Newsletter July 2016

In vitro CVS and CNS ion channel platform

Bulle 1 early identificationMany promising drugs have either been withdrawn from the market or classified into ‘restricted use’ categories due to unforeseen adverse effects on either the heart or central nervous system. In light of this, Citoxlab is now happy to offer a variety of cardiovascular (CVS) and central nervous system (CNS) in vitro electrophysiology services aimed at identifying these potential CVS and CNS liabilities early in the drug development process.

One of the most important CVS liabilities in safety pharmacology is the life-threatening condition known as Torsades de pointes (TdP). This condition is principally mediated by alterations in the activity of the hERG (Kv11.1) channel. However, other important cardiac ion channels (Cav1.2, Nav1.5, Kv4.3, Kir2.1 and Kv7.1) involved in the cardiac action potential have also been implicated. Accordingly, Citoxlab North America has developed a GLP-compliant, in vitro electrophysiological service which offers clients a robust ion channel platform from which to characterize the mechanism of action and safety of a lead compound. This in vitro cardiac profile screen provides a rigorous safety analysis including comprehensive inhibition tests and IC50 metrics.

Adverse CNS effects have also played an important role in the failure of some drug candidates within preclinical safety assessments. To help address these potential liabilities, Citoxlab offers a range of in vitro electrophysiological assays to evaluate seizure risk, drug induced cognitive dysfunction risk, dorsal root ganglia responses or nerve conduction effects. These assays have been developed, and are offered, in several preclinical animal models such as the mouse, rat, minipig, dog and nonhuman primate to allow species comparison or follow-up investigations. Our team of pharmacologists dedicated to drug safety testing is available to help progress your program to the next stage.

Citoxlab uses a QT interval correction formula for drug-induced changes in body temperature when incorporating cardiovascular safety pharmacology in regulatory toxicology studies

Bulle 4 QT intervals 2We recently encountered a case study where a new drug candidate produced significant and dose-dependent increases in QT interval duration in a four-week GLP regulatory toxicology study involving cardiovascular safety assessment by jacketed external telemetry in dogs. These effects on QT interval were observed without any effect in the hERG channel test. However, dose-dependent decreases in body temperature, were observed, along with increases in QT interval duration, corrected for heart rate according to the Van de Water formula (QTcV), but the effect was no longer observed after correction for changes in BT [QTcVcT = QTcV – 14(37.5 – BT)] according to the Van der Linde formula.

We are happy to share with you these findings and to inform you that these data have been included in a full paper, recently accepted for publication in the ‘Journal of Pharmacological and Toxicological Methods’, ‘QT interval correction for drug-induced changes in body temperature during integrated cardiovascular safety assessment in regulatory toxicology studies in dogs: a case study by El Amrani et al. 2016.

In pre-clinical drug development, and especially during regulatory toxicology studies, it seems prudent to recommend that the evaluation of any potential effects of drug candidates on body temperature should be performed on a regular basis. Subsequently ignoring correction for body temperature may lead to a misinterpretation of changes in corrected QT for heart rate in pre-clinical or clinical investigations.

Citoxlab has successfully developed approaches and methodologies of measuring body temperature, blood samplings and additional investigations during telemetry recordings without affecting the quality of the collected data.

Validation of in vitro sensitization package for cosmetics and chemicals

SiBulle 2 in vitro sensitizationnce the ban of animal use for cosmetics testing in 2009 following enforcement of the 7th amendment of the European Cosmetic Directive (Directive 2003/15/EC), the cosmetics industry must perform only in vitro assays for the safety testing of their ingredients and mixtures. A number of in vitro tests have been developed during the last decade in the field of skin sensitization. To date, there are two assays that have been fully validated by the European Center for Validation of Alternative Methods (ECVAM) and published into an OECD guideline and which can therefore be used for regulatory purposes: the Direct Peptide Reactivity Assay (DPRA) (OECD442C, 2015) and KeratinoSens assay (OECD442D, 2015). A third assay, the human cell line activation test (h-CLAT), has been validated by the ECVAM Scientific Advisory Committee and an OECD guideline is currently in draft stage with expected final adopted version by the end of the year. Each of these three assays covers one of the successive key events in the skin sensitization mechanism, as published by the OECD in the Adverse Outcome Pathway (AOP) for Skin Sensitization (OECD No. 168, 2012). The DPRA addresses the first key event, corresponding to the molecular initiating event, i.e. hapten binding to endogenous proteins, and is assessed by quantifying the reactivity of test chemicals towards model synthetic peptides containing either lysine or cysteine amino-acids. The KeratinoSens assay, which is an ARE-Nrf2 luciferase test method, addresses the second key event which takes place in the keratinocytes and includes inflammatory responses as well as gene expression associated with specific cell signaling pathways such as the antioxidant/electrophile response element (ARE)-dependent pathways. The third key event is addressed by the h-CLAT assay and is the activation of dendritic cells leading to expression of specific cell surface markers, chemokines and cytokines.

