surgeons of England and Imperial College of Science and Technology at University of London. He was a professor  of pharmacology for 31 years and Chair of the Department of Pharmacology of Université de Sherbrooke Medical School for 12 years.

Gobeil F Jr, Sirois P, Regoli D. Preclinical pharmacology, metabolic stability, pharmacokinetics and toxicology of the peptidic kinin B1 receptor antagonist R-954. Peptides. 2014 Feb;52:82-9. doi: 10.1016/j.peptides.2013.12.009. Epub 2013 Dec 18.

We previously showed that R-954 (AcOrn[Oic(2),(αMe)Phe(5),dβNal(7),Ile(8)]desArg(9)-bradykinin) is a potent, selective and stable peptide antagonist of the inducible GPCR kinin B1 receptor. This compound shows potential applications for the treatment of several diseases, including cancer and neurological disturbances of diabetes. To enable clinical translation, more information regarding its pharmacological, pharmacokinetics (PK) and toxicological properties at preclinical stage is warranted. This was the principal objective of the present study. Herein, specificity of R-954 was characterized in binding studies on 133 human molecular targets to reveal minor cross-reactivities against the angiotensin AT2 and the bombesin receptors (110- and 330-fold lower affinity than for B1R, respectively). The pharmacokinetic of R-954 was studied in both normal and streptozotocin-diabetic anaesthetized rats providing half-lives of 1.9-2.7h. R-954 does not appear to be metabolized in the rat circulation and in several rat tissue homogenates, as the kidney, lung and liver. It appears to be excreted as parent drug in the bile (21%) and in urine. A preliminary toxicological profile of R-954 was obtained in rats under various administration routes. R-954 appears to be well tolerated. Overall, these results indicate that R-954 exhibits favorable preclinical pharmacological/PK characteristics and encouraging safety profiles, suitable for early studies in humans.

Catanzaro O, Capponi JA, Michieli J, Labal E, Di Martino I, Sirois P. Bradykinin B₁ antagonism inhibits oxidative stress and restores Na+K+ ATPase activity in diabetic rat peripheral nervous system. Peptides. 2013 Jun;44:100-4. doi: 10.1016/j.peptides.2013.01.019. Epub 2013 Mar 23

Diabetic peripheral neuropathy is one the most common complications of diabetes mellitus and frequently results in clinically significant morbidities such as pain, foot ulcers and amputations. The diabetic condition progresses from early functional changes to late, poorly reversible structural changes. The chronic hyperglycemia measured alongside diabetes development is associated with significant damage and failure of various organs. In the present study diabetes was induced in male Wistar rats by a single dose of streptozotocin (STZ) and the association between the BKB1-R and the oxidative stress and Na+-K+ ATPase activity in nervous tissues was analysed. The results showed that the resulting hyperglycemia induced a reduction of the neuronal electrical function integrity and increased oxidative stress in the sciatic nerve homogenates of 30 days diabetic rats. Malondialdehyde (MDA) used as a marker of oxidative stress was elevated whereas Biological Antioxidant Potential (BAP), glutathion (GSH) levels and superoxide dismutase (SOD) activity were decreased. Treatment of the rats 3 days before the end of the 4 week period with the BKB1 antagonist R-954 restored the neuronal activity and significantly attenuated the oxidative stress as shown by the level of the various markers returning close to levels found in control rats. Our results suggest that the BKB1-R subtype is overexpressed in sciatic nerve during the STZ-induced diabetes development as evidenced by inhibitory effects of the BKB1-R antagonist R-954. The beneficial role of BKB1-R antagonist R-954 for the treatment of diabetic neuropathy is also suggested.

Marzena Biernat is a specialist in physiology, histology, pathology and pharmacology with a Ph.D. in veterinary medicine. Her PhD was defended at the Polish Academy of Science, The Kielanowski Institute of Animal Physiology and Nutrition with post-doctoral work at the University of Sherbrooke.

Morisset J, Lainé J, Biernat M, Julien S. What are the pancreatic target cells for gastrin and its CCKB receptor? Is this a couple for cancerous cells? Med Sci Monit. 2004 Oct;10(10):RA242-6. PMID: 15448615

The objectives of this review are to summarize, analyse and discuss the roles played by the CCK receptor subtypes and their agonists on pancreatic enzyme secretion, pancreas growth and regeneration, define the receptors specific target cells and evaluate the role of gastrin in pancreatic pathologies including cancer. In rodents, it is clear that the CCKARs present on pancreatic acinar cells play a major role in enzyme secretion. In large mammals, CCK does not seem to be the final mediator of enzyme release. In rat, gastrin and its CCKBR seem responsible for foetal pancreas growth while after birth, CCK was shown to be the most potent trophic factor via occupation of its CCKAR. In pig and human, no one has yet established a direct link between CCK, gastrin and pancreas growth. In rodent’s pancreas, the CCKAR were observed on acinar cells as well as on islet’s alpha and beta cells; in six other species, the CCKAR were present only on alpha and beta cells with the CCKBR always present on delta cells. The CCKBRs were overexpressed in acute pancreatitis and in metaplastic pancreas following duct ligation. In pancreatic cancer cells, a gastrin autocrine loop involving the CCKBR was suggested. The presence of both CCKR-subtypes and gastrin was observed in many pancreatic tumors; however, their role in cancer growth remains controversial.