None of these three in vitro methods can be used as a stand-alone for classification purposes. However, combination of a global in vitro classification based on the concordance of results of at least 2 out of 3 of these in vitro assays together with other information in the context of integrated approaches such as Weight of Evidence (WoE) or testing and assessment strategies, can be used to characterize the sensitization potential of new substances.

Publication of an updated ECHA’s guidance R.7a is also expected later this year, and should include these in vitro assays as part of the testing strategy for chemicals.

With these three tests now implemented, Citoxlab provides to its customers with the full in vitro test battery for skin sensitization assessment the necessary information for safety evaluation of cosmetics ingredients.

Successful renewal of GLP and GMP certification in Denmark

Bulle 5 GLP certification DKGLP and GMP accreditation are essential to the work that we do at Citoxlab in Denmark. We have no fewer than 3 GLP certificates and one for GMP. These accreditations are expected as a minimum standard by our customers worldwide.

We have GLP certification from the Danish Medicines Agency (DMA) for undertaking non-clinical studies both for medicinal products and also for medical devices. In both cases the certification is evidence that we comply with OECD GLP principles. Furthermore, we have a separate GLP certificate (also for compliance with OECD GLP) from DANAK, the Danish National Accreditation Agency in relation to studies on non-pharmaceuticals (chemicals, agrochemicals, food products, etc.).

We have GMP certification for undertaking tests primarily in connection with batch release of finished pharmaceutical products (batch control, potency, etc.). Again, this certification is under the control of the DMA.

In September of 2015, we had a successful inspection from the DMA for our GLP certificates and for our GMP certificate as well.

Andrew Makin, Managing Director of Citoxlab Denmark commented, “As a world-leading CRO, it is important for us to meet the quality expectations of our world-wide customer base. This successful inspection underlines our commitment to quality”.

Citoxlab Denmark provides a wide range of studies, not only to our local customers in Scandinavia but around the world in participation for projetcts sequesting the minipigs where our expertise and historial data are internationally recognized.

New Veterinary Anatomic Pathologist hired at Citoxlab North-America

Bulle 6 New clin pathPritpal Malhi, DVM, MVSc, PhD, DACVP is a Doctor of Veterinary Medicine and Diplomate of the American College of Veterinary Pathologists (DACVP). Before joining Citoxlab, Pritpal worked as apathologist at the Prairie Diagnostic Services, Canada for 3 years and was Adjunct Professor in the Department of Veterinary Pathology, University of Saskatchewan, Canada. Pritpal has 14 peer-reviewed publications and has served on two graduate student committees. He joined the Citoxlab team of experienced board-certified pathologists in January 2016.

Introduction of Szabolcs Gáty, Senior Director of Operations in Hungary

Bulle 7 Szlaboczs GatySzabolcs Gáty started working at Citoxlab Hungary as a Trainee Study Director in mammalian toxicology in 1995 when the company was known as TRC.

Following a transfer to Ecotoxicology, he introduced new capabilities including non-target arthropod testing, honey bee, earthworm and bird studies. From 2006, Szabolcs moved into management initially as a technical manager and later as principal scientist, laboratory-based toxicology.

In 2010, Szabolcs was promoted to the head of the quality assurance unit and was involved in re-certification for ISO 9001, AAALAC and GLP. Under his guidance, the company also acquired the ISO 14001 environmental management system.

From 2015, Szabolcs moved back to operations when he was promoted to Senior Director of operations. Since then, he has managed two large projects together with many smaller operational changes. These projects were the relocation of the histopathology department and the development of a state of the art inhalation facility with integral animal rooms.

Szabolcs’s favourite responsibility is the management of the company soccer team, which plays in the local league.

He sincerely hopes that he can follow his family tradition, namely working at one company at least for 45 years.

Scientific Literature: Active Presence of the Citoxlab scientific team

Bulle 3 sceintific litteratureOver the last year, the scientific team at Citoxlab contributed to a number of peer-reviewed publications, sharing recent advances and innovation with the broad community. Aligned with areas of expertise, the themes that were covered in these articles included a wide range of topics:

  • Carcinogenicity and genotoxicity (Dagher et al., 2015; Kirkland et al., 2015)
  • Cardiovascular safety pharmacology (Accardi et al., 2016; El Amrani et al., 2016; Pugsley et al. 2015)
  • CNS safety pharmacology (Accardi et al., 2016; Authier et al. 2016B; Authier et al. 2016C)
  • Minipig toxicology (Weaver et al., 2016)
  • Skin sensitization methods (Alépée et al., 2015)
  • Radiation related genomic, proteomic and metabolomics (Byrum et al. 2016; Garg et al. 2016; Pannkuk et al. 2015; Zheng et al. 2015)
  • Vaccine toxicology (Destexhe et al., 2015A; Destexhe et al., 2015B; Forster et al., 2015)

Several drug safety testing disciplines have been propelled by technological advances in recent years and this exciting progress opens new horizons in non-clinical research with data rich studies.