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Linderoth A, Biernat M, Prykhodko O, Kornilovska I, Pusztai A, Pierzynowski SG, Björn W. Induced growth and maturation of the gastrointestinal tract after Phaseolus vulgaris lectin exposure in suckling rats. J Pediatr Gastroenterol Nutr. 2005 Aug;41(2):195-203. PMID:16056099

OBJECTIVES: In mammals, the postnatal development of the gastrointestinal tract is characterized by vast structural and functional changes. Using a suckling rat model, we investigated whether red kidney bean lectin, phytohemagglutinin (PHA), a potent gut mitogen in adult rats, can accelerate the growth and maturation of the gastrointestinal tract.

METHODS: At either 10 or 14 days of age, suckling rats were daily gavage fed with PHA (0.05 mg/g body weight) or saline for 3 days. At 1 or 3 days after this treatment, gastrointestinal organ growth, intestinal morphology, disaccharidase pattern, macromolecular absorption capacity, and pancreatic enzyme contents were studied.

RESULTS: After PHA exposure, increased small intestinal growth and number of crypt cells were observed, whereas the proportion of enterocytes with supranuclear vacuoles in the distal intestine was decreased. The macromolecular absorption of the markers bovine immunoglobulin (Ig)G and bovine serum albumin and plasma levels of maternal IgG decreased, and intestinal disaccharidases switched toward an adult-like pattern. The pancreas weight and pancreatic protein and trypsin contents increased. These changes were partly reversible when the PHA treatment began at 10 days of age, but they persisted when the treatment began at 14 days of age.

CONCLUSIONS: PHA induced enhanced growth and precocious functional maturation of the gastrointestinal tract in suckling rats. The effects persisted if the PHA treatment started at 14 days of age, but not before, suggesting an age dependent mechanism. These findings may lead to a better understanding of gastrointestinal maturation and constitute a basis for the treatment of mammals having an immature gut.

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Annie Bouchard has a B.Sc. in microbiology and a Masters in Physiology – both from the University of Sherbrooke.

Carr Daniel B, McDonnell Moorehead T, Bouchard A, Sprenger C.R., Hamilton D.A., Lang E, Madden D, Lacouture P.G., Wright C. Effects of Injectable HPCD-Diclofenac on the Human Delayed Rectifier Potassium Channel Current In Vitro and on Proarrhythmic QTc In Vivo. Clinical Therap, Vol 35, Number 5, 2013. Read free full text

Background: Novel formulations and administration routes of established drugs may result in higher maximum concentrations or total exposures and potentially cause previously unrecognized adverse events.

Objective: This study evaluated the proarrhythmic potential of hydroxypropyl--cyclodextrin (HPCD)- diclofenac, a novel injectable diclofenac formulation solubilized with hydroxypropyl--cyclodextrin (HPCD), on ventricular electrical conduction in preclinical and clinical models.

Methods: We assessed the effects of diclofenac, HPCD, and HPCD-diclofenac on the human delayed rectifier potassium channel (IKr) using human embryonic kidney (HEK) 293 cells transfected with a human etherà-go-go–related gene (hERG) using whole-cell patchclamp. In a single-dose, active- and placebo-controlled, 4-period crossover, thorough QT in vivo study, 70 healthy volunteers (mean age, 23.3 years; range, 18–49 years; 55.75% male) received HPCD-diclofenac at 37.5- and 75-mg doses, inactive vehicle (placebo), and an active control (moxifloxacin).

Results: In vitro, diclofenac produced no statistically significant effect on IKr. Significant, non–dose-dependent effects were observed in the presence of HPCD or HPCD-diclofenac of similar magnitude across the 300-fold dose range of concentrations tested, suggesting an artifact due to the detergent effect of HPCD in this in vitro model. In vivo, neither HPCD-diclofenac dose resulted in QTc prolongation 2ms (5 ms is the threshold of clinical concern). No correlation was evident between changes in QTc and plasma concentrations of diclofenac or HPCD. Confirming study sensitivity, moxifloxacin produced a mean QTc prolongation 10 ms.

Conclusions: The findings from the present study suggest that HPCD-diclofenac does not have a dose-dependent effect in the in vitro hERG assay system and does not produce proarrhythmic QTc prolongation in vivo. identifier: NCT01812538. (Clin Ther. 2013;35:646–658) © 2013 Elsevier HS Journals, Inc. All rights reserved. Key words: cyclodextrin, diclofenac, pain, QT interval.

Helson L, Shopp G, Bouchard A, Majeed M. Liposome mitigation of curcumin inhibition of cardiac potassium delayed-rectifier current. Journ of Receptor, Ligand & Channel Research, November 2012 Volume 2012:5 Pages 1 – 8. Download article PDF from Dovepress

Background: The duration of the QT interval on the standard electrocardiogram (ECG) is measured from the beginning of the QRS complex (depolarization of the cardiac myocyte) to the end of the T-wave (completion of the repolarization phase of the cardiac myocyte). Repolarization is a result of currents generated by the outward flow of K+ through the K+ channels. Obstruction of ion flow in the channel leads to delayed repolarization, evidenced by a prolonged QT interval. Clinically, this is known as the long QT syndrome (LQTS), which, when expressed, can lead to severe cardiac arrhythmias and sudden death. Obstruction of K+ ion flow can result from gene mutations (eg, the human ether-a-go-go-related gene [hERG]) resulting in phenotypic abnormalities in K+ channels and/or common structurally diverse drugs. These gene abnormalities or drug-induced changes result in decreased cardiac delayed-rectifier K+ current (IKr, or KV11.1) in congenital or acquired LQTS, respectively. Increased risk of LQTS is a major drug development hurdle, and many drugs have been withdrawn during preclinical development, assigned black box warnings following approval, or withdrawn from the market. Autosomal recessive or dominant LQTS based upon 500 possible mutations in ten different genes coding for K+ channels has an incidence of 1:3000 or about 100,000 persons in the USA. Prolonged QT intervals or risk of LQTS occurs in 2.5% of the asymptomatic US population. The probability of cardiac death in patients with asymptomatic congenital LQTS who are concomitantly medicated with LQTS-inducing drugs appears to have increased.

Methods: E-4031 (methanesulfanalide), terfenadine (Seldane®), curcumin, liposomal curcumin, empty liposomes, empty liposomes vortexed with E-4031, or terfenadine and empty liposomes vortexed with curcumin were assayed for their effects on the K+-selective IKr tail current inhibition using human embryonic kidney (HEK 293) cells stably transfected with the hERG gene via the whole-cell manual patch clamp technique.

Results: E-4031, terfenadine, and curcumin inhibit IKr channel following nM-to-µM exposures. Empty liposomes had no effect on IKr. Both the liposomal curcumin formulation and vortexed mixtures of empty liposomes and curcumin prevented the IKr inhibitory effect of curcumin in a dose-dependent manner. Empty liposomes vortexed with E-4031 prevented the effect of E-4031 to a lesser extent, while empty liposomes vortexed with terfenadine did not alter its IKr inhibitory activity.

Conclusion: Curcumin causes an inhibition of the hERG tail current density. The liposomal curcumin formulation, as well as a mixture of empty liposomes with curcumin or with E-4031, blocked drug-induced IKr inhibition. However, empty liposomes mixed with terfenadine did not alter terfenadine’s IKr inhibitory effects. The liposomes protected against the inhibitory effect of some compounds on the K+-selective IKr current, independent of their potency.

Dan Salvail‘s undergrad studies in Biochemistry were followed by graduate studies in Thoracic Physiology. Dan’s post-graduate work was in Cardiovascular Pharmacology and Cardiology.

Venkatesh M, Wang H, Cayer J, Leroux M, Salvail D, Das B, Wrobel J.E., Mani S. In Vivo and In Vitro Characterization of a First-in-Class Novel Azole Analog That Targets Pregnane X Receptor Activation. Mol Pharmacol 80:124–135, 2011. Read Free PMC Article

The pregnane X receptor (PXR) is a master regulator of xenobiotic clearance and is implicated in deleterious drug interactions (e.g., acetaminophen hepatotoxicity) and cancer drug resistance. However, small-molecule targeting of this receptor has been difficult; to date, directed synthesis of a relatively specific PXR inhibitor has remained elusive. Here we report the development and characterization of a first-in-class novel azole analog [1-(4-(4-(((2R,4S)-2-(2,4-difluorophenyl)-2-methyl-1,3-dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)ethanone (FLB-12)] that antagonizes the activated state of PXR with limited effects on other related nuclear receptors (i.e., liver X receptor, farnesoid X receptor, estrogen receptor , peroxisome proliferator-activated receptor , and mouse constitutive androstane receptor). We investigated the toxicity and PXR antagonist effect of FLB-12 in vivo. Compared with ketoconazole, a prototypical PXR antagonist, FLB-12 is significantly less toxic to hepatocytes. FLB-12 significantly inhibits the PXR-activated loss of righting reflex to 2,2,2-tribromoethanol (Avertin) in vivo, abrogates PXRmediated resistance to 7-ethyl-10-hydroxycamptothecin (SN-38) in colon cancer cells in vitro, and attenuates PXR-mediated acetaminophen hepatotoxicity in vivo. Thus, relatively selective targeting of PXR by antagonists is feasible and warrants further investigation. This class of agents is suitable for development as chemical probes of PXR function as well as potential PXRdirected therapeutics.