Our team has the strong conviction that the quality of our work as scientists in drug development is closely related to our knowledge of the experimental models that we routinely use. Deeper knowledge of these models is the foundation for well-balanced study data interpretations. With this premise in mind, we constantly invest to characterize these important tools for drug safety and efficacy testing and are committed to share our experience with the broad drug development community. Some of our recent publications announce another trend in research efforts with intensification of post-acquisition data analysis and representation tools. Efficient interpretation relies on well-structured data processing strategies where data mining, graphical tools and statistical analyses converge to form the important message conveyed in our study reports.

Beyond scientific enhancements, technological advances provide an opportunity to refine non-clinical research by capturing a wider array of biologically relevant endpoints, hence contributing to the important goal of the 3Rs. To this effect, our team recently launched an initiative using new CNS monitoring technologies to evaluate the impact of various factors such as housing unit size, social structures and environmental factors on animal welfare with the aim to have an evidence based animal welfare program. Results from this initiative will be presented at conferences at the end of 2016 and early 2017.

Citoxlab remains committed to play an active role within the scientific community with contribution in its fields of expertise but also with animal welfare enhancement initiatives.

References

Accardi MV, Pugsley MK, Forster R, Troncy E, Huang H, Authier S. The emerging role of in vitro electrophysiological methods in CNS safety pharmacology. J Pharmacol Toxicol Methods. 2016 Apr 4. pii: S1056-8719(16)30016-8. doi:10.1016/j.vascn.2016.03.008.

Accardi MV, Troncy E, Abtout S, Ascah A, Maghezzi S, Authier S. Rat cardiovascular telemetry: Marginal distribution applied to positive control drugs. J Pharmacol Toxicol Methods. 2016B. pii: S1056-8719(16)30014-4. doi:10.1016/j.vascn.2016.03.005.

Alépée N, Piroird C, Aujoulat M, Dreyfuss S, Hoffmann S, Hohenstein A, Meloni M, Nardelli L, Gerbeix C, Cotovio J. Prospective multicentre study of the U-SENS test method for skin sensitization testing. Toxicol In Vitro. 2015 Dec 25;30(1 PtB):373-82. doi: 10.1016/j.tiv.2015.09.028.

Authier S, Accardi MV, Paquette D, Pouliot M, Arezzo J, Stubbs RJ, Gerson RJ, Friedhoff LT, Weis H. Functional neurotoxicity evaluation of noribogaine using video-EEG in cynomolgus monkeys. J Pharmacol Toxicol Methods. 2016. pii:S1056-8719(16)30039-9. doi: 10.1016/j.vascn.2016.04.012.

Authier S, Delatte MS, Kallman MJ, Stevens J, Markgraf C. EEG in non-clinical drug safety assessments: Current and emerging considerations. J Pharmacol Toxicol Methods. 2016B. pii: S1056-8719(16)30011-9. doi:10.1016/j.vascn.2016.03.002.

Authier S, Arezzo J, Delatte MS, Kallman MJ, Markgraf C, Paquette D, Pugsley MK, Ratcliff S, Redfern WS, Stevens J, Valentin JP, Vargas HM, Curtis MJ. Safety Pharmacology Investigations on the Nervous System: An Industry Survey. J Pharmacol Toxicol Methods. 2016C, In press

Byrum SD, Burdine MS, Orr L, Moreland L, Mackintosh SG, Authier S, Pouliot M, Hauer-Jensen M, Tackett AJ. A Quantitative Proteomic Analysis of Urine from Gamma-Irradiated Non-Human Primates. J Proteomics Bioinform. 2016;9(Suppl 10). pii: 005.

Dagher R, Watzinger M, Chevalier G, Thirion-Delalande C, Gervais F, Forster R. Carcinogenicity testing of eliglustat in mice and rats. Regul Toxicol Pharmacol. 2015 Oct;73(1):401-12. doi: 10.1016/j.yrtph.2015.07.024.

Destexhe E, Stannard D, Wilby OK, Grosdidier E, Baudson N, Forster R, Gérard CM, Garçon N, Segal L. Nonclinical reproductive and developmental safety evaluation of the MAGE-A3 Cancer Immunotherapeutic, a therapeutic vaccine for cancer treatment. Reprod Toxicol. 2015A Jan;51:90-105. doi:10.1016/j.reprotox.2014.12.009.