Salvail D, Cloutier M, Rousseau E. Functional reconstitution of an eicosanoid-modulated Cl- channel from bovine tracheal smooth muscle. Am J Physiol Cell Physiol 282: C567–C577, 2002. Read free full text

We describe the biochemical properties of an eicosanoid-modulated Cl- channel and assess the mechanisms by which the epoxyeicosatrienoic acids (EETs) alter both its unitary conductance and its open probability (Po). After a purification protocol involving wheat-germ agglutinin affinity and anion-exchange chromatography, the proteins were sequentially inserted into liposomes, which were then fused into PLBs. Functional and biochemical characterization tests confirm that the Cl- channel is a 55-kDa glycosylated monomer with voltage- and Ca2+ concentration-independent activity. 5,6- and 8,9-EET decreased the conductance of the native channel (control conductance: 70 +/- 5 pS in asymmetrical 50 mM trans/250 mM cis CsCl) in a concentration-dependent manner, with respective 50% inhibitory concentration values of 0.31 and 0.42 M. These regioisomers similarly decreased the conductance of the purified channel (control conductance value: 75 +/- 5 pS in asymmetrical 50 mM trans/250 mM cis CsCl), which had been stripped of its native proteic and lipidic environment. On the other hand, 5,6- and 8,9-EETs decreased the Po of the native channel with respective 50% inhibitory concentration values of 0.27 and 0.30 M but failed to alter the Po of the purified protein. Thus we suggest that the effects of these EETs on channel conductance likely result from direct interactions of EET anions with the channel pore, whereas the alteration of Po requires a lipid environment of specific composition that is lost on solubilization and purification of the protein.

Benoit C, Renaudon B, Salvail D, Rousseau E. EETs relax airway smooth muscle via an EpDHF effect: BKca channel activation and hyperpolarization. Am J Physiol Lung Cell Mol Physiol. 280: L965–L973, 2001. Read free full text

Epoxyeicosatrienoic acids (EETs) are produced from arachidonic acid via the cytochrome P-450 epoxygenase pathway. EETs are able to modulate smooth muscle tone by increasing K1 conductance, hence generating hyperpolarization of the tissues. However, the molecular mechanisms by which EETs induce smooth muscle relaxation are not fully understood. In the present study, the effects of EETs on airway smooth muscle (ASM) were investigated using three electrophysiological techniques. 8,9-EET and 14,15-EET induced concentration-dependent relaxations of the ASM precontracted with a muscarinc agonist (carbamylcholine chloride), and these relaxations were partly inhibited by 10 nM iberiotoxin (IbTX), a specific large-conductance Ca2+-activated K1 (BKCa) channel blocker. Moreover, 3 mM 8,9- or 14,15-EET induced hyperpolarizations of -12 +/- 3.5 and -16 +/- 3 mV, with EC50 values of 0.13 and 0.14 mM, respectively, which were either reversed or blocked on addition of 10 nM IbTX. These results indicate that BKca channels are involved in hyperpolarization and participate in the relaxation of ASM. In addition, complementary experiments demonstrated that 8,9- and 14,15-EET activate reconstituted BKCa channels at low free Ca2+ concentrations without affecting their unitary conductance. These increases in channel activity were IbTX sensitive and correlated well with the IbTX-sensitive hyperpolarization and relaxation of ASM. Together these results support the view that, in ASM, the EETs act through an epithelium-derived hyperpolarizing factorlike effect.

Abderrahmane A, Salvail D, Dumoulin M, Garon J, Cadieux A, Rousseau E. Direct Activation of KCa Channel in Airway Smooth Muscle by Nitric Oxide: Involvement of a Nitrothiosylation Mechanism? Am. J. Respir. Cell Mol. Biol. Vol. 19, pp. 485–497, 1998 Read free full text

Clinically, nitric oxide (NO˙) is widely used as a pulmonary vaso- and bronchodilator agent. However, the precise molecular mechanisms by which NO˙ induces smooth muscle relaxation are not well established. It has been suggested that NO˙ relaxes airway smooth muscle (ASM) via a 3′,5′-cyclic guanosine monophosphate (cGMP)-dependent pathway, and our previous work has shown that Ca2+-activated K+ (KCa) channels are susceptible to cGMP-dependent protein kinase (PKG)-dependent phosphorylation (A. Alioua, J. P. Huggins, and E. Rousseau. Am. J. Physiol. 1995;268:L1057–L1063). To assess whether KCa channels are also directly activated by NO˙ or one of its derivatives such as peroxynitrite, the activity of these channels was measured upon fusion of sarcolemmal vesicles derived from bovine tracheal smooth muscle cells into planar lipid bilayers (PLB). It was found that in the absence of adenosine triphosphate (ATP), cGMP, and cGMP-dependent protein kinase, NO˙ donors such as 1-propanamine-3-(2-hydroxy-2-nitroso-1-propylhydrazine) (PAPA NONOate) or 3-morpholinosydnonimine hydrochloride (SIN-1) in the presence of superoxide dismutase (SOD), added on either side of the bilayer, caused a concentrationdependent increase in the open probability (Po) of KCa channels without altering their unitary conductance. Release of NO˙, which was measured by chemiluminescence analysis in parallel experiments, affected the gating behavior of KCa channels in the presence of SOD and ethyleneglycol-bis-(b-aminoethyl ether)-N,N’-tetraacetic acid (EGTA) by reducing the mean closed times and increasing the number and duration of short open events. PAPA NONOate, a true NO˙ donor, had similar effects in the presence of ethylenediaminetetraacetic acid (EDTA), a heavy-metal chelator, and K-urate, a peroxynitrite scavenger. Addition of either 5 mM dithiothreitol (DTT) or 5 mM reduced glutathione (GSH), as well as 5 mM N-ethylmaleimide (NEM)—an alkylating agent—to the trans (intracellular) side of an experimental chamber slightly increased channel Po but prevented further channel activation by NO˙ donors. However, neither DTT nor GSH was able to reverse the effect of NO˙. In contrast to SIN-1, DTT had no effect when added to the cis (extracellular) side of the chamber. This suggests that the effect of NO˙ is most likely due to a chemical modification (nitrothiosylation) of intracellular sulfhydryl group(s). Neither PAPA NONOate (NO˙), nor SIN-1 had any effect on sarcolemmal Cl- channels reconstituted from the same membrane preparations. Pharmacomechanical measurements made on epithelium-denuded rat bronchus showed that 100 nM charybdotoxin decreased the sensitivity of bronchial smooth muscle to SIN-1-induced relaxations. Altogether, our data suggest that NO-induced bronchorelaxation occurs partly via a direct activation of KCa channels, possibly through a covalent interaction with the cytoplasmic side of their a subunit.

Salvail D, Dumoulin M, Rousseau E, Direct modulation of tracheal Cl- channel activity by 5,6- and 11,12-EET. Am J Physiol Lung Cell Mol Physiol 275:L432-L441, 1998 Read free full text

Using microelectrode potential measurements, we tested the involvement of Cl- conductances in the hyperpolarization induced by 5,6- and 11,12-epoxyeicosatrienoic acid (EET) in airway smooth muscle (ASM) cells. 5,6-EET and 11,12-EET (0.75 μM) caused -5.4 +/- 1.1- and -3.34 +/- 0.95-mV hyperpolarizations, respectively, of rabbit tracheal cells (from a resting membrane potential of -53.25 +/- 0.44 mV), with significant residual repolarizations remaining after the Ca2+ -activated K+ channels had been blocked by 10 nM iberiotoxin. In bilayer reconstitution experiments, we demonstrated that the EETs directly inhibit a Ca2+-insensitive Cl- channel from bovine ASM; 1 μM 5,6-EET and 1.5 μM 11,12-EET lowered the unitary current amplitude by 40 (n=6 experiments) and 44.7% (n=4 experiments), respectively. Concentration-dependent decreases in channel open probability were observed, with estimated IC50 values of 0.26 μM for 5,6- and 1.15 μM for 11,12-EET. Furthermore, pharmacomechanical tension measurements showed that both regioisomers induced significant bronchorelaxations in epithelium-denuded ASM strips. These results suggest that 5,6- and 11,12-EET can act in ASM as epithelium-derived hyperpolarizing factors.

Charles E. Laurent is one of the senior scientists at iPST. Charles earned his B.Sc. in Biochemistry at the Université du Québec à Montréal, his M.Sc. in Pharmacology and his Ph.D. in Pharmacology with Cardiovascular Applications at the Université de Montréal. He completed four years of post-doc studies in Molecular Biology at the University of Pittsburgh Medical Center.

Laurent C.E., Cardinal R., Rousseau G., Vermeulen M., Wilkinson M., Armour J.A., and Bouvier M. Functional Desensitization to Isoproterenol Without Reducing cAMP Production in Canine Failing Cardiocytes. Am. J.Physiol. 2001 Feb;280(2):R355-64. Read free full text

To corroborate alterations in the functional responses to beta-adrenergic receptor (beta-AR) stimulation with changes in beta-AR signaling in failing cardiomyocytes, contractile and L-type Ca(2+) current responses to isoproterenol along with stimulated cAMP generation were compared among cardiomyocytes isolated from canines with tachycardia-induced heart failure or healthy hearts. The magnitude of shortening of failing cardiomyocytes was significantly depressed (by 22 +/- 4.4%) under basal conditions, and the maximal response to isoproterenol was significantly reduced (by 45 +/- 18%). Similar results were obtained when the responses in the rate of contraction and rate of relaxation to isoproterenol were considered. The L-type Ca(2+) current amplitude measured in failing cardiomyocytes under basal conditions was unchanged, but the responses to isoproterenol were significantly reduced compared with healthy cells. Isoproterenol-stimulated cAMP generation was similar in sarcolemmal membranes derived from the homogenates of failing (45 +/- 6.8) and healthy cardiomyocytes (52 +/- 8.5 pmol cAMP. mg protein(-1). min(-1)). However, stimulated cAMP generation was found to be significantly reduced when the membranes were derived from the homogenates of whole tissue (failing: 67 +/- 8.1 vs. healthy: 140 +/- 27.8 pmol cAMP. mg protein(-1). min(-1)). Total beta-AR density was not reduced in membranes derived from either whole tissue or isolated cardiomyocyte homogenates, but the beta(1)/beta(2) ratio was significantly reduced in the former (failing: 45/55 vs. healthy: 72/28) without being altered in the latter (failing: 72/28, healthy: 77/23). We thus conclude that, in tachycardia-induced heart failure, reduction in the functional responses of isolated cardiomyocytes to beta-AR stimulation may be attributed to alterations in the excitation-contraction machinery rather than to limitation of cAMP generation.