Destexhe E, Grosdidier E, Baudson N, Forster R, Gerard C, Garçon N, Segal L. Non-clinical safety evaluation of single and repeated intramuscular administrations of MAGE-A3 Cancer Immunotherapeutic in rabbits and cynomolgus monkeys. J Appl Toxicol. 2015B Jul;35(7):717-28. doi: 10.1002/jat.3025.

El Amrani AI, El Amrani-Callens F, Loriot S, Singh P, Forster R. QT interval correction for drug-induced changes in body temperature during integrated cardiovascular safety assessment in regulatory toxicology studies in dogs: A case study. J Pharmacol Toxicol Methods. 2016 Apr 14. pii: S1056-8719(16)30035-1. doi:10.1016/j.vascn.2016.04.008.

Forster R, Spézia F, Papineau D, Sabadie C, Erdelmeier I, Moutet M, Yadan JC. Reproductive safety evaluation of L-Ergothioneine. Food Chem Toxicol. 2015 Jun;80:85-91. doi: 10.1016/j.fct.2015.02.019.

Garçon N, Silvano J, Kuper CF, Baudson N, Gérard C, Forster R, Segal L. Non-clinical safety evaluation of repeated intramuscular administration of the AS15 immunostimulant combined with various antigens in rabbits and cynomolgus monkeys. J Appl Toxicol. 2016 Feb;36(2):238-56. doi: 10.1002/jat.3167.

Garg S, Zheng J, Wang J, Authier S, Pouliot M, Hauer-Jensen M. Segmental Differences in Radiation-Induced Alterations of Tight Junction-Related Proteins in Non-Human Primate Jejunum, Ileum and Colon. Radiat Res. 2016 Jan;185(1):50-9. doi: 10.1667/RR14157.1.

Kirkland D, Brock T, Haddouk H, Hargeaves V, Lloyd M, Mc Garry S, Proudlock R, Sarlang S, Sewald K, Sire G, Sokolowski A, Ziemann C. New investigations into the genotoxicity of cobalt compounds and their impact on overall assessment of genotoxic risk. Regul Toxicol Pharmacol. 2015 Oct;73(1):311-38. doi:10.1016/j.yrtph.2015.07.016.

Zheng J, Wang J, Pouliot M, Authier S, Zhou D, Loose DS, Hauer-Jensen M. Gene expression profiling in non-human primate jejunum, ileum and colon after total-body irradiation: a comparative study of segment-specific molecular and cellular responses. BMC Genomics. 2015 Nov 21;16:984. doi:10.1186/s12864-015-2168-y.

Wang J, Shao L, Hendrickson HP, Liu L, Chang J, Luo Y, Seng J, Pouliot M, Authier S, Zhou D, Allaben W, Hauer-Jensen M. Total Body Irradiation in the “Hematopoietic” Dose Range Induces Substantial Intestinal Injury in Non-Human Primates. Radiat Res. 2015 Nov;184(5):545-53. doi: 10.1667/RR14191.1.

Weaver ML, Grossi AB, Schützsack J, Parish J, Løgsted J, Bøgh IB, Cameron D, Harvey W, Festag M, Downes N, Venturella S, Schlichtiger J, Mhedhbi S, Ross V, Kissner T, Stark C, Milano S, Heining P, Sanchez-Felix M. Vehicle Systems and Excipients Used in Minipig Drug Development Studies. Toxicol Pathol. 2016 Apr;44(3):367-72. doi: 10.1177/0192623315613088.

Pugsley MK, Authier S, Curtis MJ. Charge is an important determinant of hemodynamic and adverse cardiovascular effects of cationic drugs. Pharmacol Res. 2015 Dec;102:46-52. doi:10.1016/j.phrs.2015.09.008.

Pannkuk EL, Laiakis EC, Authier S, Wong K, Fornace AJ Jr. Global Metabolomic Identification of Long-Term Dose-Dependent Urinary Biomarkers in Nonhuman Primates Exposed to Ionizing Radiation. Radiat Res. 2015 Aug;184(2):121-33.

Career

Bulle 9 career> Citoxlab North-America :

> Citoxlab France :

Conferences

Citoxlab Team will be delighted to meet you at the following scientific meetings:

> Japenese Society of Toxicology 2016:
June 29 – July 01
Nagoya, Japan
Booth
CLICKING HEREBulle 10 Conferences

> RITN/NIH Conference:
July 18 – 19
Rockville Maryland
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> 52th Eurotox Meeting:
September 4-7
Seville, Spain
Booth 15 and 24
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> Chemical Industry Regulation CIR:
September 7-8
Nice, France
Booth
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> European Teratology Meeting (ETS):
September 11-14
Dublin, Ireland
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> Safety Pharmacology Society (SPS):
September 18-21
Vancouver British Columbia
Booth 301
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> European Society of Toxicologic Patholoy (ESTP):
September 20-23
Barcelona, Spain
Booth
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