Cardinal R., Nadeau R., Laurent C., Boudreau G., and Armour J.A., Reduced Capacity of Cardiac Efferent Sympathetic Neurons to Release Noradrenaline and Modify Cardiac Function in Tachycardia-Induced Canine Heart Failure. Can.J.Physiol.Pharmacol. 1996 Sep;74(9):1070-8.

To investigate the capacity of efferent sympathetic neurons to modulate the failing heart, stellate ganglion stimulation was performed in dogs with biventricular heart failure induced by rapid ventricular pacing (240 beats/min) for 4-6 weeks. Less noradrenaline was released from cardiac myoneural junctions into coronary sinus blood in response to left stellate ganglion stimulation in anesthetized failing heart preparations (582 pg/mL, lower and upper 95% confidence intervals of 288 and 1174 pg/mL, n = 19) compared with healthy heart preparations (6391 pg/mL, 95% confidence intervals of 4180 and 9770 pg/mL, n = 14; p < 0.001). There was substantial adrenaline extraction by failing hearts (49 +/- 6%), although it was slightly lower than in healthy heart preparations (65 +/- 9%, p = 0.055). In contrast with healthy heart preparations, no net release of adrenaline occurred during stellate ganglion stimulation in any of the failing heart preparations, and ventricular tissue levels of adrenaline fell below the sensitivity limit of the HPLC technique. In failing heart preparations, maximal electrical stimulation of right or left stellate ganglia resulted in minimal augmentation of left ventricular intramyocardial (17%) and chamber (12%) systolic pressures. These indices were augmented by 145 and 97%, respectively, following exogenous noradrenaline administration. Thus, the cardiac efferent sympathetic neurons’ reduced capacity to release noradrenaline and modify cardiac function can contribute to reduction of sympathetic support to the failing heart.

Nakhostine N., Laurent C., Nadeau R., Cardinal R., and Lamontagne D.. Hypoxia-Induced Release of Prostacyclin : A role for L-Type Calcium Channels. Can.J.Physiol.Pharmacol. 1995 Dec;73(12):1742-9.

The mechanism of hypoxia-induced prostacyclin (PGI2) release was studied in isolated, modified Krebs-Henseleit buffer perfused rabbit hearts under constant-flow conditions. The contribution of vascular endothelium and the role of catecholamines and calcium influx through L-type calcium (Ca2+) channels in hypoxic release of PGI2 were investigated. Reduction in the perfusion solution PO2 (from 476 +/- 13 to 127 +/- 24 mmHg; 1 mmHg = 133.3 Pa) in the presence of glucose (5 mM) and pyruvate (2 mM) caused a significant release of 6-keto-PGF1 alpha, the stable metabolite of PGI2 (from 3.0 +/- 0.4 to 7.3 +/- 2.0 pmol.min-1.g-1, p < 0.05, n = 12), whereas no variation in washout of catecholamines in the perfusate was observed. Electrolysis of the perfusion buffer solution was used to destroy the endothelium. Endothelium impairment by electrolysis almost completely abolished the vasodilation induced by serotonin (5HT) and acetylcholine (ACh), without affecting that caused by papaverine. The basal release of 6-keto-PGF1 alpha was significantly enhanced after electrolysis. However, its release during hypoxia was completely abolished. In a separate group of normal hearts, verapamil (10(-7) M) completely blocked the release of 6-keto-PGF1 alpha during hypoxia. Similar results were obtained in 15 mM KCl arrested hearts challenged with hypoxia. Under normoxic conditions, isoproterenol (10(-7) M) induced a significant release of 6-keto-PGF1 alpha (from 2.9 +/- 0.5 to 5.3 +/- 0.8 pmol.min-1.g-1, p < 0.05, n = 9). To stimulate endogenous catecholamine release, hearts perfused with glucose-free buffer (pyruvate 14.8 mM) were submitted to hypoxia. Under these conditions, hypoxia was accompanied by an enhanced release of noradrenaline (from 1.3 +/- 0.5 to 19.7 +/- 7.8 pmol.min-1.g-1). Despite the increased noradrenaline washout in effluent, the hypoxia-induced release of 6-keto-PGF1 alpha in glucose-free perfused hearts was not enhanced. Our results suggest that endothelial cells are the major site of PGI2 synthesis during hypoxia and that myocardial contractility is a prime factor involved in this process. Verapamil reduction of PGI2 release is most probably through its negative inotropic effect. Although exogenous beta-adrenergic stimulation can induce PGI2 release, endogenous catecholamines do not appear to contribute to the hypoxia-induced release of PGI2.

Boudeau ML, Laplante I, Laurent CE, Lacaille JC. KChIP1 modulation of Kv4.3-mediated A-type K(+) currents and repetitive firing in hippocampal interneurons. Neuroscience. 2011 Mar 10;176:173-87

Neuronal A-type K(+) channels regulate action potential waveform, back-propagation and firing frequency. In hippocampal CA1 interneurons located at the stratum lacunosum-moleculare/radiatum junction (LM/RAD), Kv4.3 mediates A-type K(+) currents and a Kv4 β-subunit of the Kv channel interacting protein (KChIP) family, KChIP1, appears specifically expressed in these cells. However, the functional role of this accessory subunit in A-type K(+) currents and interneuron excitability remains largely unknown. Thus, first we studied KChIP1 and Kv4.3 channel interactions in human embryonic kidney 293 (HEK293) cells and determined that KChIP1 coexpression modulated the biophysical properties of Kv4.3 A-type currents (faster recovery from inactivation, leftward shift of activation curve, faster rise time and slower decay) and this modulation was selectively prevented by KChIP1 short interfering RNA (siRNA) knockdown. Next, we evaluated the effects of KChIP1 down-regulation by siRNA on A-type K(+) currents in LM/RAD interneurons in slice cultures. Recovery from inactivation of A-type K(+) currents was slower after KChIP1 down-regulation but other properties were unchanged. In addition, down-regulation of KChIP1 levels did not affect action potential waveform and firing, but increased firing frequency during suprathreshold depolarizations, indicating that KChIP1 regulates interneuron excitability. The effects of KChIP1 down-regulation were cell-specific since CA1 pyramidal cells that do not express KChIP1 were unaffected. Overall, our findings suggest that KChIP1 interacts with Kv4.3 in LM/RAD interneurons, enabling faster recovery from inactivation of A-type currents and thus promoting stronger inhibitory control of firing during sustained activity.

Lebeau G, Maher-Laporte M, Topolnik L, Laurent C.E, Sossin W, Desgroseillers L, Lacaille JC. Staufen1 regulation of protein synthesis-dependent long-term potentiation and synaptic function in hippocampal pyramidal cells. Mol Cell Biol. 2008 May;28(9):2896-907. doi: 10.1128/MCB.01844-07. Epub 2008 Mar 3. Read free PMC article

Staufen1 (Stau1) is an RNA-binding protein involved in transport, localization, decay, and translational control of mRNA. In neurons, it is present in cell bodies and also in RNA granules which are transported along dendrites. Dendritic mRNA localization might be involved in long-term synaptic plasticity and memory. To determine the role of Stau1 in synaptic function, we examined the effects of Stau1 down-regulation in hippocampal slice cultures using small interfering RNA (siRNA). Biolistic transfection of Stau1 siRNA resulted in selective down-regulation of Stau1 in slice cultures. Consistent with a role of Stau1 in transporting mRNAs required for synaptic plasticity, Stau1 down-regulation impaired the late form of chemically induced long-term potentiation (L-LTP) without affecting early-LTP, mGluR1/5-mediated long-term depression, or basal evoked synaptic transmission. Stau1 down-regulation decreased the amplitude and frequency of miniature excitatory postsynaptic currents, suggesting a role in maintaining efficacy at hippocampal synapses. At the cellular level, Stau1 down-regulation shifted spine shape from regular to elongated spines, without changes in spine density. The change in spine shape could be rescued by an RNA interference-resistant Stau1 isoform. Therefore, Stau1 is important for processing and/or transporting in dendrites mRNAs that are critical in regulation of synaptic strength and maintenance of functional connectivity changes underlying hippocampus-dependent learning and memory.

Bourdeau M.L., Morin F., Laurent C.E., Azzi M., et Lacaille J-C. Kv4.3-mediated A-type K+ currents underlie rhythmic activity in hippocampal interneurons. J. Neurosc 2007 Feb 21;27(8):1942-53. Read free full text

Hippocampal-dependent learning and memory processes are associated with theta frequency rhythmic activity. Interneuron and pyramidal cell network interactions underlie this activity, but contributions of interneuron voltage-gated membrane conductances remain unclear. We show that interneurons at the CA1 lacunosum-moleculare (LM) and radiatum (RAD) junction (LM/RAD) display voltage-dependent subthreshold membrane potential oscillations (MPOs) generated by voltage-gated tetrodotoxin-sensitive Na+ and 4-aminopyridine (4-AP)-sensitive K+ currents. They also exhibit prominent 4-AP-sensitive A-type K+ currents, with gating properties showing activation at subthreshold membrane potentials. We found that LM/RAD cells are part of specific interneuron subpopulations expressing the K+ channel subunit Kv4.3 and their transfection with Kv4.3 small interfering RNA selectively impaired A-type K+ currents and MPOs. Thus, our findings reveal a novel function of Kv4.3-mediated A-type K+ currents in the generation of intrinsic MPOs in specific subpopulations of interneurons that may participate in hippocampal theta-related rhythmic activity.

Laurent C.E., Delfino F.J., Chang H.Y., and Smithgall T.E. The Human c-Fes Tyrosine Kinase Binds Tubulin and Microtubules through Separate Domains and Promotes Microtubule Assembly. Mol Cell. Biol. 2004 Nov;24(21):9351-8. Read free PMC article

The c-Fes protein-tyrosine kinase (Fes) has been implicated in the differentiation of vascular endothelial, myeloid hematopoietic, and neuronal cells, promoting substantial morphological changes in these cell types. The mechanism by which Fes promotes morphological aspects of cellular differentiation is unknown. Using COS-7 cells as a model system, we observed that Fes strongly colocalizes with microtubules in vivo when activated via coiled-coil mutation or by coexpression with an active Src family kinase. In contrast, wild-type Fes showed a diffuse cytoplasmic localization in this system, which correlated with undetectable kinase activity. Coimmunoprecipitation and immunofluorescence microscopy showed that the N-terminal Fes/CIP4 homology (FCH) domain is involved in Fes interaction with soluble unpolymerized tubulin. However, the FCH domain was not required for colocalization with polymerized microtubules in vivo. In contrast, a functional SH2 domain was essential for microtubule localization of Fes, consistent with the strong tyrosine phosphorylation of purified tubulin by Fes in vitro. Using a microtubule nucleation assay, we observed that purified c-Fes also catalyzed extensive tubulin polymerization in vitro. Taken together, these results identify c-Fes as a regulator of the tubulin cytoskeleton that may contribute to Fes-induced morphological changes in myeloid hematopoietic and neuronal cells.

Laurent C.E., and Smithgall T.E. The c-Fes Tyrosine Kinase Cooperates with the Breakpoint Cluster Region Protein (Bcr) to Induce Neurite Extension in a Rac- and Cdc42- Dependent Manner. Exp. Cell Res. 2004 Sep 10;299(1):188-98.

The c-fes locus encodes a cytoplasmic protein-tyrosine kinase (Fes) previously shown to accelerate nerve growth factor (NGF)-induced neurite outgrowth in rat PC12 cells. Here, we investigated the role of the Rho family small GTPases Rac1 and Cdc42 in Fes-mediated neuritogenesis, which have been implicated in neuronal differentiation in other systems. Fes-induced acceleration of neurite outgrowth in response to NGF treatment was completely blocked by the expression of dominant-negative Rac1 or Cdc42. Expression of a kinase-active mutant of Fes induced constitutive relocalization of endogenous Rac1 to the cell periphery in the absence of NGF, and led to dramatic actin reorganization and spontaneous neurite extension. We also investigated the breakpoint cluster region protein (Bcr), which possesses the Dbl and PH domains characteristic of guanine nucleotide exchange factors for Rho family GTPases, as a possible link between Fes, Rac/Cdc42 activation, and neuritogenesis. Coexpression of a GFP-Bcr fusion protein containing the Fes binding and tyrosine phosphorylation sites (amino acids 162-413) completely suppressed neurite outgrowth triggered by Fes. Conversely, coexpression of full-length Bcr with wild-type Fes in PC12 cells induced NGF-independent neurite formation. Taken together, these data suggest that Fes and Bcr cooperate to activate Rho family GTPases as part of a novel pathway regulating neurite extension in PC12 cells, and provide more evidence for an emerging role for Fes in neuronal differentiation.

Shibata A., Laurent C.E., and Smithgall T.E. The c-Fes Protein-Tyrosine Kinase Accelerates NGF-Induced Differentiation of PC12 Cells Through a PI3K-Dependent Mechanism. Cell. Signal. 2003 Mar;15(3):279-88.

The c-fes protooncogene encodes a non-receptor protein-tyrosine kinase (Fes) that has been implicated in the differentiation of myeloid haematopoietic cells. Fes is also expressed in several neuronal cell types and the vascular endothelium, suggestive of a more general function in development. To examine the role of Fes in neuronal differentiation, we investigated the effect of Fes expression on process outgrowth in PC12 cells following stimulation with nerve growth factor (NGF). PC12 cells expressing wild-type and activated mutants of Fes extended processes faster and of greater length than control cells. In contrast, expression of kinase-inactive Fes was without effect, indicating that cooperation with NGF requires Fes kinase activity. Short-term treatment of PC12-Fes cells with NGF enhanced tyrosine phosphorylation of Fes, suggesting upstream regulation by the NGF receptor. Fes-mediated acceleration of neurite outgrowth was blocked by wortmannin and LY294002, implicating phosphatidylinositol 3-kinase (PI3K) activation in the Fes-induced response. In contrast, the MEK inhibitor PD98059 was without effect, suggesting that the Ras-Erk pathway is not involved. These data provide the first evidence that Fes may contribute to morphological differentiation of neuronal cells by enhancing NGF signalling through the PI3K pathway.